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Despite the intense CD8+ T-cell infiltration in the tumor microenvironment of nasopharyngeal carcinoma, anti-PD-1 immunotherapy shows an unsatisfactory response rate in clinical trials, hindered by immunosuppressive signals. To understand how microenvironmental characteristics alter immune homeostasis and limit immunotherapy efficacy in nasopharyngeal carcinoma, here we establish a multi-center single-cell cohort based on public data, containing 357,206 cells from 50 patient samples. We reveal that nasopharyngeal carcinoma cells enhance development and suppressive activity of regulatory T cells via CD70-CD27 interaction. CD70 blocking reverts Treg-mediated suppression and thus reinvigorate CD8+ T-cell immunity. Anti-CD70+ anti-PD-1 therapy is evaluated in xenograft-derived organoids and humanized mice, exhibiting an improved tumor-killing efficacy. Mechanistically, CD70 knockout inhibits a collective lipid signaling network in CD4+ naïve and regulatory T cells involving mitochondrial integrity, cholesterol homeostasis, and fatty acid metabolism. Furthermore, ATAC-Seq delineates that CD70 is transcriptionally upregulated by NFKB2 via an Epstein-Barr virus-dependent epigenetic modification. Our findings identify CD70+ nasopharyngeal carcinoma cells as a metabolic switch that enforces the lipid-driven development, functional specialization and homeostasis of Tregs, leading to immune evasion. This study also demonstrates that CD70 blockade can act synergistically with anti-PD-1 treatment to reinvigorate T-cell immunity against nasopharyngeal carcinoma. Response rates to immunotherapies in patients with nasopharyngeal carcinoma (NPC) are still limited. Here the authors show that tumor-restricted CD70 correlates with regulatory T cell abundance and suppressive activity in NPC and that CD70 blockade improves response to anti-PD1 in preclinical models.

Tolerance to self-antigens prevents the elimination of cancer by the immune system 1 , 2 . We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27 + CD45RO – CD8 + T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27 + PD-1 – CD8 + CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies. An IL-6/STAT3 signature and memory CD8 T cell subset in preinfusion chimeric antigen receptor–expressing T cells associate with response in patients with high-risk chronic lymphocytic leukemia.

Exhausted T cells in chronic infections and cancer have sustained expression of the inhibitory receptor programmed cell death 1 (PD-1). Therapies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. Nonetheless, a better understanding of the immunological responses induced by PD-1 blockade in cancer patients is lacking. Identification of predictive biomarkers is a priority in the field, but whether peripheral blood analysis can provide biomarkers to monitor or predict patients’ responses to treatment remains to be resolved. In this study, we analyzed longitudinal blood samples from advanced stage non–small cell lung cancer (NSCLC) patients (n = 29) receiving PD-1–targeted therapies. We detected an increase in Ki-67+ PD-1+ CD8 T cells following therapy in ∼70% of patients, and most responses were induced after the first or second treatment cycle. This T-cell activation was not indiscriminate because we observed only minimal effects on EBV-specific CD8 T cells, suggesting that responding cells may be tumor specific. These proliferating CD8 T cells had an effector-like phenotype (HLA-DR⁺, CD38⁺, Bcl-2lo), expressed costimulatory molecules (CD28, CD27, ICOS), and had high levels of PD-1 and coexpression of CTLA-4. We found that 70% of patients with disease progression had either a delayed or absent PD-1+ CD8 T-cell response, whereas 80% of patients with clinical benefit exhibited PD-1+ CD8 T-cell responses within 4 wk of treatment initiation. Our results suggest that peripheral blood analysis may provide valuable insights into NSCLC patients’ responses to PD-1–targeted therapies.

