T cells play a vital role in the anti-tumoural response, and the presence of tumour-infiltrating lymphocytes has shown to be directly correlated with a good prognosis in several cancer types. Nevertheless, some patients presenting tumour-infiltrating lymphocytes do not have favourable outcomes. The TCR determines the specificities of T cells, so the analysis of the TCR repertoire has been recently considered to be a potential biomarker for patients’ progression and response to therapies with immune checkpoint inhibitors. The TCR repertoire is one of the multiple elements comprising the immune system and is conditioned by several factors, including tissue type, tumour mutational burden, and patients’ immunogenetics. Its study is crucial to understanding the anti-tumoural response, how to beneficially modulate the immune response with current or new treatments, and how to better predict the prognosis. Here, we present a critical review including essential studies on TCR repertoire conducted in patients with cancer with the aim to draw the current conclusions and try to elucidate whether it is better to encounter higher clonality with few TCRs at higher frequencies, or higher diversity with many different TCRs at lower frequencies.

EBV-specific T cells have been recently described to be involved in fatal encephalitis and myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) high-throughput sequencing was performed to monitor the T-cell clonotypes present in the different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after three passes in mice along 6 months. Eighteen of these (42%) were also found in the TNBC biopsy. TCR infiltrating the XABCL tissue showed a very restricted T-cell repertoire as compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL-LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain clonotypes were selected by the B cells. A number of these TCRs had previously been reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides that might have been expanding these T cells. The HLA class I and class II-associated peptides from XABCL-LCL were then compared with published repertoires from LCL of different HLA typing. Proteins from the antigen processing and presentation pathway remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II-presented peptides were derived from cancer-related proteins. These results suggest that bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor-infiltrating T cells and influence the TCR repertoire in the tumor site.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal mature B lymphocytes in peripheral blood, bone marrow, lymphoid tissues, and extranodal sites. Genes involved in RNA splicing such as SF3B1 and U1 are frequently mutated in CLL, leading to altered splicing and generation of tumor neoepitopes. To study the impact of these mutations on the tumor microenvironment (TME), we have developed a comprehensive single-cell atlas of unmutated CLL encompassing 26 bone marrow and lymph node tumor samples from 23 U-CLL patients with mutations in U1 (n=7), SF3B1 (n=8), or without mutations in splicing genes (n=10). We observed high intra-tumor heterogeneity, discerning 12 transcriptional programs, one linked to the U1 g.A3>C mutation and characterized by NFKB hyperactivation. T cell and NK compartments exhibited site- and mutation-specific enrichment, with increased CD4 ⁺ regulatory cells (Treg) and CD8 ⁺ exhausted cells in lymph nodes, while U1 -mutant tumors showed increased CD8 ⁺ cytotoxic activity, with a predominance of effector-like CD8 ⁺ cells. Single-cell T cell receptor sequencing revealed clonotype expansion in U1 -mutated tumors, particularly in CD8 ⁺ effector and exhausted cells, suggesting a neoantigen-driven immune response. Cell-to-cell interaction analysis identified CD44 as a key mediator in U1 -mutated tumors, showing pro-B survival interactions as those involving MIF-CD44-CD74. Furthermore, interactions between CD80 on CLL cells and CTLA4 on Tregs and CD8 ⁺ exhausted were upregulated, reflecting an immunosuppressive phenotype associated with U1 mutated CLL. These findings highlight the complex interplay between mutations in CLL and the TME, offering novel avenues for alternative therapeutic strategies for U-CLL with mutations in U1 .

The anti‐tumour immune response plays a pivotal role in eliminating tumour cells, with the presence of tumour‐infiltrating lymphocytes (TILs) often correlated with improved patient outcomes. Among these, CD4+ T lymphocytes act as key orchestrators of the immune response, functioning as effector and regulatory cells, and are essential for establishing immunological memory. To better understand the role of CD4+ T cells in anti‐tumour immunity, we analysed the HLA‐II immunopeptidome of dendritic cells (DCs) from HLA‐heterozygous donors pulsed with a protein extract from the MCF‐7 tumour cell line. Our objective was to identify differences in the arrays of peptides binding distinct HLA‐DRB1 allele combinations and the effect of DC pulsing on peptide presentation. We found that presented peptide repertoires are strongly influenced by HLA‐DR heterozygosity in an allele‐specific manner. Alleles with high binding strength (e.g., DRB1*01:01 , DRB1*03:01 and DRB1*04:04 ) tended to dominate peptide presentation; however, this dominance is significantly modulated by the allelic combination, suggesting that antigen presentation is shaped not only by individual allele properties but also by their combinations. Pulsing DCs with MCF‐7 extracts increased peptide overlap between donors and enabled the identification of 58 proteins putatively derived from the tumour cell line lysates. Interestingly, peptide presentation from these proteins reinforced allele‐specific features of dominance and weakness previously observed across the entire immunopeptidome. Gaining insights into the peptide repertoire presented by distinct HLA‐DR combinations could inform the design of personalised immunotherapies based on peptide‐pulsed DCs, ultimately enhancing CD4+ TIL responses across diverse patient populations.

Open-access immunosequencing data | Aran, Andrea | Frontiers in Immunology | Adaptive Biotechnologies | Coming soon.

Introduction: The analysis of tumor-infiltrating lymphocytes often requires techniques that expand their numbers, potentially introducing bias. To address this, we performed a detailed analysis of minimally cultured TILs to evaluate whether this approach better preserves their characteristics. Methods: The TIL culture method was based solely on tumor tissue with low IL-2 supplementation to minimize artificial alterations. The validity of this approach was confirmed by the correlation between CD3+ T cell percentages in cultures and infiltration patterns observed by immunohistochemistry. Immunophenotyping, cytokine release, and TCR repertoire analysis were used to characterize CD4+ and CD8+ T cell subsets and their molecular features during minimal expansions. Results: High TIL infiltration areas did not consistently correspond to an increased presence of any T cell subset; both CD4+ and CD8+ T cells frequently coexisted in these regions. In contrast, low TIL infiltration sections often displayed a higher proportion of CD4+ T cells. An inverse correlation between CD4+ T cell percentages and cytotoxic molecules was observed, indicating reduced cytotoxic activity in low-TIL sections with abundant CD4+ T cells. TCR repertoire analysis revealed differences between T cell subsets: CD4+ T cells were associated with longer TRA CDR3 nt and shorter TRB N(D)N nt lengths, along with lower diversity, while CD8+ T cells did not exhibit significant correlation with any TCR feature. Discussion: This study highlights the distinct biological features of CD4⁺ and CD8⁺ TIL populations within the tumor microenvironment that can be preserved using a minimally expanded TIL approach. The observed associations between IHC patterns, T cell subset composition, cytotoxic potential, and TCR repertoire diversity help identify which biopsy regions yield TILs with greater therapeutic potential, thus providing guidance for TIL selection in immunotherapy.