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Purpose: A phase Ib/II clinical trial was conducted to evaluate the safety and efficacy of the combination of all-trans retinoic acid (ATRA) with pembrolizumab in patients with stage IV melanoma. Patients and Methods: Anti–PD-1 naïve patients with stage IV melanoma were treated with pembrolizumab plus supplemental ATRA for three days surrounding each of the first four pembrolizumab infusions. The primary objective was to establish the MTD and recommended phase II dose (RP2D) of the combination. The secondary objectives were to describe the safety and toxicity of the combined treatment and to assess antitumor activity in terms of (i) the reduction in circulating myeloid-derived suppressor cell (MDSC) frequency and (ii) progression-free survival (PFS). Results: Twenty-four patients were enrolled, 46% diagnosed with M1a and 29% with M1c stage disease at enrollment. All patients had an ECOG status ≤1, and 75% had received no prior therapies. The combination was well tolerated, with the most common ATRA-related adverse events being headache, fatigue, and nausea. The RP2D was established at 150 mg/m2 ATRA + 200 mg Q3W pembrolizumab. Median PFS was 20.3 months, and the overall response rate was 71%, with 50% of patients experiencing a complete response, and the 1-year overall survival was 80%. The combination effectively lowered the frequency of circulating MDSCs. Conclusions: With a favorable tolerability and high response rate, this combination is a promising frontline treatment strategy for advanced melanoma. Targeting MDSCs remains an attractive mechanism to enhance the efficacy of immunotherapies, and this combination merits further investigation. See related commentary by Olson and Luke, p. 1167

Purpose:A phase Ib/II clinical trial was conducted to evaluate the safety and efficacy of the combination of all-trans retinoic acid (ATRA) with pembrolizumab in patients with stage IV melanoma.Patients and Methods:Anti–PD-1 naïve patients with stage IV melanoma were treated with pembrolizumab plus supplemental ATRA for three days surrounding each of the first four pembrolizumab infusions. The primary objective was to establish the MTD and recommended phase II dose (RP2D) of the combination. The secondary objectives were to describe the safety and toxicity of the combined treatment and to assess antitumor activity in terms of (i) the reduction in circulating myeloid-derived suppressor cell (MDSC) frequency and (ii) progression-free survival (PFS).Results:Twenty-four patients were enrolled, 46% diagnosed with M1a and 29% with M1c stage disease at enrollment. All patients had an ECOG status ≤1, and 75% had received no prior therapies. The combination was well tolerated, with the most common ATRA-related adverse events being headache, fatigue, and nausea. The RP2D was established at 150 mg/m2 ATRA + 200 mg Q3W pembrolizumab. Median PFS was 20.3 months, and the overall response rate was 71%, with 50% of patients experiencing a complete response, and the 1-year overall survival was 80%. The combination effectively lowered the frequency of circulating MDSCs.Conclusions:With a favorable tolerability and high response rate, this combination is a promising frontline treatment strategy for advanced melanoma. Targeting MDSCs remains an attractive mechanism to enhance the efficacy of immunotherapies, and this combination merits further investigation.See related commentary by Olson and Luke, p. 1167

Characteristics of patients experiencing clinical benefit

Supplementary figure 1. (A) Changes in other circulating cytokines. Colored box denotes the time when the patients were being treated with ATRA. (B) example gating strategy for CD8+ T cell activation.

Representativeness of study participants

All adverse events experienced by patients, including those unrelated to treatment

Trial inclusion/exclusion criteria

Establishing recommended phase II dose (RP2D) and dose limiting toxicities (DLTs)

