The primordial differentiation of tumor-specific memory CD8+ T cells as bona fide responders to PD-1/PD-L1 blockade in draining lymph nodes.

Blocking PD-1/PD-L1 signaling transforms cancer therapy and is assumed to unleash exhausted tumor-reactive CD8+ T cells in the tumor microenvironment (TME). However, recent studies have also indicated that the systemic tumor-reactive CD8+ T cells may respond to PD-1/PD-L1 immunotherapy. These discrepancies highlight the importance of further defining tumor-specific CD8+ T cell responders to PD-1/PD-L1 blockade. Here, using multiple preclinical tumor models, we revealed that a subset of tumor-specific CD8+ cells in the tumor draining lymph nodes (TdLNs) was not functionally exhausted but exhibited canonical memory characteristics. TdLN-derived tumor-specific memory (TTSM) cells established memory-associated epigenetic program early during tumorigenesis. More importantly, TdLN-TTSM cells exhibited superior anti-tumor therapeutic efficacy after adoptive transfer and were characterized as bona fide responders to PD-1/PD-L1 blockade. These findings highlight that TdLN-TTSM cells could be harnessed to potentiate anti-tumor immunotherapy.

Do patients with cN0 oral squamous cell carcinoma benefit from elective neck dissection? A large-scale population-based study.

BACKGROUND: The neck management of clinical-nodal negative (cN0) oral squamous cell carcinoma (OSCC) remains controversial. Elective neck dissection (END) and observation are the main strategies, but it is still not clear who could benefit the most from END. The purpose of this study was to clarify the potential clinical factors that affect the therapeutic value of END and to explore the actual characteristics associated with benefit from END. METHODS: Patients with cN0 OSCC were identified in the SEER database from 2000 to 2019. 5-year Overall survival (OS) and disease-specific survival (DSS) were analyzed using the Kaplan‒Meier method, and the hazard ratios (HRs) for survival were estimated using the Cox regression model. Multiple subgroup analyses of DSS and OS among different factors, comparing END and No END, were performed. RESULTS: A total of 17,019 patients with cN0 OSCC were included. The basic survival analysis and Cox regression model showed that END increased the probability of 5-year DSS and OS and was an independent prognostic factor. However, among patients who underwent only primary tumor surgery, no significant differences were found between the END and No END groups in 5-year DSS (P = 0. 585) and OS (P = 0.465). Further subgroup analysis showed that primary sites and T stage, but not other factors, might influence the benefit of END. Significant differences were found for T1 (P < 0.001 for OS) and T2 (P = 0.001 for DSS and < 0.001 for OS) tongue squamous cell carcinoma (TSCC) but not for other primary tumor sites. CONCLUSION: This large-scale retrospective population-based cohort study suggests that not all patients with cN0 OSCC could benefit from END. Patients with cN0 TSCC are recommended to undergo END, especially with early-stage tumors.

The interaction of CD300lf and ceramide reduces the development of periodontitis by inhibiting osteoclast differentiation.

AIM: The regulation of osteoclasts (OCs) by inhibitory immunoreceptors maintains bone homeostasis and is considered an important determinant of the extent of periodontal pathology. The aim of this study was to investigate the role of the inhibitory immunoreceptor CD300lf and its ligand ceramide in osteoclastogenesis in periodontitis. MATERIALS AND METHODS: The expression of CD300lf was measured in vitro and in a ligature-induced periodontitis model. The effect of CD300lf ablation on osteoclastogenesis was examined in ligature-retained and ligature removal periodontitis models. The effect of ceramide, the ligand of CD300lf, was examined in osteoclastogenesis in vitro and in vivo by smearing 20 µg of ceramide dissolved in carboxymethylcellulose on teeth and gingiva every other day in an experimental periodontitis model and ligature removal model. RESULTS: CD300lf expression was downregulated during osteoclastogenesis. Ablation of CD300lf in the ligature-induced periodontitis model increased the number of OCs and exacerbated bone damage. Bone resorption caused by CD300lf ablation was reversible following ligature removal. CD300lf-ceramide binding suppressed osteoclastogenesis in vitro and inhibited alveolar bone loss in a mouse periodontitis model. CONCLUSIONS: Our findings reveal that CD300lf-ceramide binding plays a critical negative role in alveolar bone loss in periodontitis by inhibiting OCs differentiation.

