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Acute cytotoxic T lymphocyte killing capacity

Acute cytotoxic T lymphocyte killing capacity



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how many cells can a CTL eliminate sequentially?

Cytotoxic T lymphocytes (CTLs) are a subset of the adaptive immune system which can target cells for apoptotic elimination. This elimination begins with identification leading to a transfer of the CTLs cytotoxic concoction (perforin and CT granules) into the target cell and subsequently inducing apoptosis. A natural acute limit to this elimination seems obvious as the CTL does not have an infinite supply of apoptosis inducing ammunition. However, after a CTL becomes activated it begins to produce and store these constituents in the cytoplasm - resulting in multiple cytotoxic (CT) granules being stored in the cytoplasm as is visible under a microscope. Even, the activation of these cells cytotoxic capabilities can be indicated by the presence of the cytotoxic granules.

I would like to assume that the CTL has had 'enough time' to build a fully stocked store house of CT granules, and will not add to this store house during its sequential elimination. If this question is incapable of being answered due to this assumption outright then I would think it is ok to ignore it.

Generally speaking, this question pertains to acute in situ T lymphocyte exhaustion. Discussion of general T lymphocyte exhaustion may have value, but also may be unneeded.


Activated γδ T cells exhibit cytotoxicity and the capacity for viral clearance in patients with acute hepatitis B

γδ T cells are a unique population of lymphocytes that have regulatory roles in patients with chronic hepatitis B (CHB) however, their role in acute hepatitis B (AHB) infection remains unclear. Phenotype and function of γδ T cells were analyzed in 29 AHB patients, 28 CHB patients, and 30 healthy controls (HCs) using immunofunctional assays. Compared with HCs and CHB patients, decreased peripheral and increased hepatic γδ T cells were found in AHB patients. Increased hepatic γδ T cells in AHB patients were attributed to elevated hepatic chemokine levels. Peripheral γδ T cells exhibited highly activated and terminally differentiated memory phenotype in AHB patients. Consistently, peripheral γδ T cells in AHB patients showed increased cytotoxic capacity and enhanced antiviral activity which was further proved in longitudinal study. Activated γδ T cells in AHB patients exhibited increased cytotoxicity and capacity for viral clearance associated with liver injury and the control of infection.

Keywords: Antiviral activity Cytokine Cytotoxity Longitudinal study γδ T cells.


Molecular Biology of the Cell. 4th edition.

Lymphocytes are responsible for the astonishing specificity of adaptive immune responses. They occur in large numbers in the blood and lymph (the colorless fluid in the lymphatic vessels that connect the lymph nodes in the body to each other and to the bloodstream) and in lymphoid organs, such as the thymus, lymph nodes, spleen, and appendix (Figure 24-3).

Figure 24-3

Human lymphoid organs. Lymphocytes develop in the thymus and bone marrow (yellow), which are therefore called central (or primary) lymphoid organs. The newly formed lymphocytes migrate from these primary organs to peripheral (or secondary) lymphoid organs (more. )

In this section, we discuss the general properties of lymphocytes that apply to both B cells and T cells. We shall see that each lymphocyte is committed to respond to a specific antigen and that its response during its first encounter with an antigen ensures that a more rapid and effective response occurs on subsequent encounters with the same antigen. We consider how lymphocytes avoid responding to self antigens and how they continuously recirculate between the blood and lymphoid organs, ensuring that a lymphocyte will find its specific foreign antigen no matter where the anitgen enters the body.


Centrioles control the capacity, but not the specificity, of cytotoxic T cell killing

Immunological synapse formation between cytotoxic T lymphocytes (CTLs) and the target cells they aim to destroy is accompanied by reorientation of the CTL centrosome to a position beneath the synaptic membrane. Centrosome polarization is thought to enhance the potency and specificity of killing by driving lytic granule fusion at the synapse and thereby the release of perforin and granzymes toward the target cell. To test this model, we employed a genetic strategy to delete centrioles, the core structural components of the centrosome. Centriole deletion altered microtubule architecture as expected but surprisingly had no effect on lytic granule polarization and directional secretion. Nevertheless, CTLs lacking centrioles did display substantially reduced killing potential, which was associated with defects in both lytic granule biogenesis and synaptic actin remodeling. These results reveal an unexpected role for the intact centrosome in controlling the capacity but not the specificity of cytotoxic killing.

Keywords: T cell centriole centrosome cytotoxicity microtubule.

Conflict of interest statement

The authors declare no competing interest.

Figures

Centriole loss affects killing capacity…

Centriole loss affects killing capacity but not killing specificity. ( A ) Experimental…

Microtubules are dispensable for synaptic…

Microtubules are dispensable for synaptic degranulation. ( A ) pHluorin-Lamp1–based detection of degranulation…

Centriole loss disrupts lytic granule…

Centriole loss disrupts lytic granule biogenesis. ( A ) Sas4 fl/fl Trp53 fl/fl…

Centriole loss alters synaptic F-actin…

Centriole loss alters synaptic F-actin configuration and mechanical force exertion. ( A )…


Immune Biology of Acute Myeloid Leukemia: Implications for Immunotherapy

Immune surveillance of incipient tumor cells is important for defense against cancer development. However, active immune evasion is a cancer hallmark. 1 In acute myeloid leukemia (AML), a complex hematologic malignancy, AML blasts and leukemic stem cells (LSCs) evade and suppress host immune systems. Traditional AML therapies, such as allogeneic hematopoietic stem cell (HSC) transplantation (allo-HSCT) and donor lymphocyte infusions (DLIs), rely on T-cell–mediated effects, demonstrating AML cell sensitivity to functional immune cell cytotoxicity. 2,3 These observations support immunotherapy to evoke anti-AML immunity.

What are the current data and gaps in knowledge regarding the immune biology of AML, and what are the opportunities for translational research?

Current data highlight the potential role for immunotherapy in AML. Gaps in knowledge include limited studies on the AML immune landscape and leukemic stem cell–specific mechanisms of immune escape, and lack of consensus in defining the AML tumor microenvironment.

Addressing these knowledge gaps in AML immunobiology may translate to faster clinical development of immunotherapies for patients with AML.

Clinical translation and AML immunotherapy development have been relatively slow. Intrinsic AML features complicate translation from basic immunobiology to effective immunotherapy. First, AML is not a single disease, but a family of unique malignancies thus, genetic/epigenetic heterogeneity 4,5 and subclonality 6 contribute to biologic variation. Second, AML has two main compartments: peripheral blood (PB) and bone marrow (BM). BM is composed of endothelial cells, stromal cells (eg, mesenchymal stromal/stem cells [MSCs]), and most immune cell types, including cytotoxic T lymphocytes (CTLs), regulatory T cells (Tregs), natural killer (NK) cells, and myeloid subsets (eg, myeloid-derived suppressor cells [MDSCs]). 7 Global changes in BM immune cell profiles are associated with AML microenvironment immunosuppression. 8 Furthermore, the microenvironment protects LSCs, potential drivers of relapse, from treatment- or immune-mediated destruction. 9,10

Increased knowledge of AML immune escape is needed to translate immunotherapy from bench to bedside. To date, a systematic analysis of basic science addressing this fundamental gap is lacking. We conducted this review to identify knowledge gaps and opportunities for AML immunotherapy development.

AML cells evade or suppress the immune system through five main mechanisms: reduced expression of major histocompatibility complex (MHC) molecules, enhanced inhibitory ligand expression, reduced activating ligand/receptor expression, ligand shedding, and manipulation of soluble factors within the microenvironment ( Fig 1 ). 2,7,11 Genetic mutation or immuno-editing reduces expression of HLA class II molecules and regulators required for T-cell AML recognition. Because endogenous or allogeneic immune cells eliminate high HLA/MHC-expressing cancerous cells, those with reduced or lost expression survive. 2,12 Without immunostimulation, little evidence exists of endogenous T-cell responses against AML. Reduced or lost HLA/MHC expression is predominantly observed after allo-HSCT. 13,14 In a study comparing samples from patients with AML obtained at diagnosis and relapse post–allo-HSCT, AML cells showed decreased expression of MHC class II proteins after transplantation in 17 of 34 patients. Interferon gamma (IFN-γ) administration reversed this downregulation, suggesting an epigenetic mechanism. 14 Another study of AML posttransplantation relapse samples reported transcriptional silencing of HLA class II molecules. 15 These findings suggest epigenetic mechanisms of immuno-editing may reduce HLA/MHC expression after allo-HSCT. Defective processing and loading of leukemia-associated antigens onto HLA class II proteins may also contribute to immune escape. 16

FIG 1. Cellular and molecular mechanisms of immune evasion in acute myeloid leukemia (AML) in the vascular and bone marrow microenvironments. 11,34 Cells that inhibit anti-AML immune function include regulatory T cells (Tregs), myeloid-derived suppressor cell (MDSCs), and mesenchymal stromal cells (MSCs). On the molecular level, activation of immune checkpoint pathways (eg, programmed death receptor-1 [PD-1], cytotoxic T-lymphocyte–associated protein 4 [CTLA-4]) and induction of immunosuppressive soluble factors (eg, kynurenine) by AML tumor microenvironment (TME) interactions fosters immune escape in AML. Additionally, under the pressure of allogeneic T cells, immuno-editing driven by epigenetic mechanisms may result in downregulation of major histocompatibility complex (MHC) expression on AML cells, leading to immune escape and relapse in AML. ATP, adenosine triphosphate CCR4, C-C chemokine receptor type 4 GAL9, galectin-9 IDO, indoleamine-2,3 dioxygenase IFN-γ, interferon gamma IL, interleukin iNOS, inducible nitric oxide synthase LSC, leukemic stem cell MPS, metalloproteinases NK, natural killer NOS-2, nitric oxide synthase 2 ROS, reactive oxygen species SDF1, stromal cell-derived factor 1 SIRPα, signal regulatory protein alpha TCR, T-cell receptor TGFβ, transforming growth factor beta TIM-3, T-cell immunoglobulin and mucin-domain containing-3 VEGF, vascular endothelial growth factor VISTA, V-domain immunoglobulin suppressor of T-cell activation.

