Cancer immunotherapy has experienced remarkable advances in the last decades. Striking clinical responses have been achieved for several solid cancers, particularly cancer types with a high... Show moreCancer immunotherapy has experienced remarkable advances in the last decades. Striking clinical responses have been achieved for several solid cancers, particularly cancer types with a high mutation burden, which placed tumour-mutated antigens (neoantigens) centre stage as targets of tumour immunity and cancer immunotherapy. Neoantigens can be presented in complex with HLA molecules on the tumour cell surface, where T cells with the correct specificity can recognize the neoantigen as ‘non-self’ which will trigger killing of the tumour cell by the T cell. In theory, cancers with a low/moderate mutation burden that present neoantigens in complex with HLA class molecules could still be eligible for T cell-mediated immunotherapy. This thesis, describes the finding that neoantigen-specific T cells are present in mismatch-repair proficient (MMR-p) colorectal cancer patients, a low mutation burden cancer type. Moreover, CD39 and CD103 were found as cell surface markers that pinpoint the T cell population that contains the neoantigen-specific T cells. In addition, subsequent metastasis of a melanoma patient cohort were studied and revealed that also at advanced, late-stage disease, neoantigen-directed T cell therapy is, in theory, still applicable. Taken together, the studies reveal potential for the development of neoantigen-directed cancer immunotherapy for a broader patient population. Show less
Bulk, J. van den; Ploeg, M. van der; Ijsselsteijn, M.E.; Ruano, D.; Breggen, R. van der; Duhen, R.; ... ; Miranda, N.F.C.C. de 2023
Background Expression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+ T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify... Show moreBackground Expression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+ T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify neoantigen-specific T cells in colorectal cancers (CRCs) with low mutation burden.Experimental design Whole-exome and RNA sequencing of 11 mismatch repair-proficient (MMR-proficient) CRCs and corresponding healthy tissues were performed to determine the presence of putative neoantigens. In parallel, tumor-infiltrating lymphocytes (TILs) were cultured from the tumor fragments and, in parallel, CD8+ T cells were flow-sorted from their respective tumor digests based on single or combined expression of CD103 and CD39. Each subset was expanded and subsequently interrogated for neoantigen-directed reactivity with synthetic peptides. Neoantigen-directed reactivity was determined by flow cytometric analyses of T cell activation markers and ELISA-based detection of IFN-γ and granzyme B release. Additionally, imaging mass cytometry was applied to investigate the localization of CD103+CD39+ cytotoxic T cells in tumors.Results Neoantigen-directed reactivity was only encountered in bulk TIL populations and CD103+CD39+ (double positive, DP) CD8+ T cell subsets but never in double-negative or single-positive subsets. Neoantigen-reactivity detected in bulk TIL but not in DP CD8+ T cells could be attributed to CD4+ T cells. CD8+ T cells that were located in direct contact with cancer cells in tumor tissues were enriched for CD103 and CD39 expression.Conclusion Coexpression of CD103 and CD39 is characteristic of neoantigen-specific CD8+ T cells in MMR-proficient CRCs with low mutation burden. The exploitation of these subsets in the context of adoptive T cell transfer or engineered T cell receptor therapies is a promising avenue to extend the benefits of immunotherapy to an increasing number of CRC patients. Show less
Vries, N.L. de; Haar, J. van de; Veninga, V.; Chalabi, M.; Ijsselsteijn, M.E.; Ploeg, M. van der; ... ; Voest, E.E. 2023
DNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)(1,2). Here, in... Show moreDNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)(1,2). Here, in contrast to other cancer types(3-5), we observed that 20 out of 21 (95%) MMR-d cancers with genomic inactivation of beta 2-microglobulin (encoded by B2M) retained responsiveness to ICB, suggesting the involvement of immune effector cells other than CD8(+) T cells in this context. We next identified a strong association between B2M inactivation and increased infiltration by gamma delta T cells in MMR-d cancers. These gamma delta T cells mainly comprised the V delta 1 and V delta 3 subsets, and expressed high levels of PD-1, other activation markers, including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors. In vitro, PD-1(+) gamma delta T cells that were isolated from MMR-d colon cancers exhibited enhanced reactivity to human leukocyte antigen (HLA)-class-I-negative MMR-d colon cancer cell lines and B2M-knockout patient-derived tumour organoids compared with antigen-presentation-proficient cells. By comparing paired tumour samples from patients with MMR-d colon cancer that were obtained before and after dual PD-1 and CTLA-4 blockade, we found that immune checkpoint blockade substantially increased the frequency of gamma delta T cells in B2M-deficient cancers. Taken together, these data indicate that gamma delta T cells contribute to the response to immune checkpoint blockade in patients with HLA-class-I-negative MMR-d colon cancers, and underline the potential of gamma delta T cells in cancer immunotherapy. Show less
