ABSTRACT 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.

In order to stay circulating in the human population over many years, viruses need to adapt to the environment, mainly to the host's immune response, allowing for reinfection despite preexisting immunity. These different viral strategies are clearly important for guiding vaccine design. SARS-CoV-2 is no exception, using three main strategies to avoid protective and long-lived antibody responses: one very common and two rather unique tactics, consisting of 1) random mutation to partially escape existing antibody responses, 2) increasing affinity of the receptor-binding domain (RBD) of the spike protein to it's receptor Angiotensin-converting enzyme 2 (ACE 2), and 3) diluting out neutralizing epitopes on the viral surface to avoid strong and enduring antibody responses. As the correlate of protection from SARS-CoV-2 is neutralizing antibody response, this review focuses on B cells, the major player in protecting against COVID-19.

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-1123-137/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.

T stem cell-like memory cells (TSCM cells) are considered to be essential for the maintenance of immune memory. The TSCM population has been shown to have the key properties of a stem cell population: multipotency, self-renewal and clonal longevity. Here we show that no single population has all these stem cell properties, instead the properties are distributed. We show that the human TSCM population consists of two distinct cell subpopulations which can be distinguished by the level of their CD95 expression (CD95int and CD95hi). Crucially, using long-term in vivo labelling of human volunteers, we establish that these are distinct populations rather than transient states of the same population. These two subpopulations have different functional profiles ex vivo, different transcriptional patterns, and different tissue distributions. They also have significantly different TREC content indicating different division histories and we find that the frequency of CD95hi TSCM increases with age. Most importantly, CD95hi and CD95int TSCM cells also have very different dynamics in vivo with CD95hi cells showing considerably higher proliferation but significantly reduced clonal longevity compared with CD95int TSCM. While both TSCM subpopulations exhibit considerable multipotency, no single population of TSCM cells has both the properties of self-renewal and clonal longevity. Instead, the "stemness" of the TSCM population is generated by the complementary dynamic properties of the two subpopulations: CD95int TSCM which have the property of clonal longevity and CD95hi TSCM which have the properties of expansion and self-renewal. We suggest that together, these two populations function as a stem cell population.

TCR repertoires against tumors lack high-affinity TCRs and are further suppressed by Tregs. We hypothesized that Treg depletion enhances the antitumor efficacy of low-affinity T cells. Using the weak agonistic peptide A4Y derived from LCMV glycoprotein peptide p33 as a model antigen and VLPs as a vaccine platform, we tested this approach. In a separate low-affinity model, we targeted B16F10 melanoma with our multi-target vaccine. Results revealed limited in vivo lytic cross-reactivity between A4Y and p33 peptides, and the A4Y-vaccine alone failed to inhibit B16F10p33 tumor progression. However, combining A4Y-vaccine with Treg depletion triggered a robust immune response, characterized by increased CD8+ T cell infiltration, enhanced T cell functionality, and tumor-free survival. Infiltrating T cells also exhibited closer spatial proximity and heightened migration from blood vessels. Similarly, combining low-affinity vaccine with Treg depletion enhanced antitumor responses. These findings highlight the potential of Treg depletion to advance vaccination strategies targeting TAAs with low-affinity T cells.

In melanoma, lymphangiogenesis correlates with metastasis and poor prognosis and promotes immunosuppression. However, it also potentiates immunotherapy by supporting immune cell trafficking. We show in a lymphangiogenic murine melanoma that lymphatic endothelial cells (LECs) upregulate the enzyme Ch25h, which catalyzes the formation of 25-hydroxycholesterol (25-HC) from cholesterol and plays important roles in lipid metabolism, gene regulation, and immune activation. We identify a role for LECs as a source of extracellular 25-HC in tumors inhibiting PPAR-γ in intra-tumoral macrophages and monocytes, preventing their immunosuppressive function and instead promoting their conversion into proinflammatory myeloid cells that support effector T cell functions. In human melanoma, LECs also upregulate Ch25h, and its expression correlates with the lymphatic vessel signature, infiltration of pro-inflammatory macrophages, better patient survival, and better response to immunotherapy. We identify here in mechanistic detail an important LEC function that supports anti-tumor immunity, which can be therapeutically exploited in combination with immunotherapy. VEGF-C induced lymphangiogenesis combined with immunotherapy approaches can promote anti-tumor immune responses. Here the authors report that lymphaticderived oxysterols promote anti-tumor immunity and response to immunotherapy in preclinical melanoma models