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis.

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. However, in many cases, cancer cells form metastatic loci in lymph nodes before further metastasizing to distant organs. To what extent the local and systematic CD8+ T cell responses were influenced by LN metastasis remains obscure. To this end, we set up a murine LN metastasis model combined with a B16F10-GP melanoma cell line expressing the surrogate neoantigen derived from lymphocytic choriomeningitis virus (LCMV), glycoprotein (GP), and P14 transgenic mice harboring T cell receptors (TCRs) specific to GP-derived peptide GP33-41 presented by the class I major histocompatibility complex (MHC) molecule H-2Db. This protocol enables the study of antigen-specific CD8+ T cell responses during LN metastasis. In this protocol, C57BL/6J mice were subcutaneously implanted with B16F10-GP cells, followed by adoptive transfer with naive P14 cells. When the subcutaneous tumor grew to approximately 5 mm in diameter, the primary tumor was excised, and B16F10-GP cells were directly injected into the tumor draining lymph node (TdLN). Then, the dynamics of CD8+ T cells were monitored during the process of LN metastasis. Collectively, this model has provided an approach to precisely investigate the antigen-specific CD8+ T cell immune responses during LN metastasis.

Focused ultrasound-mediated small-molecule delivery to potentiate immune checkpoint blockade in solid tumors.

In the last decade, immune checkpoint blockade (ICB) has revolutionized the standard of treatment for solid tumors. Despite success in several immunogenic tumor types evidenced by improved survival, ICB remains largely unresponsive, especially in "cold tumors" with poor lymphocyte infiltration. In addition, side effects such as immune-related adverse events (irAEs) are also obstacles for the clinical translation of ICB. Recent studies have shown that focused ultrasound (FUS), a non-invasive technology proven to be effective and safe for tumor treatment in clinical settings, could boost the therapeutic effect of ICB while alleviating the potential side effects. Most importantly, the application of FUS to ultrasound-sensitive small particles, such as microbubbles (MBs) or nanoparticles (NPs), allows for precise delivery and release of genetic materials, catalysts and chemotherapeutic agents to tumor sites, thus enhancing the anti-tumor effects of ICB while minimizing toxicity. In this review, we provide an updated overview of the progress made in recent years concerning ICB therapy assisted by FUS-controlled small-molecule delivery systems. We highlight the value of different FUS-augmented small-molecules delivery systems to ICB and describe the synergetic effects and underlying mechanisms of these combination strategies. Furthermore, we discuss the limitations of the current strategies and the possible ways that FUS-mediated small-molecule delivery systems could boost novel personalized ICB treatments for solid tumors.

Dynamic changes of exhaustion features in T cells during oral carcinogenesis.

OBJECTIVES: This study aimed to clarify the dynamic changes of exhaustion features in T cells during oral carcinogenesis. MATERIALS AND METHODS: Mice were randomly divided into 4NQO group and control group. The exhaustion features of CD4+ and CD8+ T cells of both groups were detected by flow cytometry. Furthermore, multiplex immunohistochemistry was used to evaluate the expression of inhibitory receptors in human normal, dysplastic, and carcinogenesis tissues. Finally, anti-PD-1 antibody treatment was performed at the early premalignant phase of oral carcinogenesis. RESULTS: The proportion of naive T cells in 4NQO group was lower than those in control group, while the proportion of effector memory T cells was higher in 4NQO group. The expression of inhibitory receptors on CD4+ and CD8+ T cells increased gradually during carcinogenesis. In contrast, the secretion of cytokines by CD4+ and CD8+ T cells decreased gradually with the progression stage. Strikingly, those changes occurred before the onset of oral carcinogenesis. The expression of inhibitory receptors on T cells increased gradually as the human tissues progressed from normal, dysplasia to carcinoma. Interestingly, PD-1 blockade at the early premalignant phase could reverse carcinogenesis progression by restoring T cell function. CONCLUSIONS: T-cell dysfunction was established at the early premalignant phase of oral carcinogenesis; PD-1 blockade at the early premalignant phase can effectively reverse T-cell exhaustion features and then prevent carcinogenesis progression.

