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The thymus is the central organ involved with T-cell development and the production of naïve T cells. During normal aging, the thymus undergoes marked involution, reducing naïve T-cell output and resulting in a predominance of long-lived memory T cells in the periphery. Outside of aging, systemic stress responses that induce corticosteroids (CS), or other insults such as radiation exposure, induce thymocyte apoptosis, resulting in a transient acute thymic involution with subsequent recovery occurring after cessation of the stimulus. Despite the increasing utilization of immunostimulatory regimens in cancer, effects on the thymus and naïve T cell output have not been well characterized. Using both mouse and human systems, the thymic effects of systemic immunostimulatory regimens, such as high dose IL-2 (HD IL-2) with or without agonistic anti-CD40 mAbs and acute primary viral infection, were investigated. These regimens produced a marked acute thymic involution in mice, which correlated with elevated serum glucocorticoid levels and a diminishment of naïve T cells in the periphery. This effect was transient and followed with a rapid thymic “rebound” effect, in which an even greater quantity of thymocytes was observed compared to controls. Similar results were observed in humans, as patients receiving HD IL-2 treatment for cancer demonstrated significantly increased cortisol levels, accompanied by decreased peripheral blood naïve T cells and reduced T-cell receptor excision circles (TRECs), a marker indicative of recent thymic emigrants. Mice adrenalectomized prior to receiving immunotherapy or viral infection demonstrated protection from this glucocorticoid-mediated thymic involution, despite experiencing a substantially higher inflammatory cytokine response and increased immunopathology. Investigation into the effects of immunostimulation on middle aged (7-12 months) and advance aged (22-24 months) mice, which had already undergone significant thymic involution and had a diminished naïve T cell population in the periphery at baseline, revealed that even further involution was incurred. Thymic rebound hyperplasia, however, only occurred in young and middle-aged recipients, while advance aged not only lacked this rebound hyperplasia, but were entirely absent of any indication of thymic restoration. This coincided with prolonged deficits in naïve T cell numbers in advanced aged recipients, further skewing the already memory dominant T cell pool. These results demonstrate that, in both mice and humans, systemic immunostimulatory cancer therapies, as well as immune challenges like subacute viral infections, have the potential to induce profound, but transient, glucocorticoid-mediated thymic involution and substantially reduced thymic output, resulting in the reduction of peripheral naive T cells. This can then be followed by a marked rebound effect with naïve T cell restoration, events that were shown not to occur in advanced-aged mice.

Abstract Stereotactic ablative radiotherapy (SABR) is a standard-of-care for medically-inoperable-early-stage non-small cell lung cancer (NSCLC). One third of patients progress and chemotherapy is rarely used in this population. We questioned if addition of the immune-checkpoint-inhibitor (ICI) atezolizumab to standard-of-care SABR can improve outcomes. We initiated a multi-institutional single-arm phase I study (NCT02599454) enrolling twenty patients with the primary endpoint of maximum tolerated dose (MTD); secondary endpoints of safety and efficacy; and exploratory mechanistic correlatives. Treatment is well tolerated and full dose atezolizumab (1200 mg) is the MTD. Efficacy signals include early responses (after 2 cycles of ICI, before initiation of SABR) in 17% of patients. Biomarkers of functional adaptive immunity, including T cell activation in the tumor and response to ex-vivo stimulation by circulating T cells, are highly predictive of benefit. These results require validation and are being tested in a phase III randomized trial.

Bystander activation of memory T cells occurs via cytokine signaling alone in the absence of T cell receptor (TCR) signaling and provides a means of amplifying T cell effector responses in an antigen-nonspecific manner. While the role of Programmed Cell Death Protein 1 (PD-1) on antigen-specific T cell responses is extensively characterized, its role in bystander T cell responses is less clear. We examined the role of the PD-1 pathway during human and mouse non–antigen-specific memory T cell bystander activation and observed that PD-1+ T cells demonstrated less activation and proliferation than activated PD-1– populations in vitro. Higher activation and proliferative responses were also observed in the PD-1– memory population in both mice and patients with cancer receiving high-dose IL-2, mirroring the in vitro phenotypes. This inhibitory effect of PD-1 could be reversed by PD-1 blockade in vivo or observed using memory T cells from PD-1–/– mice. Interestingly, increased activation through abrogation of PD-1 signaling in bystander-activated T cells also resulted in increased apoptosis due to activation-induced cell death (AICD) and eventual T cell loss in vivo. These results demonstrate that the PD-1/PD-Ligand 1 (PD-L1) pathway inhibited bystander-activated memory T cell responses but also protected cells from AICD.

