Idiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting... Show moreIdiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting of a course of anti-thymocyte globulin (ATG) followed by long-term use of ciclosporin. However, the immune response that underlies the pathogenesis of AA remains poorly understood. In this study, we applied high-dimensional mass cytometry on bone marrow aspirates of AA patients pre-ATG, AA patients post-ATG and healthy donors to decipher which immune cells may be implicated in the pathogenesis of AA. We show that the bone marrow of AA patients features an immune cell composition distinct from healthy donors, with significant differences in the myeloid, B-cell, CD4+ and CD8+ T-cells lineages. Specifically, we discovered that AA pre-ATG is characterized by a disease-specific immune cell network with high frequencies of CD16+ myeloid cells, CCR6++ B-cells, Th17-like CCR6+ memory CD4+ T-cells, CD45RA+CCR7+CD38+ CD8+ T-cells and KLRG1+ terminally differentiated effector memory (EMRA) CD8+ T-cells, compatible with a state of chronic inflammation. Successful treatment with IST strongly reduced the levels of CD16+ myeloid cells and showed a trend toward normalization of the frequencies of CCR6++ B-cells, CCR6+ memory CD4+ T-cells and KLRG1+EMRA CD8+ T-cells. Altogether, our study provides a unique overview of the immune landscape in bone marrow in AA at a single-cell level and proposes CCR6 as a potential new therapeutic target in AA. Show less
The immune checkpoint VISTA is upregulated on endothelial cells in cancer and selectively prevents T-cell extravasation into tumors. The data highlight a mechanism behind T-cell exclusion and... Show moreThe immune checkpoint VISTA is upregulated on endothelial cells in cancer and selectively prevents T-cell extravasation into tumors. The data highlight a mechanism behind T-cell exclusion and provide a deeper understanding of factors shaping the tumor immune microenvironment.Cancers evade T-cell immunity by several mechanisms such as secretion of anti-inflammatory cytokines, down regulation of antigen presentation machinery, upregulation of immune checkpoint molecules, and exclusion of T cells from tumor tissues. The distribution and function of immune checkpoint molecules on tumor cells and tumor-infiltrating leukocytes is well established, but less is known about their impact on intratumoral endothelial cells. Here, we demonstrated that V-domain Ig suppressor of T-cell activation (VISTA), a PD-L1 homolog, was highly expressed on endothelial cells in synovial sarcoma, subsets of different carcinomas, and immune-privileged tissues. We created an ex vivo model of the human vasculature and demonstrated that expression of VISTA on endothelial cells selectively prevented T-cell transmigration over endothelial layers under physiologic flow conditions, whereas it does not affect migration of other immune cell types. Furthermore, endothelial VISTA correlated with reduced infiltration of T cells and poor prognosis in metastatic synovial sarcoma. In endothelial cells, we detected VISTA on the plasma membrane and in recycling endosomes, and its expression was upregulated by cancer cell-secreted factors in a VEGF-A-dependent manner. Our study reveals that endothelial VISTA is upregulated by cancer-secreted factors and that it regulates T-cell accessibility to cancer and healthy tissues. This newly identified mechanism should be considered when using immunotherapeutic approaches aimed at unleashing T cell-mediated cancer immunity. Show less
Koster, E.A.S.; Bonneville, E.F.; Borne, P.A.V.; Balen, P. van; Marijt, E.W.A.; Tjon, J.M.L.; ... ; Wreede, L.C. de 2023
Alloreactive donor-derived T-cells play a pivotal role in alloimmune responses after allogeneic hematopoietic stem cell transplantation (alloSCT); both in the relapse-preventing Graft-versus... Show moreAlloreactive donor-derived T-cells play a pivotal role in alloimmune responses after allogeneic hematopoietic stem cell transplantation (alloSCT); both in the relapse-preventing Graft-versus-Leukemia (GvL) effect and the potentially lethal complication Graft-versus-Host-Disease (GvHD). The balance between GvL and GvHD can be shifted by removing T-cells via T-cell depletion (TCD) to reduce the risk of GvHD, and by introducing additional donor T-cells (donor lymphocyte infusions [DLI]) to boost the GvL effect. However, the association between T-cell kinetics and the occurrence of allo-immunological events has not been clearly demonstrated yet. Therefore, we investigated the complex associations between the T-cell kinetics and alloimmune responses in a cohort of 166 acute leukemia patients receiving alemtuzumab-based TCD alloSCT. Of these patients, 62 with an anticipated high risk of relapse were scheduled to receive a prophylactic DLI at 3 months after transplant. In this setting, we applied joint modelling which allowed us to better capture the complex interplay between DLI, T-cell kinetics, GvHD and relapse than traditional statistical methods. We demonstrate that DLI can induce detectable T-cell expansion, leading to an increase in total, CD4+ and CD8+ T-cell counts starting at 3 months after alloSCT. CD4+ T-cells showed the strongest association with the development of alloimmune responses: higher CD4 counts increased the risk of GvHD (hazard ratio 2.44, 95% confidence interval 1.45-4.12) and decreased the risk of relapse (hazard ratio 0.65, 95% confidence interval 0.45-0.92). Similar models showed that natural killer cells recovered rapidly after alloSCT and were associated with a lower risk of relapse (HR 0.62, 95%-CI 0.41-0.93). The results of this study advocate the use of joint models to further study immune cell kinetics in different settings. Show less
After allogeneic stem cell transplantation (alloSCT), patient-derived stem cells that survived the pretransplantation conditioning compete with engrafting donor stem cells for bone marrow (BM)... Show moreAfter allogeneic stem cell transplantation (alloSCT), patient-derived stem cells that survived the pretransplantation conditioning compete with engrafting donor stem cells for bone marrow (BM) repopulation. In addition, donor-derived alloreactive T cells present in the stem cell product may favor establishment of complete donor-derived hematopoiesis by eliminating patient-derived lymphohematopoietic cells. T cell-depleted alloSCT with sequential transfer of potentially alloreactive T cells by donor lymphocyte infusion (DLI) provides a unique opportunity to selectively study how competitive repopulation and allo-immunologic pressure influence lymphohematopoietic recovery. This study aimed to determine the relative contribution of competitive repopulation and donor-derived anti-recipient alloimmunologic pressure on the establishment of lymphohematopoietic chimerism after alloSCT. In this retrospective cohort study of 281 acute leukemia patients treated according to a protocol combining alemtuzumab-based T cell-depleted alloSCT with prophy-lactic DLI, we investigated engraftment and quantitative donor chimerism in the BM and immune cell subsets. DLI-induced increase of chimerism and development of graft-versus-host disease (GVHD) were analyzed as complementary indicators for donor-derived anti-recipient alloimmunologic pressure. Profound suppression of patient immune cells by conditioning sufficed for sustained engraftment without necessity for myeloablative conditioning or development of clinically significant GVHD. Although 61% of the patients without any DLI or GVHD showed full donor chimerism (FDC) in the BM at 6 months after alloSCT, only 24% showed FDC in the CD4+ T cell compartment. In contrast, 75% of the patients who had received DLI and 83% of the patients with clinically significant GVHD had FDC in this compartment. In addition, 72% of the patients with mixed hematopoiesis receiving DLI converted to complete donor-derived hematopoiesis, of whom only 34% developed clinically significant GVHD. Our data show that competitive repopulation can be sufficient to reach complete donor-derived hematopoiesis, but that some alloimmunologic pressure is needed for the establishment of a completely donor-derived T cell compartment, either by the development of GVHD or by administration of DLI. We illustrate that it is possible to separate the graft-versus-leukemia effect from GVHD, as conversion to durable complete donor-derived hematopoiesis following DLI did not require induction of clinically significant GVHD. (c) 2023 The American Society for Transplantation and Cellular Therapy. Show less
Amerongen, R.A. van; Tuit, S.; Wouters, A.K.; Meent, M. van de; Siekman, S.L.; Meeuwsen, M.H.; ... ; Heemskerk, M.H.M. 2023