Tumor-Specific CD4+ T Cells Restrain Established Metastatic Melanoma by Developing Into Cytotoxic CD4- T Cells.

Cytotoxic CD8+ T cells are the main focus of efforts to understand anti-tumor immunity and immunotherapy. The adoptive transfer of tumor-reactive cytotoxic CD8+ T lymphocytes expanded and differentiated in vitro has long been considered the primary strategy in adaptive anti-tumor immunity, however, the majority of the transferred tumor antigen-specific CD8+ T cells differentiated into CD39+CD69+ exhausted progenies, limiting its effects in repressing tumor growth. Contrarily, less attention has been addressed to the role of CD4+ T cells during tumorigenesis. Using a mouse model of metastatic melanoma, we found that transferring tumor-specific CD4+ T cells into recipients induces substantial regression of the established metastatic tumors. Notably, in vitro activated CD4+ T cells developed into cytotoxic CD4- T cells in vivo and get exhausted gradually. The blockade of PD-L1 signaling resulted in an expansion of tumor specific CD4+ T cells, which could better control the established metastatic melanoma. Moreover, the tumor-specific memory CD4+ T cell can prevent mice from tumor metastasis, and the tumor-specific effector CD4+ T cells can also mitigate the established metastatic tumor. Overall, our findings suggest a novel mechanism of CD4+ T cells in curtailing tumor metastasis and confirm their therapeutic role in combination with PD-L1 blockade in cancer immunotherapy. Hence, a better understanding of cytotoxic CD4- T cell-mediated tumor regression could provide an alternative choice for patients exhibiting suboptimal or no response to CD8+ T cell-based immunotherapies.

A novel strategy to investigate the factors regulating the Treg to Tfr transition during acute viral infection.

Follicular regulatory T cells (Tfrs), a specialized subset of regulatory T cells (Tregs), have a particular role in the control of follicular helper T cell-driven germinal center (GC) responses. Following differentiation signals similar to those received by follicular helper T cells (Tfhs), Tfrs gain expression of characteristic chemokine receptors and transcription factors, such as CXCR5 and Bcl-6, allowing them to migrate into the B-cell follicle and perform in situ suppression. Thus, together with Tfhs, Tfrs help maintaining an optimized GC-reaction. However, the mechanism underlying the Treg-to-Tfr transition remains obscure. Here, we established a highly reproducible protocol for investigating the differentiation of Tregs into Tfrs by constructing spleen-chimeric mice combined with retrovirus transduction. We demonstrated that using this strategy, over 4 folds of Tregs could differentiate into Tfrs in Bcl-6 overexpression group compared to control counterparts (Migr1), and Bcl-6 could efficiently promote Tfr differentiation during acute viral infection. Hence, this method provides us an easy access to investigate the factors that regulate the differentiation program that converts Tregs into Tfrs, which will enhance our understanding of the networks regulating GC-reaction and shed new light on the molecular basis of immune homeostasis.

A comprehensive profile of TCF1+ progenitor and TCF1- terminally exhausted PD-1+CD8+ T cells in head and neck squamous cell carcinoma: implications for prognosis and immunotherapy.