IntroductionThe incidence of obesity, a condition characterized by systemic chronic inflammation, has reached pandemic proportions and is a poor prognostic factor in many pathologic states. However, its role on immune parameters has been diverse and at times contradictory. We have previously demonstrated that obesity can result in what has been called the “obesity paradox” which results in increased T cell exhaustion, but also greater efficacy of immune checkpoint blockade in cancer treatment.MethodsThe role of obesity, particularly in the context of aging, has not been robustly explored using preclinical models. We therefore evaluated how age impacts the immune environment on T cell development and function using diet-induced obese (DIO) mice.ResultsWe observed that DIO mice initially displayed greater thymopoiesis but then developed greater thymic involution over time compared to their lean counterparts. Both aging and obesity resulted in increased T cell memory conversion combined with increased expression of T cell exhaustion markers and Treg expansion. This increased T cell immunosuppression with age then resulted in a loss of anti-tumor efficacy by immune checkpoint inhibitors (ICIs) in older DIO mice compared to the younger DIO counterparts.DiscussionThese results suggest that both aging and obesity contribute to T cell dysfunction resulting in increased thymic involution. This combined with increased T cell exhaustion and immunosuppressive parameters affects immunotherapy efficacy reducing the advantage of obesity in cancer immunotherapy responses.

PurposeClinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS.Experimental DesignUsing fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3-CD56+ NK and CD3+ T cells and CD45- viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression.ResultsSorted intra-tumoral CD3+ T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45- tumor cells. TCGA analysis revealed that tumors with high TLR4 expression (P = 0.03) and low expression of STMN1 involved in microtubule polymerization (P < 0.001) were associated with significantly improved survival.ConclusionsUnlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS.

The efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by the occurrence of acute and chronic graft-versus-host disease (GVHD). We have recently demonstrated that obesity results in exacerbated acute gastrointestinal GVHD in both mouse models and clinical outcomes due to increased pro-inflammatory cytokine responses and microbiota alterations. We therefore wanted to delineate the role of the various parameters in obesity, adiposity, effects of high-fat (HF) diet, and the role of microbiome on GVHD pathogenesis, by taking advantage of a mouse strain resistant to diet-induced obesity (DIO). Female BALB/c mice are resistant to DIO phenotype with approximately 50% becoming DIO under HF diets. The DIO-susceptible recipients rapidly succumb to acute gut GVHD, whereas the DIO-resistant recipient littermates, which do not become obese, are partially protected from GVHD, indicating that being on HF diet alone contributes to but is not the primary driver of GVHD. Microbiome assessment revealed restricted diversity in both cohorts of mice, but coprophagy normalizes the microbiota in mice housed together. We then individually housed DIO-resistant, DIO-susceptible, and lean control mice. Notably, each of the individually housed groups demonstrates marked restricted diversity that has been shown to occur from the stress of single housing. Despite the restricted microbiome diversity, the GVHD pathogenesis profile remains consistent in the group-housed mice, with the lean control single-housed mice exhibiting no acute GVHD and DIO-resistant recipients showing again partial protection. These results demonstrate that the deleterious effects of obesity on acute gut GVHD are critically dependent on adiposity with the HF diet also playing a lesser role, and the microbiome alterations with obesity instead appear to fuel ongoing acute GVHD processes.