Recurrent disease emerges in the majority of patients with ovarian cancer (OVCA). Adoptive T-cell therapies with T-cell receptors (TCRs) targeting tumor-associated antigens (TAAs) are considered... Show moreRecurrent disease emerges in the majority of patients with ovarian cancer (OVCA). Adoptive T-cell therapies with T-cell receptors (TCRs) targeting tumor-associated antigens (TAAs) are considered promising solutions for less-immunogenic 'cold' ovarian tumors. In order to treat a broader patient population, more TCRs targeting peptides derived from different TAAs binding in various HLA class I molecules are essential. By performing a differential gene expression analysis using mRNA-seq datasets, PRAME, CTCFL and CLDN6 were selected as strictly tumor-specific TAAs, with high expression in ovarian cancer and at least 20-fold lower expression in all healthy tissues of risk. In primary OVCA patient samples and cell lines we confirmed expression and identified naturally expressed TAA-derived peptides in the HLA class I ligandome. Subsequently, high-avidity T-cell clones recognizing these peptides were isolated from the allo-HLA T-cell repertoire of healthy individuals. Three PRAME TCRs and one CTCFL TCR of the most promising T-cell clones were sequenced, and transferred to CD8+ T cells. The PRAME TCR-T cells demonstrated potent and specific antitumor reactivity in vitro and in vivo. The CTCFL TCR-T cells efficiently recognized primary patient-derived OVCA cells, and OVCA cell lines treated with demethylating agent 5-aza-2 '-deoxycytidine (DAC). The identified PRAME and CTCFL TCRs are promising candidates for the treatment of patients with ovarian cancer, and are an essential addition to the currently used HLA-A*02:01 restricted PRAME TCRs. Our selection of differentially expressed genes, naturally expressed TAA peptides and potent TCRs can improve and broaden the use of T-cell therapies for patients with ovarian cancer or other PRAME or CTCFL expressing cancers. Show less
To increase the number of cancer patients that can be treated with T cell receptor (TCR) gene therapy, we aimed to identify a set of high-affinity cancer-specific TCRs targeting different melanoma... Show moreTo increase the number of cancer patients that can be treated with T cell receptor (TCR) gene therapy, we aimed to identify a set of high-affinity cancer-specific TCRs targeting different melanoma-associated antigens (MAGEs). In this study, pep-tides derived from MAGE genes with tumor-specific expression pattern were identified by human leukocyte antigen (HLA) peptidomics. Next, peptide-HLA tetramers were generated, and used to sort MAGE-specific CD8+ T cell clones from the allogeneic (allo) HLA repertoire of healthy donors. To evaluate the clinical potential, most potent TCRs were sequenced, trans-ferred into peripheral blood-derived CD8+ T cells, and tested for antitumor efficacy. In total we identified, seven MAGE-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6, and MAGE-A9 in the context of HLA-A*01:01,-A*02:01, -A*03:01, -B*07:02, -B*35:01, or -C*07:02. TCR gene transfer into CD8' T cells resulted in efficient reactivity against a variety of different tumor types, while no cross-reac-tivity was detected. In addition, major in vivo antitumor effects of MAGE-A1 specific TCR engineered CD8' T cells were observed in the orthotopic xenograft model for established multiple myeloma. The identification of seven MAGE-specific TCRs expands the pool of cancer patients eligible for TCR gene therapy and increases possibilities for personalized TCR gene therapy. Show less
Adoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase 1/2 trial, we prophylactically administered multivirusspecific T... Show moreAdoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase 1/2 trial, we prophylactically administered multivirusspecific T-cell products to protect recipients of T-cell-depleted allogeneic stem cell grafts against viral reactivation. To establish treatment efficacy, it is important to determine the fate of the individual transferred T-cell populations. However, it is difficult to unequivocally distinguish progeny of the transferred T-cell products from recipient- or stem cell graft-derived T cells that survived T-cell depletion during conditioning or stem cell graft manipulation. Using messenger RNA sequencing of the T-cell receptor beta-chains of the individual virus-specific T-cell populations within these T-cell products, we were able to track the multiple clonal virus-specific subpopulations in peripheral blood and distinguish recipient- and stem cell graft-derived virus-specific T cells from the progeny of the infused T-cell products. We observed in vivo expansion of virus-specific T cells that were exclusively derived from the T-cell products with similar kinetics as the expansion of virus-specific T cells that could also be detected before the T-cell product infusion. In addition, we demonstrated persistence of virus-specific T cells derived from the T-cell products in most patients who did not show viral reactivation. This study demonstrates that virus-specific T cells from prophylactically infused multiantigen-specific T-cell products can expand in response to antigen encounter in vivo and even persist in the absence of early viral reactivation. Show less
