Allogeneic hematopoietic cell transplantation (alloHCT) provides a potential curative treatment for haematological malignancies. The therapeutic Graft-versus-Leukaemia (GvL) effect is induced by... Show moreAllogeneic hematopoietic cell transplantation (alloHCT) provides a potential curative treatment for haematological malignancies. The therapeutic Graft-versus-Leukaemia (GvL) effect is induced by donor T cells attacking patient hematopoietic (malignant) cells. However, if healthy non-hematopoietic tissues are targeted, Graft-versus-Disease (GvHD) may develop. After HLA-matched alloHCT, GvL and GvHD are induced by donor T cells recognizing polymorphic peptides presented by HLA on patient cells, so-called minor histocompatibility antigens (MiHAs). The balance between GvL and GvHD depends on the tissue distribution of MiHAs and T-cell frequencies targeting these MiHAs. T cells against broadly expressed MiHAs induce GvL and GvHD, whereas those targeting MiHAs with hematopoietic-restricted expression induce GvL without GvHD. Recently, the MiHA repertoire identified in natural immune responses after alloHCT was expanded to 159 total HLA-I-restricted MiHAs, including 14 hematopoietic-restricted MiHAs. This review explores their potential relevance to predict, monitor, and manipulate GvL and GvHD for improving clinical outcome after HLA-matched alloHCT. Show less
Struckman, N.E.; Jong, R.C.M. de; Honders, M.W.; Smith, S.A.I.; Lee, D.I. van der; Koutsoumpli, G.; ... ; Griffioen, M. 2024
DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1 and 2 (IDH1/2) are genes involved in epigenetic regulation, each mutated in 7–23% of patients with acute myeloid leukemia. Here, we... Show moreDNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1 and 2 (IDH1/2) are genes involved in epigenetic regulation, each mutated in 7–23% of patients with acute myeloid leukemia. Here, we investigated whether hotspot mutations in these genes encode neoantigens that can be targeted by immunotherapy. Five human B-lymphoblastoid cell lines expressing common HLA class I alleles were transduced with a minigene construct containing mutations that often occur in DNMT3A or IDH1/2. From these minigene-transduced cell lines, peptides were eluted from HLA class I alleles and analyzed using tandem mass spectrometry. The resulting data are available via ProteomeXchange under the identifier PXD050560. Mass spectrometry revealed an HLA-A*01:01-binding DNMT3AR882H peptide and an HLA-B*07:02-binding IDH2R140Q peptide as potential neoantigens. For these neopeptides, peptide–HLA tetramers were produced to search for specific T-cells in healthy individuals. Various T-cell clones were isolated showing specific reactivity against cell lines transduced with full-length DNMT3AR882H or IDH2R140Q genes, while cell lines transduced with wildtype genes were not recognized. One T-cell clone for DNMT3AR882H also reacted against patient-derived acute myeloid leukemia cells with the mutation, while patient samples without the mutation were not recognized, thereby validating the surface presentation of a DNMT3AR882H neoantigen that can potentially be targeted in acute myeloid leukemia via immunotherapy. Show less
Karakaslar, E.O.; Severens, J.F.; Sánchez-López, E.; Veelen, P.A. van; Zlei, M.; Dongen, J.J.M. van; ... ; Akker, E.B. van den 2024
The diagnostic spectrum for AML patients is increasingly based on genetic abnormalities due to their prognostic and predictive value. However, information on the AML blast phenotype regarding... Show moreThe diagnostic spectrum for AML patients is increasingly based on genetic abnormalities due to their prognostic and predictive value. However, information on the AML blast phenotype regarding their maturational arrest has started to regain importance due to its predictive power for drug responses. Here, we deconvolute 1350 bulk RNA-seq samples from five independent AML cohorts on a single-cell healthy BM reference and demonstrate that the morphological differentiation stages (FAB) could be faithfully reconstituted using estimated cell compositions (ECCs). Moreover, we show that the ECCs reliably predict ex-vivo drug resistances as demonstrated for Venetoclax, a BCL-2 inhibitor, resistance specifically in AML with CD14+ monocyte phenotype. We validate these predictions using LUMC proteomics data by showing that BCL-2 protein abundance is split into two distinct clusters for NPM1-mutated AML at the extremes of CD14+ monocyte percentages, which could be crucial for the Venetoclax dosing patients. Our results suggest that Venetoclax resistance predictions can also be extended to AML without recurrent genetic abnormalities and possibly to MDS-related and secondary AML. Lastly, we show that CD14+ monocytic dominated Ven/Aza treated patients have significantly lower overall survival. Collectively, we propose a framework for allowing a joint mutation and maturation stage modeling that could be used as a blueprint for testing sensitivity for new agents across the various subtypes of AML. Show less
