TRAF3 Acts as a Checkpoint of B Cell Receptor Signaling to Control Antibody Class Switch Recombination and Anergy.

The BCR recognizes foreign Ags to initiate humoral immunity that needs isotype-switched Abs generated via class switch recombination (CSR); however, stimulating the BCR in the absence of costimulation (e.g., CD40) does not induce CSR; thus, it remains elusive whether and how the BCR induces CSR mechanistically. Autoreactive B cells can maintain anergy via unresponsiveness of their BCRs to self-antigens. However, it remains unknown what molecule(s) restrict BCR signaling strength for licensing BCR-induced CSR and whether deficiency of such molecule(s) disrupts autoreactive B cell anergy and causes B cell-mediated diseases by modulating BCR signaling. In this study, we employ mouse models to show that the BCR's capacity to induce CSR is restrained by B cell-intrinsic checkpoints TRAF3 and TRAF2, whose deletion in B cells enables the BCR to induce CSR in the absence of costimulation. TRAF3 deficiency permits BCR-induced CSR by elevating BCR-proximal signaling intensity. Furthermore, NF-κB2 is required for BCR-induced CSR in TRAF3-deficient B cells but not for CD40-induced or LPS-induced CSR, suggesting that TRAF3 restricts NF-κB2 activation to specifically limit the BCR's ability to induce CSR. TRAF3 deficiency also disrupts autoreactive B cell anergy by elevating calcium influx in response to BCR stimulation, leading to lymphoid organ disorders and autoimmune manifestations. We showed that TRAF3 deficiency-associated autoimmune phenotypes can be rectified by limiting BCR repertoires or attenuating BCR signaling strength. Thus, our studies highlight the importance of TRAF3-mediated restraint on BCR signaling strength for controlling CSR, B cell homeostasis, and B cell-mediated disorders.

Tumor immune microenvironment in head and neck cancers.

Head and neck cancers are a heterogeneous group of tumors that are highly aggressive and collectively represent the sixth most common cancer worldwide. Ninety percent of head and neck cancers are squamous cell carcinomas (HNSCCs). The tumor microenvironment (TME) of HNSCCs consists of many different subsets of cells that infiltrate the tumors and interact with the tumor cells or with each other through various networks. Both innate and adaptive immune cells play a crucial role in mediating immune surveillance and controlling tumor growth. Here, we discuss the different subsets of immune cells and how they contribute to an immunosuppressive TME of HNSCCs. We also briefly summarize recent advances in immunotherapeutic approaches for HNSCC treatment. A better understanding of the multiple factors that play pivotal roles in HNSCC tumorigenesis and tumor progression may help define novel targets to develop more effective immunotherapies for patients with HNSCC.

TRAF2 Deficiency in B Cells Impairs CD40-Induced Isotype Switching That Can Be Rescued by Restoring NF-κB1 Activation.

Effective humoral immunity requires class switch recombination (CSR) catalyzed by activation-induced cytidine deaminase (AID). In response to T cell-dependent (TD) Ags, CSR can be induced by CD40 signaling in B cells. TNFR-associated factors 2 and 3 (TRAF2/TRAF3) function as adaptors of the CD40 signaling pathway. B cell-intrinsic TRAF2 or TRAF3 (B-TRAF2 or B-TRAF3) knockout mice were previously reported to have indistinguishable phenotypes in gene expression, B cell survival and development, and enlarged peripheral lymphoid organs. However, it remains unknown whether deficiency of B-TRAF2 or B-TRAF3 differentially affects TD humoral immune responses and CD40-induced CSR. In this article, we show that B-TRAF2 is essential for optimal isotype switching induced by in vivo TD Ag immunization or by engaging CD40 in vitro. Our data clarify the controversial role of B-TRAF3 and confirm its dispensability in CD40-induced CSR. Mechanistically, CD40-induced AID expression was markedly impaired by B-TRAF2, but not B-TRAF3, deficiency. Moreover, B-TRAF2 deficiency causes defective activation of the NF-κB1 complex in a CD40-autonomous manner, and restoring CD40-induced NF-κB1 activation in TRAF2-deficient B cells rescues AID expression and CSR. We conclude that TRAF2 is essential but TRAF3 is dispensable for TD humoral immunity and CD40-induced CSR. Our studies provide significant biological bases for optimizing treatment of B cell-associated immune disorders by targeting CD40 signaling.

