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
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
MHC class I (MHC-I) molecules present a blueprint of the intracellular proteome to T cells allowing them to control infection or malignant transformation. As a response, pathogens and tumor cells... Show moreMHC class I (MHC-I) molecules present a blueprint of the intracellular proteome to T cells allowing them to control infection or malignant transformation. As a response, pathogens and tumor cells often downmodulate MHC-I mediated antigen presentation to escape from immune surveillance. Although the fundamental rules of antigen presentation are known in detail, the players in this system are not saturated and new modules of regulation have recently been uncovered. Here, we update the understanding of antigen presentation by MHC-I molecules and how this can be exploited by tumors to prevent exposure of the intracellular proteome. This knowledge can provide new ways to improve immune responses against tumors and pathogens. 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
A single model system for integrative studies on multiple facets of antigen presentation is lacking. PAKC is a novel panel of ten cell lines knocked out for individual components of the HLA class I... Show moreA single model system for integrative studies on multiple facets of antigen presentation is lacking. PAKC is a novel panel of ten cell lines knocked out for individual components of the HLA class I antigen presentation pathway. PAKC will accelerate HLA-I research in the fields of oncology, infectiology, and autoimmunity. 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
Metastatic renal cell carcinoma (RCC) has a poor prognosis. Recent advances have shown beneficial responses to immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies. As only a subset of... Show moreMetastatic renal cell carcinoma (RCC) has a poor prognosis. Recent advances have shown beneficial responses to immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies. As only a subset of RCC patients respond, alternative strategies should be explored. Patients refractory to anti-PD-1 therapy may benefit from autologous tumor-infiltrating lymphocyte (TIL) therapy. Even though efficient TIL expansion was reported from RCC lesions, it is not well established how many RCC TIL products are tumor-reactive, how well they produce pro-inflammatory cytokines in response to autologous tumors, and whether their response correlates with the presence of specific immune cells in the tumor lesions. We here compared the immune infiltrate composition of RCC lesions with that of autologous kidney tissue of 18 RCC patients. Tcell infiltrates were increased in the tumor lesions, and CD8(+) Tcell infiltrates were primarily of effector memory phenotype. Nine out of 16 (56%) tested TIL products we generated were tumor-reactive, as defined by CD137 upregulation after exposure to autologous tumor digest. Tumor reactivity was found in particular in TIL products originating from tumors with ahigh percentage of infiltrated Tcells compared to autologous kidney, and increased CD25 expression on CD8(+) Tcells. Importantly, although TIL products had the capacity to produce the key effector cytokines IFN-gamma, TNF-alpha or IL-2, they failed to produce significant amounts in response to autologous tumor digests. In conclusion, TIL products from RCC lesions contain tumor-reactive Tcells. Their restricted tumor-specific cytokine production requires further investigation of immunosuppressive factors in RCC and subsequent optimization of RCC-derived TIL culture conditions. 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 CD8+ 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
Pont, M.J.; Oostvogels, R.; Bergen, C.A.M. van; Meijden, E. van der; Honders, M.W.; Bliss, S.; ... ; Spaapen, R.M. 2019
MHC-bound peptides from aberrant proteins may be a specific immunotherapeutic target on cancer cells. Because of difficulties in identifying such antigens, viral or model antigens have so far been... Show moreMHC-bound peptides from aberrant proteins may be a specific immunotherapeutic target on cancer cells. Because of difficulties in identifying such antigens, viral or model antigens have so far been used to study their biological relevance. We here identify a naturally existing human T-cell epitope derived from a truncated protein. The antigenic peptide is derived from the gene TTK only through an alternative transcript containing a premature termination codon that may target the transcript for nonsense-mediated decay (NMD). This antigen is recognized by HLA-A(+) 02:01-restricted CD8 thorn T cells derived from an allotransplanted leukemia patient. Functional analyses showed that these T cells failed to recognize several HLA-matched primary leukemic cells that expressed the alternative TTK transcript. Conventional antigen processing and presentation were not affected, suggesting that leukemic cells modify the generation of antigens processed from aberrant proteins. This natural TTK epitope provides insights in the source of transcripts producing antigenic epitopes in healthy and leukemic cells. Our data underscore potential pitfalls of targeting NMD-derived or other unconventionally generated epitopes as immunotherapeutic approach. Show less
Zhang, T.; Waard, A.A. de; Wuhrer, M.; Spaapen, R.M. 2019
