Cytomegalovirus-specific T cells restricted for shared and donor human leukocyte antigens differentially impact on cytomegalovirus reactivation risk after allogeneic hematopoietic stem cell transplantation

After allogeneic hematopoietic stem cell transplantation (HSCT), the emergence of circulating cytomegalovirus (CMV)- specific T cells correlates with protection from CMV reactivation, an important risk factor for non-relapse mortality. However, functional assays measuring CMV-specific cells are time-consuming and often inaccurate at early time-points. We report the results of a prospective single-center, non-interventional study that identified the enumeration of Dextramerpositive CMV-specific lymphocytes as a reliable and early predictor of viral reactivation. We longitudinally monitored 75 consecutive patients for 1 year after allogeneic HSCT (n=630 samples). The presence of ≥0.5 CMV-specific CD8+ cells/mL at day +45 was an independent protective factor from subsequent clinically relevant reactivation in univariate (P<0.01) and multivariate (P<0.05) analyses. Dextramer quantification correlated with functional assays measuring interferon-γ production, and allowed earlier identification of high-risk patients. In mismatched transplants, the comparative analysis of lymphocytes restricted by shared, donor- and host-specific HLA revealed the dominant role of thymic-independent CMV-specific reconstitution. Shared and donor-restricted CMV-specific T cells reconstituted with similar kinetics in recipients of CMV-seropositive donors, while donor-restricted T-cell reconstitution from CMV-seronegative grafts was impaired, indicating that in primary immunological responses the emergence of viral-specific T cells is largely sustained by antigen encounter on host infected cells rather than by cross-priming/presentation by non-infected donor-derived antigen-presenting cells. Multiparametric flow cytometry and high-dimensional analysis showed that shared-restricted CMV-specific lymphocytes display a more differentiated phenotype and increased persistence than donor-restricted counterparts. In this study, monitoring CMV-specific cells by Dextramer assay after allogeneic HSCT shed light on mechanisms of immune reconstitution and enabled risk stratification of patients, which could improve the clinical management of post-transplant CMV reactivations

Epithelial ovarian cancer is infiltrated by activated effector T cells co-expressing CD39, PD-1, TIM-3, CD137 and interacting with cancer cells and myeloid cells

IntroductionDespite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity.MethodsIn this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs).ResultsActivated T cells showing features of partial exhaustion with a CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ signature.ConclusionThese data demonstrate that EOC is enriched in CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches

DataSheet_2_TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.pdf

BackgroundIn adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells.MethodsWe simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models.ResultsWe show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells.ConclusionThese results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.</p

DataSheet_1_5TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.pdf

BackgroundIn adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells.MethodsWe simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models.ResultsWe show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells.ConclusionThese results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.</p

DataSheet_2_5TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.pdf

BackgroundIn adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells.MethodsWe simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models.ResultsWe show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells.ConclusionThese results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.</p

DataSheet_1_TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.pdf

BackgroundIn adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells.MethodsWe simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models.ResultsWe show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells.ConclusionThese results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.</p