Identification of minor histocompatibility antigens by reverse immunology

T-cell recognition of MiHA plays an important role in the GVT effect of allo-SCT. Selective infusion of T-cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA-class I molecules may help to separate the beneficial GVT effects from GVHD after allo-SCT. To date, only a few MiHA that form attractive targets for adoptive immunotherapy have been characterized and the number of patients that can be treated with such MiHA-selective cell therapy remains limited. In this thesis we focused on __reverse immunology__ as an attractive strategy to identify clinically relevant MiHA and other T-cell epitopes. In this approach peptide predictions are the starting point and peptide candidates are subsequently screened for their capacity to induce a T-cell response. We investigated the feasibility of computational genome-wide prediction of hematopoietic MiHA and alternatively implemented mass spectrometry based HLA-peptidomics as source for candidate peptides. T-cells that reacted with these antigens were collectively isolated by MHC-tetramer pull down. Subsequently, the composition of MHC-tetramer positive T-cell populations was characterized and tested for reactivity against any of the predicted epitopes that was included in the initial MHC-tetramer panel. We generated an algorithm that could be exploited to selectively target T-cells specific for clinically relevant MiHA.Dutch Cancer Society eBioscienceUBL - phd migration 201

Blimp-1 Rather Than Hobit Drives the Formation of Tissue-Resident Memory CD8+ T Cells in the Lungs

Tissue-resident memory CD8+ T (TRM) cells that develop in the epithelia at portals of pathogen entry are important for improved protection against re-infection. CD8+ TRM cells within the skin and the small intestine are long-lived and maintained independently of circulating memory CD8+ T cells. In contrast to CD8+ TRM cells at these sites, CD8+ TRM cells that arise after influenza virus infection within the lungs display high turnover and require constant recruitment from the circulating memory pool for long-term persistence. The distinct characteristics of CD8+ TRM cell maintenance within the lungs may suggest a unique program of transcriptional regulation of influenza-specific CD8+ TRM cells. We have previously demonstrated that the transcription factors Hobit and Blimp-1 are essential for the formation of CD8+ TRM cells across several tissues, including skin, liver, kidneys, and the small intestine. Here, we addressed the roles of Hobit and Blimp-1 in CD8+ TRM cell differentiation in the lungs after influenza infection using mice deficient for these transcription factors. Hobit was not required for the formation of influenza-specific CD8+ TRM cells in the lungs. In contrast, Blimp-1 was essential for the differentiation of lung CD8+ TRM cells and inhibited the differentiation of central memory CD8+ T (TCM) cells. We conclude that Blimp-1 rather than Hobit mediates the formation of CD8+ TRM cells in the lungs, potentially through control of the lineage choice between TCM and TRM cells during the differentiation of influenza-specific CD8+ T cells

High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations

T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T-cell infusion difficult. This study represents the first attempt of genome-wide prediction of MiHA, coupled to the isolation of T-cell populations that react with these antigens. In this unbiased high-throughput MiHA screen, both the possibilities and pitfalls of this approach were investigated. First, 973 polymorphic peptides expressed by hematopoietic stem cells were predicted and screened for HLA-A2 binding. Subsequently a set of 333 high affinity HLA-A2 ligands was identified and post transplantation samples from allo-SCT patients were screened for T-cell reactivity by a combination of pMHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1IMA antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent

Pull down and expansion of naive CMV-specific T-cells covering multiple HLA class I alleles

Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Pull down and expansion of naive CMV-specific T-cells covering multiple HLA class I alleles

Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Two step selection protocol based on the expression of T cell activation marker CD137 increases efficacy in generating high-affinity allo-HLA restricted T cells

Transplantation and autoimmunit

Tissue-resident memory T cells at the center of immunity to solid tumors

Immune responses in tissues are constrained by the physiological properties of the tissue involved. Tissue-resident memory T cells (TRM cells) are a recently discovered lineage of T cells specialized for life and function within tissues. Emerging evidence has shown that TRM cells have a special role in the control of solid tumors. A high frequency of TRM cells in tumors correlates with favorable disease progression in patients with cancer, and studies of mice have shown that TRM cells are necessary for optimal immunological control of solid tumors. Here we describe what defines TRM cells as a separate lineage and how these cells are generated. Furthermore, we discuss the properties that allow TRM cells to operate in normal and transformed tissues, as well as implications for the treatment of patients with cancer

HIGH-AFFINITY CD20-SPECIFIC TCRS SUITABLE FOR ADOPTIVE IMMUNOTHERAPY CAN BE READILY ISOLATED FROM THE ALLO-REPERTOIRE USING REVERSE IMMUNOLOGY

Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations

T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T-cell infusion difficult. This study represents the first attempt of genome-wide prediction of MiHA, coupled to the isolation of T-cell populations that react with these antigens. In this unbiased high-throughput MiHA screen, both the possibilities and pitfalls of this approach were investigated. First, 973 polymorphic peptides expressed by hematopoietic stem cells were predicted and screened for HLA-A2 binding. Subsequently a set of 333 high affinity HLA-A2 ligands was identified and post transplantation samples from allo-SCT patients were screened for T-cell reactivity by a combination of pMHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1(IMA) antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent. Collectively these results demonstrate the technical feasibility of high-throughput analysis of antigen-specific T-cell responses in small patient samples. However, the high-sensitivity of this approach requires the use of potential epitope sets that are not solely based on MHC binding, to prevent the frequent detection of T-cell responses that lack biological relevance.Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease