Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity.

DNA mismatch repair deficiency (MMRd) is associated with a high tumor mutational burden (TMB) and sensitivity to immune checkpoint blockade (ICB) therapy. Nevertheless, most MMRd tumors do not durably respond to ICB and critical questions remain about immunosurveillance and TMB in these tumors. In the present study, we developed autochthonous mouse models of MMRd lung and colon cancer. Surprisingly, these models did not display increased T cell infiltration or ICB response, which we showed to be the result of substantial intratumor heterogeneity of mutations. Furthermore, we found that immunosurveillance shapes the clonal architecture but not the overall burden of neoantigens, and T cell responses against subclonal neoantigens are blunted. Finally, we showed that clonal, but not subclonal, neoantigen burden predicts ICB response in clinical trials of MMRd gastric and colorectal cancer. These results provide important context for understanding immune evasion in cancers with a high TMB and have major implications for therapies aimed at increasing TMB.

Chronic antigen in solid tumors drives a distinct program of T cell residence.

Analyses of healthy tissue reveal signatures that identify resident memory CD8+ T cells (TRM), which survey tissues without recirculating. The density of TRM phenotype cells within solid tumors correlates favorably with prognosis, suggesting that intratumoral residents control cancer. However, residence has not been directly tested, and intratumoral TRM phenotype cells could instead reflect aspects of the microenvironment that correlate with prognosis. Using a breast cancer model in mice, we found that conventional TRM markers do not inform the tumor residence of either bystander or tumor-specific cells, which exhibit further distinct phenotypes in the tumor microenvironment and healthy mammary tissue. Rather, tumor-specific, stem progenitor CD8+ T cells migrate to tumors and become resident while acquiring select markers of exhaustion. These data indicate that tonic antigen stimulation and the tumor environment drive distinct programs of residence compared with healthy tissues and that tumor immunity is sustained by continued migration of tumor-specific stem cells.

Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes.

In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.

Emergence of a High-Plasticity Cell State during Lung Cancer Evolution.

Tumor evolution from a single cell into a malignant, heterogeneous tissue remains poorly understood. Here, we profile single-cell transcriptomes of genetically engineered mouse lung tumors at seven stages, from pre-neoplastic hyperplasia to adenocarcinoma. The diversity of transcriptional states increases over time and is reproducible across tumors and mice. Cancer cells progressively adopt alternate lineage identities, computationally predicted to be mediated through a common transitional, high-plasticity cell state (HPCS). Accordingly, HPCS cells prospectively isolated from mouse tumors and human patient-derived xenografts display high capacity for differentiation and proliferation. The HPCS program is associated with poor survival across human cancers and demonstrates chemoresistance in mice. Our study reveals a central principle underpinning intra-tumoral heterogeneity and motivates therapeutic targeting of the HPCS.

IL-33 Signaling Alters Regulatory T Cell Diversity in Support of Tumor Development.

Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we use single-cell RNA sequencing (RNA-seq) to longitudinally profile dynamic shifts in the distribution of Tregs in a genetically engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive Tregs are more prevalent early in tumor development, whereas a specialized effector phenotype characterized by enhanced expression of the interleukin-33 receptor ST2 is predominant in advanced disease. Treg-specific deletion of ST2 alters the evolution of effector Treg diversity, increases infiltration of CD8+ T cells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting potential paths for therapeutic intervention.

Speed of exposure to rapid cold hardening and genotype drive the level of acclimation response in Drosophila melanogaster.

Comparing methodologies that attempt to mimic natural conditions is important when evaluating thermal tolerances of ectotherms, as exposing animals to different artificial thermal regimes may provide conflicting information of an insect's thermal profile. Rapid cold hardening (RCH) occurs in ectotherms and typically increases survivorship to extreme cold exposure through a short, pre-treatment to a non-lethal cold temperature. Here we assess survivorship in a set of genotypes from the Drosophila melanogaster Reference Panel for direct and ramping RCH pre-treatments at cooling rates occurring under more natural conditions (0.1 °C/min and 0.5 °C/min) in combination with a direct and ramped rewarming treatment post cold exposure. We find that all three pre-treatment exposures alone significantly increase survivorship. We find significant correlations in survivorship among treatments across genotypes, suggesting that regardless of the pre-treatment, individuals of a given genotype have an innate level of acclimation. When rewarming is introduced, survivorship significantly decreased relative to pre-treatment alone and correlations of survival between phenotypes were not significant. Our results suggest that rewarming and slow RCH are costly to survival while a quicker RCH may impart physiological benefits more consistently across genotypes.