Cancer immunotherapy using dendritic cells (DC) pulsed ex vivo with tumour antigens is considered safe, but its clinical efficacy is generally modest. Here we engineer DC progenitors (DCP), which can replenish conventional type 1 DCs (cDC1) in mice, to constitutively express IL-12 together with a non-signalling chimeric receptor, termed extracellular vesicle-internalizing receptor (EVIR). By binding to a bait molecule (GD2 disialoganglioside) expressed on cancer cells and their EVs, the EVIR enforces EV internalization by cDC1 to promote their cross-dressing with preformed, tumour-derived MHCI-peptide complexes. Upon systemic deployment to mice, the engineered DCPs cause only mild and transient elevation of liver enzymes, acquire tumour-derived material, engage tumour-specific T cells, and enhance the efficacy of PD-1 blockade in an immunotherapy-resistant melanoma model comprising both GD2-positive and -negative cancer cells, without the need for ex vivo antigen pulsing. These results indicate that EVIR-engineered DCPs may avert the positive selection of antigen-negative cancer cells, potentially addressing a critical limitation of immunotherapies targeting defined tumour antigens.

The global incidence of human papillomavirus (HPV) associated head and neck carcinoma is on the rise, in response to this a tetravalent therapeutic vaccine named Qβ-HPVag was developed. This vaccine, utilizing virus-like particles (VLPs) loaded with toll-like receptor ligands and chemically coupled to four HPV16-derived peptides, demonstrated strong anti-tumor effects in a murine head and neck cancer model. Qβ-HPVag impeded tumor progression, increased infiltration of HPV-specific T cells, and significantly improved survival. The vaccine`s efficacy was associated with immune repolarization in the tumor microenvironment, characterized by expanded activated dendritic cell subsets (cDC1, cDC2, DC3). Notably, mice responding to treatment exhibited a higher percentage of migratory DC3 cells expressing CCR7. These findings suggest promising prospects for optimized VLP-based vaccines in treating HPV-associated head and neck cancer.

Circular RNAs (circRNAs) are covalently closed non-coding RNAs lacking the 5’ cap and the poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed circRNAs in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents an immunopeptidomics workflow with a specific focus on generating a circRNA-specific protein fasta reference. The main goal of this workflow is to streamline the process of identifying and validating human leukocyte antigen (HLA) bound peptides potentially originating from circRNAs. We increase the analytical stringency of our workflow by retaining peptides identified independently by two mass spectrometry search engines and/or by applying a group-specific FDR for canonical-derived and circRNA-derived peptides. A subset of circRNA-derived peptides specifically encoded by the region spanning the back-splice junction (BSJ) are validated with targeted MS, and with direct Sanger sequencing of the respective source transcripts. Our workflow identifies 54 unique BSJ-spanning circRNA-derived peptides in the immunopeptidome of melanoma and lung cancer samples. Our approach enlarges the catalog of source proteins that can be explored for immunotherapy.

Tick‐borne encephalitis (TBE) virus is the most prevalent tick‐transmitted orthoflavivirus in Europe. Due to the nonspecific nature of its symptoms, TBE is primarily diagnosed by ELISA‐based detection of specific antibodies in the patient serum. However, cross‐reactivity between orthoflaviviruses complicates the diagnosis. Specificity issues may be mitigated by serum neutralization assays (SNT), although the handling of clinically relevant orthoflaviviruses requires biosafety level (BSL) 3 conditions and they have highly divergent viral kinetics and cell tropisms. In the present study, we established a reporter virus particle (RVP)‐based SNT in which the infectivity is measured by luminescence and that can be performed under BSL‐2 conditions. The RVP‐based SNT for TBEV exhibited a highly significant correlation with the traditional virus‐based SNT (R2 = 0.8637, p < 0.0001). The RVP‐based assay demonstrated a sensitivity of 92.3% (95% CI: 79.7%–97.4%) and specificity of 100% (95% CI: 81.6%–100%). We also tested the cross‐reactivity of serum samples in RVP‐based assays against other orthoflaviviruses (yellow fever virus, dengue virus type 2, Zika virus, West Nile virus and Japanese encephalitis virus). Interestingly, all serum samples which had tested TBEV‐positive by ELISA but negative by RVP‐based SNT were reactive for antibodies against other orthoflaviviruses. Thus, the RVP‐based seroneutralization assay provides an added value in clinical diagnostics as well as in epidemiological studies.

