TRAF2/3 deficient B cells resist DNA damage-induced apoptosis via NF-κB2/XIAP/cIAP2 axis and IAP antagonist sensitizes mutant lymphomas to chemotherapeutic drugs.

Deletion of TRAF2 or TRAF3 in B cells prolongs their survival. However, it remains unknown whether deletion of such factors affects B cells' ability to tolerate DNA damage, which can be induced by chemotherapeutics and cause apoptosis. Genetic alterations of TRAF2 or TRAF3 are observed in subsets of human B-cell lymphomas and B cell-specific deletion of TRAF3 led to lymphoma development in aged mice. However, it remains unknown whether double deficiency of TRAF2 and TRAF3 accelerates B-cell lymphomagenesis. Here, we showed that B cell-specific TRAF2/3 double deficient (B-TRAF2/3-DKO) B cells were remarkably more resistant to DNA damage-induced apoptosis via upregulating cIAP2 and XIAP, which in turn attenuates caspase-3 activation. Mechanistically, resistance to DNA damage-induced apoptosis required NF-κB2, which effects by upregulating XIAP and cIAP2 transcription. B-TRAF2/3-DKO mice exhibited a shorter lifespan and succumbed to splenomegaly and lymphadenopathy. Unexpectedly, the incidence of B-cell lymphoma development in B-TRAF2/3-DKO mice was relatively rare (∼10%). Sequencing B cell receptor repertoire of diseased B cells revealed that TRAF2/3 deficiency caused abnormal oligoclonal or clonal expansion of B cells. While a fraction of mutant B cells (25-43%) from aged diseased mice harbored recurrent chromosomal translocations, primary B cells isolated from young B-TRAF2/3-DKO mice had no detectable chromosomal alterations, suggesting that TRAF2/3 deficiency per se does not cause evident genomic instability in B cells. Chemo-resistant TRAF3-deficient B-cell lymphomas were sensitized to chemotherapeutic drugs by blocking IAP activity using IAP antagonist. We conclude that double deficiency of TRAF2 and TRAF3 does not accelerate B-cell lymphomagenesis. Our studies provide insight into mechanisms regulating DNA damage-induced apoptosis and may help develop effective therapies targeting mutant B-cell lymphomas using IAP antagonist.

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.

Why responses to immune checkpoint inhibitors are heterogeneous in head and neck cancers: Contributions from tumor-intrinsic and host-intrinsic factors.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment including in head and neck squamous cell carcinomas (HNSCCs); however, only a fraction of HNSCC patients respond to ICI, whereas the majority fail to do so. The mechanisms underlying such variable responses remain incompletely understood. A better understanding of such mechanisms may broaden the spectrum of responding patients and enhance the rate of ICI response. HNSCCs exhibit a high level of genetic heterogeneity, manifested as mutations or amplifications of oncogenes (e.g., PIK3CA) and mutations of tumor suppressor genes (e.g., TP53). The immune tumor microenvironment (TME) of HNSCCs also varies significantly in composition and in relative abundance of distinct immune subsets such as CD8 tumor-infiltrating lymphocytes (TILs) or tumor-associated macrophages (TAMs), which represents a high degree of immunological heterogeneity. Here, we briefly discuss how heterogeneous ICI responses may be attributed to tumor-intrinsic factors, including genetic, transcriptional, and functional variations in tumor cells, and host-intrinsic factors, including cellular composition of the TME (e.g., CD8 TILs and TAMs), and host-intrinsic differences in the T cell receptor (TCR) repertoire of CD8 TILs. We also discuss the potential impact of these factors on designing strategies for personalized immunotherapy of HNSCCs.

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.

Sesamol, a lipid lowering agent, ameliorates aluminium chloride induced behavioral and biochemical alterations in rats.

BACKGROUND: Sesame oil from the seeds of Sesamum indicum Linn. (Pedaliaceae) has been used traditionally in Indian medical practice of Ayurveda in the treatment of central nervous system disorders and insomnia. A few published reports favor the anti-dementia effect of sesamol (SML), an active constituent of sesame oil. OBJECTIVE: Thus, the present study was aimed to explore the anti-dementia effect and possible mechanism (s) of SML in aluminium chloride (AlCl3)-induced cognitive dysfunction model in rodents with special emphasis on memory centers viz., hippocampus and frontal cortex. METHODS: Male Wistar rats were exposed to AlCl3 (175 mg/kg p.o.) for 60 days. SML (10 and 20 mg/kg) and rivastigmine (1 mg/kg) were administered orally 45 min before administration of AlCl3 for 60 days. Spatial memory was assessed using Morris water maze test. After 60 days of treatment animals were sacrificed, hippocampus and frontal cortex were collected and analyzed for acetylcholinesterase (AChE) activity, tumor necrosis factor (TNF-α) level, antioxidant enzymes (Glutathione, catalase), lipid peroxidation, and nitrite level. The circulating triglycerides, total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were also analyzed. RESULTS: SML significantly prevented behavioral impairments in aluminium-exposed rats. Treatment with SML reversed the increased cholesterol, triglycerides and LDL while raised the HDL levels. SML significantly corrected the effect of AlCl3 on AChE activity. Further, SML reversed the elevated nitric oxide, TNF-α and reduced antioxidant enzymes in hippocampus and frontal cortex. CONCLUSION: The present study suggests the neuro-protection by SML against cognitive dysfunction induced by environmental toxin (AlCl3) in hippocampus and frontal cortex.

Insulin blocks glutamate-induced neurotoxicity in differentiated SH-SY5Y neuronal cells.

Insulin is a cytokine which promotes cell growth. Recently, a few published reports on insulin in different cell lines support the antiapoptotic effect of insulin. But the reports fail to explain the role of insulin in modulating glutamate-mediated neuronal cell death through excitotoxicity. Thus, we examined the neuroprotective effect of insulin on glutamate-induced toxicity on differentiated SH-SY5Y neuronal cells. Changes in cell viability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) based assay, while apoptotic damage was detected by acridine orange/ethidium bromide and Hoechst staining. Intracellular reactive oxygen species (ROS) accumulation and morphological alterations were also measured. Treatment with glutamate induced apoptosis, elevated ROS levels and caused damage to neurons. Insulin was able to attenuate the glutamate-induced excitotoxic damage to neuronal cells.