Immune checkpoint pathways inhibit T- or NK-cell function. Enhanced expression of ligands for T-cell–regulating checkpoints, including cytotoxic T-lymphocyte antigen-4 (CTLA-4 surface protein expression), 17 programmed cell death protein 1 (PD-1 RNA and surface protein expression), 15,18 B7-H3 (surface protein expression), 19 and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3 RNA and surface protein expression) 20 were reported in AML and correlated with inferior outcomes. 21-23 In AML, the most extensively studied immune checkpoint pathway is PD-1/programmed death-ligand 1 (PD-L1). Several studies found absent or restricted surface protein expression of PD-L1 in de novo AML, but increased expression at relapse. 24-26 PD-L1 expression on AML blasts during disease progression could be adaptive for antitumor immunity and the associated inflamed microenvironment. 7 Supporting this hypothesis, ex vivo addition of IFN-γ and interleukin-6, inflammatory cytokines associated with activated T cells, upregulated PD-L1 on AML blasts. 24-26

Downregulation of NK-cell receptor DNAX accessory molecule-1 (DNAM-1) 27 and NK group 2D ligand (NKG2DL), the ligand for the NK-cell immunostimulatory receptor NKG2D, 11 are potential AML mechanisms of NK-cell evasion. Although DNAM-1 cross-linking with its ligands on AML cells (CD112 and CD155) results in NK-cell–mediated killing, chronic cross-linking downregulates DNAM-1. Additionally, AML cells lacking NKG2DL because of intrinsically low expression, 28 ligand shedding, 29 and/or epigenetic silencing 30 escape NK cells. 11,28,31 Along with expressing immune checkpoint ligands, AML cells secrete or shed soluble factors, receptors, and ligands into the microenvironment to create an immunosuppressive milieu as summarized in Table 1.

TABLE 1. Effects of Select Molecules Secreted or Shed by AML Cells on the Immune System

AML cell interactions with the immune system create an immunosuppressive microenvironment with reductions in population and/or function of CTLs and NK cells and accumulation of Tregs, macrophages, and MDSCs. This is significant because AML LSCs preferentially reside in BM where they are protected from the immune system.

AML LSCs, or leukemia-initiating cells, were discovered in xenotransplantation experiments. 42,43 LSCs are capable of self-renewal and give rise to more differentiated bulk blast cells. 44 Analyses of LSC populations show that they are generally quiescent, 45 are chemotherapy resistant, 46 and evade immunosurveillance. 28,47 AML LSCs have elevated expression levels of CD47, a ligand for signal regulatory protein alpha located on macrophages and some dendritic cells (DCs). Activation via CD47 ligation inhibits phagocytosis. Thus, increased CD47 on LSCs enables evasion from macrophage-mediated phagocytosis. 47 Promising preclinical data showed that an anti-CD47 monoclonal antibody (mAb) induced macrophage-mediated phagocytosis of AML cells. 48 AML LSCs lack NKG2DL, resulting in escape from NK-cell–mediated lysis. 28 Poly(ADP-ribose) polymerase 1 (PARP1) is enriched in NKG2DL-negative AML cells, providing a possible therapeutic target for this population. 28

A bioinformatics approach identified two LSC-specific immunosuppressive targets, galectin-1 and CD200, with enhanced expression on LSCs compared with normal HSCs. 49,50 CD200 positivity was associated with reduced immune-specific apoptosis and downregulation of inflammatory immune response-associated genes in AML cell lines. 50

These studies provide candidate targets for immune-mediated eradication of AML LSCs. Some LSC-directed agents are already under investigation. An anti-CD47 mAb is being evaluated in clinical trials for patients with relapsed/refractory AML (ClinicalTrials.gov identifiers: NCT02678338, NCT03248479). 51 A CD47×CD33 bispecific antibody (bsAb) is also being evaluated preclinically. 51

In patient xenograft models, PARP1 inhibition induced NKG2DL expression on LSCs, sensitizing them to NK-cell–mediated clearance. 28 Another promising approach is disruption of AML-niche interactions (eg, E-selectin inhibition 52 ) to free LSCs from the microenvironment and render them more vulnerable to immunosurveillance.

Although spontaneous T-cell reactivity against defined AML antigens has been described, 53 no consensus has emerged regarding number, distribution, and functional status of T cells in the AML microenvironment (Table 2). 7 The contrasting findings may reflect underlying biologic differences between assessed AML compartments (PB or BM), disease heterogeneity, disease stage, prior therapy effects, limited patients evaluated, and/or patient differences. 7,54-56

TABLE 2. Studies of the T-Cell Repertoire and Function in AML

Increased inhibitory checkpoint molecule expression is consistently found on BM T cells 55,57-59 however, the timing of increase (diagnosis or relapse) is unclear. 55,58,60 It is also unclear whether this reflects T-cell exhaustion or a population shift to differentiated effector T cells. 8,55,60

In AML, Tregs dampen effector cell activity via secretion of cytokines and adenosine and increased adenosine triphosphate hydrolysis. 7,11 Higher PB and BM Treg frequencies were reported in patients with AML versus healthy controls, possibly because of increased indoleamine 2,3-dioxygenase 1 (IDO1) expression. 54,55,61 In AML, Tregs may preferentially accumulate in BM because of CXCL12/CXCR4 signaling. 7,62,63 Higher BM Treg frequencies were observed at diagnosis, with additional increases at relapse, suggesting Treg induction is an early event that is further modulated by the immunosuppressive AML microenvironment. 55

Functional T cells appear to infiltrate the AML microenvironment, but their efficacy is limited by immunosuppressive factors, including Tregs, impaired antigen recognition, and upregulation of immune checkpoints. Some therapies target immunosuppressive factors (eg, IDO1), whereas others are T-cell directed. Several immunotherapies have been developed to overcome AML immune evasion of T cells (Table 3).

TABLE 3. T-Cell–Directed Treatment Strategies in AML: Advantages and Limitations

Peptide- and cell-based vaccines induce expansion of AML-specific T cells via tumor-associated antigen recognition. 53,81 In a phase II study of patients with AML in first complete remission (CR) treated with a Wilms tumor 1 peptide vaccine (n = 22), 64% had an immunologic response, and median disease-free survival since first CR was 16.9 months the vaccine was well tolerated. 82 Promising data were also reported in trials of DC-based vaccines and patient-derived AML cell vaccines, with some patients achieving prolonged remission. 51,83

Although checkpoint inhibitors (CPIs) targeting PD-1 and CTLA-4 have been approved for various solid tumors, the activity of these inhibitors in AML seems to be relatively less potent based on available data. In a phase Ib study of ipilimumab (anti–CTLA-4) 10 mg/kg in patients with hematologic malignancies relapsing post–allo-HSCT (n = 22), the CR rate was 23% (AML, n = 4 myelodysplastic syndrome [MDS], n = 1). Immune-related adverse events and graft-versus-host disease were reported. 84 In a phase II study, patients with relapsed/refractory AML (n = 70) were treated with nivolumab (anti-PD-1) plus azacitidine. The rate of CR/CR with incomplete hematologic recovery (CRi) was 22%. Overall, the combination was safe, although immune-related toxicities were reported. 66 CPIs targeting other molecules are also under investigation. 51

An antibody platform recently developed is the bsAb/antibody construct, which includes bispecific T-cell engager (BiTE) molecules and dual-affinity retargeting antibodies. BsAbs bind CD3 receptors on an endogenous T cell and a target antigen on a malignant cell. Simultaneous binding results in a cytolytic synapse with consequent lysis of the cancer cell. BsAb treatment was clinically validated for patients with B-cell precursor acute lymphoblastic leukemia (ALL). 85 In AML, bsAbs demonstrated promising antileukemic activity (including CRs) in phase I studies, supporting their potential role in this disease. 86-88 Their targets are primarily CD123, CD33, CLL1 (CLEC12A), and FLT3.

