Hanny Fredrix

Bi-directional allelic recognition of the human minor histocompatibility antigen HB-1 by cytotoxic T lymphocytes

Human minor histocompatibility antigens (mHag) are target antigens of the graft‐versus‐leukemia response observed after allogeneic HLA‐identical stem cell transplantation. We previously defined the molecular nature of the B cell lineage‐specific mHag HB‐1. The CTL epitope was identified as the decamer peptide EEKRGSLHVW presented in the context of HLA‐B44. The HB‐1 antigen is encoded by a locus of yet unknown function on chromosome 5q32. A single nucleotide polymorphism within this locus results in an amino acid change from histidine (H) to tyrosine (Y) at position P8 within the CTL epitope. Based on genomic information, we have developed a PCR‐RFLP assay to perform HB‐1 typing at the DNA level. We determined that the allelic frequency for the H and Y variant is 0.79 and 0.21, respectively. From these data, we calculated that the expected recipient disparity between HLA‐B44‐matched sibling pairs for HB‐1H is 2.8%, whereas recipient disparity for HB‐1Y is expected to be 12.4%. Therefore, we addressed whether the HB‐1Y peptide is reciprocally immunogenic. We revealed that both peptide variants bind equally efficient to HLA‐B44 molecules and that the H/Y substitution has noinfluence on formation of epitope precursor peptides by 20 S proteasome‐mediated degradation. More directly, CTL recognizing the naturally presented HB‐1Y peptide could be generated from a HB‐1H homozygous donor using peptide‐pulsed dendritic cells. Using a set of synthetic structurally related peptide variants, we found that the H/Y substitution has a major impact on TCR recognition by CTL specific for either of the HB‐1 allelic homologues. HB‐1 is the first human mHag described that induces bi‐directional allogeneic CTL responses that may contribute to a specific graft‐versus‐leukemiaresponse following allogeneic stem cell transplantation.

Improving dendritic cell vaccine immunogenicity by silencing PD-1 ligands using siRNA-lipid nanoparticles combined with antigen mRNA electroporation

Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8+ T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.

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.

TCR gamma delta cytotoxic T lymphocytes expressing the killer cell-inhibitory receptor p58.2 (CD158b) selectively lyse acute myeloid leukemia cells.

Cytotoxic T lymphocytes (CTL) are thought to play an important role in the graft-versus-leukemia (GVL) response. Unfortunately, GVL reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Characterization of CTL that selectively attack leukemic cells but not normal cells may lead to the development of adjuvant immunotherapy that separates GVL from GVHD. Here, we describe TCRγδ (Vγ9/Vδ1) CTL, isolated from the peripheral blood of an AML patient after stem cell transplantation (SCT), that very efficiently lysed freshly isolated acute myeloid leukemia (AML) cells and AML cell lines. Interestingly, HLA-matched non-malignant hematopoietic cells were not killed. We revealed that the killer cell-inhibitory receptor (KIR) p58.2 (CD158b) specific for group 2 HLA-C molecules negatively regulates the cytotoxic effector function displayed by these TCRγδ CTL. First, an antibody against HLA-C enhances lysis of non-malignant cells. Secondly, stable transfection of HLA-Cw*0304 into the class I-negative cell line 721.221 inhibited lysis. Finally, engagement of p58.2 by antibodies immobilized on FcγR-expressing murine P815 cells inhibits CD3- and TCRγδ-directed lysis. Compared to non-malignant hematopoietic cells, AML cells express much lower levels of MHC class I molecules making them susceptible to lysis by p58.2+ TCRγδ CTL. Such KIR-regulated CTL reactivity may have a role in the GVL response without affecting normal tissues of the host and leading to GVHD. Bone Marrow Transplantation (2001) 27, 1087–1093.

