Cytotoxic T cells are important effectors of anti-tumor immunity. While tumor killing is ascribed to CD8+ T cell function, pre-clinical and clinical studies have identified intra-tumoral CD4+ T cells ...that possess cytotoxic programs and can directly kill cancer cells. Cytotoxic CD4+ T cells are found in other disease settings including infection and autoimmunity. Here, we review the phenotypic and functional characteristics of cytotoxic CD4+ T cells in non-cancer and cancer contexts. We conduct a comparative examination of cytolytic mechanisms of cytotoxic CD4+ T cells across disease states and synthesize features that define these cells independent of context. We discuss regulatory mechanisms driving ontogeny and effector function and evidence for the clinical relevance of cytotoxic CD4+ T cells in cancer. In this context, we highlight important gaps in understanding in the biology of cytotoxic CD4+ T cells as well as the potential use of these cells in immunotherapies for specific cancers.
CD4+ T cells with cytotoxic features and potential are found in various human cancers. Oh and Fong review the phenotypic and functional characteristics of cytotoxic CD4+ T cells in non-cancer and cancer contexts and discuss the current understanding of the mechanism that regulate the differentiation and function of these cells as well as the evidence for their clinical relevance.
Immunotherapy using chimeric antigen receptor (CAR)‐engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models ...are needed that allow to test CAR‐mediated cytotoxicity in a tissue‐like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3‐dimensional (3D) patient‐derived colon organoids. Luciferase‐based measurement served as a quantitative read‐out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR‐engineered NK‐92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen‐specific cytotoxicity was studied with CAR‐NK‐92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor‐specific activity. Taken together, we report a sensitive in vitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner.
Synopsis
Establishment of a preclinical in vitro organoid model allows testing the efficacy and safety of solid tumor cancer immunotherapy with chimeric antigen receptor (CAR)‐engineered lymphocytes in a tissue‐like environment. 3D live‐imaging of single colorectal cancer (CRC) organoids is highly sensitive and allows to validate treatment assumptions to minimize therapy side effects.
Co‐culture of human natural killer (NK) cells with normal or CRC organoids on an ECM layer allows stable effector‐target cell interaction.
Luciferase‐based cytotoxicity assessment is benchmarked with EPCAM‐directed effector NK cells.
Low expression of EGFRvIII neoantigen is sufficient to render CRC organoids susceptible to lysis.
CAR targeting of FRIZZLED receptors in intestinal organoids reveals off‐target cytotoxicity against wild‐type epithelia of human origin.
A preclinical organoid co‐culture allows testing of immunotherapy with chimeric antigen receptor (CAR)‐engineered lymphocytes in a personalised tissue‐like environment.
Summary Checkpoint blockade immunotherapy has received mainstream attention as a result of striking and durable clinical responses in some patients with metastatic disease and a reasonable response ...rate in many tumour types. The activity of checkpoint blockade immunotherapy is not restricted to melanoma or lung cancer, and additional indications are expected in the future, with responses already reported in renal cancer, bladder cancer, and Hodgkin's lymphoma among many others. Additionally, the interactions between radiation and the immune system have been investigated, with several studies describing the synergistic effects on local and distant tumour control when radiation therapy is combined with immunotherapy. Clinical enthusiasm for this approach is strengthened by the many ongoing trials combining immunotherapy with definitive and palliative radiation. Herein, we discuss the biological and mechanistic rationale behind combining radiation with checkpoint blockade immunotherapy, with a focus on the preclinical data supporting this potentially synergistic combination. We explore potential hypotheses and important considerations for clinical trial designs. Finally, we reintroduce the notion of radiosensitising immunotherapy, akin to radiosensitising chemotherapy, as a potential definitive therapeutic modality.
Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition. Primary HLH occurs early in life as a result of monogenic biallelic mutations affecting lymphocyte ...cytotoxicity. Secondary HLH occurs mostly in adults secondary to infection, lymphoma, or rheumatic disease. In this latter setting, lymphocyte cytotoxicity status is not known. We conducted a systematic evaluation of natural killer (NK) cell cytotoxicity in adult patients with secondary HLH. Adult patients with secondary HLH were prospectively studied ex vivo for total lymphocyte count and subtype, NK cell phenotype, perforin expression and degranulation, and natural or antibody-dependent cell cytotoxicity, in comparison with patients affected by the same underlying disease without HLH (disease controls DCs) and with healthy controls (HCs). Screening for variants of cytotoxity genes was systematically performed. 68 patients were included in the HLH group and 34 each in the DC and HC groups. In HLH patients, severe and transient lymphopenia, activated NK cell phenotype (eg, increased CD69, ICAM-1, HLADR, and CCR5 expression), and decreased capacity of interferon γ production were observed; mean perforin expression was normal; and degranulation tests and NK cell cytotoxicity were not different from those in DCs. A monoallelic variant of uncertain significance affecting a lymphocyte cytotoxicity gene or the perforin variant A91V was observed in almost 50% of the patients. We detected no major intrinsic cytotoxicity dysfunction in secondary HLH patients compared with DCs and no predicted pathogenic gene variant. The activated NK phenotype profile associated with decreased interferon γ production seems similar to those of other hyperinflammatory diseases such as sepsis or systemic juvenile idiopathic arthritis.
•Secondary HLH is not due to primary cytotoxicity defects, but mimics other hyperinflammatory syndromes associated with a cytokine storm.
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CD73 inhibits antitumor immunity through the activation of adenosine receptors expressed on multiple immune subsets. CD73 also enhances tumor metastasis, although the nature of the immune subsets and ...adenosine receptor subtypes involved in this process are largely unknown. In this study, we revealed that A ₂A/A ₂B receptor antagonists were effective in reducing the metastasis of tumors expressing CD73 endogenously (4T1.2 breast tumors) and when CD73 was ectopically expressed (B16F10 melanoma). A ₂A⁻/⁻ mice were strongly protected against tumor metastasis, indicating that host A ₂A receptors enhanced tumor metastasis. A ₂A blockade enhanced natural killer (NK) cell maturation and cytotoxic function in vitro, reduced metastasis in a perforin-dependent manner, and enhanced NK cell expression of granzyme B in vivo, strongly suggesting that the antimetastatic effect of A ₂A blockade was due to enhanced NK cell function. Interestingly, A ₂B blockade had no effect on NK cell cytotoxicity, indicating that an NK cell-independent mechanism also contributed to the increased metastasis of CD73 ⁺ tumors. Our results thus revealed that CD73 promotes tumor metastasis through multiple mechanisms, including suppression of NK cell function. Furthermore, our data strongly suggest that A ₂A or A ₂B antagonists may be useful for the treatment of metastatic disease. Overall, our study has potential therapeutic implications given that A ₂A/A ₂B receptor antagonists have already entered clinical trials in other therapeutic settings.
Isatuximab is a monoclonal antibody targeting the transmembrane receptor and ectoenzyme CD38, a protein highly expressed on hematological malignant cells, including those in multiple myeloma (MM). ...Upon binding to CD38-expressing MM cells, isatuximab is thought to induce tumor cell killing via fragment crystallizable (Fc)-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), as well as via direct Fc-independent mechanisms. Here, these mechanisms of action were investigated in MM and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in peripheral blood mononuclear cells derived from healthy donors, and in MM patient-derived samples. Our findings show that isatuximab-mediated cytotoxicity occurred primarily via ADCC and ADCP in MM cell lines and via ADCC and apoptosis in DLBCL cell lines expressing high levels of CD38. We identified the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and MM cell-secreted transforming growth factor-beta (TGF-β) as tumor cell-related features that could suppress CD38-mediated ADCC. Furthermore, we established that isatuximab can directly activate natural killer (NK) cells and promote NK cell-mediated cytotoxicity via crosslinking of CD38 and CD16. Finally, isatuximab-induced CDC was observed in cell lines with high CD38 receptor density (>250,000 molecules/cell) and limited expression of inhibitory complement regulatory proteins (CD46, CD55, and CD59; <50,000 molecules/cell). Taken together, our findings highlight mechanistic insights for isatuximab and provide support for a range of combination therapy approaches that could be tested for isatuximab in the future.