Potential Mechanisms Underlying Centralized Pain and Emerging Therapeutic Interventions.

Centralized pain syndromes are associated with changes within the central nervous system that amplify peripheral input and/or generate the perception of pain in the absence of a noxious stimulus. Examples of idiopathic functional disorders that are often categorized as centralized pain syndromes include fibromyalgia, chronic pelvic pain syndromes, migraine, and temporomandibular disorder. Patients often suffer from widespread pain, associated with more than one specific syndrome, and report fatigue, mood and sleep disturbances, and poor quality of life. The high degree of symptom comorbidity and a lack of definitive underlying etiology make these syndromes notoriously difficult to treat. The main purpose of this review article is to discuss potential mechanisms of centrally-driven pain amplification and how they may contribute to increased comorbidity, poorer pain outcomes, and decreased quality of life in patients diagnosed with centralized pain syndromes, as well as discuss emerging non-pharmacological therapies that improve symptomology associated with these syndromes. Abnormal regulation and output of the hypothalamic-pituitary-adrenal (HPA) axis is commonly associated with centralized pain disorders. The HPA axis is the primary stress response system and its activation results in downstream production of cortisol and a dampening of the immune response. Patients with centralized pain syndromes often present with hyper- or hypocortisolism and evidence of altered downstream signaling from the HPA axis including increased Mast cell (MC) infiltration and activation, which can lead to sensitization of nearby nociceptive afferents. Increased peripheral input via nociceptor activation can lead to "hyperalgesic priming" and/or "wind-up" and eventually to central sensitization through long term potentiation in the central nervous system. Other evidence of central modifications has been observed through brain imaging studies of functional connectivity and magnetic resonance spectroscopy and are shown to contribute to the widespreadness of pain and poor mood in patients with fibromyalgia and chronic urological pain. Non-pharmacological therapeutics, including exercise and cognitive behavioral therapy (CBT), have shown great promise in treating symptoms of centralized pain.

Voluntary wheel running attenuates urinary bladder hypersensitivity and dysfunction following neonatal maternal separation in female mice.

AIMS: Patients with interstitial cystitis/painful bladder syndrome (IC/PBS) commonly suffer from widespread pain and mood disorder, which has been attributed to improper functioning of the hypothalamic-pituitary-adrenal (HPA) axis. Voluntary exercise has been shown to improve HPA axis function, therefore we are determining whether voluntary wheel running can attenuate urological pain and dysfunction following neonatal maternal separation (NMS) in female mice. METHODS: Mice underwent NMS for 3 h/day from postnatal Day 1-21, were caged with free access to running wheels at 4 weeks of age, and assessed 4 weeks later for bladder sensitivity, micturition, reward behavior, mast cell degranulation, and HPA axis-related in vitro analysis. RESULTS: Increased bladder sensitivity, void frequency, and mast cell degranulation was observed in adult sedentary (-Sed) NMS mice, compared to naïve-Sed controls. Sucrose preference was increased in NMS-Sed mice and corticotropin-releasing factor receptor 1 (CRF1 ) and glucocorticoid receptor mRNA levels were significantly reduced in the hippocampus. Exercise normalized bladder sensitivity, micturition output, and increased brain-derived neurotrophic factor (BDNF) mRNA levels in the hippocampus of NMS mice. Mast cell degranulation was also normalized in NMS bladders following exercise. CONCLUSIONS: Voluntary exercise normalized behavioral outcomes resulting from NMS in female mice, increased hippocampal BDNF mRNA levels, and decreased mast cell degranulation in the bladder. Together these results provide novel insight into the efficacy of voluntary exercise to attenuate comorbid outcomes resulting from exposure to early life stress.