Expression of P2X5 in lymphoid malignancies results in LRH-1-specific cytotoxic T-cell-mediated lysis

Minor histocompatibility antigens (MiHA) selectively expressed by haematopoietic cells are attractive targets for specific immunotherapy after allogeneic stem cell transplantation (SCT). Previously, we described LRH‐1 as a haematopoietic‐lineage restricted MiHA that is capable of eliciting an allogeneic cytotoxic T‐lymphocyte (CTL) response after SCT and donor lymphocyte infusion. Importantly, the gene encoding LRH‐1, P2X5, is not expressed in prominent graft‐versus‐host‐disease target tissues such as skin, liver and gut. Here, we investigate whether LRH‐1‐specific immunotherapy may be exploited for the treatment of lymphoid malignancies. We examined P2X5 mRNA expression in a large panel of patient samples and cell lines from different types of lymphoid malignancies by real‐time quantitative reverse transcription polymerase chain reaction. P2X5 mRNA was highly expressed in malignant cells from all stages of lymphoid development. Furthermore, all LRH‐1‐positive lymphoid tumour cell lines were susceptible to LRH‐1 CTL‐mediated lysis in flow cytometry‐based cytotoxicity assays. However, interferon‐γ production was low or absent after stimulation with some cell lines, possibly due to differences in activation thresholds for CTL effector functions. Importantly, primary cells from patients with lymphoid malignancies were effectively lysed by LRH‐1‐specific CTL. These findings indicate that MiHA LRH‐1 is a potential therapeutic target for cellular immunotherapy of lymphoid malignancies.

Partial T cell-depleted allogeneic stem cell transplantation following reduced-intensity conditioning creates a platform for immunotherapy with donor lymphocyte infusion and recipient dendritic cell vaccination in multiple myeloma.

Allogeneic stem cell transplantation (SCT) in multiple myeloma (MM) may induce a curative graft-versus-myeloma (GVM) effect. Major drawback in unmanipulated reduced-intensity conditioning (RIC) SCT is the risk of severe and longstanding graft-versus-host-disease (GVHD). This study demonstrates that transplantation with a partial T cell-depleted graft creates a platform for boosting GVM immunity by preemptive donor lymphocyte infusion (DLI) and recipient dendritic cell (DC) vaccination, with limited GVHD. All 20MM patients engrafted successfully. Chimerism analysis in 19 patients evaluable at 3 months revealed that 7 patients were complete donor, whereas 12 patients were mixed chimeric. Grade II acute GVHD (aGVHD) occurred in 7 patients (35%) and only 4 patients (21%) developed chronic GVHD (cGVHD). Fourteen patients received posttransplantation immunotherapy, 8 preemptive DLI, 5 patients both DLI and DC vaccination, and 1 patient DC vaccination only. DC vaccination was associated with limited toxicity, and none of these patients developed GVHD. Importantly, overall treatment-related mortality (TRM) at 1 year was low (10%). Moreover, the overall survival (OS) is 84% with median follow-up of 27 months, and none of the patients died from progressive disease. These findings illustrate that this novel approach is associated with limited GVHD and mortality, thus creating an ideal platform for adjuvant immunotherapy.

siRNA silencing of PD-1 ligands on dendritic cell vaccines boosts the expansion of minor histocompatibility antigen-specific CD8(+) T cells in NOD/SCID/IL2Rg(null) mice

Abstract Allogeneic stem cell transplantation (allo-SCT) can be a curative therapy for patients suffering from hematological malignancies. The therapeutic efficacy is based on donor-derived CD8+ T cells that recognize minor histocompatibility antigens (MiHAs) expressed by patient’s tumor cells. However, these responses are not always sufficient, and persistence and recurrence of the malignant disease are often observed. Therefore, application of additive therapy targeting hematopoietic-restricted MiHAs is essential. Adoptive transfer of MiHA-specific CD8+ T cells in combination with dendritic cell (DC) vaccination could be a promising strategy. Though effects of DC vaccination in anti-cancer therapy have been demonstrated, improvement in DC vaccination therapy is needed, as clinical responses are limited. In this study, we investigated the potency of program death ligand (PD-L) 1 and 2 silenced DC vaccines for ex vivo priming and in vivo boosting of MiHA-specific CD8+ T cell responses. Co-culturing CD8+ T cells with MiHA-loaded DCs resulted in priming and expansion of functional MiHA-specific CD8+ T cells from the naive repertoire, which was augmented upon silencing of PD-L1 and PD-L2. Furthermore, DC vaccination supported and expanded adoptively transferred antigen-specific CD8+ T cells in vivo. Importantly, the use of PD-L silenced DCs improved boosting and further expansion of ex vivo primed MiHA-specific CD8+ T cells in immunodeficient mice. In conclusion, adoptive transfer of ex vivo primed MiHA-specific CD8+ T cells in combination with PD-L silenced DC vaccination, targeting MiHAs restricted to the hematopoietic system, is an interesting approach to boost GVT immunity in allo-SCT patients and thereby prevent relapse.

