Fully functional, tumor-specific, SLAMF7-expressing cytotoxic CD4 T cells directly mediate immunity against human cancer.

Vectors that facilitate the engineering of T cells that can better harness endogenous immunity and overcome suppressive barriers in the tumour microenvironment would help improve the safety and efficacy of T-cell therapies for more patients. Here we report the design, production and applicability, in T-cell engineering, of a lentiviral vector leveraging an antisense configuration and comprising a promoter driving the constitutive expression of a tumour-directed receptor and a second promoter enabling the efficient activation-inducible expression of a genetic payload. The vector allows for the delivery of a variety of genes to human T cells, as we show for interleukin-2 and a microRNA-based short hairpin RNA for the knockdown of the gene coding for haematopoietic progenitor kinase 1, a negative regulator of T-cell-receptor signalling. We also show that a gene encoded under an activation-inducible promoter is specifically expressed by tumour-redirected T cells on encountering a target antigen in the tumour microenvironment. The single two-gene-encoding vector can be produced at high titres under an optimized protocol adaptable to good manufacturing practices.

Immunotherapy has become a standard treatment in many cancers and it is based on three main therapeutic axes: immune checkpoint blockade (ICB), vaccination and adoptive cell transfer (ACT). If originally these therapies mainly focused on exploiting CD8 T cells given their role in the direct elimination of tumor cells, increasing evidence highlights the crucial role CD4 T cells play in the antitumor immune response. Indeed, these cells can profoundly modulate the tumor microenvironment (TME) by secreting different types of cytokine or by directly eliminating cancer cells. In this review, we describe how different CD4 T cell subsets can contribute to tumor immune responses during immunotherapy and the novel high-throughput immune monitoring tools that are expected to facilitate the study of CD4 T cells, at antigen-specific and single cell level, thus accelerating bench-to-bed translational research in cancer.

While cancer immunotherapy has primarily focused on CD8 T cells, CD4 T cells are increasingly recognized for their role in antitumor immunity. The HLA-DRB3*02:02 allele is found in 50% of Caucasians. In this study, we screened HLA-DRB3*02:02 patients with melanoma for tumor-specific CD4 T cells and identified robust New York esophageal squamous cell carcinoma 1 (NY-ESO-1) _123–137 / HLA-DRB3*02:02 CD4 T cell activity in both peripheral blood and tumor tissue. By analyzing NY-ESO-1 _123–137 / HLA-DRB3*02:02 –restricted CD4 T cell clones, we uncovered an unexpectedly high cytotoxicity, strong T helper 1 polarization, and recurrent αβ T cell receptor (TCRαβ) usage across patients and anatomical sites. These responses were also present in other NY-ESO-1–expressing cancers. TCRs from these clones, when transduced into primary CD4 T cells, showed direct antitumor efficacy both in vitro and in vivo. Our findings suggest that these TCRs are promising for adoptive T cell transfer therapy, enabling broader targeting of NY-ESO-1–expressing adult and pediatric cancers in clinical settings.

Given the critical role of CD4 T cells in anti-tumor immunity, strategies to harness these cells for cancer immunotherapy are gaining increasing interest. Historically overshadowed by CD8 T cells, cytotoxic CD4 T cells can directly kill MHC class II-expressing tumor cells. However, the defining molecular signature and the mechanisms underlying their cytolytic activity remain poorly understood, particularly in cancer patients. Here, using ex vivo single-cell transcriptomic and spatial analyses of CD4 T cells from paired blood and tumor samples of melanoma patients, we identified Killer Cell Lectin-Like Receptor G1 (KLRG1) as a defining surface marker of cytotoxic CD4 T cells. The CD4 ⁺ KLRG1 ⁺ T cell subset was notably enriched among circulating cells compared with tumor-infiltrating populations, which were instead enriched in T follicular helper (Tfh) states. Functionally, KLRG1 ⁺ CD4 T cells expressed elevated levels of cytotoxic genes and exhibited superior tumor-killing capacity compared with their KLRG1 ^- counterparts. We demonstrated that their cytotoxicity is granulysin-dependent, as confirmed by CRISPR/Cas9-mediated gene deletion. Mechanistically, CD4 T cells spared MHC class II ⁺ cells lacking the KLRG1 ligands CD324 and CD325, such as professional antigen-presenting cells (APCs), indicating that cytotoxicity was selectively directed towards tumor cells while preserving immune cells. Finally, by investigating how the tumor microenvironment may impair CD4 T cell cytotoxicity, we showed that tumor-derived factors, including interleukin-6 (IL-6), are key drivers promoting the transition of cytotoxic CD4 T cells toward a Tfh phenotype. In summary, our findings define KLRG1 as a defining cell surface marker of cytotoxic CD4 T cells in cancer patients, as well as a key regulator that protects MHC class II ⁺ APCs. Moreover, targeting the IL-6 signalling pathway may enhance CD4 T cell anti-tumor cytotoxicity, offering new avenues for cancer immunotherapy.