Adoptive transfer of T cells is another method of T-cell–mediated immunotherapy. Chimeric antigen receptor (CAR) T cells are modified T cells that express receptors engineered to engage target antigens on malignant cells 68 in an MHC-independent manner. CAR T-cell therapy has revolutionized treatment of hematologic malignances such as ALL and non-Hodgkin lymphoma. Translation to AML has been slower, with only early-phase data in small populations. 51,68 Slow uptake of CAR T-cell therapy reflects several challenges associated with this strategy in AML (Table 3). Alternative approaches, such as CAR NK cells and T-cell receptor (TCR) gene therapy, are being evaluated. 51,89 Activation of immunoregulatory checkpoints can hamper the efficacy of T-cell adoptive immunotherapy, and lack of costimulation is a TCR gene therapy limitation. 90 Studies to improve clinical translation of T-cell adoptive immunotherapy and TCR gene therapy are ongoing. 90 A phase I/II study of patients with high-risk relapsed AML, MDS, or chronic myelogenous leukemia previously treated with allo-HSCT is underway (ClinicalTrials.gov identifier: NCT01640301).

NK cells from patients with AML often present with an unfavorable phenotype, including downregulation of natural cytotoxicity receptors, 31 reduced capacity to produce and secrete IFN-γ, and inhibited activity via Tregs and soluble factors in the microenvironment.

Although AML cells are sensitive to NK-cell–mediated cytotoxicity, the immunosuppressive microenvironment fosters immune escape. Several strategies have been developed to overcome immune evasion of NK-cell surveillance.

Fragment crystallizable (Fc)-optimized antibodies are mAbs where the Fc region has been engineered to optimize therapeutic activity. In a recent phase Ib trial, an anti-CD33 Fc-optimized mAb (BI 836858) with azacitidine was evaluated in patients with previously untreated AML (n = 31). The combination demonstrated acceptable tolerability and was active, with five of 28 evaluable patients having CR/CRi. 91 Coengagement of AML target cells via CD33 and NK cells via CD16 through bispecific/trispecific killer cell engager antibodies are also promising strategies to enhance AML-specific targeting by NK cells. 51,92 Bispecific fusion proteins targeting NKG2DL on AML cells to activate NK cells are under investigation. 93

NK-cell–mediated killing of cancer cells is not MHC presentation dependent. NK cells can be donor-derived haploidentical and transferred in conjunction with haploidentical transplantation. Promising efficacy and safety from phase I AML trials have been observed. 51

MDSCs, heterogeneous CD33+ immature myeloid cells, act as a major immunosuppressive factor, and MDSC expansion is linked with poor outcomes. 94,95 MDSCs exert their immunosuppressive activity via arginase-1, inducible nitric oxide synthase expression, and NOX2-derived reactive oxygen species (ROS) production. 96,97 Although the AML MDSC immunobiology is not well understood, 7 evidence suggests MDSCs accumulate in the AML microenvironment, contribute to immunosuppression, and could be immunotherapy targets.

One study found increased BM MDSC frequencies in patients with active AML versus normal controls engraftment of mice with AML led to MDSC expansion in BM and spleen. 97 Two other studies found increased BM and PB MDSC frequencies in newly diagnosed AML versus controls, 98,99 suggesting MDSC expansion occurs early. Interactions between AML cells and the microenvironment likely play a role. In one study, AML cells released c-myc–containing extracellular vesicles that trafficked to myeloid accessory cells in the BM microenvironment, resulting in MUC1-mediated upregulation of proproliferative cyclins E1 and D2 in MDSCs. 97

MDSCs inhibit T-cell proliferation. 78,97,98 One study found this inhibition was partially mediated by immune checkpoint V-domain immunoglobulin suppressor of T-cell activation (VISTA). VISTA expression was greater on MDSCs from patients with AML versus healthy controls VISTA knockdown reduced MDSC-associated T-cell inhibition. Furthermore, the proportion of VISTA-expressing MDSCs correlated with the proportion of PD-1–expressing T cells. 98 In another study, IDO upregulation in MDSCs through cell-to-cell contact with AML blasts was observed. 78

One study reported increased frequency of immunosuppressive M2-like macrophages in BM and spleen of patients with AML versus healthy patients. 100 AML cells also polarized nonleukemic macrophages into an AML-supporting state and induced their proliferation and infiltration into BM of human AML mouse models. 100

Preclinical evidence shows that immunosuppressive MDSC populations are expanded in AML, supporting evaluation of MDSC-targeted therapies. CD33-directed therapies hold promise for MDSC elimination. In ex vivo studies, CD33-directed bispecific molecules engaged T cells to MDSCs to achieve antileukemic effects and restore immune homeostasis in AML 78 and MDS. 101 Immunodepletion of MDSCs was accompanied by T-cell expansion and activation and improved hematopoiesis. 101 Gemtuzumab ozogamicin, a CD33-directed antibody-drug conjugate, restored T-cell and CAR T-cell immunity against various cancers via MDSC targeting. 102

Phase 1 studies of the BiTE AMG 330 in AML (ClinicalTrials.gov identifier: NCT02520427) and the bispecific T-cell engager AMV564 in AML (ClinicalTrials.gov identifier: NCT03144245) and MDS (ClinicalTrials.gov identifier: NCT03516591) are currently underway, and multiple trials investigating gemtuzumab ozogamicin in AML are recruiting or completed. Another strategy relies on pharmacologic blockade of ROS production, an important mechanism of MDSC immunosuppression and differentiation into macrophages and DCs. In a phase III study, postconsolidation treatment with the NOX2 inhibitor histamine dihydrochloride in combination with interleukin-2 improved leukemia-free survival versus no treatment of patients with AML in CR. 103,104 Histamine dihydrochloride significantly reduced PB MDSCs in patients with AML, with strong reductions associated with longer leukemia-free survival. 96 Other strategies for selective targeting of MDSCs in AML include MUC1 inhibition, 97,105 granulocyte colony-stimulating factor therapy, 106 and HO-1 protein inhibition 107 these treatments may synergize with CPIs. 98,107 Association of MDSC levels with clinical outcomes is unclear, because two small studies (N = 27 and N = 6) reported a correlation with minimal residual disease (MRD) 99 or relapse, respectively, 9 whereas one large study (N = 341) did not find a statistically significant relationship with MRD. 108

MSCs are fundamental BM regulators, with potent immunosuppressive functions that affect innate and adaptive cellular immunity however, relatively little is known about their effects in hematologic malignancies. 109-111 This is likely because of a lack of standardized methods for isolating and characterizing MSCs, and the heterogeneity of AML and MSCs. 110,111 Preclinical data show a dynamic interplay among MSCs, AML cells, and the immunosuppressive microenvironment (Table 4).

TABLE 4. Preclinical Studies Assessing the Role of Immunosuppressive MSCs in AML

MSCs are critical BM components, supporting long-term maintenance and quiescence of LSCs 115,116 and protecting them from anti-AML therapy. 115-117 Future immunotherapy research should consider MSC-to-AML cell cross talk in shaping the immune microenvironment. Targeting individual pathways to reduce MSC-associated immunosuppression or interfering with MSC-to-AML cell cross talk may be effective strategies.

Tumor immune microenvironments vary substantially between patients. In solid tumors, immunologically hot versus cold tumor microenvironments may have prognostic implications for CPI therapy. However, little is known about AML microenvironment heterogeneity.

There have been several attempts to define the AML immune microenvironment. Using the Cancer Genome Atlas, a pan-cancer Tumor Inflammation Signature (TIS) was developed to characterize the immune microenvironment in a variety of cancers. TIS includes 18 genes related to abundance of antigen-presenting cells, T- and NK-cell levels, IFN activity, and T-cell exhaustion. Higher scores indicate inflamed or hot tumors that may be more recognizable by the immune system. The TIS score for patients with AML was approximately 4—lower than the median of 5.5 observed in the entire cancer dataset, indicating AML tumor microenvironments are generally cold. 117a Another AML study investigated T-cell repertoires at diagnosis and relapse and identified a dual checkpoint-positive T-cell population (PD-1+ and TIM-3+ or LAG3+). 55 The authors proposed this population demarcated two types of patients with AML: those with and those without an exhausted immune microenvironment. The proportion of double-positive T cells increased from diagnosis to relapse, indicating greater immune system exhaustion at later stages. 55 Finally, transcriptomic and proteomic profiling identified two types of immune microenvironments in AML BM samples: an immune-enriched and IFN-γ dominant type (elevated expression of lymphocyte-associated genes, IFN-γ, and immune checkpoint molecules) and an immune-depleted type (elevated expression of mast cell function– and T-cell exhaustion–associated genes, low expression of T-cell and B-cell genes). 118 The immune-enriched profile was observed in approximately 30% of patients, indicating that most AML tumors are cold. 119