Chimeric antigen receptors (CARs) can redirect T cells to target abnormal cells, but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here, we show that CARs have a >100-fold lower antigen sensitivity compared to theTcell receptor (TCR) when antigen is presented on antigen-presenting cells (APCs) but nearly identical sensitivity when antigen is presented as purified protein. We next systematically measured the impact of engaging important T cell accessory receptors (CD2, LFA-1, CD28, CD27, and 4-1BB) on antigen sensitivity by adding their purified ligands. Unexpectedly, we found that engaging CD2 or LFA-1 improved the antigen sensitivity of the TCR by 125- and 22-fold, respectively, but improved CAR sensitivity by only <5-fold. This differential effect of CD2 and LFA-1 engagement on the TCR vs. CAR was confirmed using APCs. We found that sensitivity to antigen can be partially restored by fusing the CAR variable domains to the TCR CD3ε subunit (also known as a TRuC) and fully restored by exchanging the TCRαβ variable domains for those of the CAR (also known as STAR or HIT). Importantly, these improvements in TRuC and STAR/HIT sensitivity can be predicted by their enhanced ability to exploit CD2 and LFA-1. These findings demonstrate that the CAR sensitivity defect is a result of their inefficient exploitation of accessory receptors and suggest approaches to increase sensitivity.

Lymphocytes have a critical role in visceral adipose tissue (AT) inflammation. The CD28 costimulatory molecule is required for lymphocyte activation and for the development of a functional regulatory T cells (Tregs) compartment; however, its role during obesity is unknown.During diet-induced obesity, we investigated the effects of selective interference with CD28 signaling using knockout mice (Cd28KO) and a CTLA4-Ig fusion protein inhibiting CD28-B7 interactions.Cd28 deficiency decreased pathogenic T cells and Treg content within AT without changing the macrophages number. Cd28KO epididymal but not subcutaneous fat was characterized by enlarged adipocytes, reduced levels of inflammatory cytokines and increased Glut4, adiponectin and lipogenic enzyme mRNA levels. This was associated with reduced inflammation, fat accumulation and enhanced glucose metabolism in liver. Weight gain and fasting glucose tolerance were not affected. CTLA4-Ig injections reduced the number of T cells in epididymal AT (epiAT) but not the inflammatory cytokines levels and failed to improve liver fat accumulation.Deletion of CD28 creates a new pro/anti-inflammatory balance in epiAT and liver and exerts a protective effect against hepatic steatosis.

High-throughput quantification of protein and gene expression in single cells is achieved using DNA-barcoded antibodies. We present a tool to measure gene and protein expression levels in single cells with DNA-labeled antibodies and droplet microfluidics. Using the RNA expression and protein sequencing assay (REAP-seq), we quantified proteins with 82 barcoded antibodies and >20,000 genes in a single workflow. We used REAP-seq to assess the costimulatory effects of a CD27 agonist on human CD8 + lymphocytes and to identify and characterize an unknown cell type.

An unprecedented global monkeypox outbreak started in May, 2022. No data are yet available about the dynamics of the immune response against monkeypox virus. The aim of this study was to describe kinetics of T-cell response, inflammatory profile, and pox-specific T-cell induction in patients with laboratory-confirmed monkeypox. 17 patients with laboratory-confirmed monkeypox admitted at the Lazzaro Spallanzani National Institute for Infectious Diseases (Rome, Italy), from May 19, to July 7, 2022, were tested for differentiation and activation profile of CD4 and CD8 T (expression of CD38, PD-1, and CD57 assessed by flow cytometry), frequency of pox-specific T cells (by standard interferon-γ ELISpot), and release of interleukin (IL)-1β, IL-6, IL-8, and tumour necrosis factor (TNF) in plasma (by ELISA). All patients were tested 10–12 days after symptoms onset. In a subgroup of nine patients with a laboratory-confirmed monkeypox, the kinetics of the immune response were analysed longitudinally according to timing from symptoms onset and compared with ten healthy donors (ie, health-care workers recruited from the same institution). Among the 17 patients, ten were HIV negative and seven HIV positive, all with good viro-immunological status. On days 0–3 from symptom onset, patients with laboratory-confirmed monkeypox were characterised by a statistically significant reduction in CD4+ T cells (p=0·0011) and a concurrent increase of CD8+ T cells (p=0·0057) compared with healthy donors. A lower proportion of naive (CD45RA+CD27+) CD4+ T cells was observed in six (67%) of nine patients and a concomitant higher proportion of effector memory (CD45RA-CD27-) CD4+ T cells in all patients; this skewed immune profile tended to normalise over time. A similar differentiated profile was also observed in CD8+ T cells with a consistent expansion of terminally differentiated CD8+ T cells. Patients with monkeypox had a higher proportion of CD4+CD38+ and CD38+CD8+ T-cells than healthy donors, which normalised after 12–20 days from symptom onset. The expression of PD-1 and CD57 on CD4+ and CD8+ T-cells showed kinetics similar to that observed for CD38. Furthermore, the inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF) were higher in patients with monkeypox than in healthy donors and, although they decreased over time, they remained elevated after recovery. Almost all patients (15 [94%] of 16) developed a pox-specific Th1 response. No differences in immune cells profile were observed between patients with and without HIV, whereas paucysimptomatic patients (without systemic symptoms, with less than five skin lesions, and no other mucosal localisation of monkeypox) showed a less perturbed immune profile early after symptom onset. Our data showed the immunological signature of monkeypox virus infection, characterised by an early expansion of activated effector CD4+ and CD8+ T cells that persisted over time. Almost all patients, even regardless of HIV infection, developed a poxvirus-specific Th1 cell response. These results might have implications on the expected immunogenicity of monkeypox vaccination, suggesting that it might not be necessary to vaccinate people who have already been infected. Italian Ministry of Health. For the Italian translation of the abstract see Supplementary Materials section.

