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.

UM171-Expanded Cord Blood Transplants Support Robust T Cell Reconstitution with Low Rates of Severe Infections.

Rapid T cell reconstitution following hematopoietic stem cell transplantation (HSCT) is essential for protection against infections and has been associated with lower incidence of chronic graft-versus-host disease (cGVHD), relapse, and transplant-related mortality (TRM). While cord blood (CB) transplants are associated with lower rates of cGVHD and relapse, their low stem cell content results in slower immune reconstitution and higher risk of graft failure, severe infections, and TRM. Recently, results of a phase I/II trial revealed that single UM171-expanded CB transplant allowed the use of smaller CB units without compromising engraftment (www.clinicaltrials.gov, NCT02668315). We assessed T cell reconstitution in patients who underwent transplantation with UM171-expanded CB grafts and retrospectively compared it to that of patients receiving unmanipulated CB transplants. While median T cell dose infused was at least 2 to 3 times lower than that of unmanipulated CB, numbers and phenotype of T cells at 3, 6, and 12 months post-transplant were similar between the 2 cohorts. T cell receptor sequencing analyses revealed that UM171 patients had greater T cell diversity and higher numbers of clonotypes at 12 months post-transplant. This was associated with higher counts of naive T cells and recent thymic emigrants, suggesting active thymopoiesis and correlating with the demonstration that UM171 expands common lymphoid progenitors in vitro. UM171 patients also showed rapid virus-specific T cell reactivity and significantly reduced incidence of severe infections. These results suggest that UM171 patients benefit from rapid T cell reconstitution, which likely contributes to the absence of moderate/severe cGVHD, infection-related mortality, and late TRM observed in this cohort.

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.

Leukoreduction system chambers are a reliable cellular source for the manufacturing of T-cell therapeutics.

BACKGROUND: Following solid organ or hematopoietic cell transplantation, refractory opportunistic viral reactivations are a significant cause of morbidity and mortality but can effectively be controlled by virus-specific T-cell transfer. Among effective and safe strategies is the use of "third-party" (neither from the transplant donor nor recipient) virus-specific T cells that can be manufactured from healthy donors and used as "off-the-shelf" therapies. Leukoreduction system chambers (LRSCs), recovered after routine plateletpheresis, were evaluated as a potential source of peripheral blood mononuclear cells (PBMCs) for the manufacturing of clinical-scale virus-specific T cell. STUDY DESIGN AND METHODS: PBMCs from the same donors obtained either from LRSCs or peripheral blood were compared, focusing on T-cell function and phenotype as well as the potential to generate cytomegalovirus (CMV)-specific T-cell lines from both CMV seropositive and seronegative donors. RESULTS: PBMCs from both sources were comparable except for a transient downregulation of CD62L expression on freshly extracted PBMCs from LRSCs. Both nonspecific stimulation using anti-CD3/CD28 antibodies and CMV peptides revealed that LRSCs or blood T cells were equivalent in terms of expansion, differentiation, and function. Moreover, PBMCs from LRSCs can be used to generate autologous monocyte-derived dendritic cells to prime and expand CMV-specific T cells from seronegative donors. CONCLUSION: LRSCs are a reliable source of PBMCs for the generation of virus-specific T cells for immunotherapy. These findings have implications for the development of third-party therapeutic T-cell products from well-characterized blood product donors.

Clinical-Scale Rapid Autologous BK Virus-Specific T Cell Line Generation From Kidney Transplant Recipients With Active Viremia for Adoptive Immunotherapy.

BACKGROUND: Polyomavirus-associated nephropathy (PVAN) after BK virus reactivation in kidney transplant recipients (KTR) can compromise graft survival. Lowering immunosuppression is the only established approach to prevent or treat PVAN but nonspecifically increasing host immune competence also augments rejection risk. Ex vivo T cell stimulation/expansion offers the possibility to generate BK-specific T cell lines for adoptive immunotherapy. The objective of this study was to develop and characterize a clinical-scale protocol to generate BK-specific T cell lines from viremic KTR. METHODS: Peripheral blood mononuclear cells from healthy controls and viremic KTR were stimulated using BK virus peptide libraries loaded or not on monocytes-derived dendritic cells. Cell counts, flow cytometry, and next-generation sequencing were used to assess T cell expansion, differentiation, and clonal diversity. Enzyme-linked immunospots, cytotoxicity assays as well as adoptive transfer in NOD/SCID/IL2Rγ mice were used to assess for pathogen-specificity and evidence of nonspecific alloreactivity. RESULTS: T cell lines from KTR and healthy control showed similar characteristics, implying that ongoing immunosuppression and chronic virus exposure do not compromise the differentiation, specificity, or clonal diversity of T cell lines after ex vivo production. Using antigen-loaded dendritic cells improved T cell expansion and favored central memory T cell differentiation. The T cell lines were antigen-specific and showed no nonspecific alloreactivity in vitro and in vivo. CONCLUSIONS: Using a rapid, clinically compliant culture system, we show that autologous BK virus-specific T cell lines can be reliably generated from viremic KTR. Our results pave the way for the treatment or prevention of PVAN with adoptive immunotherapy.