Future studies evaluating AML BM heterogeneity are important for personalized immunotherapy. Agent (eg, CPIs) efficacy may be compromised by immune-depleted microenvironments, whereas hot tumors may be more susceptible to CPIs. Combination therapies incorporating CPIs may thus be more effective for tumors with immune-enriched microenvironments. Understanding how immunotherapies can modulate the microenvironment immune signature represents an additional research avenue. A preliminary study reported that treatment with a bsAb shifted the AML BM microenvironment signature to a more inflamed type as evidenced by increased immune cell infiltration, a higher TIS score, and enhanced IFN-γ signaling. 120

To deliver optimal immunotherapy to patients relapsing after allo-HSCT or chemotherapy, it is important to understand immune-related effects of these treatments. Allo-HSCT efficacy depends on an immune-mediated graft-versus-leukemia (GVL) effect to confer anti-AML alloimmunity. The GVL effect is mediated primarily by alloreactive T cells, although there is increasing recognition of alloreactive NK-cell responses and tumor-specific T-cell and anti‐body responses. 121 Understanding immune escape mechanisms during post–allo-HSCT relapse can inform immunotherapeutic strategies. A comprehensive study of immune signatures from patients relapsing after allo-HSCT found that significant downregulation of HLA class II transcripts commonly occurred posttransplantation and may be driven by epigenetic mechanisms. 15 The same study described another modality of relapse after allo-HSCT, which was characterized by impaired T-cell costimulation by AML blasts. 15 These effects were not observed at relapse after chemotherapy only. 15 Another study showed that compared with healthy donors, patients with AML relapsing after allo-HSCT had reduced T-cell frequencies characterized by an exhausted phenotype and altered cytokine profile. 59 These studies assessed circulating T cells but not BM-infiltrating T cells. A recent study reported early-differentiated T memory stem cells and central memory T cells from the BM of patients relapsing after allo-HSCT had an exhausted phenotype characterized by expression of multiple inhibitory receptors. 122 These findings show systemic T-cell impairment and exhaustion present at relapse after allo-HSCT.

The percentage of donor-derived T lymphocytes (chimerism) after allo-HSCT is correlated with disease outcomes, with mixed chimerism predictive of relapse and shorter survival. 123,124 Mixed chimerism via residual host Tregs and DCs may inhibit activation of donor-derived DCs and alloreactive T-cell induction. 125

Profound immunodeficiency occurs after T-cell–depleted HLA-haploidentical allo-HSCT (hHSCT). 126 Reconstitution kinetics of various immune cell populations may be related to post-hHSCT relapse. NK cells reconstitute early posttransplantation, and their rapid recovery is associated with lower relapse risk in certain hematologic malignancies. 127 Furthermore, transplantation from NK alloreactive donors has been associated with better outcomes. 128 Another immune cell population of interest is invariant NK T (iNKT) cells. One study found that patients with hematologic malignancies who maintained remission after hHSCT had full reconstitution of iNKT cells, whereas patients who relapsed did not. 126 IFN-γ production 126 or direct killing of AML blasts in a CD1d-dependent manner 129 may explain the role of iNKT cells in maintaining antitumor immunity after allo-HSCT.

Chemotherapy affects humoral immunity to a greater degree than cellular immunity. In one study, T-cell frequency and function recovered to normal levels after consolidation chemotherapy, whereas B-cell immunity was impaired. 130

The role of chimerism is well recognized, and chimerism analysis remains an important tool to predict relapse post–allo-HSCT. 131 Several ongoing clinical trials focus on chimerism in AML post–allo-HSCT, including early detection and treatment of mixed chimerism (eg, ClinicalTrials.gov identifiers: NCT03850418, NCT03128034, NCT02724163). MRD measurements in the posttransplantation period can be performed however, MRD monitoring remains challenging because suitable molecular markers are not available for all patients with AML. 132

AML immune evasion mechanisms post–allo-HSCT suggest epigenetic therapies that reverse HLA expression loss may be effective for posttransplantation relapse. In the phase II RELAZA trial, azacitidine (hypomethylator) treatment of MRD post–allo-HSCT led to long-term responses in patients with MDS or AML. 133 Azacitidine combined with immune therapy (DLIs or lenalidomide) was also effective for patients with AML relapsing post–allo-HSCT. 59,134 Given impaired T-cell costimulation observed at relapse post–allo-HSCT, CPIs may be beneficial. 15 The preserved T-cell population and function after chemotherapy suggests T-cell–directed immunotherapies, such as bsAbs and CPIs, will not be compromised by prior chemotherapy.

In conclusion, AML is a complex, heterogeneous disease, and fundamental understanding of its immunobiology is critical for immunotherapy. Immune dysfunction is important for AML pathogenesis, supporting the role of immunotherapies to restore local immunity to eradicate the disease. An improved understanding of how LSCs evade the immune system and how they can be eradicated is needed. Moreover, cellular components of the AML immune microenvironment, such as MSCs and B cells, have not been well characterized. These understudied populations may have important roles to play for example, B cells from high-risk patients with durable GVL responses produce anti-AML antibodies. 135 Standardized methods for characterizing the tumor microenvironment are important for translational development of patient-specific immunotherapies.

Allo-HSCT remains a cornerstone of immune-mediated AML therapy. Development of optimal strategies should be informed by lessons learned from allo-HSCT and the mechanisms of AML immune dysfunction. Allo-HSCT findings suggest combination therapies targeting multiple pathways and cellular effectors (including T cells and NK cells) will likely be optimal for developing alternative immunotherapeutic strategies. Post–allo-HSCT, the GVL effect enables donor immune cells to eliminate host leukemic cells by engaging a multicellular (T cells, NK cells, antibodies, antigen-presenting cells) response against multiple antigens, including leukemia-associated antigens and nonleukemia-specific antigens overexpressed in leukemia. 136,137 This broad immune response is likely important in overcoming the multiple immunosuppressive mechanisms and clonal heterogeneity observed in AML.

The implications of allo-HSCT for target identification and cellular effectors are important. Allo-HSCT can target multiple intracellular (presented by HLA) and/or surface antigens, whereas current AML therapies can only target limited surface antigens. Furthermore, although minor histocompatibility antigen-specific T cells can induce a potent GVL effect, translating this observation into AML clinical trials is difficult. 138 Prognosis for patients with relapsed/refractory AML remains suboptimal even with allo-HSCT, 139 an important consideration when investigating combination therapies in relapsed patients.

Being cognizant of optimal early- and late-phase trial design with regard to endpoints and molecular inclusion criteria is critical. 140 The allo-HSCT experience provides rationale for personalized immunotherapy based on immunologic profiles. For example, the modes of immune evasion after allo-HSCT (genomic loss of HLA v loss of HLA class II expression v T-cell exhaustion) could be targeted by distinct salvage immunotherapies. 12,15 Early-phase trial endpoints can also include dose-dependent changes in immunity and stratification of responders versus nonresponders using immune readouts. For early- and late-phase trials, biomarkers should be collected and analyzed using modern platforms (eg, immunology panels), and retrospective stratifications should be made and published regardless of outcome. Given the prognostic significance of MRD in AML and the potential for immunotherapy to eliminate LSCs, MRD-negative CR could be a useful endpoint in late-stage anti-AML immunotherapy trials. Late-stage trials can implement findings from earlier phases to better identify responder populations and develop inclusion/exclusion criteria. Additional understanding of the optimal sequencing of immunotherapies—specifically, which immunotherapies can be reserved for later lines of therapy and retain activity in patients refractory to other agents—is also important.

Overall, the gaps and opportunities highlighted in this review reiterate the importance of a strong foundation in basic immune biology that can be harnessed for clinical translation. We expect current and future advances in our knowledge of immune escape mechanisms will translate to powerful immunotherapies for patients with AML.

Medical writing assistance was funded by Amgen.

Conception and design: All authors

Collection and assembly of data: Sophia Khaldoyanidi, Marion Subklewe

Data analysis and interpretation: Sophia Khaldoyanidi, Marion Subklewe

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/jco/authors/author-center.

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Stock and Other Ownership Interests: Bristol Myers Squibb, Amgen

Stock and Other Ownership Interests: Amgen

Patents, Royalties, Other Intellectual Property: Inventor on patents related to the use of CD3 bispecifics

Consulting or Advisory Role: Stemline Therapeutics, Amgen

Speakers' Bureau: Amgen, Stemline Therapeutics

Consulting or Advisory Role: Celgene, Janssen, Novartis, Amgen, Pfizer, Roche, Bristol Myers Squibb, AGIOS, AbbVie/Genentech, Astellas Pharma, Otsuka, Daiichi Sankyo

Research Funding: Janssen (Inst), Celgene (Inst), Novartis (Inst)

Travel, Accommodations, Expenses: Roche

Consulting or Advisory Role: Amgen, Celgene, Gilead Sciences, Janssen, Novartis, Pfizer, Seattle Genetics

Speakers' Bureau: Amgen, Celgene, Gilead Sciences, Janssen, Pfizer, Octapharm, Novartis

Research Funding: Amgen, Gilead Sciences, Miltenyi Biotec, MorphoSys, Seattle Genetics, Novartis, Celgene

Patents, Royalties, Other Intellectual Property: Administration of a bispecific construct binding to CD33 and CD3 for use in a method for the treatment of myeloid leukemia, PCT/EP2017/059108. Trispecific molecules combining specific tumor targeting and local immune checkpoint inhibition, PTC/EP2016/07717. Combination of epigenetic factors and bispecific compounds targeting CD33 and CD3 in the treatment of myeloid leukemia, PCT/EP2014/069575

Travel, Accommodations, Expenses: Celgene, Gilead Sciences, Amgen

No other potential conflicts of interest were reported.