Supplementary Tables S1, S2 and S3

Gating strategy of combinatorial tetramer staining

It is with deep sadness that we report the passing away of Hugh Robson (Rob) MacDonald on March 16th, 2023, aged 76, following a complicated recovery from a fall last December. Rob was born on September 16th, 1946, and grew up in Willowdale, a neighborhood in Toronto, Canada. After graduating from the University of Toronto (B.Sc. 1968 in Astrophysics), he went on to earn a Ph.D. in Medical Biophysics (1972) with a dissertation on the response of murine lymphocytes to histocompatibility antigens under the direction of Richard G. Miller. He then joined the Swiss Institute for Experimental Cancer Research in Lausanne, Switzerland, where he performed postdoctoral work with Theodore (Teddy) Brunner on the differentiation of cytolytic T cells. Upon his return to Canada in 1975, he took up a joint assistant professorship appointment at the Departments of Bacteriology and Immunology and Therapeutic Radiology of the University of Western Ontario, London, Canada, and performed experimental work in immune-oncology at the Ontario Cancer Treatment and Research Foundation, London, Canada. In 1977, Rob joined the Lausanne Branch of the Ludwig Institute for Cancer Research in Epalinges, Switzerland, where he had a distinguished research career as a developmental T cell immunologist until his retirement in 2015. He became the Associate Director from 1989 and was the Director from 2007–2012. Rob was a pioneer in driving our understanding of how the immune system ensures that T cells are useful and not harmful. He made numerous fundamental discoveries on T cell biology, including T cell development, lineage commitment, repertoire selection, tolerance, and immune memory. He authored more than 430 scientific articles published in peer-reviewed journals that had over 34,000 citations as of 2018. In 1989, he was presented with the prestigious Swiss Max Cloetta Award. In 2001, he was recognized as a Highly Cited Researcher by the Institute for Scientific Information. Some of the key biological insights during his tenure at the Lausanne Branch of the Ludwig Institute came at a time when T cells were defined as thymus-dependent lymphocytes whose function was hard to measure, that were difficult to maintain in vitro, and whose antigen receptors were unknown. By the early 1970s, Jean-Charles Cerottini and Teddy Brunner had devised and painstakingly refined a functional assay to measure T cell-mediated killing using the release of 51chromium by tumor targets. Rob's seminal contributions to further characterize and understand cytolytic T lymphocytes included the long-term growth in vitro of these cells using T cell growth factor (now known as interleukin-2 [IL-2], which was contained in homemade culture supernatants of recently activated T cells), the cloning of cytolytic T lymphocytes, the determination of their precursor frequency, and the demonstration that cytolytic activity was a function of T cells expressing Lyt2 (now known as CD8) (summarized in1MacDonald H.R. Cerottini J.C. Ryser J.E. Maryanski J.L. Taswell C. Widmer M.B. Brunner K. T. Quantitation and cloning of cytolytic T lymphocytes and their precursors. Immunol.Rev. 1980; 51: 93-123https://doi.org/10.1111/j.1600-065x.1980.tb00318.xCrossref Google Scholar). Rob's central break-through discovery in developmental immunology was made possible thanks to his visionary implementation of the technology of fluorescence flow cytometry in Europe, which had just been developed by the Herzenberg lab in Stanford University. He liked to stress how his training in biophysics allowed him to have a good understanding of the principles and mechanics involved in the early bulky fluorescence-actived cell sorting (FACS) instruments. This, together with key reagents generated by Hans Acha-Orbea (Ludwig Institute, Lausanne) and Hans Hengartner (University of Zurich), allowed him to demonstrate that the absence of T cell reactivity to self-antigens (immunological tolerance) is ensured by the physical elimination of self-reactive T cells in the thymus, a process now termed "negative selection."2MacDonald H.R. Schneider R. Lees R.K. Howe R.C. Acha-Orbea H. Festenstein H. Zinkernagel R.M. Hengartner H. T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens. Nature. 