VEGF Requires the Receptor NRP-1 To Inhibit Lipopolysaccharide-Dependent Dendritic Cell Maturation.

To stimulate a productive T cell response, dendritic cells (DC) must undergo maturation characterized by heightened cell surface expression of MHC and costimulatory molecules as well as cytokine production. Conversely, the inhibition of DC maturation is a central mechanism of immune tolerance. The control of the DC maturation process relies on the integration of several cellular stimulatory or inhibitory signals. The soluble factors and their receptors controlling this central aspect of DC biology are incompletely characterized. We show that murine bone marrow-derived DC (BMDC) maturation induced by LPS, as opposed to polyinosinic:polycytidylic acid or cytosine-phosphate-guanine, is robustly inhibited by vascular endothelial growth factor (VEGF), a previously identified immunosuppressive cytokine. Using BMDC from wild type and conditional knockout mice, we show that neuropilin-1 (NRP-1), a known receptor of VEGF, is necessary to suppress LPS-dependent BMDC maturation. The absence of NRP-1 had no ostensible effects on the biology of BMDC in the absence of VEGF. However, NRP-1-deficient BMDC remained completely insensitive to the VEGF-dependent inhibition of BMDC maturation in culture. In the presence of VEGF, NRP-1 directly interacted with the LPS receptor TLR4 and suppressed downstream signaling through ERK and NF-κß, resulting in a sharp inhibition of MHC class II and costimulatory molecules (CD40, CD86) expression as well as proinflammatory cytokine production. Consequently, we identify NRP-1 as a target to optimize DC maturation within environments that are rich in VEGF, such as tumors.

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.

Identification of multiple and distinct defects in prostaglandin biosynthetic pathways in eutopic and ectopic endometrium of women with endometriosis.

OBJECTIVE: To investigate prostaglandin (PG) biosynthesis and catabolism pathways in eutopic and ectopic endometrium of women with endometriosis. DESIGN: Retrospective study. SETTING: Human reproduction research laboratory. PATIENT(S): Forty-five women with endometriosis and 29 normal controls. INTERVENTION(S): Endometrial and endometriotic tissue samples were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): Cyclo-oxygenases (Coxs 1 and 2), PGE2 synthases (microsomal [m] PGES 1 and 2 and cytosolic [c] PGES), PGF2α synthases (aldoketoreductase [AKR]-1C3 and AKR-1B1), and the PG catabolic enzyme 15-hydroxyprostaglandin dehydrogenase messenger RNA expression by quantitative real-time polymerase chain reaction and protein localization by immunohistochemistry. RESULT(S): This study showed a marked increase in the key PG biosynthesis enzymes Cox-2, mPGES-1, mPGES-2, cPGES, and AKR-1C3 in ectopic endometrial tissue of women with endometriosis, particularly in the earliest and most active stages of the disease, without a noticeable change in the expression of the PG catabolic enzyme 15-hydroxyprostaglandin dehydrogenase. Meanwhile, the significant increase in rate-limiting Cox-2 expression upstream was correlated downstream by a significant stage- and cycle phase-dependent decrease in the terminal specific synthase mPGES-2, thereby revealing the presence of counter-regulatory mechanisms, which operate in the eutopic endometrium of women with endometrium but seem to be lacking in the ectopic implantation sites. CONCLUSION(S): This study reveals for the first time multiple defects in PG biosynthesis pathways, which differ between eutopic intrauterine and ectopic endometrial tissues and may, owing to the wide spectrum of PG properties, contribute to the initial steps of endometrial tissue growth and development and have an important role to play in the pathogenesis and symptoms of this disease.

Roles of transforming growth factor-ß in graft-versus-host and graft-versus-tumor effects.

Transforming growth factor (TGF)-ß is a pleiotropic cytokine with widespread and profound effects on immune cells. Consequently, it has generated considerable interest in relation to the immunologic outcomes after allogeneic hematopoietic cell transplantation. The TGF-ß pathway has been shown to be an important modulator of alloimmunity, with direct consequences on graft-versus-host disease pathophysiology and graft-versus-tumor response. The TGF-ß-related effects can be both beneficial and detrimental to the host, underscoring the complexity of TGF-ß biology. This article reviews the evidence linking TGF-ß to alloimmune responses in allogeneic hematopoietic cell transplantation and highlights foreseeable strategies that would maximize the beneficial effects of TGF-ß pathway modulation on both graft-versus-host disease pathophysiology and the graft-versus-tumor effect.

Macrophage migration inhibitory factor elicits an angiogenic phenotype in human ectopic endometrial cells and triggers the production of major angiogenic factors via CD44, CD74, and MAPK signaling pathways.