Background:Adrenal gland metastases (AGMs) are common in advanced-stage melanoma, occurring in up to 50% of patients. The introduction of immune checkpoint inhibitors (ICIs) has markedly altered the outcome of patients with melanoma. However, despite significant successes, anecdotal evidence has suggested that treatment responses in AGMs are significantly lower than in other metastatic sites. We sought to investigate whether having an AGM is associated with altered outcomes and whether ICI responses are dampened in the adrenal glands.Patients and Methods:We retrospectively compared ICI responses and overall survival (OS) in 68 patients with melanoma who were diagnosed with an AGM and a control group of 100 patients without AGMs at a single institution. Response was determined using RECIST 1.1. OS was calculated from time of ICI initiation, anti–PD-1 initiation, initial melanoma diagnosis, and stage IV disease diagnosis. Tumor-infiltrating immune cells were characterized in 9 resected AGMs using immunohistochemical analysis.Results:Response rates of AGMs were significantly lower compared with other metastatic sites in patients with AGMs (16% vs 22%) and compared with those without AGMs (55%). Patients with AGMs also had significantly lower median OS compared with those without AGMs (3.1 years vs not reached, respectively). We further observed that despite this, AGMs exhibited high levels of tumor-infiltrating immune cells.Conclusions:In this cohort of patients with melanoma, those diagnosed with an AGM had lower ICI response rates and OS. These results suggest that tissue-specific microenvironments of AGMs present unique challenges that may require novel, adrenal gland–directed therapies or surgical resection.

Most human killer cell immunoglobulin-like receptors (KIR) are expressed by Natural Killer (NK) cells and recognize HLA class I molecules as ligands. Uniquely, KIR3DL3 is a conserved but polymorphic inhibitory KIR recognizing a B7 family ligand, HHLA2, and is implicated for immune checkpoint targeting. Because the expression profile and biological function of KIR3DL3 remained elusive, we searched extensively for KIR3DL3 transcripts, revealing expression is highly enriched in γδ and CD8+ T cells rather than NK cells. These KIR3DL3 expressing cells are rare in the blood and thymus, but more common in the lungs and digestive tract. High resolution flow cytometry and single cell transcriptomics showed that peripheral blood KIR3DL3+ T cells have an activated transitional memory phenotype and are hypofunctional. The TCR usage is biased towards genes from early rearranged TCR-α variable segments or Vδ1 chains. Whereas we detected no impact of KIR3DL3 polymorphism on ligand binding, variants in the proximal promoter and at residue 86 can reduce expression. Together, we demonstrate that KIR3DL3 is upregulated in response to unconventional T cell stimulation and that individuals may vary in their ability to express KIR3DL3. These results have implications for the personalized targeting of KIR3DL3/HHLA2 checkpoint inhibition.

There is increasing evidence that oxidative metabolism and fatty acids play an important role in BRAF-driven tumorigenesis, yet the effect of BRAF mutation and expression on metabolism is poorly understood. We examined how BRAF mutation and expression modulates metabolite abundance. Using the non-transformed NIH3T3 cell line, we generated cells that stably overexpressed BRAF V600E or BRAF WT. We found that cells expressing BRAF V600E were enriched with immunomodulatory lipids. Further, we found a unique transcriptional signature that was exclusive to BRAF V600E expression. We also report that BRAF V600E mutation promoted accumulation of long chain polyunsaturated fatty acids (PUFAs) and rewired metabolic flux for non-Warburg behavior. This cancer promoting mutation further induced the formation of tunneling nanotube (TNT)-like protrusions in NIH3T3 cells that preferentially accumulated lipid droplets. In the plasma of melanoma patients harboring the BRAF V600E mutation, levels of lysophosphatidic acid, sphingomyelin, and long chain fatty acids were significantly increased in the cohort of patients that did not respond to BRAF inhibitor therapy. Our findings show BRAF V600 status plays an important role in regulating immunomodulatory lipid profiles and lipid trafficking, which may inform future therapy across cancers.

Although the consequences of splenectomy are well understood in mice, much less is known about the immunologic changes that occur following splenectomy in humans. We sought to characterize the circulating immune cell populations of patients before and after elective splenectomy to determine if these changes are related to postsplenectomy survival outcomes. Retrospective clinical information was collected from 95 patients undergoing elective splenectomy compared with 91 patients undergoing pancreaticoduodenectomy (Whipple procedure). We further analyzed peripheral blood from five patients in the splenectomy group, collected before and after surgery, using single-cell cytometry by time-of-flight mass spectrometry. We compared pre- and postsplenectomy data to characterize both the major and minor immune cell populations in significantly greater detail. Compared with patients undergoing a Whipple procedure, splenectomized patients had significant and long-lasting elevated counts of lymphocytes, monocytes, and basophils. Cytometry by time-of-flight mass spectroscopy analysis demonstrated that the elevated lymphocytes primarily consisted of naive CD4+ T cells and a population of activated CD25+CD56+CD4+ T cells, whereas the elevated monocyte counts were mainly mature, activated monocytes. We also observed a significant increase in the expression of the chemokine receptors CCR6 and CCR4 on several cellular populations. Taken together, these data indicate that significant immunological changes take place following splenectomy. Whereas other groups have compared splenectomized patients to healthy controls, this study compared patients undergoing elective splenectomy to those undergoing a similar major abdominal surgery. Overall, we found that splenectomy results in significant long-lasting changes in circulating immune cell populations and function.