Deletion of p53 and Hyper-Activation of PIK3CA in Keratin-15+ Stem Cells Lead to the Development of Spontaneous Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer, and SCCs make up about 90% of head and neck cancers (HNSCCs). HNSCCs harbor two frequent molecular alterations, namely, gain-of-function alterations of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and loss-of-function mutations of tumor protein p53 (TP53). However, it remains poorly understood whether HNSCCs harboring different genetic alterations exhibit differential immune tumor microenvironments (TME). It also remains unknown whether PIK3CA hyperactivation and TP53 deletion can lead to SCC development spontaneously. Here, we analyzed the Cancer Genome Atlas (TCGA) datasets of HNSCCs and found that patients with both PIK3CA and TP53 alterations exhibited worse survival, significantly lower CD8 tumor infiltrating lymphocytes (TILs) and higher M0 macrophages than other controls. To better model human tumorigenesis, we deleted TP53 and constitutively activated PIK3CA in mouse keratin-15-expressing stem cells, which leads to the spontaneous development of multilineage tumors including SCCs, termed Keratin-15-p53-PIK3CA (KPPA) tumors. KPPA tumors were heavily infiltrated with myeloid-derived suppressor cells (MDSCs), with a drastically increased ratio of polymorphonuclear-MDSC (PMN-MDSC) versus monocytic-MDSC (M-MDSC). CD8 TILs expressed more PD-1 and reduced their polyfunctionality. Overall, we established a genetic model to mimic human HNSCC pathogenesis, manifested with an immunosuppressive TME, which may help further elucidate immune evasion mechanisms and develop more effective immunotherapies for HNSCCs.

Host-specific differences in top-expanded TCR clonotypes correlate with divergent outcomes of anti-PD-L1 treatment in responders versus non-responders.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment; however, the responses to ICI treatment are highly variable in different individuals and the underlying mechanisms remain poorly understood. Here, we employed a mouse squamous cell carcinoma (SCC) model where tumor-bearing recipients diverged into responders (R) versus non-responders (NR) upon anti-PD-L1 treatment. We performed in-depth TCRß sequencing with immunoSEQ platform to delineate the differences in CD8 tumor-infiltrating lymphocytes (TILs). We found that R and NR CD8 TILs both exhibited evidence of clonal expansion, suggesting activation regardless of response status. We detected no differences in clonal expansion or clonal diversity indexes between R vs. NR. However, the top expanded (>1%) TCRß clonotypes appeared to be mutually exclusive between R and NR CD8 TILs, showing a preferential expansion of distinct TCRß clonotypes in response to the same SCC tumor in R vs. NR. Notably, the mutual exclusivity of TCR clonotypes in R vs. NR was only observed when top TCRß clonotypes were counted, because such top-expanded clonotypes are present in the opposite outcome group at a much lower frequency. Many TCRß sequences were detected in only one recipient at a high frequency, implicating highly individualized anti-tumor immune responses. We conclude that differences in the clonal frequency of top TCR clonotypes between R and NR CD8 TILs may be one of the factors underlying differential anti-PD-L1 responses. This notion may offer a novel explanation for variable ICI responses in different individuals, which may substantially impact the development of new strategies for personalized cancer immunotherapy.

Divergent outcomes of anti-PD-L1 treatment coupled with host-intrinsic differences in TCR repertoire and distinct T cell activation states in responding versus non-responding tumors.