The heterogeneity of exhausted T cells (Tex) is a critical determinant of immune checkpoint blockade therapy efficacy. However, few studies have explored exhausted T cell subpopulations in human cancers. In the present study, we examined samples from two cohorts of 175 patients with head and neck squamous cell cancer (HNSCC) by multiplex immunohistochemistry (mIHC) to investigate two subsets of Tex, CD8+PD1+TCF1+ progenitor exhausted T cells (TCF1+Texprog) and CD8+PD1+TCF1- terminally exhausted T cells (TCF1-Texterm). Moreover, fresh tumor samples from 34 patients with HNSCC were examined by flow cytometry and immunohistochemistry to further investigate their properties and cytotoxic capabilities and their correlation with regulatory T cells (Tregs) in the tumor immune microenvironment (TIME). mIHC and flow cytometry analysis showed that TCF1-Texterm represented a greater proportion of CD8+PD1+Tex than TCF1+Texprog in most patients. TCF1+Texprog produced abundant TNFα, while TCF1-Texterm expressed higher levels of CD103, TIM-3, CTLA-4, and TIGIT. TCF1-Texterm exhibited a polyfunctional TNFα+GZMB+IFNγ+ phenotype; and were associated with better overall survival and recurrence-free survival. The results also indicated that larger proportions of TCF1-Texterm were accompanied by an increase in the proportion of Tregs. Therefore, it was concluded that TCF1-Texterm was the major CD8+PD1+Tex subset in the HNSCC TIME and that these cells favor patient survival. A high proportion of TCF1-Texterm was associated with greater Treg abundance.

Regulation of autoimmune disease progression by Pik3ip1 through metabolic reprogramming in T cells and therapeutic implications.

Metabolic alterations could profoundly affect immune functions and influence the progression and outcome of autoimmune diseases. However, the detailed mechanisms and their therapeutic potential remain to be defined. Here, we show that phosphatidylinositide 3-kinase interacting protein 1 (Pik3ip1), a newly identified negative immune regulator, is notably down-regulated in several major autoimmune diseases through a previously unidentified mechanism mediated by interleukin-21/p38 mitogen-activated protein kinase/a disintegrin and metalloprotease-17 (ADAM17) pathway. Down-regulation of Pik3ip1 in T cells causes a major metabolic shift from oxidative phosphorylation toward aerobic glycolysis, leading to their overactivation and aggressive disease progression in experimental autoimmune encephalomyelitis (EAE) mouse model. Suppression of hypoxia-inducible factor 1α (Hif1α) or pharmacologic inhibition of glycolysis could reverse these phenotypes and largely mitigate EAE severity. Our study reveals a previously unrecognized role of Pik3ip1 in metabolic regulation that substantially affects the inflammatory loop in the autoimmune setting and identifies the Pik3ip1/Hif1α/glycolysis axis as a potential therapeutic target for treatment of autoimmune diseases.

TDO2+ myofibroblasts mediate immune suppression in malignant transformation of squamous cell carcinoma.

Characterization of the dynamic change in the immunological landscape during malignant transformation from precancerous lesions to cancerous lesions in squamous cell carcinoma (SCC) is critical for the application of immunotherapy. Here, we performed single-cell RNA-Seq (scRNA-Seq) of 131,702 cells from 13 cancerous tissues of oral squamous cell carcinoma (OSCC), 3 samples of precancerous oral leukoplakia, and 8 adjacent normal samples. We found that tumor-infiltrating CD4+ and CD8+ T cells were functionally inhibited by immunosuppressive ligands expressed on various types of myeloid cells or neutrophils in the process of oral carcinogenesis. Notably, we identified a subset of myofibroblasts that exclusively expressed tryptophan 2,3-dioxygenase (TDO2). These TDO2+ myofibroblasts were located distally from tumor nests, and both CD4+ and CD8+ T cells were enriched around them. Functional experiments revealed that TDO2+ myofibroblasts were more likely to possess the ability for chemotaxis toward T cells but induced the transformation of CD4+ T cells into Tregs and caused CD8+ T cell dysfunction. We further showed that use of the TDO2 inhibitor LM10 attenuated the inhibitory states of T cells, restored the T cell antitumor response, and prevented the progression of OSCC malignant transformation in murine models. Our study reveals a multistep transcriptomic landscape of OSCC and demonstrates that TDO2+ myofibroblasts are potential targets for immunotherapy.