Purpose Given the unmet need for novel immunotherapy in soft tissue sarcoma (STS), we sought to characterize the phenotype and function of intratumoral natural killer (NK) and T cells to identify novel strategies to augment tumor-infiltrating lymphocyte (TIL) function.Experimental design Using prospectively collected specimens from dogs and humans with sarcomas, archived specimens, and The Cancer Genome Atlas (TCGA) data, we evaluated blood and tumor NK and T cell phenotype and function and correlated those with outcome. We then assessed the effects of interleukin 15 (IL-15) stimulation on both NK and T cell activation and TIGIT upregulation. Finally, we evaluated cytotoxic effects of IL-15 combined with TIGIT blockade using a novel anti-TIGIT antibody.Results TILs were strongly associated with survival outcome in both archived tissue and TCGA, but higher TIL content was also associated with higher TIGIT expression. Compared with blood, intratumoral NK and T cells showed significantly higher expression of both activation and exhaustion markers, in particular TIGIT. Ex vivo stimulation of blood and tumor NK and T cells from patients with STS with IL-15 further increased both activation and exhaustion markers, including TIGIT. Dogs with metastatic osteosarcoma receiving inhaled IL-15 also exhibited upregulation of activation markers and TIGIT. Ex vivo, combined IL-15 and TIGIT blockade using STS blood and tumor specimens significantly increased cytotoxicity against STS targets.Conclusion Intratumoral NK and T cells are prognostic in STS, but their activation is marked by significant upregulation of TIGIT. Our data suggest that combined IL-15 and TIGIT blockade may be a promising clinical strategy in STS.

Despite obesity reaching pandemic proportions, its impact on antigen-specific T cell responses is still unclear. We have recently demonstrated that obesity results in increased expression of PD-1 on T cells, and checkpoint blockade targeting PD-1/PD-L1 surprisingly resulted in greater clinical efficacy in cancer therapy. Adverse events associated with this therapy center around autoimmune reactions. In this study, we examined the impact of obesity on T cell priming and on autoimmune pathogenesis using the mouse model experimental autoimmune encephalomyelitis (EAE), which is mediated by autoreactive myelin-specific T cells generated after immunization. We observed that diet-induced obese (DIO) mice had a markedly delayed EAE onset and developed milder clinical symptoms compared to mice on control diet (CD). This delay was associated with impaired generation of myelin-specific T cell numbers and concurrently correlated with increased PD-L1 upregulation on antigen-presenting cells in secondary lymphoid organs. PD-1 blockade during the priming stage of EAE restored disease onset and severity and increased numbers of pathogenic CD4+ T cells in the central nervous system (CNS) of DIO mice to similar levels to those of CD mice. Administration of anti–PD-1 after onset of clinical symptoms did not increase EAE pathogenesis demonstrating that initial priming is the critical juncture affected by obesity. These findings demonstrate that obesity impairs antigen-specific T cell priming, but this can be reversed with PD-1 blockade. Our results further suggest that PD-1 blockade may increase the risk of autoimmune toxicities, particularly in obesity.

The thymus is the central organ involved with T-cell development and the production of naïve T cells. During normal aging, the thymus undergoes marked involution, reducing naïve T-cell output and resulting in a predominance of long-lived memory T cells in the periphery. Outside of aging, systemic stress responses that induce corticosteroids (CS), or other insults such as radiation exposure, induce thymocyte apoptosis, resulting in a transient acute thymic involution with subsequent recovery occurring after cessation of the stimulus. Despite the increasing utilization of immunostimulatory regimens in cancer, effects on the thymus and naïve T cell output have not been well characterized. Using both mouse and human systems, the thymic effects of systemic immunostimulatory regimens, such as high dose IL-2 (HD IL-2) with or without agonistic anti-CD40 mAbs and acute primary viral infection, were investigated. These regimens produced a marked acute thymic involution in mice, which correlated with elevated serum glucocorticoid levels and a diminishment of naïve T cells in the periphery. This effect was transient and followed with a rapid thymic "rebound" effect, in which an even greater quantity of thymocytes was observed compared to controls. Similar results were observed in humans, as patients receiving HD IL-2 treatment for cancer demonstrated significantly increased cortisol levels, accompanied by decreased peripheral blood naïve T cells and reduced T-cell receptor excision circles (TRECs), a marker indicative of recent thymic emigrants. Mice adrenalectomized prior to receiving immunotherapy or viral infection demonstrated protection from this glucocorticoid-mediated thymic involution, despite experiencing a substantially higher inflammatory cytokine response and increased immunopathology. Investigation into the effects of immunostimulation on middle aged (7-12 months) and advance aged (22-24 months) mice, which had already undergone significant thymic involution and had a diminished naïve T cell population in the periphery at baseline, revealed that even further involution was incurred. Thymic rebound hyperplasia, however, only occurred in young and middle-aged recipients, while advance aged not only lacked this rebound hyperplasia, but were entirely absent of any indication of thymic restoration. This coincided with prolonged deficits in naïve T cell numbers in advanced aged recipients, further skewing the already memory dominant T cell pool. These results demonstrate that, in both mice and humans, systemic immunostimulatory cancer therapies, as well as immune challenges like subacute viral infections, have the potential to induce profound, but transient, glucocorticoid-mediated thymic involution and substantially reduced thymic output, resulting in the reduction of peripheral naive T cells. This can then be followed by a marked rebound effect with naïve T cell restoration, events that were shown not to occur in advanced-aged mice.