Differential responses to immune checkpoint inhibitors (ICI) may be attributed to tumor-intrinsic factors or environmental cues; however, these mechanisms cannot fully explain the variable ICI responses in different individuals. Here, we investigate the potential contribution of immunological heterogeneity with a focus on differences in T-cell receptor (TCR) repertoire to ICI responses, which has not been defined previously. To reveal additional factors underlying heterogeneous responses to ICI, we employed a squamous cell carcinoma (SCC) mouse model in which tumor-bearing recipients unambiguously diverged into responders (R) or non-responders (NR) upon anti-PD-L1 treatment. Treatment efficacy absolutely required CD8 T-cells and correlated positively with effector functions of CD8 tumor-infiltrating lymphocytes (TILs). We showed that TCR repertoires exhibited a similar magnitude of clonal expansion in R vs. NR CD8 TILs. However, the top expanded TCR clonotypes appeared to be mutually exclusive between R and NR CD8 TILs, which also occurred in a recipient-specific manner, demonstrating preferential expansion of distinct TCR clonotypes against the same SCC tumor. Unexpectedly, R vs. NR CD8 TILs reached all activation clusters and did not exhibit substantial global differences in transcriptomes. By linking single-cell transcriptomic data with unique TCR clonotypes, CD8 TILs harboring top TCR clonotypes were found to occupy distinct activation clusters and upregulate genes favoring anti-tumor immunity to different extents in R vs. NR. We conclude that stochastic differences in CD8 TIL TCR repertoire and distinct activation states of top TCR clonotypes may contribute to differential anti-PD-L1 responses. Our study suggests that host-intrinsic immunological heterogeneity may offer a new explanation for differential ICI responses in different individuals, which could impact on strategies for personalized cancer immunotherapy.

Differential responses to immune checkpoint inhibitor dictated by pre-existing differential immune profiles in squamous cell carcinomas caused by same initial oncogenic drivers.

BACKGROUND: While immune checkpoint inhibitors (ICI) were approved for head and neck squamous cell carcinomas (HNSCCs), the response rate remains relatively low. Mechanisms underlying ICI unresponsiveness versus sensitivity are not fully understood. METHOD: To better delineate differential responses to ICI treatment, we employed mouse SCC models, termed KPPA tumors that were caused by deleting p53 and hyperactivating PIK3CA, two most frequently mutated genes in human HNSCCs. We transplanted two KPPA tumor lines (TAb2 versus TCh3) into C57BL/6 recipients and examined the immune tumor microenvironment using flow cytometry. Furthermore, we employed single-cell RNA sequencing to identify the difference in tumor infiltrating lymphocytes (TILs). RESULTS: We found that different KPPA tumors exhibited heterogeneous immune profiles pre-existing treatment that dictated their sensitivity or unresponsiveness to anti-PD-L1. Unresponsive TAb2 tumors were highly enriched with functional tumor-associated macrophages (TAMs), especially M2-TAMs. In contrast, sensitive TCh3 tumors contained more CD8 TILs with better effector functions. TAb2 tumor cells drastically expanded F4/80+ TAMs from bone marrow precursors, requiring CSF1 and VEGF. Consistently, a higher combined expression of VEGF-C and CSF1 predicts worse survival in PIK3CAAmp/TP53Mutated HNSCC patients. Unresponsive TAb2 tumors upregulated distinct signaling pathways that correlate with aggressive tumor phenotypes. While anti-PD-L1 did not affect the TME of TAb2 tumors, it significantly increased the number of CD8 TILs in TCh3 tumors. CONCLUSIONS: We uncovered tumor-intrinsic differences that may underlie the differential responses to ICI by establishing and employing two SCC tumor lines, TAb2 vs. TCh3, both of which harbor TP53 deletion and PIK3CA hyperactivation. Our study indicates the limitation of stratifying cancers according to their genetic alterations and suggests that evaluating HNSCC tumor-intrinsic cues along with immune profiles in the TME may help better predict ICI responses. Our experimental models may provide a platform for pinpointing tumor-intrinsic differences underlying an immunosuppressive TME in HNSCCs and for testing combined immunotherapies targeting either tumor-specific or TAM-specific players to improve ICI efficacy.

MHC class I-independent activation of virtual memory CD8 T cells induced by chemotherapeutic agent-treated cancer cells.