CD4+ T-cell epitope-based heterologous prime-boost vaccination potentiates anti-tumor immunity and PD-1/PD-L1 immunotherapy.

BACKGROUND: Antitumor therapeutic vaccines are generally based on antigenic epitopes presented by major histocompatibility complex (MHC-I) molecules to induce tumor-specific CD8+ T cells. Paradoxically, continuous T cell receptor (TCR) stimulation from tumor-derived CD8+ T-cell epitopes can drive the functional exhaustion of tumor-specific CD8+ T cells. Tumor-specific type-I helper CD4+ T (TH1) cells play an important role in the population maintenance and cytotoxic function of exhausted tumor-specific CD8+ T cells in the tumor microenvironment. Nonetheless, whether the vaccination strategy targeting MHC-II-restricted CD4+ T-cell epitopes to induce tumor-specific TH1 responses can confer effective antitumor immunity to restrain tumor growth is not well studied. Here, we developed a heterologous prime-boost vaccination strategy to effectively induce tumor-specific TH1 cells and evaluated its antitumor efficacy and its capacity to potentiate PD-1/PD-L1 immunotherapy. METHODS: Listeria monocytogenes vector and influenza A virus (PR8 strain) vector stably expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein-specific I-Ab-restricted CD4+ T cell epitope (GP61-80) or ovalbumin-specific CD4+ T cell epitope (OVA323-339) were constructed and evaluated their efficacy against mouse models of melanoma and colorectal adenocarcinoma expressing lymphocytic choriomeningitis virus glycoprotein and ovalbumin. The impact of CD4+ T cell epitope-based heterologous prime-boost vaccination was detected by flow-cytometer, single-cell RNA sequencing and single-cell TCR sequencing. RESULTS: CD4+ T cell epitope-based heterologous prime-boost vaccination efficiently suppressed both mouse melanoma and colorectal adenocarcinoma. This vaccination primarily induced tumor-specific TH1 response, which in turn enhanced the expansion, effector function and clonal breadth of tumor-specific CD8+ T cells. Furthermore, this vaccination strategy synergized PD-L1 blockade mediated tumor suppression. Notably, prime-boost vaccination extended the duration of PD-L1 blockade induced antitumor effects by preventing the re-exhaustion of tumor-specific CD8+ T cells. CONCLUSION: CD4+ T cell epitope-based heterologous prime-boost vaccination elicited potent both tumor-specific TH1 and CTL response, leading to the efficient tumor control. This strategy can also potentiate PD-1/PD-L1 immune checkpoint blockade (ICB) against cancer.

TCF-1 maintains CD8+ T cell stemness in tumor microenvironment.

T cell factor 1 (TCF-1) is a transcription factor (TF) of the canonical Wnt signaling pathway that encoded by the Tcf7. The crucial role of TCF-1 in T cell development and memory formation has been widely recognized. Recent studies have demonstrated that exhausted CD8+ T cell with the expression of TCF-1 may have inspiring function to amplify immunoreaction and improve the response to immunotherapy in chronic viral infection and cancer. In this short review, we summarized recent progress in intratumoral exhausted CD8+ T cells expressing TCF-1 that represent a fantastic subset with stem cell-like properties that associated with improved antitumor immunity and response to immune checkpoint blockade (ICB).

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis. Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Prognostic value of tertiary lymphoid structure and tumour infiltrating lymphocytes in oral squamous cell carcinoma.

