Cancer immunotherapy activates the immune system to specifically target malignant cells. Research has often focused on CD8+ cytotoxic T cells, as those have the capacity to eliminate tumor cells after specific recognition upon TCR-MHC class I interaction. However, CD4+ T cells have gained attention in the field, as they are not only essential to promote help to CD8+ T cells, but are also able to kill tumor cells directly (via MHC-class II dependent recognition) or indirectly (e.g., via the activation of other immune cells like macrophages). Therefore, immunotherapy approaches have shifted from only stimulating CD8+ T cells to targeting and assessing both, CD4+ and CD8+ T cell subsets. Here, we discuss the various subsets of CD4+ T cells, their plasticity and functionality, their relevance in the antitumor immune response in patients affected by cancer, and their ever-growing role in therapeutic approaches for human cancer.

Elongated peptides (EPs), containing possibly one or multiple epitope/s, are increasingly used for the screening of antigen-specific CD8⁺ and CD4⁺ cell responses. Here, we present an in vitro protocol that allows the amplification of antigen-specific cells and the subsequent functional analysis of both T cell types using EPs. Known viral-derived epitopes were elongated to 20 mer EPs on the N-, C-, and both termini for HLA class I binders, or on the N- and C- termini for HLA class II binders. With EP stimulation only, the percentage of responding CD8⁺ T cells was dependent on the elongation site of the EP, whereas CD4⁺ T cell responses were completely lost in 22% of the tests performed ex vivo. A short-term amplification step plus the addition of a TLR3 agonist (Poly-ICLC) together with an increased EP concentration improved markedly the detection of CD8⁺ and CD4⁺ T cell reactivities.

Objective We investigated blood samples from fully SARS-CoV2-vaccinated subjects and from patients up to one year after infection with SARS-CoV2, and compared short and long term T cell and antibody responses, with a special focus on the recently emerged delta variant (B.1.617.2). Methods and Results In 23 vaccinated subjects, we documented high anti-SARS-CoV2 spike protein receptor binding domain (RBD) antibody titers. Average virus neutralization by antibodies, assessed as inhibition of ACE2 binding to RBD, was 2.2-fold reduced for delta mutant vs. wt RBD. Specific CD4+ T cell responses as measured using stimulation with peptides representing wt or alpha, beta, gamma and delta variant SARS-CoV2 entire spike proteins by flow cytometric intracellular cytokine staining, did not differ significantly between vaccinees. One year post infection in one of the earliest SARS-CoV2 populations in the Western world, mean specific antibody titers were lower than in vaccinees; ACE2 binding to delta mutant vs. wt RBD was 1.65-fold reduced. T cell responses, especially interferon gamma expression, to mutant SARS-CoV2 spike were significantly reduced compared to those in vaccinees. Conclusion Strong T cell responses occurred for wildtype and mutant SARS-CoV2 variants, including delta (B.1.617.2), in fully vaccinated individuals, whereas they were partly reduced 1 year after natural infection. Antibody neutralisation of delta mutant was reduced compared to wt, as assessed in a novel inhibition assay with a finger stick drop of blood. Hence, immune responses after vaccination are stronger compared to those after naturally occurring infection, pointing out the need of the vaccine to overcome the pandemic.