The Immune System

The immune system is a series of processes utilized to protect the body from infections and diseases. It is compromised of the innate and adaptive systems. The innate system is the initial response when a pathogen first enters the body. It is both quick and non-specific. Meanwhile, the adaptive system is the following immune response. It is much slower initially, but “adapts” to be specific to the threat present. Both the innate and adaptive immune responses utilize white blood cells, known as leukocytes. However, each immune response optimizes specific leukocyte types. The leukocytes commonly found in the innate and adaptive immune systems can be found in the diagram below.

The innate immune system commonly recruits basophils, masts cells, eosinophils, neutrophils, and monocytes. Monocytes can then be differentiated to become macrophages and dendritic cells. Meanwhile, the adaptive immune system commonly recruits T cells, B cells, all of their variations (which will be defined throughout this article), and natural killer cells.

The Innate Immune System

The innate immune system is the first immune response at wounds and pathogen entry points. It is a quick and generic response that is optimized to target any pathogen. Generally, this is done as the immune cells in the host’s body recognize specific proteins and/ or sugars on the membranes of the invading pathogens. Specifically, the host cells detect that these proteins/ sugars are foreign and therefore must be targeted by the immune system. In the case of pathogenic bacteria, the lipopolysaccharides (a sugar molecule) on the bacteria’s membrane are recognized by toll-like receptors, which are proteins on the host’s leukocyte membranes. Lipopolysaccharides are present on all gram- negative bacteria and do not occur in humans. They are thus a reliable target to alert the body of foreign invaders and activate the immune response.

Activation of the immune response begins with the inflammatory response. Initially in this response, macrophages are recruited to the wound site. Here, they secret chemokines, which are molecules that form a chemical gradient to recruit additional immune cells to the wound site. These additional cells can include mast cells, which release histamine to dilate blood vessels further away from the wound. The dilated vessel and chemokine combination then attracts neutrophils, which exit the dilated vessels and gravitate towards the wound site. The neutrophils “eat” the foreign materials through a process known as phagocytosis. During phagocytosis, an enzyme found inside the neutrophil- known as lysozyme- kills the invading pathogen.

The immune response does not end with phagocytosis. Additional monocytes are then recruited to the wound, where they can be differentiated into macrophages. Macrophages also have the ability to release chemokines, in addition to cytokines. Cytokines are immune markers that can cause several effects throughout the body, including fever adjustments and tissue repair activation. Macrophages also perform phagocytosis, in addition to the previously mentioned neutrophils and the next leukocyte recruited- dendritic cells.

Antigen Presentation

Once dendritic cells are recruited to the infection site, they consume additional debris through phagocytosis. Furthermore, they consume and intracellularly process antigens, which are key structures that provide pathogen identity and will be used for immune memory. After the dendritic cell consumes the antigen, the antigen is broken down into peptide fragments. The peptide fragments are attached to the major histocompatibility complex (MHC), which are glycoproteins on the dendritic cell’s membrane that hold the processed antigen (now called an epitope). The now combined MHC- epitope complex is brought to the surface of the dendritic cell, where it can then be presented to T cells. This antigen presentation is the switch from the innate to the adaptive immune system.

Before moving on to the adaptive immune system, it is important to note that there are variations in the MHC. The two major types in relation to T cells are class I and class II. Class I MHC binds to antigens from inside the endoplasmic reticulum while class II MHC binds to antigens from inside endosomes. The differentiation between class I and class II MHC will be important for T cell activation later on.

The Adaptive Immune System

Where the innate response is quick and non-specific, the adaptive response is slow, pathogen specific, and diverse. The adaptive response constitutes the recruitment of leukocytes that customize their response to foreign materials. This is where antibodies and the concept of memory are incorporated into the immune response.

In the adaptive response, specific leukocytes known as lymphocytes are attracted to the area of interest. There are two types of lymphocytes: bone marrow- derived cells (B cell lymphocytes) and thymus- derived cells (T cell lymphocytes). Where all lymphocytes are produced in the bone marrow, B cells remain in the bone marrow through maturity while T cells mature in the thymus. (The thymus is found just under the sternum in humans.) When these lymphocytes need to the activated, they are moved to the spleen and lymph nodes. They then travel through the blood, lymph nodes, and additional immune organs to disperse throughout the body.

B cells are extremely important for antibody production. Antibodies are blood proteins that are created to attack specific pathogens. They belong to a group of immunoglobulins (Ig) and are divided into five classes (IgG, IgD, IgE, IgA, and IgM). B cells must first become activated by specific T cells (which will be discussed shortly) to become plasma cells. The now mature plasma cells are then responsible for creating and releasing antibodies in the pathogen’s presence.


Cell-Mediated Immunity

T cell co-inhibition

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) or CD152 belongs to the immunoglobulin superfamily. Although approximately 30% identity has been shown in sequences of CTLA4 and CD28, and also similar ligands on the surface of APCs, CTLA4 delivers inhibitory signals to T cells compared to stimulatory signals provided by CD28. CTLA-4 is not expressed on the surface of naïve T cells however, it is expressed on the surface of regulatory T cells, and its expression increases upon activation on the surface of conventional T cells. CTLA4 is fundamentally an intracellular antigen which transferred to the surface of T lymphocyte upon activation. Also, its expression on the cell membrane is transient due to rapid internalization ( Carreno and Collins, 2002 Chambers et al., 2001 ). CTLA4 remains for a longer duration on the surface of memory and regulatory T lymphocytes ( Jago et al., 2004 ).

CTLA4 represents a co-inhibitory receptor for B7 molecules. CTLA4 inhibits T cell activation through different mechanisms. Some of these mechanisms are related to the effect of CTLA4 on the function of the same T cell that has primarily expressed CTLA4 these mechanisms are known as cell-intrinsic mechanisms. Four mechanisms were categorized as cell-intrinsic mechanisms First of all, CTLA4 can interfere with signaling pathways of CD3 and CD28. Secondly, compared to CD28, CTLA4 displays a higher affinity for B7-1 and B7-2, resulted in the competition of CTLA4 with CD28 and therefore, suppression of efficient T cell responses. Thirdly, CTLA4 strengthens the adhesion of T cells to the APCs and shortens the duration of their interaction. Finally, splice variants of CTLA4 may attach to B7-1 or B7-2 and prevents T cell activation. On the other side, CTLA4 could affect the expression of costimulatory molecules on the encountered APCs. These mechanisms are categorized as cell-extrinsic mechanisms. Three different extrinsic mechanisms have been explained up to now. First of all, CTLA4 can induce the production of Indolamine 2, 3-dioxygenase (IDO) within APCs, which in turn deprives T lymphocytes of tryptophan and inhibits their activation. The second cell-extrinsic mechanism includes downregulation of costimulatory molecules (B7-1/B7-2) on the surface of APCs and, the third cell-extrinsic mechanism involves reducing the accessibility of B7-1 and B7-2 through triggering the endocytosis of these molecules ( Schildberg et al., 2016 ).

Programmed cell death protein 1 (PD1, CD279) has the potential to appear on the surface of T lymphocytes following their activation. PD1 belongs to the Ig superfamily, and it could attach to two ligands, including programmed death-ligand 1 (PD-L1, CD274) and PD-L2 (CD273). PD-L1 and PD-L2, members of the B7 family, are expressed on the surface of a wide range of immune and non-immune cells. After T cell activation, the interaction of PD1 (on the T cell membrane) with either PD-L1 or PD-L2 (on the surface of APC) can lead to the cell cycle arrest of T cell, reducing the production of IL-2 and finally, attenuation of immune responses. This downregulation plays a fundamental role in returning the immune responses to the baseline state and establishment of a homeostasis condition in the final stages of T cell responses ( Carreno and Collins, 2002 ).


Introduction

T cells play a major role in the GVL effect that confers cure in patients who are incurable by chemotherapy and “small-molecule” drugs. However, the GVL effect is limited, especially in patients with advanced disease and those with aggressive malignancies. Adoptive T-cell therapy strategies have arisen out of the need to improve GVL. The first attempts to boost GVL used donor lymphocyte infusions (DLI). Since then, the field has diversified and the underlying biology of the interactions between T cells and tumor cells has become better understood. Here, we address the current developments in allogeneic T-cell therapy using a non-gene-transfer approach to direct specificity and describe ways in which similar strategies can be applied in the autologous setting.


