Combined PD-L1 and TIM3 blockade improves expansion of fit human CD8+ antigen-specific T cells for adoptive immunotherapy.

Antigen-specific T cell expansion ex vivo followed by adoptive transfer enables targeting of a multitude of microbial and cancer antigens. However, clinical-scale T cell expansion from rare precursors requires repeated stimulation, which may lead to T cell dysfunction and limited therapeutic potential. We used a clinically compliant protocol to expand Epstein-Barr virus (EBV) and Wilms tumor 1 (WT1) antigen-specific CD8+ T cells, and leveraged T cell exhaustion-associated inhibitory receptor blockade to improve T cell expansion. Several inhibitory receptors were expressed early by ex vivo-expanded antigen-specific CD8+ T cells, including PD-1 and TIM3, with co-expression matching evidence of T cell dysfunction as the cultures progressed. Introduction of anti-PD-L1 and anti-TIM3 blockade in combination (but not individually) to the culture led to markedly improved antigen-specific T cell expansion without inducing T cell dysfunction. Single-cell RNA sequencing (RNA-seq) and T cell receptor (TCR) repertoire profiling revealed that double blockade does not impart specific transcriptional programs in T cells or alterations in TCR repertoires. However, combined blockade may affect gene expression in a minority of clonotypes in a donor-specific fashion. We conclude that antigen-specific CD8+ T cell manufacturing can be improved by using TIM3 and PD-L1/PD-1 axis blockade in combination. This approach is readily applicable to several adoptive immunotherapy strategies.

p16INK4a Regulates Cellular Senescence in PD-1-Expressing Human T Cells.

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy.

T-Cell Dysfunction as a Limitation of Adoptive Immunotherapy: Current Concepts and Mitigation Strategies.

Over the last decades, cellular immunotherapy has revealed its curative potential. However, inherent physiological characteristics of immune cells can limit the potency of this approach. Best defined in T cells, dysfunction associated with terminal differentiation, exhaustion, senescence, and activation-induced cell death, undermine adoptive cell therapies. In this review, we concentrate on how the multiple mechanisms that articulate the various forms of immune dysfunction impact cellular therapies primarily involving conventional T cells, but also other lymphoid subtypes. The repercussions of immune cell dysfunction across the full life cycle of cell therapy, from the source material, during manufacturing, and after adoptive transfer, are discussed, with an emphasis on strategies used during ex vivo manipulations to limit T-cell dysfunction. Applicable to cellular products prepared from native and unmodified immune cells, as well as genetically engineered therapeutics, the understanding and potential modulation of dysfunctional features are key to the development of improved cellular immunotherapies.

T-Cell Immunotherapies Targeting Histocompatibility and Tumor Antigens in Hematological Malignancies.

Over the last decades, T-cell immunotherapy has revealed itself as a powerful, and often curative, strategy to treat blood cancers. In hematopoietic cell transplantation, most of the so-called graft-vs.-leukemia (GVL) effect hinges on the recognition of histocompatibility antigens that reflect immunologically relevant genetic variants between donors and recipients. Whether other variants acquired during the neoplastic transformation, or the aberrant expression of gene products can yield antigenic targets of similar relevance as the minor histocompatibility antigens is actively being pursued. Modern genomics and proteomics have enabled the high throughput identification of candidate antigens for immunotherapy in both autologous and allogeneic settings. As such, these major histocompatibility complex-associated tumor-specific (TSA) and tumor-associated antigens (TAA) can allow for the targeting of multiple blood neoplasms, which is a limitation for other immunotherapeutic approaches, such as chimeric antigen receptor (CAR)-modified T cells. We review the current strategies taken to translate these discoveries into T-cell therapies and propose how these could be introduced in clinical practice. Specifically, we discuss the criteria that are used to select the antigens with the greatest therapeutic value and we review the various T-cell manufacturing approaches in place to either expand antigen-specific T cells from the native repertoire or genetically engineer T cells with minor histocompatibility antigen or TSA/TAA-specific recombinant T-cell receptors. Finally, we elaborate on the current and future incorporation of these therapeutic T-cell products into the treatment of hematological malignancies.