Tertiary lymphoid structures (TLS) are ectopic lymphoid structures in cancers that are largely associated with favourable prognosis. However, the prognostic value of TLSs in oral squamous cell carcinoma (OSCC) is largely unknown, and the association between tumour infiltrating lymphocytes (TILs) and TLSs has been rarely explored in OSCC. In this study, associated markers of TLS, including peripheral node address (PNAd) in high endothelial venules, CD20 in B cells and CD3 in T cells, were examined in 168 OSCC patients, and survival analysis was performed between TLS-positive and TLS-negative cohorts. We detected the presence of TILs by staining CD8+ cytotoxic T cells and CD57+ NK cells as well. TLSs appeared as highly organized structures in 45 (26.8%) cases. TLS-positive patients had a better 5-year overall survival (OS) rate (88.9% vs. 56.1%, P < 0.001) and relapse-free survival (RFS) rate (88.9% vs. 63.4%, P = 0.002). Moreover, the presence of TLS was an independent prognostic factor for both the 5-year OS rate (hazard ratio [HR] = 3.784; 95% confidence interval [CI], 1.498-9.562) and RFS rate (HR = 3.296; 95% CI, 1.279-8.490) in multivariate analysis. Furthermore, a higher density of CD8+ T cells and CD57+ NK cells was found in TLS-positive sections than in TLS-negative counterparts (P < 0.001), and their combination provided a higher predictive accuracy (AUC = 0.730; 95% CI, 0.654-0.805). In conclusion, our results suggest that TLS is an independent positive prognostic factor for OSCC patients. These findings provide a theoretical basis for the future diagnostic and therapeutic value of TLSs in OSCC treatment.

High Abundance of Intratumoral γδ T Cells Favors a Better Prognosis in Head and Neck Squamous Cell Carcinoma: A Bioinformatic Analysis.

γδ T cells are a small subset of unconventional T cells that are enriched in the mucosal areas, and are responsible for pathogen clearance and maintaining integrity. However, the role of γδ T cells in head and neck squamous cell carcinoma (HNSCC) is largely unknown. Here, by using RNA-seq data from The Cancer Genome Atlas (TCGA), we discovered that HNSCC patients with higher levels of γδ T cells were positively associated with lower clinical stages and better overall survival, and high abundance of γδ T cells was positively correlated with CD8+/CD4+ T cell infiltration. Gene ontology and pathway analyses showed that genes associated with T cell activation, proliferation, effector functions, cytotoxicity, and chemokine production were enriched in the group with a higher γδ T cell abundance. Furthermore, we found that the abundance of γδ T cells was positively associated with the expression of the butyrophilin (BTN) family proteins BTN3A1/BTN3A2/BTN3A3 and BTN2A1, but only MICB, one of the ligands of NKG2D, was involved in the activation of γδ T cells, indicating that the BTN family proteins might be involved in the activation and proliferation of γδ T cells in the tumor microenvironment of HNSCC. Our results indicated that γδ T cells, along with their ligands, are promising targets in HNSCC with great prognostic values and treatment potentials.

Contributions of T cell dysfunction to the resistance against anti-PD-1 therapy in oral carcinogenesis.

BACKGROUND: Programmed death 1 (PD-1) blockade has great effect in the prevention of oral precancerous lesions, but the drug resistance has also been observed. The determinants of immune resistance during the malignant transformation are poorly understood. METHODS: Anti-PD-1 antibody was administered in the 4NQO-induced carcinogenesis mouse models. The mice were then subdivided into PD-1 resistance(PD-1R) group and PD-1 sensitive(PD-1S) group according to the efficacy. The expression of PD-1 and PD-L1, and the abundance of CD3+ T cells in tumor microenvironment between the two groups was tested by immunohistochemistry. In addition, the activation and effector functions, as well as the accumulation of immunosuppressive cells and expression of immune checkpoints of T cells in the draining lymph nodes and spleen between PD-1R and PD-1S group were analyzed by flow cytometry. RESULTS: Our results showed that T cell infiltration in tumor microenvironment, effector T cell cytokine secretion and central memory T cell accumulation in peripheral lymphoid organs were all inhibited in the anti-PD-1 resistance group. Furthermore, we found that an increase of regulatory T cell (Treg) population contributed to the resistance of the anti-PD-1 therapy. Notably, TIM-3 was found to be the only immunosuppressive molecule that mediated the resistance to anti-PD-1 therapy in the oral malignant transformation model. CONCLUSIONS: Our findings identified a novel mechanism that T cell dysfunction contributes to the immune resistance during the malignant transformation of the oral mucosa. This study provides new targets for improving the efficacy of immunotherapy for early stage of tumorigenesis.