Four Stages of the Adaptive Immune Response

B cells, Helper T cells, and Cytotoxic T cells all respond to antigens in a similar pattern subsequent sections of this chapter will address the specifics of the immune response in each cell type. In order for their immune functions to be elicited, the cells must first encounter antigens by binding specifically to them using specialized membrane proteins. This binding elicits changes in the activity of the immune cells, termed activation, which is the second step in the adaptive immune response. Activation responses vary between the three types of cells, but in general all involve both changes in gene expression and in the initiation of cell division. Third, the immune cells attack invading pathogens or infected cells. Depending on the type of lymphocyte, the specific methods used to neutralize pathogens can vary. In most infections, the attacks from many different lymphocytes, and from cells of the innate immune system, occur simultaneously and the atttacking cells often stimulate each other through chemical messenger such as cytokines. Finally, long-lived, pre-activated immune cells that wait for a subsequent infection are formed in the memory phase of the adaptive immune response. These cells are identical to the initial cells that first encountered the pathogen except that they have already undergone the activation step so are able to attack right away when activated again by an antigen.


Chapter 43 - The Immune System

  • An invading microbe must penetrate the external barrier formed by the skin and mucous membranes, which cover the surface and line the openings of an animal’s body.
  • If it succeeds, the pathogen encounters the second line of nonspecific defense, innate cellular and chemical mechanisms that defend against the attacking foreign cell.

The skin and mucous membrane provide first-line barriers to infection.

  • Intact skin is a barrier that cannot normally be penetrated by bacteria or viruses, although even minute abrasions may allow their passage.
  • Likewise, the mucous membranes that line the digestive, respiratory, and genitourinary tracts bar the entry of potentially harmful microbes.
    • Cells of these mucous membranes produce mucus, a viscous fluid that traps microbes and other particles.
    • In the trachea, ciliated epithelial cells sweep out mucus with its trapped microbes, preventing them from entering the lungs.
    • In humans, for example, secretions from sebaceous and sweat glands give the skin a pH ranging from 3 to 5, which is acidic enough to prevent colonization by many microbes.
    • Microbial colonization is also inhibited by the washing action of saliva, tears, and mucous secretions that continually bathe the exposed epithelium.
      • All these secretions contain antimicrobial proteins.
      • One of these, the enzyme lysozyme, digests the cell walls of many bacteria, destroying them.
      • The acid destroys many microbes before they can enter the intestinal tract.
      • One exception, the virus hepatitis A, can survive gastric acidity and gain access to the body via the digestive tract.

      Phagocytic cells and antimicrobial proteins function early in infection.

      • Microbes that penetrate the first line of defense face the second line of defense, which depends mainly on phagocytosis, the ingestion of invading organisms by certain types of white cells.
      • Phagocyte function is intimately associated with an effective inflammatory response and also with certain antimicrobial proteins.
      • Phagocytes attach to their prey via surface receptors found on microbes but not normal body cells.
      • After attaching to the microbe, a phagocyte engulfs it, forming a vacuole that fuses with a lysosome.
        • Microbes are destroyed within lysosomes in two ways.
          • Lysosomes contain nitric oxide and other toxic forms of oxygen, which act as potent antimicrobial agents.
          • Lysozymes and other enzymes degrade mitochondrial components.
          • The outer capsule of some bacterial cells hides their surface polysaccharides and prevents phagocytes from attaching to them.
          • Other bacteria are engulfed by phagocytes but resist digestion, growing and reproducing within the cells.
          • Cells damaged by invading microbes release chemical signals that attract neutrophils from the blood.
          • The neutrophils enter the infected tissue, engulfing and destroying microbes there.
          • Neutrophils tend to self-destruct as they destroy foreign invaders, and their average life span is only a few days.
          • After a few hours in the blood, they migrate into tissues and develop into macrophages, which are large, long-lived phagocytes.
          • Some macrophages migrate throughout the body, while others reside permanently in certain tissues, including the lungs, liver, kidneys, connective tissues, brain, and especially in lymph nodes and the spleen.
          • Microbes that enter the blood become trapped in the spleen, while microbes in interstitial fluid flow into lymph and are trapped in lymph nodes.
          • In either location, microbes soon encounter resident macrophages.
          • Eosinophils position themselves against the external wall of a parasite and discharge destructive enzymes from cytoplasmic granules.
          • In addition to lysozyme, other antimicrobial agents include about 30 serum proteins, known collectively as the complement system.
            • Substances on the surface of many microbes can trigger a cascade of steps that activate the complement system, leading to lysis of microbes.
            • These proteins are secreted by virus-infected body cells and induce uninfected neighboring cells to produce substances that inhibit viral reproduction.
            • Interferon limits cell-to-cell spread of viruses, helping to control viral infection.
            • Because they are nonspecific, interferons produced in response to one virus may confer short-term resistance to unrelated viruses.
            • One type of interferon activates phagocytes.
            • Interferons can be produced by recombinant DNA technology and are being tested for the treatment of viral infections and cancer.
            • When injured, mast cells release their histamine.
            • Histamine triggers both dilation and increased permeability of nearby capillaries.
            • Leukocytes and damaged tissue cells also discharge prostaglandins and other substances that promote blood flow to the site of injury.
            • Increased local blood supply leads to the characteristic swelling, redness, and heat of inflammation.
            • Blood-engorged leak fluid into neighboring tissue, causing swelling.
            • First, they aid in delivering clotting elements to the injured area.
              • Clotting marks the beginning of the repair process and helps block the spread of microbes elsewhere.
              • Phagocyte migration usually begins within an hour after injury.
              • Injured cells secrete chemicals that stimulate the release of additional neutrophils from the bone marrow.
              • In a severe infection, the number of white blood cells may increase significantly within hours of the initial inflammation.
              • Another systemic response to infection is fever, which may occur when substances released by activated macrophages set the body’s thermostat at a higher temperature.
                • Moderate fever may facilitate phagocytosis and hasten tissue repair.
                • Characterized by high fever and low blood pressure, septic shock is the most common cause of death in U.S. critical care units.
                • Clearly, while local inflammation is an essential step toward healing, widespread inflammation can be devastating.
                • They also attack abnormal body cells that could become cancerous.
                • NK cells attach to a target cell and release chemicals that bring about apoptosis, or programmed cell death.

                Invertebrates also have highly effective innate defenses.

                • Insect hemolymph contains circulating cells called hemocytes.
                  • Some hemocytes can phagocytose microbes, while others can form a cellular capsule around large parasites.
                  • Other hemocytes secrete antimicrobial peptides that bind to and destroy pathogens.
                  • Sponge cells can distinguish self from nonself cells.
                  • Phagocytic cells of earthworms show immunological memory, responding more quickly to a particular foreign tissue the second time it is encountered.

                  Concept 43.2 In acquired immunity, lymphocytes provide specific defenses against infection

                  • While microorganisms are under assault by phagocytic cells, the inflammatory response, and antimicrobial proteins, they inevitably encounter lymphocytes, the key cells of acquired immunity, the body’s second major kind of defense.
                  • As macrophages and dendritic cells phagocytose microbes, they secrete certain cytokines that help activate lymphocytes and other cells of the immune system.
                    • Thus the innate and acquired defenses interact and cooperate with each other.
                    • Most antigens are large molecules such as proteins or polysaccharides.
                    • Most are cell-associated molecules that protrude from the surface of pathogens or transplanted cells.
                    • A lymphocyte actually recognizes and binds to a small portion of an antigen called an epitope.

                    Lymphocytes provide the specificity and diversity of the immune system.

                    • The vertebrate body is populated by two main types of lymphocytes: B lymphocytes (B cells) and T lymphocytes (T cells).
                      • Both types of lymphocytes circulate throughout the blood and lymph and are concentrated in the spleen, lymph nodes, and other lymphatic tissue.
                      • A single B or T cell bears about 100,000 identical antigen receptors.
                      • A region in the tail portion of the molecule, the transmembrane region, anchors the receptor in the cell’s plasma membrane.
                      • A short region at the end of the tail extends into the cytoplasm.
                      • B cell receptors are often called membrane antibodies or membrane immunoglobulins.
                      • Depending on their source, peptide antigens are handled by a different class of MHC molecule and recognized by a particular subgroup of T cells.
                        • Class I MHC molecules, found on almost all nucleated cells of the body, bind peptides derived from foreign antigens that have been synthesized within the cell.
                          • ? Any body cell that becomes infected or cancerous can display such peptide antigens by virtue of its class I MHC molecules.
                          • ? Class I MHC molecules displaying bound peptide antigens are recognized by a subgroup of T cells called cytotoxic T cells.
                          • In these cells, class II MHC molecules bind peptides derived from foreign materials that have been internalized and fragmented by phagocytosis.
                          • As a result of the large number of different alleles in the human population, most of us are heterozygous for every one of our MHC genes.
                          • Moreover, it is unlikely that any two people, except identical twins, will have exactly the same set of MHC molecules.
                          • The MHC provides a biochemical fingerprint virtually unique to each individual that marks body cells as “self.”