The balance between safety and efficacy of T cell therapies re-mains challenging and T cell mediated toxicities have occurred. The stringent selection of tumor-specific targets and careful se... Show moreThe balance between safety and efficacy of T cell therapies re-mains challenging and T cell mediated toxicities have occurred. The stringent selection of tumor-specific targets and careful se-lection of tumor-specific T cells using T cell toxicity screenings are essential. In vitro screening options against vital organs or specialized cell subsets would be preferably included in preclin-ical pipelines, but options remain limited. Here, we set up preclinical models with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, epicardial cells, and kid-ney organoids to investigate toxicity risks of tumor-specific T cells more thoroughly. CD8+T cells reactive against PRAME, HA-1H, CD20, or WT1, currently used or planned to be used in phase I/II clinical studies, were included. Using these hiPSC-derived preclinical models, we demonstrated that WT1-specific T cells caused on-target toxicity that correlated with target gene expression. Multiple measures of T cell reac-tivity demonstrated this toxicity on the level of T cells and hiPSC-derived target cells. In addition, phenotypic analysis illustrated interaction and crosstalk between infiltrated T cells and kidney organoids. In summary, we demonstrated the benefit of hiPSC-derived models in determining toxicity risks of tumor-specific T cells. Furthermore, our data empha-sizes the additional value of other measures of T cell reactivity on top of the commonly used cytokine levels. Show less
Zouwen, B. van der; Koster, E.A.S.; Borne, P.A. von dem; Oosten, L.E.M.; Roza-Scholten, M.W.I.; Snijders, T.J.F.; ... ; Halkes, C.J.M. 2023
Prophylactic donor lymphocyte infusion (DLI) starting at 6 months after T cell-depleted allogeneic stem cell transplantation (TCD-alloSCT) can introduce a graft-versus-leukemia (GvL) effects with... Show moreProphylactic donor lymphocyte infusion (DLI) starting at 6 months after T cell-depleted allogeneic stem cell transplantation (TCD-alloSCT) can introduce a graft-versus-leukemia (GvL) effects with low risk of severe graft-versus-host-disease (GvHD). We established a policy to apply low-dose early DLI at 3 months after alloSCT to prevent early relapse. This study analyzes this strategy retrospectively. Of 220 consecutive acute leukemia patients undergoing TCD-alloSCT, 83 were prospectively classified to have a high relapse risk and 43 were scheduled for early DLI. 95% of these patients received freshly harvested DLI within 2 weeks of the planned date. In patients transplanted with reduced intensity conditioning and an unrelated donor, we found an increased cumulative incidence of GvHD between 3 and 6 months after TCD-alloSCT for patients receiving DLI at 3 months compared to patients who did not receive this DLI (0.42 (95%Confidence Interval (95% CI): 0.14-0.70) vs 0). Treatment success was defined as being alive without relapse or need for systemic immunosuppressive GvHD treatment. The five-year treatment success in patients with acute lymphatic leukemia was comparable between high- and non-high-risk disease (0.55 (95% CI: 0.42-0.74) and 0.59 (95% CI: 0.42-0.84)). It remained lower in high-risk acute myeloid leukemia (AML) (0.29 (95% CI: 0.18-0.46)) than in non-high-risk AML (0.47 (95% CI: 0.42-0.84)) due to an increased relapse rate despite early DLI. Show less
Background: The immunoglobulin J chain (Jchain) is highly expressed in the majority of multiple myeloma (MM), and Jchain-derived peptides presented in HLA molecules may be suitable antigens for T... Show moreBackground: The immunoglobulin J chain (Jchain) is highly expressed in the majority of multiple myeloma (MM), and Jchain-derived peptides presented in HLA molecules may be suitable antigens for T-cell therapy of MM. Methods: Using immunopeptidomics, we identified Jchain-derived epitopes presented by MM cells, and pHLA tetramer technology was used to isolate Jchain-specific T-cell clones. Results: We identified T cells specific for Jchain peptides presented in HLA-A1, -A24, -A3, and -A11 that recognized and lysed JCHAIN-positive MM cells. TCRs of the most promising T-cell clones were sequenced, cloned into retroviral vectors, and transferred to CD8 T cells. Jchain TCR T cells recognized target cells when JCHAIN and the appropriate HLA restriction alleles were expressed, while JCHAIN or HLA-negative cells, including healthy subsets, were not recognized. Patient-derived JCHAIN-positive MM samples were also lysed by Jchain TCR T cells. In a preclinical in vivo model for established MM, Jchain-A1, -A24, -A3, and -A11 TCR T cells strongly eradicated MM cells, which resulted in 100-fold lower tumor burden in Jchain TCR versus control-treated mice. Conclusions: We identified TCRs targeting Jchain-derived peptides presented in four common HLA alleles. All four TCRs demonstrated potent preclinical anti-myeloma activity, encouraging further preclinical testing and ultimately clinical development. Show less