Fuchs, K.J.; Meent, M. van de; Honders, M.W.; Khatri, I.; Kester, M.G.D.; Koster, E.A.S.; ... ; Griffioen, M. 2024
Allogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing... Show moreAllogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy nonhematopoietic tissues of patients, thereby inducing side effects known as graft-versus-host disease. Here, we aimed to identify the dominant repertoire of HLA-I-restricted MiHAs to enable strategies to predict, monitor or modulate immune responses after alloSCT. To systematically identify novel MiHAs by genome-wide association screening, T-cell clones were isolated from 39 transplanted patients and tested for reactivity against 191 Epstein-Barr virus transformed B cell lines of the 1000 Genomes Project. By discovering 81 new MiHAs, we more than doubled the antigen repertoire to 159 MiHAs and demonstrated that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients. Furthermore, we showed that one quarter of the antigens are cryptic, that is translated from unconventional open reading frames, for example long noncoding RNAs, showing that these antigen types are relevant targets in natural immune responses. Finally, using single cell RNA-seq data, we analyzed tissue expression of MiHA-encoding genes to explore their potential role in clinical outcome, and characterized 11 new hematopoietic-restricted MiHAs as potential targets for immunotherapy. In conclusion, we expanded the repertoire of HLA-I-restricted MiHAs and identified recurrent, cryptic and hematopoietic-restricted antigens, which are fundamental to predict, follow or manipulate immune responses to improve clinical outcome after alloSCT. Show less
Severens, J.F.; Karakaslar, E.O.; Reijden, B.A. van der; Sánchez-López, E.; Berg, R.R. van den; Halkes, C.J.M.; ... ; Akker, E.B. van den 2024
Subtyping of acute myeloid leukaemia (AML) is predominantly based on recurrent genetic abnormalities, but recent literature indicates that transcriptomic phenotyping holds immense potential to... Show moreSubtyping of acute myeloid leukaemia (AML) is predominantly based on recurrent genetic abnormalities, but recent literature indicates that transcriptomic phenotyping holds immense potential to further refine AML classification. Here we integrated five AML transcriptomic datasets with corresponding genetic information to provide an overview (n = 1224) of the transcriptomic AML landscape. Consensus clustering identified 17 robust patient clusters which improved identification of CEBPA-mutated patients with favourable outcomes, and uncovered transcriptomic subtypes for KMT2A rearrangements (2), NPM1 mutations (5), and AML with myelodysplasia-related changes (AML-MRC) (5). Transcriptomic subtypes of KMT2A, NPM1 and AML-MRC showed distinct mutational profiles, cell type differentiation arrests and immune properties, suggesting differences in underlying disease biology. Moreover, our transcriptomic clusters show differences in ex-vivo drug responses, even when corrected for differentiation arrest and superiorly capture differences in drug response compared to genetic classification. In conclusion, our findings underscore the importance of transcriptomics in AML subtyping and offer a basis for future research and personalised treatment strategies. Our transcriptomic compendium is publicly available and we supply an R package to project clusters to new transcriptomic studies. Show less
Giannakopoulou, E.; Lehander, M.; Culleton, S.V.; Yang, W.W.; Li, Y.Q.; Karpanen, T.; ... ; Olweus, J. 2023
Acute myeloid leukemia (AML), the most frequent leukemia in adults, is driven by recurrent somatically acquired genetic lesions in a restricted number of genes. Treatment with tyrosine kinase... Show moreAcute myeloid leukemia (AML), the most frequent leukemia in adults, is driven by recurrent somatically acquired genetic lesions in a restricted number of genes. Treatment with tyrosine kinase inhibitors has demonstrated that targeting of prevalent FMS-related receptor tyrosine kinase 3 (FLT3) gain-of-function mutations can provide significant survival benefits for patients, although the efficacy of FLT3 inhibitors in eliminating FLT3-mutated clones is variable. We identified a T cell receptor (TCR) reactive to the recurrent D835Y driver mutation in the FLT3 tyrosine kinase domain (TCRFLT3D/Y). TCRFLT3D/Y-redirected T cells selectively eliminated primary human AML cells harboring the FLT3D835Y mutation in vitro and in vivo. TCRFLT3D/Y cells rejected both CD34+ and CD34− AML in mice engrafted with primary leukemia from patients, reaching minimal residual disease-negative levels, and eliminated primary CD34+ AML leukemia-propagating cells in vivo. Thus, T cells targeting a single shared mutation can provide efficient immunotherapy toward selective elimination of clonally involved primary AML cells in vivo. Show less
