Increased Coexpression of PD-1, TIGIT, and KLRG-1 on Tumor-Reactive CD8+ T Cells During Relapse after Allogeneic Stem Cell Transplantation.

Allogeneic stem cell transplantation (allo-SCT) can be a curative treatment for patients with a hematologic malignancy due to alloreactive T cell responses recognizing minor histocompatibility antigens (MiHA). Yet tumor immune escape mechanisms can cause failure of T cell immunity, leading to relapse. Tumor cells display low expression of costimulatory molecules and can up-regulate coinhibitory molecules that inhibit T cell functionality on ligation with their counter-receptors on the tumor-reactive T cells. The aim of this explorative study was to evaluate immune checkpoint expression profiles on T cell subsets and on cytomegalovirus (CMV)- and/or MiHA-reactive CD8+ T cells of allo-SCT recipients using a 13-color flow cytometry panel, and to correlate these expression patterns to clinical outcomes. MiHA-reactive CD8+ T cells exhibited an early differentiated CD27++/CD28++ phenotype with low KLRG-1 and CD57 expression. These T cells also displayed increased expression of PD-1, TIM-3, and TIGIT compared with total effector memory T cells and CMV-specific CD8+ T cells in healthy donors and allo-SCT recipients. Remarkably, high coexpression of PD-1, TIGIT, and KLRG-1 on MiHA-reactive CD8+ T cells was associated with relapse after allo-SCT. Taken together, these findings indicate that MiHA-specific CD8+ T cells of relapsed patients have a distinctive coinhibitory expression signature compared with patients who stay in remission. This phenotype may serve as a potential monitoring tool in patients. Moreover, these findings suggest that PD-1 and TIGIT play important roles in regulating T cell-mediated tumor control, providing a rationale for immunotherapy with blocking antibodies to treat relapse after allo-SCT.

Efficient nontoxic delivery of PD-L1 and PD-L2 siRNA into dendritic cell vaccines using the cationic lipid SAINT-18.

Dendritic cell (DC)-based vaccination is an appealing strategy to boost graft-versus-tumor immunity after allogeneic stem cell transplantation (allo-SCT), and thereby prevent or counteract tumor recurrence. By exploiting minor histocompatibility antigens (MiHA) presented on hematopoietic cells, donor CD8 T-cell immunity can be selectively targeted to patient's hematological tumor cells without the risk of inducing graft-versus-host disease. Previously, we demonstrated that silencing RNA (siRNA) of programmed death-ligand 1 (PD-L1) and PD-L2 on DCs markedly augments the expansion and function of MiHA-specific CD8 T cells. However, previously applied methods based on electroporation or lipid nanoparticles were either incompatible with target antigen mRNA delivery or required complex manufacturing compliant to Good Manufacturing Practice. Here, we investigated whether transfection using lipoplexes composed of PD-L1 and PD-L2 siRNAs plus SAINT-18:DOPE (ie, SAINT-RED) is an effective and feasible clinical-grade method in DC vaccine manufacturing. We observed that a single siRNA/SAINT-RED transfection resulted in efficient and long-term knockdown of the PD-1 ligands without affecting DC maturation or viability. Furthermore, we demonstrated that SAINT-RED can be heat sterilized without loss of function, facilitating its use in aseptic DC vaccine production. Finally, we showed that the established transfection method can be combined with target antigen mRNA or peptide loading to efficiently stimulate MiHA-specific T-cell expansion and cytokine production. Together, these findings indicate that the developed PD-L siRNA/SAINT-RED transfection protocol in combination with MiHA mRNA or peptide loading can be applied in the generation of clinical-grade DC vaccines to boost antitumor immunity after allo-SCT.

B and T lymphocyte attenuator mediates inhibition of tumor-reactive CD8+ T cells in patients after allogeneic stem cell transplantation.

Allogeneic stem cell transplantation (allo-SCT) can cure hematological malignancies by inducing alloreactive T cell responses targeting minor histocompatibility antigens (MiHA) expressed on malignant cells. Despite induction of robust MiHA-specific T cell responses and long-term persistence of alloreactive memory T cells specific for the tumor, often these T cells fail to respond efficiently to tumor relapse. Previously, we demonstrated the involvement of the coinhibitory receptor programmed death-1 (PD-1) in suppressing MiHA-specific CD8(+) T cell immunity. In this study, we investigated whether B and T lymphocyte attenuator (BTLA) plays a similar role in functional impairment of MiHA-specific T cells after allo-SCT. In addition to PD-1, we observed higher BTLA expression on MiHA-specific CD8(+) T cells compared with that of the total population of CD8(+) effector-memory T cells. In addition, BTLA's ligand, herpes virus entry mediator (HVEM), was found constitutively expressed by myeloid leukemia, B cell lymphoma, and multiple myeloma cells. Interference with the BTLA-HVEM pathway, using a BTLA blocking Ab, augmented proliferation of BTLA(+)PD-1(+) MiHA-specific CD8(+) T cells by HVEM-expressing dendritic cells. Notably, we demonstrated that blocking of BTLA or PD-1 enhanced ex vivo proliferation of MiHA-specific CD8(+) T cells in respectively 7 and 9 of 11 allo-SCT patients. Notably, in 3 of 11 patients, the effect of BTLA blockade was more prominent than that of PD-1 blockade. Furthermore, these expanded MiHA-specific CD8(+) T cells competently produced effector cytokines and degranulated upon Ag reencounter. Together, these results demonstrate that BTLA-HVEM interactions impair MiHA-specific T cell functionality, providing a rationale for interfering with BTLA signaling in post-stem cell transplantation therapies.

Coinhibitory molecules in hematologic malignancies: targets for therapeutic intervention.

The adaptive immune system can be a potent defense mechanism against cancer; however, it is often hampered by immune suppressive mechanisms in the tumor microenvironment. Coinhibitory molecules expressed by tumor cells, immune cells, and stromal cells in the tumor milieu can dominantly attenuate T-cell responses against cancer cells. Today, a variety of coinhibitory molecules, including cytotoxic T lymphocyte-associated antigen-4, programmed death-1, B and T lymphocyte attenuator, LAG3, T-cell immunoglobulin and mucin domain 3, and CD200 receptor, have been implicated in immune escape of cancer cells. Sustained signaling via these coinhibitory molecules results in functional exhaustion of T cells, during which the ability to proliferate, secrete cytokines, and mediate lysis of tumor cells is sequentially lost. In this review, we discuss the influence of coinhibitory pathways in suppressing autologous and allogeneic T cell-mediated immunity against hematologic malignancies. In addition, promising preclinical and clinical data of immunotherapeutic approaches interfering with negative cosignaling, either as monotherapy or in conjunction with vaccination strategies, are reviewed. Numerous studies indicate that coinhibitory signaling hampers the clinical benefit of current immunotherapies. Therefore, manipulation of coinhibitory networks is an attractive adjuvant immunotherapeutic intervention for hematologic cancers after standard treatment with chemotherapy and hematopoietic stem cell transplantation.

PD-1/PD-L1 interactions contribute to functional T-cell impairment in patients who relapse with cancer after allogeneic stem cell transplantation.

Tumor relapses remain a serious problem after allogeneic stem cell transplantation (alloSCT), despite the long-term persistence of minor histocompatibility antigen (MiHA)-specific memory CD8(+) T cells specific for the tumor. We hypothesized that these memory T cells may lose their function over time in transplanted patients. Here, we offer functional and mechanistic support for this hypothesis, based on immune inhibition by programmed death-1 (PD-1) expressed on MiHA-specific CD8(+) T cells and the associated role of the PD-1 ligand PD-L1 on myeloid leukemia cells, especially under inflammatory conditions. PD-L1 was highly upregulated on immature human leukemic progenitor cells, whereas costimulatory molecules such as CD80 and CD86 were not expressed. Thus, immature leukemic progenitor cells seemed to evade the immune system by inhibiting T-cell function via the PD-1/PD-L1 pathway. Blocking PD-1 signaling using human antibodies led to elevated proliferation and IFN-γ production of MiHA-specific T cells cocultured with PD-L1-expressing leukemia cells. Moreover, patients with relapsed leukemia after initial MiHA-specific T-cell responses displayed high PD-L1 expression on CD34(+) leukemia cells and increased PD-1 levels on MiHA-specific CD8(+) T cells. Importantly, blocking PD-1/PD-L1 interactions augment proliferation of MiHA-specific CD8(+) memory T cells from relapsed patients. Taken together, our findings indicate that the PD-1/PD-L pathway can be hijacked as an immune escape mechanism in hematological malignancies. Furthermore, they suggest that blocking the PD-1 immune checkpoint offers an appealing immunotherapeutic strategy following alloSCT in patients with recurrent or relapsed disease.

Myeloid leukemic progenitor cells can be specifically targeted by minor histocompatibility antigen LRH-1-reactive cytotoxic T cells.

CD8(+) T cells recognizing minor histocompatibility antigens (MiHAs) on leukemic stem and progenitor cells play a pivotal role in effective graft-versus-leukemia reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified a hematopoiesis-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene. We found that P2X5 is significantly expressed in CD34(+) leukemic subpopulations from chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. Here, we demonstrate that LRH-1-specific CD8(+) T-cell responses are frequently induced in myeloid leukemia patients following donor lymphocyte infusions. Patients with high percentages of circulating LRH-1-specific CD8(+) T cells had no or only mild graft-versus-host disease. Functional analysis showed that LRH-1-specific cytotoxic T lymphocytes (CTLs) isolated from 2 different patients efficiently target LRH-1-positive leukemic CD34(+) progenitor cells from both CML and AML patients, whereas mature CML cells are only marginally lysed due to down-regulation of P2X5. Furthermore, we observed that relative resistance to LRH-1 CTL-mediated cell death due to elevated levels of antiapoptotic XIAP could be overcome by IFN-gamma prestimulation and increased CTL-target ratios. These findings provide a rationale for use of LRH-1 as immunotherapeutic target antigen to treat residual or persisting myeloid malignancies after allogeneic SCT.