Chimeric antigen receptor (CAR) T cell therapies have resulted in profound clinical responses in the treatment of CD19-positive hematological malignancies, but a significant proportion of patients... Show moreChimeric antigen receptor (CAR) T cell therapies have resulted in profound clinical responses in the treatment of CD19-positive hematological malignancies, but a significant proportion of patients do not respond or relapse eventually. As an alternative to CAR T cells, T cells can be engineered to express a tumor-targeting T cell receptor (TCR). Due to HLA restriction of TCRs, CARs have emerged as a preferred treatment moiety when targeting surface antigens, despite the fact that functional differences between engineered TCR (eTCR) T and CAR T cells remain ill-defined. Here, we compared the activity of CAR T cells versus engineered TCR T cells in targeting the B cell malignancy-associated antigen CD20 as a function of antigen exposure. We found CAR T cells to be more potent effector cells, producing higher levels of cytokines and killing more efficiently than eTCR T cells in a short time frame. However, we revealed that the increase of antigen exposure significantly impaired CAR T cell expansion, a phenotype defined by high expression of coinhibitory molecules and effector differentiation. In contrast, eTCR T cells expanded better than CAR T cells under high antigenic pressure, with lower expression of coinhibitory molecules and maintenance of an early differentiation phenotype, and comparable clearance of tumor cells. Show less
Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) specific CD4(+ )and CD8(+) T cells in SARS- CoV- 2-unexposed donors has been explained by the presence of T cells primed... Show moreDetection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) specific CD4(+ )and CD8(+) T cells in SARS- CoV- 2-unexposed donors has been explained by the presence of T cells primed by other coronaviruses. However, based on the relatively high frequency and prevalence of cross-reactive T cells, we hypothesized cytomegalovirus (CMV) may induce these cross-reactive T cells. Stimulation of pre-pandemic cryo-preserved peripheral blood mononuclear cells (PBMCs) with SARS- CoV- 2 peptides revealed that frequencies of SARS- CoV- 2-specific T cells were higher in CMVseropositive donors. Characterization of these T cells demonstrated that membrane-specific CD4(+ )and spike-specific CD8(+) T cells originate from cross-reactive CMVspecific T cells. Spike-specific CD8(+ )T cells recognize SARS- CoV- 2 spike peptide FVSNGTHWF (FVS) and dissimilar CMV pp65 peptide IPSINVHHY (IPS) presented by HLA- B*35:01. These dual IPS/FVS-reactive CD8(+) T cells were found in multiple donors as well as severe COVID- 19 patients and shared a common T cell receptor (TCR), illustrating that IPS/FVS- cross-reactivity is caused by a public TCR. In conclusion, CMVspecific T cells cross react with SARS-CoV- 2, despite low sequence homology between the two viruses, and may contribute to the pre-existing immunity against SARS-CoV- 2. Show less
Huisman, W.; Gier, M. de; Hageman, L.; Shomuradova, A.S.; Leboux, D.A.T.; Amsen, D.; ... ; Jedema, I. 2022
Anti-viral T-cell responses are usually directed against a limited set of antigens, but often contain many T cells expressing different T-cell receptors (TCRs). Identical TCRs found within virus... Show moreAnti-viral T-cell responses are usually directed against a limited set of antigens, but often contain many T cells expressing different T-cell receptors (TCRs). Identical TCRs found within virus-specific T-cell populations in different individuals are known as public TCRs, but also TCRs highly-similar to these public TCRs, with only minor variations in amino acids on specific positions in the Complementary Determining Regions (CDRs), are frequently found. However, the degree of freedom at these positions was not clear. In this study, we used the HLA-A*02:01-restricted EBV-LMP2(FLY)-specific public TCR as model and modified the highly-variable position 5 of the CDR3 beta sequence with all 20 amino acids. Our results demonstrate that amino acids at this particular position in the CDR3 beta region of this TCR are completely inter-changeable, without loss of TCR function. We show that the inability to find certain variants in individuals is explained by their lower recombination probability rather than by steric hindrance. Show less