Sluijs, J.V. van der; Ens, D. van; Brummelman, J.; Heister, D.; Sareen, A.; Truijen, L.; ... ; Hobo, W. 2023
Allogeneic stem cell transplantation (alloSCT) can be curative for hemato-oncology patients due to effective graft-versus-tumor immunity. However, relapse remains the major cause of treatment... Show moreAllogeneic stem cell transplantation (alloSCT) can be curative for hemato-oncology patients due to effective graft-versus-tumor immunity. However, relapse remains the major cause of treatment failure, emphasizing the need for adjuvant immunotherapies. In this regard, post-transplantation dendritic cell (DC) vaccination is a highly interesting strategy to boost graft-versus-tumor responses. Previously, we developed a clinically applicable protocol for simultaneous large-scale generation of end-stage blood DC subsets from donor-derived CD34(+) stem cells, including conventional type 1 and 2 DCs (cDC1s and cDC2s), and plasmacytoid DCs (pDCs). In addition, the total cultured end-product (DC-complete vaccine), also contains non-end-stage-DCs (i.e. non-DCs). In this study, we aimed to dissect the phenotypic identity of these non-DCs and their potential immune modulatory functions on the potency of cDCs and pDCs in stimulating tumor-reactive CD8(+ )T and NK cell responses, in order to obtain rationale for clinical translation of our DC-complete vaccine. The non-DC compartment was heterogeneous and comprised of myeloid progenitors and (immature) granulocyte- and monocyte-like cells. Importantly, non-DCs potentiated toll-like receptor-induced DC maturation, as reflected by increased expression of co-stimulatory molecules and enhanced cDC-derived IL-12 and pDC-derived IFN-alpha production. Additionally, antigen-specific CD8(+) T cells effectively expanded upon DC-complete vaccination in vitro and in vivo. This effect was strongly augmented by non-DCs in an antigen-independent manner. Moreover, non-DCs did not impair in vitro DC-mediated NK cell activation, degranulation nor cytotoxicity. Notably, in vivo i.p. DC-complete vaccination activated i.v. injected NK cells. Together, these data demonstrate that the non-DC compartment potentiates DC-mediated activation and expansion of antigen-specific CD8+ T cells and do not impair NK cell responses in vitro and in vivo. This underscores the rationale for further clinical translation of our CD34+-derived DC-complete vaccine in hemato-oncology patients post alloSCT. Show less
Bergen, C.A.M. van; Kloet, S.L.; Quinten, E.; Yáñez, J.H.S.; Menafra, R.; Griffioen, M.; ... ; Veelken, H. 2023
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
Analytical techniques with high sensitivity and selectivity are essential to the quantitative analysis of clinical samples. Liquid chromatography coupled to tandem mass spectrometry is the gold... Show moreAnalytical techniques with high sensitivity and selectivity are essential to the quantitative analysis of clinical samples. Liquid chromatography coupled to tandem mass spectrometry is the gold standard in clinical chemistry. However, tandem mass spectrometers come at high capital expenditure and maintenance costs. We recently showed that it is possible to generate very similar results using a much simpler single mass spectrometry detector by performing enhanced in-source fragmentation/annotation (EISA) combined with correlated ion monitoring. Here we provide a step-by-step protocol for optimizing the analytical conditions for EISA, so anyone properly trained in liquid chromatography-mass spectrometry can follow and apply this technique for any given analyte. We exemplify the approach by using 2-hydroxyglutarate (2-HG) which is a clinically relevant metabolite whose d-enantiomer is considered an 'oncometabolite', characteristic of cancers associated with mutated isocitrate dehydrogenases 1 or 2 (IDH1/2). We include procedures for determining quantitative robustness, and show results of these relating to the analysis of dl-2-hydroxyglutarate in cells, as well as in serum samples from patients with acute myeloid leukemia that contain the IDH1/2 mutation. This EISA-mass spectrometry protocol is a broadly applicable and low-cost approach for the quantification of small molecules that has been developed to work well for both single-quadrupole and time-of-flight mass analyzers.The tandem mass spectrometers used in clinical chemistry are expensive. This protocol describes how to generate similar results using a single mass spectrometry detector by optimizing in-source fragmentation and data analysis via correlated ion monitoring. Show less