Objectives While much of the research concerning factors associated with responses to immune checkpoint inhibitors (ICIs) has focussed on the contributions of conventional peptide‐specific T cells, the role of unconventional T cells, such as mucosal‐associated invariant T (MAIT) cells, in human melanoma remains largely unknown. MAIT cells are an abundant population of innate‐like T cells expressing a semi‐invariant T‐cell receptor restricted to the MHC class I‐like molecule, MR1, presenting vitamin B metabolites derived from bacteria. We sought to characterise MAIT cells in melanoma patients and determined their association with treatment responses and clinical outcomes. Methods In this prospective clinical study, we analysed the frequency and functional profile of circulating and tumor‐infiltrating MAIT cells in human melanoma patients. Using flow cytometry, we compared these across metastatic sites and between ICI responders vs. non‐responders as well as healthy donors. Results We identified tumor‐infiltrating MAIT cells in melanomas across metastatic sites and found that the number of circulating MAIT cells is reduced in melanoma patients compared to healthy donors. However, circulating MAIT cell frequencies are restored by ICI treatment in responding patients, correlating with treatment responses, in which patients with high frequencies of MAIT cells exhibited significantly improved overall survival. Conclusion Our results suggest that MAIT cells may be a potential predictive marker of responses to immunotherapies and provide rationale for testing MAIT cell‐directed therapies in combination with current and next‐generation ICIs., While the number of circulating MAIT cells is reduced in melanoma patients, circulating MAIT cell frequency is restored by immune checkpoint inhibitor treatment in responding patients, correlating with treatment responses, in which patients with high frequencies of MAIT cells exhibited significantly improved overall survival.

Myeloid-derived suppressor cells (MDSCs) are potent suppressors of antitumor immunity and are commonly associated with poor outcomes in melanoma patients treated with immune checkpoint inhibitors. Inducing the differentiation of MDSCs using all-trans retinoic acid (ATRA) alters their activity and reduces MDSC frequency. This trial seeks to assess the safety and efficacy of combining ATRA and pembrolizumab in metastatic melanoma patients. In 24 stage IV melanoma patients, treatment with pembrolizumab Q3W plus the supplemental treatment of ATRA orally for three days surrounding each of the first four pembrolizumab infusions effectively lowered the frequency of circulating PMN-MDSCs and enhanced melanoma-specific T cell activity. The combination was well tolerated. Median progression free survival was 20.3 months, and the overall response rate was 71%, with 50% of patients experiencing a complete response. Targeting MDSCs remains a promising mechanism to enhance the efficacy of anti-PD-1 therapies and this combination merits further investigation.

Uveal melanoma (UM) is a subtype of melanoma. Although they share a melanocytic origin with cutaneous melanoma (CM), patients with UM have few treatment options. BCL2 homologous 3 mimetics are small-molecule drugs that mimic proapoptotic BCL2 family members. We compared BCL2 family member expression between UM and CM using immunoblot and The Cancer Genome Atlas transcriptomic analysis. UM has a unique signature of low BFL1 and high PUMA proteins compared with CM and 30 other cancer types, making them an attractive candidate for BCL2 homologous 3 protein mimetics. We tested the efficacy of a BCL2 inhibitor and MCL1 inhibitor (MCL1i) in UM, with viability assays, live-cell imaging, sphere assays, and mouse xenograft models. UM had a higher sensitivity to MCL1i than CM. Overexpression of BFL1 or knockdown of PUMA made the UM more resistant to MCL1i. In contrast, MAPK/extracellular signal‒regulated kinase inhibitor treatment in CM made them more sensitive to MCL1i. However, MCL1i-alone treatment was not very effective to reduce the UM initiating cells; to overcome this, we employed a combination of MCL1i with BCL2 inhibitor that synergistically inhibited UM initiating cell's capacity to expand. Overall, we identify a distinct expression profile of BCL2 family members for UM that makes them susceptible to BCL2 homologous 3 mimetics.

