Biology subjects

Ramello, M. C.

Publications and source records attributed to Ramello, M. C..

4 recordsLinked to original sources

bioRxiv2026

C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

CD28 is a co-stimulatory component of several second-generation chimeric antigen receptor (CAR)-T cells, providing signals essential for T cell proliferation, survival, and cytokine secretion. However, the specific contribution of individual CD28 intracellular motifs to CAR-T cell function remains incompletely understood. Here, we identify tyrosine 218 (Y218) in the CD28 cytoplasmic domain as a critical regulatory site, and demonstrate that its phosphorylation is essential for optimal CAR-T cell activity. Using a 218F mutant, we show that loss of Y218 phosphorylation leads to impaired IL-2 production and abrogates antitumor efficacy. Transcriptomic profiling of 218F CAR-T cells revealed increased expression of IL-17A, IL-17F, and related cytokines, suggesting a shift toward a pro-inflammatory Th17-like phenotype that may contribute to dysfunction. Mechanistically, we demonstrate that the interleukin-2-inducible T-cell kinase, ITK, mediates Y218 phosphorylation. To further understand the role of this kinase, we engineered a novel CAR incorporating an ITK-binding motif (PYRP), which enhances ITK recruitment, increases Y218 phosphorylation, and boosts IL-2 secretion, and improves anti-tumor efficacy in vivo. Our findings underscore the functional relevance of Y218 phosphorylation in modulating CAR-T cell fate and reveal a strategy to fine-tune CAR signaling through targeted kinase recruitment to enhance therapeutic efficacy.

immunology↗

bioRxiv2023

CD39+ conventional CD4+ T cells with exhaustion traits and cytotoxic potential infiltrate tumors and expand upon CTLA-4 blockade

BackgroundConventional CD4+ T (Tconv) lymphocytes play important roles in tumor immunity; however, their contribution to tumor elimination remains poorly understood. MethodsHere we describe a subset of Tconv cells characterized by the expression of CD39. The phenotype, the effector function and transcriptional profile of tumor-infiltrating CD39+ Tconv lymphocytes from different mouse cancer models and breast cancer patients were studied by multiparametric flow cytometry and RNA sequencing. The impact of the in vivo CTLA-4 blockade on the tumor-infiltrating CD39+ Tconv population was assessed in mice grafted with the immunogenic MC38 colorectal tumor. Overall survival was evaluated in a cohort of patients from the TCGA consortium. ResultsIn mouse cancer models, we observed that CD39+ Tconv cells accumulated in tumors as they grew but were absent in lymphoid organs. Compared to tumor CD39- counterparts, CD39+ Tconv cells exhibited a cytotoxic and exhausted signature at the transcriptomic level, confirmed by high protein expression of inhibitory receptors and transcription factors related to the exhaustion phenotype. Additionally, CD39+ Tconv cells showed increased production of IFN gamma, granzyme B, perforin and CD107a expression, but reduced production of TNF. In vivo CTLA-4 blockade induced the expansion of tumor CD39+ Tconv cells, which maintained their cytotoxic and exhausted features. In breast cancer patients, CD39+ Tconv cells were found in tumors and in metastatic lymph nodes but were less frequent in adjacent non-tumoral mammary tissue and not detected in non-metastatic lymph nodes and blood. Human tumor CD39+ Tconv cells constituted a heterogeneous cell population with features of exhaustion, impaired TNF production, and high expression of inhibitory receptors and CD107a. We found that high CD4 and ENTPD1 (CD39) gene expression in human tumor tissues correlated with a higher overall survival rate in breast cancer patients. ConclusionsWe found that CD39 acts as a biomarker of Tconv cells with characteristics of both exhaustion and cytotoxic potential. CTLA-4 blockade expands CD39+CD4+ T cells which may contribute to the reduction of tumor development. Discovering the role of CD39-expressing CD4+ T cells in the tumor microenvironment should help design new strategies to manipulate them and improve the efficacy of current immunotherapies.

immunology↗

bioRxiv2020

PD-L1hi plasmablasts limit the T cell response during the acute phase of parasite infections

During infections with protozoan parasites or virus, T cell immunosuppression is generated simultaneously with a high B cell activation. Here, we show that in T. cruzi infection, all plasmablasts detected had higher surface expression of PD-L1, than other mononuclear cells. PD-L1hi plasmablasts were induced in vivo in an antigen-specific manner and required help from Bcl-6+CD4+T cells. PD-L1hi expression was not a characteristic of all antibody-secreting cells since plasma cells found during the chronic phase of infection express PD-L1 but at lower levels. PD-L1hi plasmablasts were also present in mice infected with Plasmodium or with lymphocytic choriomeningitis virus, but not in mice with autoimmune disorders or immunized with T cell-dependent antigens. PD-L1hi plasmablasts suppressed T cell response, via PD-L1, in vitro and in vivo. Thus, this study reveals that extrafollicular PD-L1hi plasmablasts, which precede the germinal center (CG) response, are a suppressive population in infections that may influence T cell response. Brief summaryPathogens develop different strategies to settle in the host. We identified a plasmablats population induced by pathogens in acute infections which suppress T cell response.

immunology↗

bioRxiv2018

IL-17RA-signaling modulates CD8+ T cell survival and exhaustion during Trypanosoma cruzi infection

The IL-17 family contributes to host defense against many intracellular pathogens by mechanisms not fully understood. CD8+ T lymphocytes are key elements against intracellular microbes and their survival and appropriate response is orchestrated by several cytokines. Here, we demonstrated that IL-17RA-signaling cytokines sustain pathogen-specific CD8+ T cell immunity. Absence of IL-17RA and IL-17A/F during Trypanosoma cruzi infection resulted in increased tissue parasitism and reduced frequency of parasite-specific CD8+ T cells. Impaired IL-17RA-signaling in vivo increased apoptosis of parasite-specific CD8+ T cells while recombinant IL-17 in vitro down-regulated the pro-apoptotic protein BAD and promoted activated CD8+ T cell survival. Phenotypic, functional and trancriptomic profiling showed that T. cruzi-specific CD8+ T cells arising in IL-17RA-deficient mice presented features of cell dysfunction. PD-L1 blockade partially restored the magnitude of CD8+ T cell responses and parasite control in these mice. Adoptive transfer experiments established that IL-17RA-signaling is intrinsically required for the proper maintenance of functional effector CD8+ T cells. Altogether, our results identify IL-17RA and IL-17A as critical factors for sustaining CD8+ T cell immunity to T. cruzi.

immunology↗