Bystander activation of memory T cells occurs via cytokine signaling alone in the absence of T cell receptor (TCR) signaling and provides a means of amplifying T cell effector responses in an antigen-nonspecific manner. While the role of Programmed Cell Death Protein 1 (PD-1) on antigen-specific T cell responses is extensively characterized, its role in bystander T cell responses is less clear. We examined the role of the PD-1 pathway during human and mouse non-antigen-specific memory T cell bystander activation and observed that PD-1+ T cells demonstrated less activation and proliferation than activated PD-1- populations in vitro. Higher activation and proliferative responses were also observed in the PD-1- memory population in both mice and patients with cancer receiving high-dose IL-2, mirroring the in vitro phenotypes. This inhibitory effect of PD-1 could be reversed by PD-1 blockade in vivo or observed using memory T cells from PD-1-/- mice. Interestingly, increased activation through abrogation of PD-1 signaling in bystander-activated T cells also resulted in increased apoptosis due to activation-induced cell death (AICD) and eventual T cell loss in vivo. These results demonstrate that the PD-1/PD-Ligand 1 (PD-L1) pathway inhibited bystander-activated memory T cell responses but also protected cells from AICD.

Stereotactic ablative radiotherapy (SABR) is a standard-of-care for medically-inoperable-early-stage non-small cell lung cancer (NSCLC). One third of patients progress and chemotherapy is rarely used in this population. We questioned if addition of the immune-checkpoint-inhibitor (ICI) atezolizumab to standard-of-care SABR can improve outcomes. We initiated a multi-institutional single-arm phase I study (NCT02599454) enrolling twenty patients with the primary endpoint of maximum tolerated dose (MTD); secondary endpoints of safety and efficacy; and exploratory mechanistic correlatives. Treatment is well tolerated and full dose atezolizumab (1200 mg) is the MTD. Efficacy signals include early responses (after 2 cycles of ICI, before initiation of SABR) in 17% of patients. Biomarkers of functional adaptive immunity, including T cell activation in the tumor and response to ex-vivo stimulation by circulating T cells, are highly predictive of benefit. These results require validation and are being tested in a phase III randomized trial.

IntroductionThe incidence of obesity, a condition characterized by systemic chronic inflammation, has reached pandemic proportions and is a poor prognostic factor in many pathologic states. However, its role on immune parameters has been diverse and at times contradictory. We have previously demonstrated that obesity can result in what has been called the "obesity paradox" which results in increased T cell exhaustion, but also greater efficacy of immune checkpoint blockade in cancer treatment.MethodsThe role of obesity, particularly in the context of aging, has not been robustly explored using preclinical models. We therefore evaluated how age impacts the immune environment on T cell development and function using diet-induced obese (DIO) mice.ResultsWe observed that DIO mice initially displayed greater thymopoiesis but then developed greater thymic involution over time compared to their lean counterparts. Both aging and obesity resulted in increased T cell memory conversion combined with increased expression of T cell exhaustion markers and Treg expansion. This increased T cell immunosuppression with age then resulted in a loss of anti-tumor efficacy by immune checkpoint inhibitors (ICIs) in older DIO mice compared to the younger DIO counterparts.DiscussionThese results suggest that both aging and obesity contribute to T cell dysfunction resulting in increased thymic involution. This combined with increased T cell exhaustion and immunosuppressive parameters affects immunotherapy efficacy reducing the advantage of obesity in cancer immunotherapy responses.

PurposeClinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS.Experimental designUsing fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3-CD56+ NK and CD3+ T cells and CD45- viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression.ResultsSorted intra-tumoral CD3+ T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45- tumor cells. TCGA analysis revealed that tumors with high TLR4 expression (P = 0.03) and low expression of STMN1 involved in microtubule polymerization (P < 0.001) were associated with significantly improved survival.ConclusionsUnlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS.

Natural killer (NK) cells are involved in innate defense against viral infection and cancer. NK cells can be divided into subsets based on the ability of different receptors to bind to major histocompatibility (MHC) class 1 molecules, resulting in differential responses upon activation in a process called "licensing" or "arming." NK cells expressing receptors that bind self-MHC are considered licensed due to an augmented effector lytic function capability compared with unlicensed subsets. However, we demonstrated that unlicensed NK subsets instead positively regulate the adaptive T-cell response during viral infections that are related to localization and cytokine production. In this study, the differential effects of the two types of NK subsets were contingent on the environment in viral infection and hematopoietic stem cell transplantation (HSCT) models. Infection of mice with high-dose (HD) murine cytomegalovirus (MCMC) led to a loss of licensing-associated differences, as compared with mice with low-dose (LD) infection: the unlicensed NK subset no longer localized in lymph nodes (LNs), but instead remained at the site of infection. Similarly, the patterns observed during HD infection paralleled the phenotypes of both human and mouse NK cells in an HSCT setting where NK cells exhibit an activated phenotype. However, in contrast to the effects of subset depletion in T-cell replete models, the licensed NK cell subsets still dominated antiviral responses after HSCT. Overall, our results highlight the intricate tuning of NK cells and how it affects overall immune responses with regard to licensing patterns and their dependency on the level of stimulation and activation status.

Immune checkpoint inhibition (ICI) has emerged as one of the most powerful tools to reverse cancer induced immune suppression. Monoclonal antibodies (mAbs) targeting programmed cell death 1/programmed cell death ligand 1(PD-1/PD-L1) are FDA-approved and their clinical use is rapidly expanding. As opposed to the clinical paradigm, which can result in significant responses and toxicities, it has been difficult to reproduce these effects preclinically using mouse models. In large part, this is due to models, which employ rapidly growing ex vivo cultured transplantable tumor cell lines engrafted into young naïve inbred laboratory mice. However, another issue concerns the use and repeated application of xenogeneic reagents in mice (i.e., rat or hamster mAbs directed against mouse antigens at variance with clinical use of human or humanized mAbs). Building on our previous studies demonstrating that repeated administration of commonly used xenogeneic anti-PD-1 mAbs derived from both rat and hamster can induce fatal hypersensitivity in some tumor-bearing mice, we sought to compare these result with the effects of a mouse anti-mouse PD-1 mAb. Application of a murine anti-mouse PD-1 (clone: MuDX400) did not result in lethal anaphylaxis in the 4T1 tumor model. It also displayed superior antitumor effects in this and other tumor models, as it did not induce neutralizing antibody responses against the anti-PD-1 mAb, such as were observed when using xenogeneic anti-PD1 mAbs. These results demonstrate that more accurate preclinical modeling necessitates the use of mouse reagents mirroring the clinical scenario to ascertain long-term effects or toxicities, while avoiding xenogeneic responses, which do not occur clinically. Furthermore, these studies suggest a direct mechanism, whereby preclinical murine studies have often failed to recapitulate the clinical efficacy and toxicity of single agent checkpoint inhibition.

The efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by the occurrence of acute and chronic graft-versus-host disease (GVHD). We have recently demonstrated that obesity results in exacerbated acute gastrointestinal GVHD in both mouse models and clinical outcomes due to increased pro-inflammatory cytokine responses and microbiota alterations. We therefore wanted to delineate the role of the various parameters in obesity, adiposity, effects of high-fat (HF) diet, and the role of microbiome on GVHD pathogenesis, by taking advantage of a mouse strain resistant to diet-induced obesity (DIO). Female BALB/c mice are resistant to DIO phenotype with approximately 50% becoming DIO under HF diets. The DIO-susceptible recipients rapidly succumb to acute gut GVHD, whereas the DIO-resistant recipient littermates, which do not become obese, are partially protected from GVHD, indicating that being on HF diet alone contributes to but is not the primary driver of GVHD. Microbiome assessment revealed restricted diversity in both cohorts of mice, but coprophagy normalizes the microbiota in mice housed together. We then individually housed DIO-resistant, DIO-susceptible, and lean control mice. Notably, each of the individually housed groups demonstrates marked restricted diversity that has been shown to occur from the stress of single housing. Despite the restricted microbiome diversity, the GVHD pathogenesis profile remains consistent in the group-housed mice, with the lean control single-housed mice exhibiting no acute GVHD and DIO-resistant recipients showing again partial protection. These results demonstrate that the deleterious effects of obesity on acute gut GVHD are critically dependent on adiposity with the HF diet also playing a lesser role, and the microbiome alterations with obesity instead appear to fuel ongoing acute GVHD processes.

PurposeGiven the unmet need for novel immunotherapy in soft tissue sarcoma (STS), we sought to characterize the phenotype and function of intratumoral natural killer (NK) and T cells to identify novel strategies to augment tumor-infiltrating lymphocyte (TIL) function.Experimental designUsing prospectively collected specimens from dogs and humans with sarcomas, archived specimens, and The Cancer Genome Atlas (TCGA) data, we evaluated blood and tumor NK and T cell phenotype and function and correlated those with outcome. We then assessed the effects of interleukin 15 (IL-15) stimulation on both NK and T cell activation and TIGIT upregulation. Finally, we evaluated cytotoxic effects of IL-15 combined with TIGIT blockade using a novel anti-TIGIT antibody.ResultsTILs were strongly associated with survival outcome in both archived tissue and TCGA, but higher TIL content was also associated with higher TIGIT expression. Compared with blood, intratumoral NK and T cells showed significantly higher expression of both activation and exhaustion markers, in particular TIGIT. Ex vivo stimulation of blood and tumor NK and T cells from patients with STS with IL-15 further increased both activation and exhaustion markers, including TIGIT. Dogs with metastatic osteosarcoma receiving inhaled IL-15 also exhibited upregulation of activation markers and TIGIT. Ex vivo, combined IL-15 and TIGIT blockade using STS blood and tumor specimens significantly increased cytotoxicity against STS targets.ConclusionIntratumoral NK and T cells are prognostic in STS, but their activation is marked by significant upregulation of TIGIT. Our data suggest that combined IL-15 and TIGIT blockade may be a promising clinical strategy in STS.

Despite obesity reaching pandemic proportions, its impact on antigen-specific T cell responses is still unclear. We have recently demonstrated that obesity results in increased expression of PD-1 on T cells, and checkpoint blockade targeting PD-1/PD-L1 surprisingly resulted in greater clinical efficacy in cancer therapy. Adverse events associated with this therapy center around autoimmune reactions. In this study, we examined the impact of obesity on T cell priming and on autoimmune pathogenesis using the mouse model experimental autoimmune encephalomyelitis (EAE), which is mediated by autoreactive myelin-specific T cells generated after immunization. We observed that diet-induced obese (DIO) mice had a markedly delayed EAE onset and developed milder clinical symptoms compared to mice on control diet (CD). This delay was associated with impaired generation of myelin-specific T cell numbers and concurrently correlated with increased PD-L1 upregulation on antigen-presenting cells in secondary lymphoid organs. PD-1 blockade during the priming stage of EAE restored disease onset and severity and increased numbers of pathogenic CD4+ T cells in the central nervous system (CNS) of DIO mice to similar levels to those of CD mice. Administration of anti-PD-1 after onset of clinical symptoms did not increase EAE pathogenesis demonstrating that initial priming is the critical juncture affected by obesity. These findings demonstrate that obesity impairs antigen-specific T cell priming, but this can be reversed with PD-1 blockade. Our results further suggest that PD-1 blockade may increase the risk of autoimmune toxicities, particularly in obesity.