CONTEXT: An active angiogenesis is required for ectopic endometrial tissue growth. Our previous studies led to the identification of macrophage migration inhibitory factor (MIF), which is markedly elevated in active, vascularized, and early-stage endometriotic lesions, as a potent mitogenic factor for endothelial cells. OBJECTIVE: Our objective was to study the mechanisms by which MIF may stimulate angiogenesis in ectopic endometrial implantation sites. DESIGN: Primary cultures of ectopic endometrial cells were exposed to MIF, and the release of major angiogenic factors with targeted disruption of MIF signaling pathways was assessed. PATIENTS: Patients were women found to have endometriosis during laparoscopy. SETTING: The study was conducted at a hospital and reproduction research laboratory. INTERVENTIONS: Biopsies were removed from endometriotic lesions. MAIN OUTCOME MEASURES: Vascular endothelial cell growth factor (VEGF), IL-8, and monocyte chemotactic protein-1 (MCP-1) mRNA and protein levels and expression and small interfering RNA silencing of MIF CD74/CD44 receptor complex and phosphorylation of ERK and p38 MAPKs were evaluated. RESULTS: MIF markedly up-regulated VEGF, IL-8, and MCP-1 expression in endometriotic cells. Such an effect was abolished by (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), a specific inhibitor of MIF, and significantly down-regulated after specific small interfering RNA silencing of CD44 or CD74. MIF treatment strongly activated ERK and p38 MAPKs, and specific inhibitors of both pathways completely blocked basal and MIF-induced VEGF, IL-8, and MCP-1 synthesis. CONCLUSIONS: These results show for the first time that MIF exerts a potent indirect angiogenic effect by interacting with ectopic endometrial cells and inducing the secretion of major angiogenic factors via CD44, CD74, and MAPK signaling pathways and provide evidence for a possible new mechanism underlying endometriosis development and pathophysiology.

Up-regulation of cyclooxygenase-2 expression and prostaglandin E2 production in human endometriotic cells by macrophage migration inhibitory factor: involvement of novel kinase signaling pathways.

Cyclooxygenase (COX) is the rate-limiting enzyme in the metabolic conversion of arachidonic acid to prostaglandins (PGs), including prostaglandin E(2) (PGE(2)), a major mediator of inflammation and angiogenesis. Herein, we report that macrophage migration inhibitory factor (MIF), a potent proinflammatory and growth-promoting factor found at elevated concentrations in the peritoneal fluid of women with endometriosis and active endometriosis lesions, acts directly on ectopic endometrial cells to stimulate the synthesis of COX-2, the inducible form of COX, and the release of PGE(2). MIF treatment strongly activated p38 and ERK MAPK, and specific inhibitors of both pathways completely blocked basal and MIF-induced PGE(2) synthesis. Whereas p38 inhibitors negatively affected the stimulated synthesis of COX-2 and that of PGE(2), ERK inhibitors only decreased the production of PGE(2). These findings show for the first time a direct role for MIF in the up-regulation of COX-2 synthesis and PGE(2) secretion in ectopic endometrial cells. They further indicate that whereas p38 and ERK MAPK signaling pathways both play a significant role in the regulation of basal and MIF-induced synthesis of PGE(2) by ectopic endometrial cells, only p38 kinase is involved in the regulation of COX-2 expression in these cells. This suggests that MIF acts at more than one level to stimulate the synthesis of PGE(2) and triggers the coordinate activation of multiple enzymes in the biosynthesis pathway. Our data provide evidence for a novel mechanism by which MIF can induce a proinflammatory phenotype in ectopic endometrial cells, and favor the establishment of endometriosis and its related clinical symptoms.

Direct effect of macrophage migration inhibitory factor on sperm function: possible involvement in endometriosis-associated infertility.

OBJECTIVE: To evaluate the effect of macrophage migration inhibitory factor (MIF) on sperm capacitation, a maturational process that occurs in the female reproductive tract and enables spermatozoa to become fully competent at fertilizing the oocyte. DESIGN: Incubation of Percoll-washed spermatozoa with varying concentrations of human recombinant MIF or fetal cord serum (positive control). SETTING: Human reproduction research laboratories. INTERVENTION(S): Fresh semen samples obtained from healthy volunteers after a minimum of 2 days of sexual abstinence. MAIN OUTCOME MEASURE(S): Protein tyrosine phosphorylation by Western blotting, the acrosomal status upon binding to the Pisum sativum agglutinin conjugated to fluorescein isothiocyanate, and sperm motility by computer-assisted sperm analysis. RESULTS: MIF displayed a dose-dependent effect on the phosphotyrosine content of p105 and p81, the two major tyrosine-phosphorylated proteins associated with human sperm capacitation. A significant induction of tyrosine phosphorylation was seen at 2 ng/mL of MIF for both p105 and p81, but a trend for a down-regulation of the basal tyrosine phosphorylation level was noted at elevated concentrations (12-24 ng/mL). MIF pretreatment of spermatozoa resulted in a dose-dependent change in the acrosome reaction induced by the Ca(2+) ionophore A23187. After being increased at 1-4 ng/mL MIF with a statistically significant effect observed at 4 ng/mL, the acrosome reaction gradually decreased and fell below the control levels at higher concentrations. Furthermore, a significant decrease in the motility of spermatozoa was observed after exposure to an elevated concentration of MIF (12 ng/mL). CONCLUSION(S): The present data indicate that MIF may play a physiological role in sperm capacitation but may have deleterious effects on sperm function at abnormal pathophysiological levels, which suggests a role in endometriosis-associated infertility.