While much of the research concerning factors associated with responses to immunotherapies focuses on the contributions of conventional peptide-specific T cells, the role of unconventional T cells, such as mucosalassociated invariant T (MAIT) cells, in human melanoma remains largely unknown. MAIT cells are innate-like T cells expressing a semi-invariant T cell receptor restricted to the non-classical MHC class I molecule MR1 presenting vitamin metabolites derived from bacteria. In this prospective clinical study, we sought to characterize MAIT cells in melanoma patients and determine their association with clinical outcomes. We identified tumor-infiltrating MAIT cells in melanomas across metastatic sites and found that the number of circulating MAIT cells is reduced in melanoma patients. However, circulating MAIT cell frequency is restored by anti-PD1 treatment in responding patients, correlating with treatment responses in which patients with high frequencies of MAIT cells exhibited improved overall survival. These data provide evidence for leveraging MAIT cells and their functions as novel targets for future therapies.

There is an urgent need to develop treatments for patients with melanoma who are refractory to or ineligible for immune checkpoint blockade, including patients who lack BRAF-V600E/K mutations. This is often the case in patients diagnosed with rare melanoma subtypes such as mucosal and acral melanoma. Here, we analyzed data from the cutaneous melanoma The Cancer Genome Atlas Network (TCGA) transcriptomic and proteomic databases for differential expression of apoptosis molecules between melanomas with or without BRAF hotspot mutations. Our data indicated higher B-cell CLL/lymphoma 2 (BCL2) expression in melanoma without BRAF hotspot mutations, suggesting that BH3 mimetics, such as ABT-199 (venetoclax, a small molecule against BCL2), may be a potential therapeutic option for these patients. We explored the efficacy of combining two BH3 mimetics, ABT-199 and a myeloid cell leukemia sequence 1 (MCL1) inhibitor (S63845 or S64315/MIK665) in cutaneous, mucosal and acral melanomas, in vitro and in vivo. Our data indicate this combination induced cell death in a broad range of melanoma cell lines, including melanoma initiating cell populations, and was more potent in melanoma cells without BRAF-V600E/K mutations. Our knockdown/knockout experiments suggest that several pro-apoptotic BCL2 family members, BCL2-like 11 (apoptosis facilitator) (BIM), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) or BID, play a role in the combination-induced effects. Overall, our study supports the rationale for combining an MCL1 inhibitor with a BCL2 inhibitor as a therapeutic option in patients with advanced melanoma.

Oncogenic BRAF fusions have emerged as an alternate mechanism for BRAF activation in melanomas and other cancers. A number of BRAF fusions with different 5' gene partners and BRAF exon breakpoints have been described, but the effects of different partners and breakpoints on cancer phenotypes and treatment responses has not been well characterized. Targeted RNA sequencing was used to screen 60 melanoma patient-derived xenograft (PDX) models for BRAF fusions. We identified three unique BRAF fusions, including a novel SEPT3-BRAF fusion, occurring in four tumors (4/60, 6.7%), all of which were "pan-negative" (lacking other common mutations) (4/18, 22.2%). The BRAF fusion PDX models showed variable growth rates and responses to MAPK inhibitors in vivo. Overexpression of BRAF fusions identified in our study, as well as other BRAF fusions previously identified in melanomas, resulted in a high degree of variability in 2D proliferation and 3D invasion between the different fusions. While exogenously expressed BRAF fusions all responded to MAPK inhibition in vitro, we observed potential differences in signaling and feedback mechanisms. In summary, BRAF fusions are actionable therapeutic targets, however there are significant differences in phenotypes, treatment responses, and signaling which may be clinically relevant.