TGFß Programs Central Memory Differentiation in Ex Vivo-Stimulated Human T Cells.

The adoptive transfer of ex vivo-expanded T cells is a promising approach to treat several malignancies. Several lines of evidence support that the infusion of T cells with early memory features, capable of expanding and persisting after transfer, are associated with better outcomes. We report herein that exposure to exogenous TGFß during human T-cell stimulation ex vivo leads to the accumulation of early/central memory (Tcm) cells. Exposure to TGFß suppressed the expression of BLIMP-1, a key orchestrator of effector T-cell differentiation, and led to the upregulation of the memory-associated transcription factor ID3. Accordingly, this was associated with an early memory transcriptional signature in both CD4+ and CD8+ T-cell subsets. The T cells stimulated in the presence of TGFß expanded normally, and displayed polyfunctional features and no suppressive activity. The adoptive transfer of ex vivo-stimulated T cells into immunodeficient mice confirmed that TGFß-conditioned cells had an enhanced capacity to persist and mediate xenogeneic graft-versus-host disease, as predicted by their early T-cell memory phenotype. Chimeric antigen receptor-expressing T cells generated in the presence of exogenous TGFß were cytotoxic and more effective at controlling tumor growth in immunodeficient animals. This work unveils a new role for TGFß in memory T-cell differentiation and indicates that TGFß signaling may be harnessed to program Tcm differentiation in the context of ex vivo T-cell stimulation for adoptive immunotherapy in humans.

Defining novel parameters for the optimal priming and expansion of minor histocompatibility antigen-specific T cells in culture.

BACKGROUND: Adoptive transfer of minor histocompatibility antigen (MiHA)-specific T cells is a promising therapy for patients with hematological cancers. However, the efficacy of the transferred cells is hampered by the acquisition of terminal effector differentiation and exhaustion features during expansion in vitro thus preventing their function and persistence in vivo. Yet, the factors that induce T-cell differentiation and functional impairment in culture remain poorly defined and are likely to vary depending on the method used for expansion. METHODS: Using the clinically relevant HLA-A0201-restricted MiHA HA-1 as well as reagents and procedures that are readily transferable to a clinical environment, we designed a novel culture protocol and defined how exhaustion features appeared in function of time. The optimal time points for the expansion of "fit" MiHA-specific T cells were delineated using phenotypic and functional assessments including KLRG-1 and PD-1 surface markers as well as Ki67 staining and cytokine secretion assays. RESULTS: Following a priming phase, an enrichment step and a rapid expansion stage, our method generates MiHA-specific T-cell lines. Evidence of phenotypic and functional dysfunction appear in function of culture duration, but display different characteristics following the extension of the priming or rapid expansion phases. While repeated antigen exposure during the priming phase induced the decline of the antigen-specific population and the expression of PD-1 and KLRG-1 on antigen-specific CD8+ T cells, the prolongation of an antigen-free expansion phase induced proliferation arrest and the relative loss of antigen-specific cells without impairing polyfunctional cytokine secretion or inducing PD-1 and KLRG-1 expression. A similar pattern was also observed after stimulating a virus-specific memory repertoire, except for the more rapid acquisition of exhaustion features upon repeated antigen exposure. CONCLUSION: Our results offer novel insights on the impact of culture duration on the acquisition of T-cell exhaustion features. Using a new clinical-compliant protocol, we define critical parameters to monitor in order to optimally differentiate and expand MiHA-specific T cells in culture prior to adoptive transfer.

Early exposure to interleukin-21 limits rapidly generated anti-Epstein-Barr virus T-cell line differentiation.