Huisman, W.; Gille, I.; Maarel, L.E. van der; Hageman, L.; Morton, L.T.; Jong, R.C.M. de; ... ; Jedema, I. 2022
Background: Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated... Show moreBackground: Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated malignancies of latency type II/III that express EBV antigens (LMP1/2). Patients who are HLA-A*01:01 positive could benefit from such products, since no T cells recognizing any EBV-derived peptide in this common HLA allele have been found thus far. Methods: HLA-A*01:01-restricted EBV-LMP2-specific T cells were isolated using peptide major histocompatibility complex (pMHC) tetramers. Functionality was assessed by production of interferon gamma (IFN-gamma) and cytotoxicity when stimulated with EBV-LMP2-expressing cell lines. Functionality of primary T cells transduced with HLA-A*01:01-restricted EBV-LMP2-specific TCRs was optimized by knocking out the endogenous TCRs of primary T cells ( increment TCR) using CRISPR-Cas9 technology. Results: EBV-LMP2-specific T cells were successfully isolated and their TCRs were characterized. TCR gene transfer in primary T cells resulted in specific pMHC tetramer binding and reactivity against EBV-LMP2-expressing cell lines. The mean fluorescence intensity of pMHC-tetramer binding was increased 1.5-2 fold when the endogenous TCRs of CD8(+) T cells was knocked out. CD8(+/ increment TCR) T cells modified to express EBV-LMP2-specific TCRs showed IFN-gamma secretion and cytotoxicity toward EBV-LMP2-expressing malignant cell lines. Conclusions: We isolated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can potentially be used in future TCR gene therapy to treat EBV-associated latency type II/III malignancies.Here we identify the first HLA-A*01:01-restricted Epstein-Barr virus Latent Membrane Protein 2 (EBV-LMP2)-specific T-cell population and show that these T-cell populations and T cells modified to express the LMP2-specific T-cell receptor showed IFN-gamma secretion and cytotoxicity toward EBV-LMP2-expressing malignant cell lines. Show less
Background Transcription factor Wilms' tumor gene 1 (WT1) is an ideal tumor target based on its expression in a wide range of tumors, low-level expression in normal tissues and promoting role in... Show moreBackground Transcription factor Wilms' tumor gene 1 (WT1) is an ideal tumor target based on its expression in a wide range of tumors, low-level expression in normal tissues and promoting role in cancer progression. In clinical trials, WT1 is targeted using peptide-based or dendritic cell-based vaccines and T-cell receptor (TCR)-based therapies. Antitumor reactivities were reported, but T-cell reactivity is hampered by self-tolerance to WT1 and limited number of WT1 peptides, which were thus far selected based on HLA peptide binding algorithms. Methods In this study, we have overcome both limitations by searching in the allogeneic T-cell repertoire of healthy donors for high-avidity WT1-specific T cells, specific for WT1 peptides derived from the HLA class I associated ligandome of primary leukemia and ovarian carcinoma samples. Results Using broad panels of malignant cells and healthy cell subsets, T-cell clones were selected that demonstrated potent and specific anti-WT1 T-cell reactivity against five of the eight newly identified WT1 peptides. Notably, T-cell clones for WT1 peptides previously used in clinical trials lacked reactivity against tumor cells, suggesting limited processing and presentation of these peptides. The TCR sequences of four T-cell clones were analyzed and TCR gene transfer into CD8+ T cells installed antitumor reactivity against WT1-expressing solid tumor cell lines, primary acute myeloid leukemia (AML) blasts, and ovarian carcinoma patient samples. Conclusions Our approach resulted in a set of naturally expressed WT1 peptides and four TCRs that are promising candidates for TCR gene transfer strategies in patients with WT1-expressing tumors, including AML and ovarian carcinoma. Show less