1988; 332: 40-50https://doi.org/10.1038/332040a0Crossref Scopus (857) Google Scholar Moreover, he showed that the proper maturation of T cells in the thymus depends on the interaction of their specific T cell receptor with major histocompatibility complex (MHC) antigens expressed by the thymic stroma, a process referred to as "positive selection."3MacDonald H.R. Lees R.K. Schneider R. Zinkernagel R.M. Hengartner H. Positive selection of CD4+ thymocytes controlled by MHC class II gene products.Nature. 1988; 336: 471-473https://doi.org/10.1038/336471a0Crossref PubMed Scopus (157) Google Scholar These seminal findings are part of the foundation of modern immunology. The above discoveries relied on following the fate of T cells with reactivity toward the mysterious minor lymphocyte-stimulating (Mls) antigens. Further work in collaboration with virologist Heidi Diggelmann (Swiss Institute for Cancer Research, ISREC, Lausanne) molecularly identified Mls antigens as mouse mammary tumor virus (MMTV)-derived superantigens and defined the importance of superantigens in MMTV infection. His long-standing interest in T cell development further led to the discovery of unconventional T cells that are characterized by a very restricted T cell receptor repertoire (with Ralph Budd)4Budd R.C. Miescher G.C. Howe R.C. Lees R.K. Bron C. MacDonald H.R. Developmentally regulated expression of T cell receptor beta chain variable domains in immature thymocytes..J Exp Med. 1987; 166: 577-582https://doi.org/10.1084/jem.166.2.577Crossref PubMed Scopus (202) Google Scholar and whose maturation in the thymus requires interactions with hematopoietic rather than stromal MHCs (with T. Ohteki). These unconventional T cells are now known as invariant natural killer T (NKT) cells and understood to play important roles in bridging innate and adaptive immune responses. Finally, to come back to developmental immunology and lineage commitment, together with Freddy Radtke, Anne Wilson, and Michel Aguet, Rob identified the Notch dependent signaling pathway that commits very early thymic precursor cells to the T cell lineage.5Radtke F. Wilson A. Stark G. Bauer M. van Meerwijk J. MacDonald H.R. Aguet M. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity. 1999; 10: 547-558https://doi.org/10.1016/s1074-7613(00)80054-0Abstract Full Text Full Text PDF Google Scholar Rob was loved by the scientific community for his bright intellect, phenomenal memory, great sense of humor, eclectic spirit, insatiable curiosity, and his trademark humility. Rob's sharp and critical thinking about results and manuscripts alike was invariably delivered to trainees and peers in measured, nuanced, and balanced critiques. He was always engaged in supporting his trainees and collaborators. He both fostered and relied on the quiet and diligent laboratory management and unfailing experimental support of Rosemary Lees throughout his tenure at the Ludwig Institute. He leaves a large alumni family, many of whom are leading research in immunology at centers of academic excellence worldwide. He is remembered by his beloved wife, Lana; his sisters, Chris and Kathy; his brothers-in-law, John and Peter; his sister-in-law, Rita; and his many nieces and nephews and their families.

Supplementary FACS and sorting procedures

Direct ex vivo analysis of circulating Melan-A-specific CD8 T-cell frequencies

<p>Supplementary Figure S3. Monomeric dissociation half-lives of naturally occurring or vaccine-induced tumor-specific CD8+ T cell clones from three melanoma patients.</p>

Supplementary Methods - PDF file 55K, This file contains Supplementary Methods.

Specific CD8 T-cell frequencies before and after each cycle of vaccination

<p>Supplementary Figure S5. Impact of rapid association on-rates on cell surface dissociation kinetics by reversible NTAmers.</p>