In the current era of T cell-based immunotherapies, it is crucial to understand which types of MHC-presented T cell antigens are produced by tumor cells. In addition to linear peptide antigens,... Show moreIn the current era of T cell-based immunotherapies, it is crucial to understand which types of MHC-presented T cell antigens are produced by tumor cells. In addition to linear peptide antigens, chimeric peptides are generated through proteasome-catalyzed peptide splicing (PCPS). Whether such spliced peptides are abundantly presented by MHC is highly disputed because of disagreement in computational analyses of mass spectrometry data of MHC-eluted peptides. Moreover, such mass spectrometric analyses cannot elucidate how much spliced peptides contribute to the pool of immunogenic antigens. In this Perspective, we explain the significance of knowing the contribution of spliced peptides for accurate analyses of peptidomes on one hand, and to serve as a potential source of targetable tumor antigens on the other hand. Toward a strategy for mass spectrometry independent estimation of the contribution of PCPS to the immunopeptidome, we first reviewed methodologies to identify MHC-presented spliced peptide antigens expressed by tumors. Data from these identifications allowed us to compile three independent datasets containing 103, 74, and 83 confirmed T cell antigens from cancer patients. Only 3.9%, 1.4%, and between 0% and 7.2% of these truly immunogenic antigens are produced by PCPS, therefore providing a marginal contribution to the pool of immunogenic tumor antigens. We conclude that spliced peptides will not serve as a comprehensive source to expand the number of targetable antigens for immunotherapies. 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
Aberrant expression of certain glycosphingolipids (GSLs) is associated withthe differentiation of acute myeloid leukemia (AML) cells. However, the expressionpatterns of GSLs in AML are still poorly... Show moreAberrant expression of certain glycosphingolipids (GSLs) is associated withthe differentiation of acute myeloid leukemia (AML) cells. However, the expressionpatterns of GSLs in AML are still poorly explored because of their complexity, the presenceof multiple isomeric structures, and tedious analytical procedures. In this study, weperformed an in-depth GSL glycan analysis of 19 AML cell lines using porous graphitizedcarbon liquid chromatography-mass spectrometry revealing strikingly different GSL glycanprofiles between the various AML cell lines. The cell lines of the M6 subtype showed a highexpression of gangliosides with alpha 2,3-sialylation and Neu5Gc, while the M2 and M5subtypes were characterized by high expression of (neo)lacto-series glycans and Lewis A/Xantigens. Integrated analysis of glycomics and available transcriptomics data revealed theassociation of GSL glycan abundances with the transcriptomics expression of certainglycosyltransferases (GTs) and transcription factors (TFs). In addition, correlations werefound between specific GTs and TFs. Our data reveal TFsGATA2,GATA1, andRUNX1as candidate inducers of the expression of gangliosides and sialylation via regulation of the GTsST3GAL2andST8SIA1.Inconclusion, we show that GSL glycan expression levels are associated with hematopoietic AML classifications and TF and GT geneexpression. Further research is needed to dissect the regulation of GSL expression and its role in hematopoiesis and associated malignancies. 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
Ali, M.; Giannakopoulou, E.; Li, Y.Q.; Lehander, M.; Culleton, S.V.; Yang, W.W.; ... ; Olweus, J. 2021
Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs... Show moreUnlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.Engineered T cells kill leukemic cells with little off-target toxicity. Show less
Lee, D.I. van der; Koutsoumpli, G.; Reijmers, R.M.; Honders, M.W.; Jong, R.C.M. de; Remst, D.F.G.; ... ; Griffioen, M. 2021
Simple Summary: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis. For AML relapses after chemotherapy, new and effective therapies are needed. In 30-35% of... Show moreSimple Summary: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis. For AML relapses after chemotherapy, new and effective therapies are needed. In 30-35% of AMLs, a frameshift mutation in the nucleophosmin 1 gene (dNPM1) creates potential neoantigens that are attractive targets for immunotherapy. We previously isolated a T-cell receptor (TCR) that targets an HLA-A*02:01-binding dNPM1 neoantigen on primary AML. Here, we investigated whether AVEEVSLRK is another dNPM1 neoantigen that can be targeted by TCR gene transfer. We isolated various T-cells, cloned the HLA-A*11:01-restricted TCR from one T-cell clone and, upon transfer to CD8 cells, demonstrated targeting of dNPM1 primary AMLs in vitro. However, the TCR failed to mediate an anti-tumor effect