Pik3ip1 Is a Negative Immune Regulator that Inhibits Antitumor T-Cell Immunity.

PURPOSE: Multiple negative regulators restrict the ability of T cells to attack tumors. This work demonstrates the role of PI3K-interacting protein 1 (Pik3ip1) in restraining T-cell responses and antitumor immunity. EXPERIMENTAL DESIGN: An anti-Pik3ip1 mAb was generated to identify the Pik3ip1 expression pattern of hematopoietic cells. Pik3ip1 -/- mice and a Pik3ip1 fusion protein were generated to investigate the effect of Pik3ip1 on T-cell-mediated antitumor immunity in MC38 and B16-F10 tumor models. Immunoblotting and confocal microscopy were used to identify inhibitory effects of Pik3ip1 on T-cell receptor (TCR) signaling. Pik3ip1 expression was quantified, and its impact on T-cell function in human tumors was measured. RESULTS: We demonstrated that Pik3ip1 was predominantly expressed on T cells and served as an essential rheostat for T-cell-mediated immunity. A Pik3ip1 genetic deficiency led to enhanced T-cell responsiveness upon immunization with a neoantigen. Pik3ip1 -/- mice exhibited a marked increase in antitumor immunity and were resistant to tumor growth. Furthermore, Pik3ip1 extracellular domain fusion protein enhanced MC38 tumor growth was observed. Mechanistically, we found that Pik3ip1 inhibited TCR signaling by mediating the degradation of SLP76 through Pik3ip1 oligomerization via its extracellular region. Consistent with the results from the mouse models, PIK3IP1 expression correlated with T-cell dysfunction in human tumors. CONCLUSIONS: Our data reveal a critical role for Pik3ip1 as a novel inhibitory immune regulator of T-cell responses and provide a potential molecular target for cancer immunotherapy.

Calnexin Impairs the Antitumor Immunity of CD4+ and CD8+ T Cells.

Elucidation of the mechanisms of T-cell-mediated antitumor responses will provide information for the rational design and development of cancer immunotherapies. Here, we found that calnexin, an endoplasmic reticulum (ER) chaperone protein, is significantly upregulated in oral squamous cell carcinoma (OSCC). Upregulation of its membranous expression on OSCC cells is associated with inhibited T-cell infiltration in tumor tissues and correlates with poor survival of patients with OSCC. We found that calnexin inhibits the proliferation of CD4+ and CD8+ T cells isolated from the whole blood of healthy donors and patients with OSCC and inhibits the secretion of IFNγ, TNFα, and IL2 from these cells. Furthermore, in a melanoma model, knockdown of calnexin enhanced the infiltration and effector functions of T cells in the tumor microenvironment and conferred better control of tumor growth, whereas treatment with a recombinant calnexin protein impaired the infiltration and effector functions of T cells and promoted tumor growth. We also found that calnexin enhanced the expression of PD-1 on CD4+ and CD8+ T cells by restraining the DNA methylation status of a CpG island in the PD-1 promoter. Thus, this work uncovers a mechanism by which T-cell antitumor responses are regulated by calnexin in tumor cells and suggests that calnexin might serve as a potential target for the improvement of antitumor immunotherapy.