Vries, N.L. de; Haar, J. van de; Veninga, V.; Chalabi, M.; Ijsselsteijn, M.E.; Ploeg, M. van der; ... ; Voest, E.E. 2023
DNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. Here, in... Show moreDNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. Here, in contrast to other cancer types3,4,5, we observed that 20 out of 21 (95%) MMR-d cancers with genomic inactivation of β2-microglobulin (encoded by B2M) retained responsiveness to ICB, suggesting the involvement of immune effector cells other than CD8+ T cells in this context. We next identified a strong association between B2M inactivation and increased infiltration by γδ T cells in MMR-d cancers. These γδ T cells mainly comprised the Vδ1 and Vδ3 subsets, and expressed high levels of PD-1, other activation markers, including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors. In vitro, PD-1+ γδ T cells that were isolated from MMR-d colon cancers exhibited enhanced reactivity to human leukocyte antigen (HLA)-class-I-negative MMR-d colon cancer cell lines and B2M-knockout patient-derived tumour organoids compared with antigen-presentation-proficient cells. By comparing paired tumour samples from patients with MMR-d colon cancer that were obtained before and after dual PD-1 and CTLA-4 blockade, we found that immune checkpoint blockade substantially increased the frequency of γδ T cells in B2M-deficient cancers. Taken together, these data indicate that γδ T cells contribute to the response to immune checkpoint blockade in patients with HLA-class-I-negative MMR-d colon cancers, and underline the potential of γδ T cells in cancer immunotherapy. Show less
Hos, B.J.; Tondini, E.; Camps, M.G.M.; Rademaker, W.; Bulk, J. van den; Ruano, D.; ... ; Ossendorp, F. 2022
We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides... Show moreWe report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides. MHC class II -presented peptide sequences are identified by introducing the MHC class II transactivator (CIITA) in tumor cells that were originally MHC class II negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals oncoviral-and neo-epitopes as well as shared epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses, which helped tu-mor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes pre-sented by any MHC class II allele that would be otherwise very difficult to predict and reveals important tar-gets for cancer immunotherapy. Show less
Hos, B.J.; Tondini, E.; Camps, M.G.M.; Rademaker, W.; Bulk, J. van den; Ruano, D.; ... ; Ossendorp, F. 2022
We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of MHC class II-eluted natural peptides. MHC class II-presented peptide... Show moreWe report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of MHC class II-eluted natural peptides. MHC class II-presented peptide sequences are identified by introducing the MHC class II transactivator CIITA in tumor cells that were originally MHC class II-negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals many novel oncoviral-, shared and neo-epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses which helped tumor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes presented by any MHC class II allele that would be otherwise very difficult to predict and reveals important new targets for cancer immunotherapy. Show less
Bulk, J. van den; Miranda, N.F.C.C. de; Dijke, P. ten 2021
Cancers may escape elimination by the host immune system by rewiring the tumour microenvironment towards an immune suppressive state. Transforming growth factor-β (TGF-β) is a secreted... Show moreCancers may escape elimination by the host immune system by rewiring the tumour microenvironment towards an immune suppressive state. Transforming growth factor-β (TGF-β) is a secreted multifunctional cytokine that strongly regulates the activity of immune cells while, in parallel, can promote malignant features such as cancer cell invasion and migration, angiogenesis, and the emergence of cancer-associated fibroblasts. TGF-β is abundantly expressed in cancers and, most often, its abundance associated with poor clinical outcomes. Immunotherapeutic strategies, particularly T cell checkpoint blockade therapies, so far, only produce clinical benefit in a minority of cancer patients. The inhibition of TGF-β activity is a promising approach to increase the efficacy of T cell checkpoint blockade therapies. In this review, we briefly outline the immunoregulatory functions of TGF-β in physiological and malignant contexts. We then deliberate on how the therapeutic targeting of TGF-β may lead to a broadened applicability and success of state-of-the-art immunotherapies. Show less
Bulk, J. van den; Miranda, N.F.C.C. de; Dijke, P. ten 2021