The thymus is the central organ involved with T-cell development and the production of naïve T cells. During normal aging, the thymus undergoes marked involution, reducing naïve T-cell output and resulting in a predominance of longlived memory T cells in the periphery. Outside of aging, systemic stress responses that induce corticosteroids (CS), or other insults such as radiation exposure, induce thymocyte apoptosis, resulting in a transient acute thymic involution with subsequent recovery occurring after cessation of the stimulus. Despite the increasing utilization of immunostimulatory regimens in cancer, effects on the thymus and naïve T cell output have not been well characterized. Using both mouse and human systems, the thymic effects of systemic immunostimulatory regimens, such as high dose IL-2 (HD IL-2) with or without agonistic anti-CD40 mAbs and acute primary viral infection, were investigated. These regimens produced a marked acute thymic involution in mice, which correlated with elevated serum glucocorticoid levels and a diminishment of naïve T cells in the periphery. This effect was transient and followed with a rapid thymic "rebound" effect, in which an even greater quantity of thymocytes was observed compared to controls. Similar results were observed in humans, as patients receiving HD IL-2 treatment for cancer demonstrated significantly increased cortisol levels, accompanied by decreased peripheral blood naïve T cells and reduced T-cell receptor excision circles (TRECs), a marker indicative of recent thymic emigrants. Mice adrenalectomized prior to receiving immunotherapy or viral infection demonstrated protection from this glucocorticoid-mediated thymic involution, despite experiencing a substantially higher inflammatory cytokine response and increased immunopathology. Investigation into the effects of immunostimulation on middle aged (7-12 months) and advance aged (22-24 months) mice, which had already undergone significant thymic involution and had a diminished naïve T cell population in the periphery at baseline, revealed that even further involution was incurred. Thymic rebound hyperplasia, however, only occurred in young and middle-aged recipients, while advance aged not only lacked this rebound hyperplasia, but were entirely absent of any indication of thymic restoration. This coincided with prolonged deficits in naïve T cell numbers in advanced aged recipients, further skewing the already memory dominant T cell pool. These results demonstrate that, in both mice and humans, systemic immunostimulatory cancer therapies, as well as immune challenges like subacute viral infections, have the potential to induce profound, but transient, glucocorticoid-mediated thymic involution and substantially reduced thymic output, resulting in the reduction of peripheral naive T cells. This can then be followed by a marked rebound effect with naïve T cell restoration, events that were shown not to occur in advanced-aged mice. [ABSTRACT FROM AUTHOR]