Unconventional HLA class I-restricted CD8+ T cell epitopes, longer than 10 aa, have been implicated to play a role in human immunity against viruses and cancer. T cell recognition of long peptides... Show moreUnconventional HLA class I-restricted CD8+ T cell epitopes, longer than 10 aa, have been implicated to play a role in human immunity against viruses and cancer. T cell recognition of long peptides, centrally bulging from the HLA cleft, has been described previously. Alternatively, long peptides can contain a linear HLA-bound core peptide, with a N- or C-terminal peptide "tail" extending from the HLA peptide binding groove. The role of such a peptide "tail" in CD8+ T cell recognition remains unclear. In this study, we identified a 20mer peptide (FLPTPEELGLLGPPRPQVLA [FLP]) derived from the IL-27R subunit α gene restricted to HLA-A*02:01, for which we solved the crystal structure and demonstrated a long C-terminal "tail" extension. FLP-specific T cell clones demonstrated various recognition modes, some T cells recognized the FLP core peptide, while for other T cells the peptide tail was essential for recognition. These results demonstrate a crucial role for a C-terminal peptide tail in immunogenicity. Show less
Since multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of... Show moreSince multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of T cells in a human body, the likelihood that many public identical (PUB-I) TCR-sequences frequently contribute to immune responses has been estimated to be low. Here, we quantitatively analyzed the TCR-repertoires of 190 purified virus-specific memory T-cell populations, directed against 21 epitopes of Cytomegalovirus, Epstein-Barr virus and Adenovirus isolated from 29 healthy individuals, and determined the magnitude, defined as prevalence within the population and frequencies within individuals, of PUB-I TCR and of TCR-sequences that are highly-similar (PUB-HS) to these PUB-I TCR-sequences. We found that almost one third of all TCR nucleotide-sequences represented PUB-I TCR amino-acid (AA) sequences and found an additional 12% of PUB-HS TCRs differing by maximally 3 AAs. We illustrate that these PUB-I and PUB-HS TCRs were structurally related and contained shared core-sequences in their TCR-sequences. We found a prevalence of PUB-I and PUB-HS TCRs of up to 50% among individuals and showed frequencies of virus-specific PUB-I and PUB-HS TCRs making up more than 10% of each virus-specific T-cell population. These findings were confirmed by using an independent TCR-database of virus-specific TCRs. We therefore conclude that the magnitude of the contribution of PUB-I and PUB-HS TCRs to these virus-specific T-cell responses is high. Because the T cells from these virus-specific memory TCR-repertoires were the result of successful control of the virus in these healthy individuals, these PUB-HS TCRs and PUB-I TCRs may be attractive candidates for immunotherapy in immunocompromised patients that lack virus-specific T cells to control viral reactivation. Show less
Background T cell receptor (TCR)-engineered cells can be powerful tools in the treatment of malignancies. However, tumor resistance by Human Leukocyte antigen (HLA) class I downregulation can... Show moreBackground T cell receptor (TCR)-engineered cells can be powerful tools in the treatment of malignancies. However, tumor resistance by Human Leukocyte antigen (HLA) class I downregulation can negatively impact the success of any TCR-mediated cell therapy. Allogeneic natural killer (NK) cells have demonstrated efficacy and safety against malignancies without inducing graft-versus-host-disease, highlighting the feasibility for an 'off the shelf' cellular therapeutic. Furthermore, primary NK cells can target tumors using a broad array of intrinsic activation mechanisms. In this study, we combined the antitumor effector functions of NK cells with TCR engineering (NK-TCR), creating a novel therapeutic strategy to avoid TCR-associated immune resistance. Methods BOB1, is a transcription factor highly expressed in all healthy and malignant B cell lineages, including multiple myeloma (MM). Expression of an HLA-B*07:02 restricted BOB1-specifc TCR in peripheral blood-derived NK cells was achieved following a two-step retroviral transduction protocol. NK-TCR was then compared with TCR-negative NK cells and CD8-T cells expressing the same TCR for effector function against HLA-B*07:02+ B-cell derived lymphoblastoid cell lines (B-LCL), B-cell acute lymphoblastic leukemia and MM cell lines in vitro and in vivo. Results Firstly, TCR could be reproducibly expressed in NK cells isolated from the peripheral blood of multiple healthy donors generating pure NK-TCR cell products. Secondly, NK-TCR demonstrated antigen-specific