Cancer cells can evade immune recognition by losing major histocompatibility complex (MHC) class I. Hence, MHC class I-negative cancers represent the most challenging cancers to treat. Chemotherapeutic drugs not only directly kill tumors but also modulate the tumor immune microenvironment. However, it remains unknown whether chemotherapy-treated cancer cells can activate CD8 T cells independent of tumor-derived MHC class I and whether such MHC class I-independent CD8 T-cell activation can be exploited for cancer immunotherapy. Here, we showed that chemotherapy-treated cancer cells directly activated CD8 T cells in an MHC class I-independent manner and that these activated CD8 T cells exhibit virtual memory (VM) phenotypes. Consistently, in vivo chemotherapeutic treatment preferentially increased tumor-infiltrating VM CD8 T cells. Mechanistically, MHC class I-independent activation of CD8 T cells requires cell-cell contact and activation of the PI3K pathway. VM CD8 T cells contribute to a superior therapeutic effect on MHC class I-deficient tumors. Using humanized mouse models or primary human CD8 T cells, we also demonstrated that chemotherapy-treated human lymphomas activated VM CD8 T cells independent of tumor-derived MHC class I. In conclusion, CD8 T cells can be directly activated in an MHC class I-independent manner by chemotherapy-treated cancers, and these activated CD8 T cells may be exploited for developing new strategies to treat MHC class I-deficient cancers.

Differences in TCR repertoire and T cell activation underlie the divergent outcomes of antitumor immune responses in tumor-eradicating versus tumor-progressing hosts.

BACKGROUND: Antitumor immunity is highly heterogeneous between individuals; however, underlying mechanisms remain elusive, despite their potential to improve personalized cancer immunotherapy. Head and neck squamous cell carcinomas (HNSCCs) vary significantly in immune infiltration and therapeutic responses between patients, demanding a mouse model with appropriate heterogeneity to investigate mechanistic differences. METHODS: We developed a unique HNSCC mouse model to investigate underlying mechanisms of heterogeneous antitumor immunity. This model system may provide a better control for tumor-intrinsic and host-genetic variables, thereby uncovering the contribution of the adaptive immunity to tumor eradication. We employed single-cell T-cell receptor (TCR) sequencing coupled with single-cell RNA sequencing to identify the difference in TCR repertoire of CD8 tumor-infiltrating lymphocytes (TILs) and the unique activation states linked with different TCR clonotypes. RESULTS: We discovered that genetically identical wild-type recipient mice responded heterogeneously to the same squamous cell carcinoma tumors orthotopically transplanted into the buccal mucosa. While tumors initially grew in 100% of recipients and most developed aggressive tumors, ~25% of recipients reproducibly eradicated tumors without intervention. Heterogeneous antitumor responses were dependent on CD8 T cells. Consistently, CD8 TILs in regressing tumors were significantly increased and more activated. Single-cell TCR-sequencing revealed that CD8 TILs from both growing and regressing tumors displayed evidence of clonal expansion compared with splenic controls. However, top TCR clonotypes and TCR specificity groups appear to be mutually exclusive between regressing and growing TILs. Furthermore, many TCRα/TCRß sequences only occur in one recipient. By coupling single-cell transcriptomic analysis with unique TCR clonotypes, we found that top TCR clonotypes clustered in distinct activation states in regressing versus growing TILs. Intriguingly, the few TCR clonotypes shared between regressors and progressors differed greatly in their activation states, suggesting a more dominant influence from tumor microenvironment than TCR itself on T cell activation status. CONCLUSIONS: We reveal that intrinsic differences in the TCR repertoire of TILs and their different transcriptional trajectories may underlie the heterogeneous antitumor immune responses in different hosts. We suggest that antitumor immune responses are highly individualized and different hosts employ different TCR specificities against the same tumors, which may have important implications for developing personalized cancer immunotherapy.

Distinct immune microenvironment profiles of therapeutic responders emerge in combined TGFß/PD-L1 blockade-treated squamous cell carcinoma.

Transforming growth factor beta (TGFß) and programmed death-ligand 1 (PD-L1) are often overproduced in refractory squamous cell carcinoma (SCC). We examined spatial patterns of PD-L1+ cells in mouse and human SCCs and found that PD-L1 was primarily expressed on infiltrating leukocytes. Although combined TGFß and PD-L1 blockade are undergoing cancer clinical trials, there are no predictive markers for therapeutic responders. To address this, we used both a small molecule TGFß inhibitor in combination with anti-PD-L1 and a bifunctional fusion protein targeting both TGFß and PD-L1 to treat mouse SCCs and found TGFß inhibition enhanced PD-L1 blockade-induced tumor eradication in multiple tumor models. Furthermore, we identified distinct cell populations of responders and non-responders to bintrafusp alfa, with responders showing a shift toward a more immune-permissive microenvironment. The cellular and molecular signatures of responders versus non-responders to combined TGFß and PD-L1 blockade provide important insights into future personalized immunotherapy in SCC.