BACKGROUND AIMS: The adoptive transfer of ex vivo-expanded Epstein-Barr virus (EBV)-specific T-cell lines is an attractive strategy to treat EBV-related neoplasms. Current evidence suggests that for adoptive immunotherapy in general, clinical responses are superior if the transferred cells have not reached a late or terminal effector differentiation phenotype before infusion. The cytokine interleukin (IL)-21 has shown great promise at limiting late T-cell differentiation in vitro, but this remains to be demonstrated in anti-viral T-cell lines. METHODS: We adapted a clinically validated protocol to rapidly generate EBV-specific T-cell lines in 12 to 14 days and tested whether the addition of IL-21 at the initiation of the culture would affect T-cell expansion and differentiation. RESULTS: We generated clinical-scale EBV-restricted T-cell line expansion with balanced T-cell subset ratios. The addition of IL-21 at the beginning of the culture decreased both T-cell expansion and effector memory T-cell accumulation, with a relative increase in less-differentiated T cells. Within CD4 T-cell subsets, exogenous IL-21 was notably associated with the cell surface expression of CD27 and high KLF2 transcript levels, further arguing for a role of IL-21 in the control of late T-cell differentiation. CONCLUSIONS: Our results show that IL-21 has profound effects on T-cell differentiation in a rapid T-cell line generation protocol and as such should be further explored as a novel approach to program anti-viral T cells with features associated with early differentiation and optimal therapeutic efficacy.

Expression of Viral Antigen by the Liver Leads to Chronic Infection Through the Generation of Regulatory T Cells.

BACKGROUND & AIMS: The constant exposure of the liver to food and bacterial antigens through the mesenteric circulation requires it to maintain tolerance while preserving the ability to mount an effective immune response against pathogens. We investigated the contribution of the liver's tolerogenic nature on the establishment of chronic viral infections. METHODS: TTR-NP mice, which express the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) specifically in hepatocytes under control of a modified transthyretin (TTR) promoter, were infected with the Armstrong (Arm) or WE acute strains of LCMV. RESULTS: The infection persisted for at least 147 days in TTR-NP mice. Expression of NP by the liver induced a strong peripheral tolerance against NP that was mediated by interleukin-10-secreting CD4+ regulatory T cells, leading to high PD-1 (programmed death-1) expression and reduced effector function of virus-specific T cells. Despite an active immune response against LCMV, peripheral tolerance against a single viral protein was sufficient to induce T-cell exhaustion and chronic LCMV Armstrong (Arm) or WE infection by limiting the antiviral T-cell response in an otherwise immunocompetent host. Regulatory T-cell depletion of chronically infected TTR-NP mice led to functional restoration of LCMV-specific CD4+ and CD8+ T cell responses and viral clearance. CONCLUSIONS: Expression of a viral antigen by hepatocytes can induce a state of peripheral tolerance mediated by regulatory T cells that can lead to the establishment of a chronic viral infection. Strategies targeting regulatory T cells in patients chronically infected with hepatotropic viruses could represent a promising approach to restore functional antiviral immunity and clear infection.

Role of the complement system in NK cell-mediated antitumor T-cell responses.

The role of the complement system in oncogenesis and tumor progression remains poorly understood. We have recently demonstrated that the induction of a tumor-specific CD8+ T-cell response is improved upon transient inhibition of the complement system, which is coupled to an increased availability of natural killer cells. The complement system may therefore turn out to constitute a promising target for the development of novel anticancer therapeutics.

Transient complement inhibition promotes a tumor-specific immune response through the implication of natural killer cells.

Although the role of the complement system in cancer development has been studied, its involvement in the development of an antitumoral immune response remains poorly understood. Using cobra venom factor (CVF) to inhibit the complement cascade via C3 molecule exhaustion in immunocompetent mice bearing B16gp33 melanoma tumors, we show that transient inhibition of the complement system allowed for the development of a more robust gp33-specific antitumoral CD8(+) T-cell response. This immune response proved to be natural killer (NK) dependent, suggesting an interaction of complement proteins with this cellular subset leading to T lymphocyte activation and enhanced cytotoxic T-cell activity against tumor cells. This study demonstrates for the first time the implication of the complement system in the development of NK-mediated cytotoxic T-cell-dependent antitumoral immune responses. The complement pathway could therefore be a potent therapeutic target to improve NK-dependent antitumoral immune responses in patients with cancer.