                          Lymphocyte development gives rise to an immune system that distinguishes self from nonself.

                          • Lymphocytes, like all blood cells, originate from pluripotent stem cells in the bone marrow or liver of a developing fetus.
                          • Early lymphocytes are all alike, but they later develop into T cells or B cells, depending on where they continue their maturation.
                          • Lymphocytes that migrate from the bone marrow to the thymus develop into T cells.
                          • Lymphocytes that remain in the bone marrow and continue their maturation there become B cells.
                          • There are three key events in the life of a lymphocyte.
                            • The first two events take place as a lymphocyte matures, before it has contact with any antigen.
                            • The third event occurs when a mature lymphocyte encounters and binds a specific antigen, leading to its activation, proliferation, and differentiation—a process called clonal selection.
                            • The variability of these regions is enormous.
                            • Each person has as many as a million different B cells and 10 million different T cells, each with a specific antigen-binding ability.
                            • These genes consist of numerous coding gene segments that undergo random, permanent rearrangement, forming functional genes that can be expressed as receptor chains.
                            • Genes for the light chain of the B cell receptor and for the alpha and beta chains of the T cell receptor undergo similar rearrangements, but we will consider only the gene coding for the light chain of the B cell receptor.
                            • The immunoglobulin light-chain gene contains a series of 40 variable (V) gene segments separated by a long stretch of DNA from 5 joining (J) gene segments.
                            • Beyond the J gene segments is an intron, followed by a single exon that codes for the constant region of the light chain.
                            • In this state, the light-chain gene is not functional.
                            • However, early in B cell development, a set of enzymes called recombinase link one V gene segment to one J gene segment, forming a single exon that is part V and part J.
                              • Recombinase acts randomly and can link any one of 40 V gene segments to any one of 5 J gene segments.
                              • For the light-chain gene, there are 200 possible gene products (20 V × 5 J).
                              • Once V-J rearrangement has occurred, the gene is transcribed and translated into a light chain with a variable and constant region. The light chains combine randomly with the heavy chains that are similarly produced.
                              • Failure to do this can lead to autoimmune diseases such as multiple sclerosis.

                              Antigens interact with specific lymphocytes, inducing immune responses and immunological memory.

                              • Although it encounters a large repertoire of B cells and T cells, a microorganism interacts only with lymphocytes bearing receptors specific for its various antigenic molecules.
                              • A lymphocyte is “selected” when it encounters a microbe with epitopes matching its receptors.
                                • Selection activates the lymphocyte, stimulating it to divide and differentiate, and eventually to produce two clones of cells.
                                • One clone consists of a large number of effector cells, short-lived cells that combat the same antigen.
                                • The other clone consists of memory cells, long-lived cells bearing receptors for the same antigen.
                                • Each antigen, by binding selectively to specific receptors, activates a tiny fraction of cells from the body’s diverse pool of lymphocytes.
                                • This relatively small number of selected cells gives rise to clones of thousands of cells, all specific for and dedicated to eliminating that antigen.
                                • About 10 to 17 days are required from the initial exposure for the maximum effector cell response.
                                • During this period, selected B cells and T cells generate antibody-producing effector B cells called plasma cells, and effector T cells, respectively.
                                • While this response is developing, a stricken individual may become ill, but symptoms of the illness diminish and disappear as antibodies and effector T cells clear the antigen from the body.
                                • This response is faster (only 2 to 7 days), of greater magnitude, and more prolonged.
                                • In addition, the antibodies produced in the secondary response tend to have greater affinity for the antigen than those secreted in the primary response.
                                • The immune system’s capacity to generate secondary immune responses is called immunological memory, based not only on effector cells, but also on clones of long-lived T and B memory cells.
                                  • These memory cells proliferate and differentiate rapidly when they later contact the same antigen.

                                  Concept 43.3 Humoral and cell-mediated immunity defend against different types of threats

                                  • The immune system can mount two types of responses to antigens: a humoral response and a cell-mediated response.
                                    • Humoral immunity involves B cell activation and clonal selection and results in the production of antibodies that circulate in the blood plasma and lymph.
                                      • Circulating antibodies defend mainly against free bacteria, toxins, and viruses in the body fluids.

                                      Helper T lymphocytes function in both humoral and cell-mediated immunity.

                                      • When a helper T cell recognizes a class II MHC molecule-antigen complex on an antigen-presenting cell, the helper T cell proliferates and differentiates into a clone of activated helper T cells and memory helper T cells.
                                      • A surface protein called CD4 binds the side of the class II MHC molecule.
                                      • This interaction helps keep the helper T cell and the antigen-presenting cell joined while activation of the helper T cell proceeds.
                                      • Activated helper T cells secrete several different cytokines that stimulate other lymphocytes, thereby promoting cell-mediated and humoral responses.
                                      • Dendritic cells are important in triggering a primary immune response.
                                        • They capture antigens, migrate to the lymphoid tissues, and present antigens, via class II MHC molecules, to helper T cells.

                                        In the cell-mediated response, cytotoxic T cells counter intracellular pathogens.

                                        • Antigen-activated cytotoxic T lymphocytes kill cancer cells and cells infected by viruses and other intracellular pathogens.
                                        • Fragments of nonself proteins synthesized in such target cells associate with class I MHC molecules and are displayed on the cell surface, where they can be recognized by cytotoxic T cells.
                                          • This interaction is greatly enhanced by the T surface protein CD8 that helps keep the cells together while the cytotoxic T cell is activated.
                                          • The death of the infected cell not only deprives the pathogen of a place to reproduce, but also exposes it to circulating antibodies, which mark it for disposal.
                                          • Once activated, cytotoxic T cells kill other cells infected with the same pathogen.
                                          • Because tumor cells carry distinctive molecules not found on normal cells, they are identified as foreign by the immune system.
                                          • Class I MHC molecules on a tumor cell present fragments of tumor antigens to cytotoxic T cells.
                                          • Interestingly, certain cancers and viruses actively reduce the amount of class I MHC protein on affected cells so that they escape detection by cytotoxic T cells.
                                          • The body has a backup defense in the form of natural killer cells, part of the nonspecific defenses, which lyse virus-infected and cancer cells.

                                          In the humoral response, B cells make antibodies against extracellular pathogens.

                                          • Antigens that elicit a humoral immune response are typically proteins and polysaccharides present on the surface of bacteria or transplanted tissue.
                                          • The activation of B cells is aided by cytokines secreted by helper T cells activated by the same antigen.
                                            • These B cells proliferate and differentiate into a clone of antibody-secreting plasma cells and a clone of memory B cells.
                                            • These include the polysaccharides of many bacterial capsules and the proteins of the bacterial flagella.
                                            • These antigens bind simultaneously to a number of membrane antibodies on the B cell surface.
                                            • This stimulates the B cell to generate antibody-secreting plasma cells without the help of cytokines.
                                            • While this response is an important defense against many bacteria, it generates a weaker response than T-dependent antigens and generates no memory cells.
                                            • Each plasma cell is estimated to secrete about 2,000 antibody molecules per second over the cell’s 4- to 5-day life span.
                                            • A secreted antibody has the same general Y-shaped structure as a B cell receptor, but lacks a transmembrane region that would anchor it to a plasma membrane.
                                            • In addition, for some humans, the proteins of foreign substances such as pollen or bee venom act as antigens that induce an allergic, or hypersensitive, humoral response.
                                            • Two classes exist primarily as polymers of the basic antibody molecule: IgM as a pentamer and IgA as a dimmer.
                                            • The other three classes—IgG, IgE, and IgD—exist exclusively as monomers,
                                            • Some antibody tools are polyclonal, the products of many different clones of B cells, each specific for a different epitope.
                                            • Others are monoclonal, prepared from a single clone of B cells grown in culture.
                                              • These cells produce monoclonal antibodies, specific for the same epitope on an antigen.
                                              • These have been used to tag specific molecules.
                                              • For example, toxin-linked antibodies search and destroy tumor cells.
                                              • In viral neutralization, antibodies bind to proteins on the surface of a virus, blocking the virus’s ability to infect a host cell.
                                              • In opsonization, the bound antibodies enhance macrophage attachment to and phagocytosis of the microbes. Neither the B cell receptor for an antigen nor the secreted antibody actually binds to an entire antigen molecule.
                                              • Agglutination is possible because each antibody molecule has at least two antigen-binding sites.
                                              • IgM can link together five or more viruses or bacteria.
                                              • These large complexes are readily phagocytosed by macrophages.
                                              • The first complement component links two bound antibodies and is activated, initiating the cascade.
                                                • Ultimately, complement proteins generate a membrane attack complex (MAC), which forms a pore in the bacterial membrane, resulting in cell lysis.

                                                Immunity can be achieved naturally or artificially.