Declining soil fertility is a major threat to agricultural productivity and livelihoods in sub-Saharan Africa, particularly in Niger, where most farmers have few resources and depend on degraded lands. Large-scale adoption of soil fertility management technologies (SFMT) by small-scale farmers is among the proposed solutions, but this remains low because of various constraints. A better understanding of the socio-economic factors that influence farming practices is needed to adapt SFMT to farmers' use. A survey using semi-structured interviews with 101 household heads was conducted in 2013 in Karabedji, Niger. The influence of household socio-economic factors on soil fertility management practices (SFMP) was assessed through farmers that were involved in demonstration activities as part of a long-term research project (13 years) on the restoration of soil fertility on farmers' fields. The results showed that the farmer's household size was between 9 and 14 people and that the majority of farmers did not attain formal education. Fifty percent (50%) of the non-participating farmers and 69% of the participants of the demonstration were affiliated with farmers' organizations. The proportion of demonstration participants and non-participants having an average farm income of less than 300,000 FCFA (about US $ 600) was 70 to 75%. Logistic regression analysis showed a significant positive effect (P Key words: Livelihood, Farmer-Association, Resource-Endowment, Pearl Millet, Fertilizer, Soil fertility, sub-Saharan Africa La baisse de la fertilite des sols est une menace majeure pour la productivite agricole et les moyens de subsistance en Afrique subsaharienne, en particulier au Niger, ou la plupart des agriculteurs disposent de peu de ressources et dependent de terres degradees. L'adoption a grande echelle de technologies de gestion de la fertilite des sols (SFMT) par les petits agriculteurs fait partie des solutions proposees, mais reste faible en raison de diverses contraintes. Une meilleure comprehension des facteurs socio-economiques qui influencent les pratiques agricoles est necessaire pour adapter SFMT a l'utilisation par les agriculteurs. Une enquete utilisant des entretiens semi-structures avec 101 chefs de menage a ete menee en 2013 a Karabedji, au Niger. L'influence des facteurs socioeconomiques des menages sur les pratiques de gestion de la fertilite des sols (SFMP) a ete evaluee a travers des agriculteurs participant a des activites de demonstration dans le cadre d'un projet de recherche a long terme (13 ans) sur la restauration de la fertilite des sols dans les champs des agriculteurs. Les resultats ont montre que le menage de l'agriculteur comprenait entre 9 et 14 personnes et que la majorite des agriculteurs n'assistaient pas a l'education formelle. Cinquante pour cent (50%) des agriculteurs non participants et 69% des participants a la demonstration etaient affilies a des organisations d'agriculteurs. Les participants a la demonstration et les non-participants ayant un revenu agricole moyen inferieur a 300 000 FCFA (environ 600 USD) representaient entre 70 et 75%. Une analyse de regression logistique a montre un effet positif significatif (P Mots-cles: Subsistance, Organisation paysanne, Ressources, Mil, Engrais, Fertilite sol, Afrique Subsaharienne, INTRODUCTION Soil fertility decline is a major concern in sub-Saharan Africa, particularly in Niger where agricultural activities generally depend on rainfall [1-4]. The rainfall pattern is unimodal and spread over [...]

Highlights A new and compact device configuration was created with two interpenetrated, individually addressable electrodes, allowing precise control over the geometric features and interactions between the electrodes. The interpenetrated electrode design improves ion diffusion kinetics in electrochemical energy storage devices by shortening the ion diffusion length and reducing ion concentration inhomogeneity. The device with interpenetrated electrodes outperformed the traditional separate electrode configuration, enhancing both volumetric energy density and capacity retention rate.

Background Previous studies have examined social media habits and utilization patterns among various groups of healthcare professionals. However, very few studies have evaluated the use of social media to support continuing professional development activities. The goal of the 2023 Clinical Education Alliance social media survey was to explore how HCPs interact professionally with social media, describe utilization trends, and identify barriers to using social media to disseminate CPD content.Methods We conducted an online anonymous, voluntary survey of healthcare professionals contained in the Clinical Education Alliance learner database from January to March 2023. The survey was distributed via email and all learners were invited to participate regardless of profession or specialty. This survey consisted of 16 questions and collected demographic information and social media utilization and habits of healthcare professionals.Results Of the 2,615 healthcare professionals who completed the survey, 71.2% use social media. Most respondents were physicians (50.6%) practicing in an urban setting (59.6%) and have been practicing for more than 15 years (70.5%). The most widely used platform was Facebook (70.7%), but there were no significant differences among the different professions. Of the respondents who use social media, 44.5% used social media to access continuing professional development-certified activities. Surveyed learners preferred passive participation with social media content. Participant-reported concerns include issues with legitimacy of the information, privacy, time constraints, and institutional barriers.Discussion As the continuing professional development community continues to evolve and seek new innovative strategies to reach healthcare professionals, the findings of this survey highlight the need to identify and enact social media-based strategies aimed to engage healthcare professionals and provide them with unbiased evidence-based education.

Fourth and fifth grade boys who were or were not rejected by peers reported how they would handle conflictual interactions described in vignettes. Rejected boys who were aggressive or residual, that is, neither aggressive nor submissive, exhibited less integration of personal and relational goals than did nonrejected boys. (BC)