Immune system-mediated eradication of neoplastic cells requires induction of a strong long-lasting antitumor T-cell response. However, generation of tumor-specific effector T cells does not ...necessarily result in tumor clearance. CTL must first be able to migrate to the tumor site, infiltrate the tumor tissue, and interact with the target to finally trigger effector functions indispensable for tumor destruction. Chemokines are involved in circulation, homing, retention, and activation of immunocompetent cells. Although some of them are known to contribute to tumor growth and metastasis, others are responsible for changes in the tumor microenvironment that lead to extensive infiltration of lymphocytes, resulting in tumor eradication. Given their chemoattractive and activating properties, a role for chemokines in the development of the effector phase of the antitumor immune response has been suggested. Here, we emphasize the role of the chemokine-chemokine receptor network at multiple levels of the T-cell-mediated antitumor immune response. The identification of chemokine-dependent molecular mechanisms implicated in tumor-specific CTL trafficking, retention, and regulation of their in situ effector functions may offer new perspectives for development of innovative immunotherapeutic approaches to cancer treatment.
T cell receptor activation of naïve CD8
T lymphocytes initiates their maturation into effector cytotoxic T lymphocytes (CTLs), which can kill cancer and virally infected cells. Although CTLs show an ...increased reliance on glycolysis upon acquisition of effector function, we found an essential requirement for mitochondria in target cell–killing. Acute mitochondrial depletion in USP30 (ubiquitin carboxyl-terminal hydrolase 30)–deficient CTLs markedly diminished killing capacity, although motility, signaling, and secretion were all intact. Unexpectedly, the mitochondrial requirement was linked to mitochondrial translation, inhibition of which impaired CTL killing. Impaired mitochondrial translation triggered attenuated cytosolic translation, precluded replenishment of secreted killing effectors, and reduced the capacity of CTLs to carry out sustained killing. Thus, mitochondria emerge as a previously unappreciated homeostatic regulator of protein translation required for serial CTL killing.
Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals ...during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (
Prf1) transcription, and repressed re-expression of memory CTL markers
Bcl6 and IL-7Rα. Binding of Eomes and STAT5 to
Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the
Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Rα and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Rα repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Rα-deficient effector CD8
+ T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1
+ and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.
► Strong, sustained IL-2 signals promote effector and repress memory CTL development ► IL-2Rα is required for effective effector CTL differentiation during viral infection ► IL-2 induces eomesodermin and perforin gene transcription by recruiting RNA Pol II ► Complex interplay between inflammation and IL-2 for gene regulation in CD8
+ T cells
Synonymous with secondary hemophagocytic lymphohistiocytosis, macrophage activation syndrome (MAS) is a term used by rheumatologists to describe a potentially life-threatening complication of ...systemic inflammatory disorders, most commonly systemic juvenile idiopathic arthritis (sJIA) and systemic lupus erythematosus (SLE). Clinical and laboratory features of MAS include sustained fever, hyperferritinemia, pancytopenia, fibrinolytic coagulopathy, and liver dysfunction. Soluble interleukin-2 receptor alpha chain (sCD25) and sCD163 may be elevated, and histopathology often reveals characteristic increased hemophagocytic activity in the bone marrow (and other tissues), with positive CD163 (histiocyte) staining. A common hypothesis as to the pathophysiology of many cases of MAS proposes a defect in lymphocyte cytolytic activity. Specific heterozygous gene mutations in familial HLH-associated cytolytic pathway genes (e.g.,
) have been linked to a substantial subset of MAS patients. In addition, the pro-inflammatory cytokine environment, particularly IL-6, has been shown to decrease NK cell cytolytic function. The inability of NK cells and cytolytic CD8 T cells to lyse infected and otherwise activated antigen presenting cells results in prolonged cell-to-cell (innate and adaptive immune cells) interactions and amplification of a pro-inflammatory cytokine cascade. The cytokine storm results in activation of macrophages, causing hemophagocytosis, as well as contributing to multi-organ dysfunction. In addition to macrophages, dendritic cells likely play a critical role in antigen presentation to cytolytic lymphocytes, as well as contributing to cytokine expression. Several cytokines, including tumor necrosis factor, interferon-gamma, and numerous interleukins (i.e., IL-1, IL-6, IL-18, IL-33), have been implicated in the cytokine cascade. In addition to broadly immunosuppressive therapies, novel cytokine targeted treatments are being explored to dampen the overly active immune response that is responsible for much of the pathology seen in MAS.
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