in immunodeficient mice engrafted with dNPM1 OCI-AML3 cells. Our results demonstrate that AVEEVSLRK is an HLA-A*11:01-binding neoantigen on dNPM1 AML. Whether the isolated TCR is of sufficient affinity to treat patients remains uncertain.Acute myeloid leukemia (AML) is a hematological malignancy caused by clonal expansion of myeloid progenitor cells. Most patients with AML respond to chemotherapy, but relapses often occur and infer a very poor prognosis. Thirty to thirty-five percent of AMLs carry a four base pair insertion in the nucleophosmin 1 gene (NPM1) with a C-terminal alternative reading frame of 11 amino acids. We previously identified various neopeptides from the alternative reading frame of mutant NPM1 (dNPM1) on primary AML and isolated an HLA-A*02:01-restricted T-cell receptor (TCR) that enables human T-cells to kill AML cells upon retroviral gene transfer. Here, we isolated T-cells recognizing the dNPM1 peptide AVEEVSLRK presented in HLA-A*11:01. The TCR cloned from a T-cell clone recognizing HLA-A*11:01+ primary AML cells conferred in vitro recognition and lysis of AML upon transfer to CD8 cells, but failed to induce an anti-tumor effect in immunodeficient NSG mice engrafted with dNPM1 OCI-AML3 cells. In conclusion, our data show that AVEEVSLRK is a dNPM1 neoantigen on HLA-A*11:01+ primary AMLs. CD8 cells transduced with an HLA-A*11:01-restricted TCR for dNPM1 were reactive against AML in vitro. The absence of reactivity in a preclinical mouse model requires further preclinical testing to predict the potential efficacy of this TCR in clinical development. Show less
Tumors with an impaired transporter associated with antigen processing (TAP) present several endoplasmic reticulum-derived self-antigens on HLA class I (HLA-I) which are absent on healthy cells.... Show moreTumors with an impaired transporter associated with antigen processing (TAP) present several endoplasmic reticulum-derived self-antigens on HLA class I (HLA-I) which are absent on healthy cells. Selection of such TAP-independent antigens for T cell-based immunotherapy should include analysis of their expression on healthy cells to prevent therapy-induced adverse toxicities. However, it is unknown how the absence of clinically relevant antigens on healthy cells needs to be validated. Here, we monitored TAP-independent antigen presentation on various healthy cells after establishing a T cell tool recognizing a TAP-independent signal sequence receptor 1-derived antigen. We found that most but not all healthy cells present this antigen under normal and inflammatory conditions, indicating that TAP-independent antigen presentation is a variable phenomenon. Our data emphasize the necessity of extensive testing of a wide variety of healthy cell types to define clinically relevant TAP-independent antigens that can be safely targeted by immunotherapy. Show less
Meurer, T.; Crivello, P.; Metzing, M.; Kester, M.; Megger, D.A.; Chen, W.Q.; ... ; Fleischhauer, K. 2021
In hematopoietic cell transplantation (HCT), permissive HLA-DPB1 mismatches between patients and their unrelated donors are associated with improved outcomes compared with nonpermissive mismatches,... Show moreIn hematopoietic cell transplantation (HCT), permissive HLA-DPB1 mismatches between patients and their unrelated donors are associated with improved outcomes compared with nonpermissive mismatches, but the underlying mechanism is incompletely understood. Here, we used mass spectrometry, T-cell receptor-beta (TCR beta) deep sequencing, and cellular in vitro models of alloreactivity to interrogate the HLA-DP immunopeptidome and its role in alloreactive T-cell responses. We find that permissive HLA-DPB1 mismatches display significantly higher peptide repertoire overlaps compared with their nonpermissive counterparts, resulting in lower frequency and diversity of alloreactive TCR beta clonotypes in healthy individuals and transplanted patients. Permissiveness can be reversed by the absence of the peptide editor HLA-DM or the presence of its antagonist, HLA-DO, through significant broadening of the peptide repertoire. Our data establish the degree of immunopeptidome divergence between donor and recipient as the mechanistic basis for the clinically relevant permissive HLA-DPB1 mismatches in HCT and show that permissiveness is dependent on HLA-DM-mediated peptide editing. Its key role for harnessing T-cell alloreactivity to HLA-DP highlights HLA-DM as a potential novel target for cellular and immunotherapy of leukemia. Show less
HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8(+) T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options... Show moreHLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8(+) T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CDS+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity. Show less