The strength of the T cell response against a surrogate tumor antigen induced by oncolytic VSV therapy does not correlate with tumor control.

Cancer therapy using oncolytic viruses has gained interest in the last decade. Vesicular stomatitis virus is an attractive candidate for this alternative treatment approach. The importance of the immune response against tumor antigens in virotherapy efficacy is now well recognized, however, its relative contribution versus the intrinsic oncolytic capacity of viruses has been difficult to evaluate. To start addressing this question, we compared glycoprotein and matrix mutants of vesicular stomatitis virus (VSV), showing different oncolytic potentials for B16/B16gp33 melanoma tumor cells in vitro, with the wild-type virus in their ability to induce tumor-specific CD8(+) T cell responses and control tumor progression in vivo. Despite the fact that wild-type and G mutants induced a stronger gp33-specific immune response compared to the MM51R mutant, all VSV strains showed a similar capacity to slow down tumor progression. The effectiveness of the matrix mutant treatment proved to be CD8(+) dependent and directed against tumor antigens other than gp33 since adoptive transfer of isolated CD8(+) T lymphocytes from treated B16gp33-bearing mice resulted in significant protection of naive mice against challenge with the parental tumor. Remarkably, the VSV matrix mutant induced the upregulation of major histocompatibility class-I antigen at the tumor cell surface thus favoring recognition by CD8(+) T cells. These results demonstrate that VSV mutants induce an antitumor immune response using several mechanisms. A better understanding of these mechanisms will prove useful for the rational design of viruses with improved therapeutic efficacy.

Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5' triphosphate (5'ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5'pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5'pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5'pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5'pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5'pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.

[Vesicular stomatitis virus in the fight against cancer]. / Le virus de la stomatite vésiculaire dans la lutte contre le cancer.

Cancer is a complex disease that affects more and more people around the world. Unfortunately, existing treatments are only partially efficient and often induce major side effects. Thus, the use of viruses to selectively kill cancer cells is a new promising therapeutic approach. Recently, VSV has been used in oncolytic virotherapy because of its capacity to preferentially infect most human tumor cells. However, despite the availability of good oncolytic VSV mutants, the large variability of tumor cell types and the multiple ways in which they can evade viral infection suggests that therapeutic combinations of various viruses will be necessary to efficiently treat most cancers. A better understanding of the infection mechanisms and immune system recruitment by oncolytic viruses will be of great value for the development of safe and efficient strategies for cancer treatment.

Mutations in the glycoprotein of vesicular stomatitis virus affect cytopathogenicity: potential for oncolytic virotherapy.

Vesicular stomatitis virus (VSV) has been widely used to characterize cellular processes, viral resistance, and cytopathogenicity. Recently, VSV has also been used for oncolytic virotherapy due to its capacity to selectively lyse tumor cells. Mutants of the matrix (M) protein of VSV have generally been preferred to the wild-type virus for oncolysis because of their ability to induce type I interferon (IFN) despite causing weaker cytopathic effects. However, due to the large variability of tumor types, it is quite clear that various approaches and combinations of multiple oncolytic viruses will be needed to effectively treat most cancers. With this in mind, our work focused on characterizing the cytopathogenic profiles of four replicative envelope glycoprotein (G) VSV mutants. In contrast to the prototypic M mutant, VSV G mutants are as efficient as wild-type virus at inhibiting cellular transcription and host protein translation. Despite being highly cytopathic, the mutant G(6R) triggers type I interferon secretion as efficiently as the M mutant. Importantly, most VSV G mutants are more effective at killing B16 and MC57 tumor cells in vitro than the M mutant or wild-type virus through apoptosis induction. Taken together, our results demonstrate that VSV G mutants retain the high cytopathogenicity of wild-type VSV, with G(6R) inducing type I IFN secretion at levels similar to that of the M mutant. VSV G protein mutants could therefore prove to be highly valuable for the development of novel oncolytic virotherapy strategies that are both safe and efficient for the treatment of various types of cancer.