                                                • Immunity conferred by recovering from an infectious disease such as chicken pox is called active immunity because it depends on the response of the infected person’s own immune system.
                                                  • Active immunity can be acquired naturally or artificially, by immunization, also known as vaccination.
                                                  • Vaccines include inactivated bacterial toxins, killed microbes, parts of microbes, viable but weakened microbes, and even genes encoding microbial proteins.
                                                  • These agents can act as antigens, stimulating an immune response and, more important, producing immunological memory.
                                                  • Routine immunization of infants and children has dramatically reduced the incidence of infectious diseases such as measles and whooping cough, and has led to the eradication of smallpox, a viral disease.
                                                  • Unfortunately, not all infectious agents are easily managed by vaccination.
                                                    • For example, the emergence of new strains of pathogens with slightly altered surface antigens complicates development of vaccines against some microbes, such as the parasite that causes malaria.
                                                    • This occurs naturally when IgG antibodies of a pregnant woman cross the placenta to her fetus.
                                                    • In addition, IgA antibodies are passed from mother to nursing infant in breast milk.
                                                    • Passive immunity persists as long as these antibodies last, a few weeks to a few months.
                                                      • This protects the infant from infections until the baby’s own immune system has matured.
                                                      • This confers short-term, but immediate, protection against that disease.
                                                      • For example, a person bitten by a rabid animal may be injected with antibodies against rabies virus because rabies may progress rapidly, and the response to an active immunization could take too long to save the life of the victim.
                                                        • Most people infected with rabies virus are given both passive immunizations (the immediate defense) and active immunizations (a longer-term defense).

                                                        Concept 43.4 The immune system’s ability to distinguish self from nonself limits tissue transplantation

                                                        • In addition to attacking pathogens, the immune system will also attack cells from other individuals.
                                                          • For example, a skin graft from one person to a nonidentical individual will look healthy for a day or two, but it will then be destroyed by immune responses.
                                                          • Interestingly, a pregnant woman does not reject the fetus as a foreign body. Apparently, the structure of the placenta is the key to this acceptance.
                                                          • In the ABO blood groups, an individual with type A blood has A antigens on the surface of red blood cells.
                                                            • This is not recognized as an antigen by the “owner,” but it can be identified as foreign if placed in the body of another individual.
                                                            • These antibodies arise in response to bacteria (normal flora) that have epitopes very similar to blood group antigens.
                                                            • Thus, an individual with type A blood does not make antibodies to A-like bacterial epitopes—these are considered self—but that person does make antibodies to B-like bacterial epitopes.
                                                            • If a person with type A blood receives a transfusion of type B blood, the preexisting anti-B antibodies will induce an immediate and devastating transfusion reaction.
                                                            • Each response is like a primary response, and it generates IgM anti-blood-group antibodies, not IgG.
                                                            • This is fortunate, because IgM antibodies do not cross the placenta, where they may harm a developing fetus with a blood type different from its mother’s.
                                                            • This situation arises when a mother that is Rh-negative (lacks the Rh factor) has a fetus that is Rh-positive, having inherited the factor from the father.
                                                            • If small amounts of fetal blood cross the placenta late in pregnancy or during delivery, the mother mounts a humoral response against the Rh factor.
                                                            • The danger occurs in subsequent Rh-positive pregnancies, when the mother’s Rh-specific memory B cells produce IgG antibodies that can cross the placenta and destroy the red blood cells of the fetus.
                                                            • She is, in effect, passively immunized (artificially) to eliminate the Rh antigen before her own immune system responds and generates immunological memory against the Rh factor, endangering her future Rh-positive babies.
                                                            • Because MHC creates a unique protein fingerprint for each individual, foreign MHC molecules are antigenic, inducing immune responses against the donated tissue or organ.
                                                            • To minimize rejection, attempts are made to match MHC of tissue donor and recipient as closely as possible.
                                                              • In the absence of identical twins, siblings usually provide the closest tissue-type match.
                                                              • However, this strategy leaves the recipient more susceptible to infection and cancer during the course of treatment.
                                                              • More selective drugs, which suppress helper T cell activation without crippling nonspecific defense or T-independent humoral responses, have greatly improved the success of organ transplants.
                                                              • Bone marrow transplants are used to treat leukemia and other cancers as well as various hematological diseases.
                                                              • Prior to the transplant, the recipient is typically treated with irradiation to eliminate the recipient’s immune system, eliminating all abnormal cells and leaving little chance of graft rejection.
                                                              • However, the donated marrow, containing lymphocytes, may react against the recipient, producing graft versus host reaction, unless well matched.

                                                              Concept 43.5 Exaggerated, self-directed, or diminished immune responses can cause disease

                                                              • Malfunctions of the immune system can produce effects ranging from the minor inconvenience of some allergies to the serious and often fatal consequences of certain autoimmune and immunodeficiency diseases.
                                                              • Allergies are hypersensitive (exaggerated) responses to certain environmental antigens, called allergens.
                                                                • One hypothesis to explain the origin of allergies is that they are evolutionary remnants of the immune system’s response to parasitic worms.
                                                                • The humoral mechanism that combats worms is similar to the allergic response that causes such disorders as hay fever and allergic asthma.
                                                                • Hay fever, for example, occurs when plasma cells secrete IgE specific for pollen allergens.
                                                                • Some IgE antibodies attach by their tails to mast cells present in connective tissue, without binding to the pollen.
                                                                • Later, when pollen grains enter the body, they attach to the antigen-binding sites of mast cell-associated IgE, cross-linking adjacent antibody molecules.
                                                                • These inflammatory events lead to typical allergy symptoms: sneezing, runny nose, tearing eyes, and smooth muscle contractions that can result in breathing difficulty.
                                                                • Antihistamines diminish allergy symptoms by blocking receptors for histamine.
                                                                • Anaphylactic shock results when widespread mast cell degranulation triggers abrupt dilation of peripheral blood vessels, causing a precipitous drop in blood pressure.
                                                                  • Death may occur within minutes.
                                                                  • In systemic lupus erythematosus (lupus), the immune system generates antibodies against various self-molecules, including histones and DNA released by the normal breakdown of body cells.
                                                                    • Lupus is characterized by skin rashes, fever, arthritis, and kidney dysfunction.
                                                                    • In MS, T cells reactive against myelin infiltrate the central nervous system and destroy the myelin sheath that surrounds some neurons.
                                                                    • People with MS experience a number of serious neurological abnormalities.
                                                                    • It was thought that people with autoimmune diseases had self-reactive lymphocytes that escaped elimination during their development.
                                                                    • We now know that healthy people also have lymphocytes with the capacity to react against self, but these cells are inhibited from inducing an autoimmune reaction by several regulatory mechanisms.
                                                                    • Autoimmune disease likely arises from some failure in immune regulation, perhaps linked with particular MHC alleles.
                                                                    • For individuals with this disease, long-term survival requires a bone marrow transplant that will continue to supply functional lymphocytes.
                                                                    • Several gene therapy approaches are in clinical trials to attempt to reverse SCID.
                                                                    • Recent successes include a child with SCID who received gene therapy in 2002 when she was 2 years old. In 2004, her T cells and B cells were still functioning normally.
                                                                    • For example, certain cancers suppress the immune system. An example is Hodgkin’s disease, which damages the lymphatic system.
                                                                    • For example, hormones secreted by the adrenal glands during stress affect the number of white blood cells and may suppress the immune system in other ways.
                                                                    • Similarly, some neurotransmitters secreted when we are relaxed and happy may enhance immunity.
                                                                    • Physiological evidence also points to an immune system–nervous system link based on the presence of neurotransmitter receptors on the surfaces of lymphocytes and a network of nerve fibers that penetrates deep into the thymus.

                                                                    AIDS is an immunodeficiency disease caused by a virus.

                                                                    • In 1981, increased rates of two rare diseases, Kaposi’s sarcoma, a cancer of the skin and blood vessels, and pneumonia caused by the protozoan Pneumocystis carinii, were the first signals to the medical community of a new threat to humans, later known as acquired immunodeficiency syndrome, or AIDS.
                                                                      • Both conditions were previously known to occur mainly in severely immunosuppressed individuals.
                                                                      • People with AIDS are susceptible to opportunistic diseases.
                                                                      • Because AIDS arises from the loss of helper T cells, both humoral and cell-mediated immune responses are impaired.
                                                                      • The main receptor for HIV on helper T cells is the cell’s CD4 molecule.
                                                                      • In addition to CD4, HIV requires a second cell-surface protein, a coreceptor.
                                                                      • However, these drugs are very expensive and not available to all infected people, especially in developing countries.
                                                                      • In addition, the mutational changes that occur with each round of virus reproduction can generate drug-resistant strains of HIV.
                                                                      • Transmission of HIV requires the transfer of body fluids containing infected cells, such as semen or blood, from person to person.
                                                                      • In December 2003, the Joint UN Program on AIDS estimated that 40 million people worldwide are living with HIV/AIDS. The best approach for slowing the spread of HIV is to educate people about the practices that lead to transmission, such as using dirty needles or having unprotected intercourse.

                                                                      Lecture Outline for Campbell/Reece Biology, 7th Edition, © Pearson Education, Inc. 43-9


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