effector functions against malignancies which were previously resistant to NK-mediated lysis and enhanced NK efficacy in vivo using a preclinical xenograft model of MM. Moreover, antigen-specific cytotoxicity and cytokine production of NK-TCR was comparable to CD8 T cells expressing the same TCR. Finally, in a model of HLA-class I loss, tumor cells with B2M KO were lysed by NK-TCR in an NK-mediated manner but were resistant to T-cell based killing. Conclusion NK-TCR cell therapy enhances NK cell efficacy against tumors through additional TCR-mediated lysis. Furthermore, the dual efficacy of NK-TCR permits the specific targeting of tumors and the associated TCR-associated immune resistance, making NK-TCR a unique cellular therapeutic. Show less
Laghmouchi, A.; Kester, M.G.D.; Hoogstraten, C.; Hageman, L.; Klerk, W. de; Huisman, W.; ... ; Jedema, I. 2022
In the context of HLA-DP-mismatched allogeneic stem cell transplantation, mismatched HLA-DP alleles can provoke profound allo-HLA-DP-specific immune responses from the donor T-cell repertoire... Show moreIn the context of HLA-DP-mismatched allogeneic stem cell transplantation, mismatched HLA-DP alleles can provoke profound allo-HLA-DP-specific immune responses from the donor T-cell repertoire leading to graft-versus-leukemia effect and/or graft-versus-host disease in the patient. The magnitude of allo-HLA-DP-specific immune responses has been shown to depend on the specific HLA-DP disparity between donor and patient and the immunogenicity of the mismatched HLA-DP allele(s). HLA-DP peptidome clustering (DPC) was developed to classify the HLA-DP molecules based on similarities and differences in their peptide-binding motifs. To investigate a possible categorization of HLA-DP molecules based on overlap of presented peptides, we identified and compared the peptidomes of the thirteen most frequently expressed HLA-DP molecules. Our categorization based on shared peptides was in line with the DPC classification. We found that the HLA-DP molecules within the previously defined groups DPC-1 or DPC-3 shared the largest numbers of presented peptides. However, the HLA-DP molecules in DPC-2 segregated into two subgroups based on the overlap in presented peptides. Besides overlap in presented peptides within the DPC groups, a substantial number of peptides was also found to be shared between HLA-DP molecules from different DPC groups, especially for groups DPC-1 and -2. The functional relevance of these findings was illustrated by demonstration of cross-reactivity of allo-HLA-DP-reactive T-cell clones not only against HLA-DP molecules within one DPC group, but also across different DPC groups. The promiscuity of peptides presented in various HLA-DP molecules and the cross-reactivity against different HLA-DP molecules demonstrate that these molecules cannot be strictly categorized in immunogenicity groups. Show less
CAR T cell therapy has shown great promise for the treatment of B cell malignancies. However, antigen-negative escape variants often cause disease relapse, necessitating the development of multi... Show moreCAR T cell therapy has shown great promise for the treatment of B cell malignancies. However, antigen-negative escape variants often cause disease relapse, necessitating the development of multi-antigen-targeting approaches. We propose that a T cell receptor (TCR)-based strategy would increase the number of potential antigenic targets, as peptides from both intracellular and extracellular proteins can be recognized. Here, we aimed to isolate a broad range of promising TCRs targeting multiple antigens for treatment of B cell malignancies. As a first step, 28 target genes for B cell malignancies were selected based on gene expression profiles. Twenty target peptides presented in human leukocyte antigen (HLA)-A*01:01, -A*24:02, -B*08:01, or -B*35:01 were identified from the immunopeptidome of B cell malignancies and used to form peptide-HLA (pHLA)-tetramers for T cell isolation. Target-peptide-specific CD8 T cells were isolated from HLA-mismatched healthy donors and subjected to a stringent stepwise selection procedure to ensure potency and eliminate cross-reactivity. In total, five T cell clones specific for FCRL5 in HLA-A*01:01, VPREB3 in HLA-A*24:02, and BOB1 in HLA-B*35:01 recognized B cell malignancies. For all three specificities, TCR gene transfer into CD8 T cells resulted in cytokine production and efficient killing of multiple B cell malignancies. In conclusion, using this systematic approach we successfully identified three promising TCRs for T cell therapy against B cell malignancies. Show less