Efficient Activation of LRH-1―specific CD8+ T-cell Responses From Transplanted Leukemia Patients by Stimulation With P2X5 mRNA-electroporated Dendritic Cells

Alloreactive CD8+ T cells targeting minor histocompatibility antigens (MiHA) on malignant cells of the recipient play a pivotal role in graft-versus-tumor responses observed after allogeneic stem cell transplantation and donor lymphocyte infusion (DLI). However, these MiHA-specific CD8+ T-cell responses do not result in complete eradication of tumor cells in all patients. Furthermore, CD8+ memory T cells persisting after DLI do not always efficiently expand with recurrence of the disease. Adjuvant immunotherapy using dendritic cells (DC) loaded with hematopoietic-restricted MiHA may boost antitumor CD8+ T-cell immunity without inducing graft-versus-host disease. Here, we explored the use of mRNA-electroporated DC to stimulate MiHA-specific CD8+ T-cell responses. We demonstrate that electroporation of mature DC with P2X5 mRNA encoding for hematopoietic-restricted MiHA LRH-1 results in high expression of both mRNA and protein, and has no negative effect on the mature phenotype and migratory capacity of the DC. Furthermore, these DC can efficiently stimulate LRH-1–specific CD8+ effector T cells to proliferate and produce interferon-γ. In addition, LRH-1–specific CD8+ memory T cells that are present in patient-derived peripheral blood mononuclear cells at long periods post-DLI can be effectively activated by stimulation with P2X5 mRNA-electroporated DC to proliferate and degranulate upon target cell recognition. These results indicate that adjuvant immunotherapy using DC electroporated with mRNA encoding hematopoietic-restricted MiHA mismatched between patients and donors may enhance the graft versus tumor response induced by stem cell transplantation and DLI.

A Polymorphism in the Splice Donor Site of ZNF419 Results in the Novel Renal Cell Carcinoma-Associated Minor Histocompatibility Antigen ZAPHIR

Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT.

CLEC12A-Mediated Antigen Uptake and Cross-Presentation by Human Dendritic Cell Subsets Efficiently Boost Tumor-Reactive T Cell Responses

Potent immunotherapies are urgently needed to boost antitumor immunity and control disease in cancer patients. As dendritic cells (DCs) are the most powerful APCs, they are an attractive means to reinvigorate T cell responses. An appealing strategy to use the effective Ag processing and presentation machinery, T cell stimulation and cross-talk capacity of natural DC subsets is in vivo tumor Ag delivery. In this context, endocytic C-type lectin receptors are attractive targeting molecules. In this study, we investigated whether CLEC12A efficiently delivers tumor Ags into human DC subsets, facilitating effective induction of CD4+ and CD8+ T cell responses. We confirmed that CLEC12A is selectively expressed by myeloid cells, including the myeloid DC subset (mDCs) and the plasmacytoid DC subset (pDCs). Moreover, we demonstrated that these DC subsets efficiently internalize CLEC12A, whereupon it quickly translocates to the early endosomes and subsequently routes to the lysosomes. Notably, CLEC12A Ab targeting did not negatively affect DC maturation or function. Furthermore, CLEC12A-mediated delivery of keyhole limpet hemocyanin resulted in enhanced proliferation and cytokine secretion by keyhole limpet hemocyanin–experienced CD4+ T cells. Most importantly, CLEC12A-targeted delivery of HA-1 long peptide resulted in efficient Ag cross-presentation by mDCs and pDCs, leading to strong ex vivo activation of HA-1–specific CD8+ T cells of patients after allogeneic stem cell transplantation. Collectively, these data indicate that CLEC12A is an effective new candidate with great potential for in vivo Ag delivery into mDCs and pDCs, thereby using the specialized functions and cross-talk capacity of these DC subsets to boost tumor-reactive T cell immunity in cancer patients.