Cancers may escape elimination by the host immune system by rewiring the tumour microenvironment towards an immune suppressive state. Transforming growth factor-beta (TGF-beta) is a secreted... Show moreCancers may escape elimination by the host immune system by rewiring the tumour microenvironment towards an immune suppressive state. Transforming growth factor-beta (TGF-beta) is a secreted multifunctional cytokine that strongly regulates the activity of immune cells while, in parallel, can promote malignant features such as cancer cell invasion and migration, angiogenesis, and the emergence of cancer-associated fibroblasts. TGF-beta is abundantly expressed in cancers and, most often, its abundance associated with poor clinical outcomes. Immunotherapeutic strategies, particularly T cell checkpoint blockade therapies, so far, only produce clinical benefit in a minority of cancer patients. The inhibition of TGF-beta activity is a promising approach to increase the efficacy of T cell checkpoint blockade therapies. In this review, we briefly outline the immunoregulatory functions of TGF-beta in physiological and malignant contexts. We then deliberate on how the therapeutic targeting of TGF-beta may lead to a broadened applicability and success of state-of-the-art immunotherapies. Show less
Hos, B.J.; Camps, M.G.M.; Bulk, J. van den; Tondini, E.; Ende, T.C. van den; Ruano, D.; ... ; Ossendorp, F. 2020
The murine MC-38 colorectal cancer model is a commonly used model for cancer with high mutational burden, which is sensitive for immune checkpoint immunotherapy. We set out to analyze endogenous... Show moreThe murine MC-38 colorectal cancer model is a commonly used model for cancer with high mutational burden, which is sensitive for immune checkpoint immunotherapy. We set out to analyze endogenous CD8(+) T cell responses to MC-38 neo-antigens in tumor-bearing mice and after anti-PD-L1 checkpoint therapy. Through combination of whole-exome sequencing analysis with mass spectrometry of MHC class I eluted peptides we could identify eight candidate epitopes. Of these, a neo-epitope encoded by a point-mutation in the sequence of the ribosomal protein L18 (Rpl18) strongly dominated the CD8(+) T cell response to our MC-38 cell-line in comparison to a previously described neo-epitope in the Adpgk protein. Therapeutic vaccination with synthetic peptides induced CD8(+) T cell responses against the mutated Rpl18 epitope, which controlled tumor growth in vivo. This immunologically dominant response to mutated Rpl18 is of great importance in the development and optimization of immunotherapeutic strategies with the MC-38 tumor model. Show less
Bulk, J. van den; Verdegaal, E.M.E.; Ruano, D.; Ijsselsteijn, M.E.; Visser, M.; Breggen, R. van der; ... ; Miranda, N.F.C.C. de 2019
Background: The efficacy of checkpoint blockade immunotherapies in colorectal cancer is currently restricted to a minority of patients diagnosed with mismatch repair-deficient tumors having high... Show moreBackground: The efficacy of checkpoint blockade immunotherapies in colorectal cancer is currently restricted to a minority of patients diagnosed with mismatch repair-deficient tumors having high mutation burden. However, this observation does not exclude the existence of neoantigen-specific T cells in colorectal cancers with low mutation burden and the exploitation of their anti-cancer potential for immunotherapy. Therefore, we investigated whether autologous neoantigen-specific T cell responses could also be observed in patients diagnosed with mismatch repair-proficient colorectal cancers.Methods: Whole-exome and transcriptome sequencing were performed on cancer and normal tissues from seven colorectal cancer patients diagnosed with mismatch repair-proficient tumors to detect putative neoantigens. Corresponding neo-epitopes were synthesized and tested for recognition by in vitro expanded T cells that were isolated from tumor tissues (tumor-infiltrating lymphocytes) and from peripheral mononuclear blood cells stimulated with tumor material.Results: Neoantigen-specific T cell reactivity was detected to several neo-epitopes in the tumor-infiltrating lymphocytes of three patients while their respective cancers expressed 15, 21, and 30 non-synonymous variants. Cell sorting of tumor-infiltrating lymphocytes based on the co-expression of CD39 and CD103 pinpointed the presence of neoantigen-specific T cells in the CD39(+)CD103(+) T cell subset. Strikingly, the tumors containing neoantigen-reactive TIL were classified as consensus molecular subtype 4 (CMS4), which is associated with TGF-beta pathway activation and worse clinical outcome.Conclusions: We have detected neoantigen-targeted reactivity by autologous T cells in mismatch repair-proficient colorectal cancers of the CMS4 subtype. These findings warrant the development of specific immunotherapeutic strategies that selectively boost the activity of neoantigen-specific T cells and target the TGF-beta pathway to reinforce T cell reactivity in this patient group. Show less
Bulk, J. van den; Verdegaal, E.M.E.; Miranda, N.F.C.C. de 2018