CD40 signaling in classical type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1s, including Cd70, Tnfsf9, Ptgs2 and Bcl2l1, and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T cell expansion. Loss of 4-1BB, alone or in Cd27−/− mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased caspase activation in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation by Cd40−/− cDC1s to CD8+ T cells, which were reversed by re-expression of Bcl2l1. Thus, CD40 signaling in cDC1s not only induces costimulatory ligands for CD8+ T cells but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses.CD40 is typically understood as a costimulatory molecule. Here, the authors show CD4+ T cell-induced CD40 signaling in conventional type 1 dendritic cells results in complicated gene expression that can enhance CD8+ T cell priming by various underappreciated and independent mechanisms.

Harnessing the power of the immune system to recognize and eliminate cancer cells is a longtime exploration. In the past decade, monoclonal antibody (mAb)-based immune checkpoint blockade (ICB) and chimeric antigen receptor T (CAR-T) cell therapy have proven to be safe and effective in hematologic malignancies. Despite the unprecedented success of ICB and CAR-T therapy, only a subset of patients can benefit partially due to immune dysfunction and lack of appropriate targets. Here, we review the preclinical and clinical advances of CTLA-4 and PD-L1/PD-1-based ICB and CD19-specific CAR-T cell therapy in hematologic malignancies. We also discuss the basic research and ongoing clinical trials on emerging immune checkpoints (Galectin-9/Tim-3, CD70/CD27, LAG-3, and LILRBs) and on new targets for CAR-T cell therapy (CD22, CD33, CD123, BCMA, CD38, and CD138) for the treatment of hematologic malignancies.

Immunosenescence is a process of immune dysfunction that occurs with age and includes remodeling of lymphoid organs, leading to changes in the immune function of the elderly, which is closely related to the development of infections, autoimmune diseases, and malignant tumors. T cell–output decline is an important feature of immunosenescence as well as the production of senescence-associated secretory phenotype, increased glycolysis, and reactive oxygen species. Senescent T cells exhibit abnormal phenotypes, including downregulation of CD27, CD28, and upregulation of CD57, killer cell lectin-like receptor subfamily G, Tim-3, Tight, and cytotoxic T-lymphocyte-associated protein 4, which are tightly related to malignant tumors. The role of immunosenescence in tumors is sophisticated: the many factors involved include cAMP, glucose competition, and oncogenic stress in the tumor microenvironment, which can induce the senescence of T cells, macrophages, natural killer cells, and dendritic cells. Accordingly, these senescent immune cells could also affect tumor progression. In addition, the effect of immunosenescence on the response to immune checkpoint blocking antibody therapy so far is ambiguous due to the low participation of elderly cancer patients in clinical trials. Furthermore, many other senescence-related interventions could be possible with genetic and pharmacological methods, including mTOR inhibition, interleukin-7 recombination, and NAD + activation. Overall, this review aims to highlight the characteristics of immunosenescence and its impact on malignant tumors and immunotherapy, especially the future directions of tumor treatment through senescence-focused strategies.