HDAC inhibitors overcome immunotherapy resistance in B-cell lymphoma.

Immunotherapy has been applied successfully to treat B-cell lymphomas in preclinical models or clinical settings. However, immunotherapy resistance is a major challenge for B-cell lymphoma treatment. To overcome this issue, combinatorial therapeutic strategies have been pursued to achieve a better efficacy for treating B-cell lymphomas. One of such strategies is to combine immunotherapy with histone deacetylase (HDAC) inhibitors. HDAC inhibitors can potentially increase tumor immunogenicity, promote anti-tumor immune responses, or reverse immunosuppressive tumor environments. Thus, the combination of HDAC inhibitors and immunotherapy has drawn much attention in current cancer treatment. However, not all HDAC inhibitors are created equal and their net effects are highly dependent on the specific inhibitors used and the HDACs they target. Hence, we suggest that optimal treatment efficacy requires personalized design and rational combination based on prognostic biomarkers and unique profiles of HDAC inhibitors. Here, we discuss the possible mechanisms by which B-cell lymphomas acquire immunotherapy resistance and the effects of HDAC inhibitors on tumor cells and immune cells that could help overcome immunotherapy resistance.

Histone Deacetylase Inhibition Sensitizes PD1 Blockade-Resistant B-cell Lymphomas.

PD1 blockade is effective in a subset of patients with B-cell lymphoma (e.g., classical-Hodgkin lymphomas); however, most patients do not respond to anti-PD1 therapy. To study PD1 resistance, we used an isoform-selective histone deacetylase inhibitor (HDACi; OKI-179), and a mouse mature B-cell lymphoma, G1XP lymphoma, immunosuppressive features of which resemble those of human B-cell lymphomas, including downregulation of MHC class I and II, exhaustion of CD8+ and CD4+ tumor-infiltrating lymphocytes (TIL), and PD1-blockade resistance. Using two lymphoma models, we show that treatment of B-cell lymphomas refractory to PD1 blockade with both OKI-179 and anti-PD1 inhibited growth; furthermore, sensitivity to single or combined treatment required tumor-derived MHC class I, and positively correlated with MHC class II expression level. We conclude that OKI-179 sensitizes lymphomas to PD1-blockade by enhancing tumor immunogenicity. In addition, we found that different HDACis exhibited distinct effects on tumors and T cells, yet the same HDACi could differentially affect HLA expression on different human B-cell lymphomas. Our study highlights the immunologic effects of HDACis on antitumor responses and suggests that optimal treatment efficacy requires personalized design and rational combination based on prognostic biomarkers (e.g., MHCs) and the individual profiles of HDACi.

Fibroblast growth factor 8 deficiency compromises the functional response of the serotonergic system to stress.

Functionally heterogeneous populations of serotonergic neurons, located within the dorsal raphe nucleus (DR), play a role in stress-related behaviors and neuropsychiatric illnesses such as anxiety and depression. Abnormal development of these neurons may permanently alter their structure and connections, making the organism more susceptible to anxiety-related disorders. A factor that critically regulates the development of serotonergic neurons is fibroblast growth factor 8 (Fgf8). In this study, we used acute restraint stress followed by behavioral testing to examine whether Fgf8 signaling during development is important for establishing functional stress- and anxiety-related DR neurocircuits in adulthood. Wild-type and heterozygous male mice globally hypomorphic for Fgf8 were exposed to acute restraint stress and then tested for anxiety-like behavior on the elevated plus-maze. Further, we measured c-Fos immunostaining as a marker of serotonergic neuronal activation and tissue 5-hydroxyindoleacetic acid concentrations as a marker of serotonin functional output. Results showed that Fgf8 hypomorphs exhibited 1) an exaggerated response of DR anxiety-promoting circuits and 2) a blunted response of a DR panic-inhibiting circuit to stress, effects that together were associated with increased baseline anxiety-like behavior. Overall, our results provide a neural substrate upon which Fgf8 deficiency could affect stress response and support the hypothesis that developmental disruptions of serotonergic neurons affect their postnatal functional integrity.