Toll-like receptor 4 (TLR4) activation contributes to vascular dysfunction in pathological conditions such as hypertension and diabetes, but the role of chronic TLR4 activation on renal autoregulatory behavior is unknown. We hypothesized that subclinical TLR4 stimulation with low-dose lipopolysaccharide (LPS) infusion increases TLR4 activation and blunts renal autoregulatory behavior. We assessed afferent arteriolar autoregulatory behavior in male Sprague-Dawley rats after prolonged LPS (0.1 mg·kg−1·day−1 sq) infusion via osmotic minipump for 8 or 14 days. Some rats also received daily cotreatment with either anti-TLR4 antibody (1 μg ip), competitive antagonist peptide (CAP; 3 mg/kg ip) or tempol (2 mmol/l, drinking water) throughout the 8-day LPS treatment period. Autoregulatory behavior was assessed using the in vitro blood-perfused juxtamedullary nephron preparation. Selected physiological measures, systolic blood pressure and baseline diameters were normal and similar across groups. Pressure-dependent vasoconstriction averaged 72 ± 2% of baseline in sham rats, indicating intact autoregulatory behavior. Eight-day LPS-treated rats exhibited significantly impaired pressure-mediated vasoconstriction (96 ± 1% of baseline), whereas it was preserved in rats that received anti-TLR4 antibody (75 ± 3%), CAP (84 ± 2%), or tempol (82 ± 2%). Using a 14-day LPS (0.1 mg·kg−1·day−1 sq) intervention protocol, CAP treatment started on day 7, where autoregulatory behavior is already impaired. Systolic blood pressures were normal across all treatment groups. Fourteen-day LPS treatment retained the autoregulatory impairment (95 ± 2% of baseline). CAP intervention starting on day 7 rescued pressure-mediated vasoconstriction with diameters decreasing to 85 ± 1% of baseline. These data demonstrate that chronic subclinical TLR4 activation impairs afferent arteriolar autoregulatory behavior through mechanisms involving reactive oxygen species and major histocompatibility complex class II activation.

9536 Background: Myeloid-derived suppressor cells (MDSCs) are potent suppressors of antitumor immunity and are commonly associated with poor outcomes in melanoma patients treated with immune checkpoint inhibitors. Inducing the differentiation of MDSCs using all-trans retinoic acid (ATRA) reduces MDSC frequency. This analysis seeks to assess the safety and efficacy of combining ATRA and pembrolizumab in advanced melanoma patients. Methods: This single arm, single institution, phase I/II study (NCT03200847) enrolled 24 patients diagnosed with stage IV melanoma. Eligible patients were over the age of 18 and had not been previously treated anti-PD-1 therapy. Treatment consisted of 200mg Q3W pembrolizumab plus the supplemental treatment of 150 mg/m2 ATRA orally for 3 days surrounding each of the first four infusions of pembrolizumab, with patients continuing pembrolizumab for up to two years until confirmed disease progression or unacceptable toxicity. The primary endpoints were safety and reduction in circulating MDSCs. Secondary endpoints were overall response rate (ORR), disease control rate (DCR), progression free survival (PFS) according to RECIST v1.1. Results: At data cut off (Feb, 2021) 22 patients were evaluable for tumor response. Median follow-up was 1.0 years (0.3-2 years). In general, the combination of pembrolizumab and ATRA was well tolerated. The most common treatment-related adverse events (AEs) were grade 1 or 2, including headache (22 pts, 92%), fatigue (18 pts, 75%), rash (16 pts, 66%), and nausea (8 pts, 33%), most of which corresponded with the 3-day course of ATRA treatment. Ten patients had grade 3 or higher AEs with most being common ICI-related AEs. The ORR was 60% and DCR was 83%. Six-month PFS rate was 62%. Excluding patients diagnosed with uveal melanoma (n = 2) the ORR was 72%, DCR was 86%, and the six-month PFS rate was 68%. Paired analysis showed sustained decreases in absolute numbers ( p = 0.002) and percentage ( p = 0.007) of circulating MDSCs (CD3-CD19-CD56-CD11b+CD33+HLA-DR-/low) 4-6 weeks after stopping ATRA. The study is ongoing and further data will be presented in the future. Conclusions: This study demonstrates that the combination of ATRA and pembrolizumab is well tolerated and suggests that reducing MDSCs with ATRA may enhance the efficacy of pembrolizumab. This strategy of targeting MDSCs in combination with pembrolizumab warrants further development. Research Funding: Merck. Clinical trial information: NCT03200847.

Cutaneous melanoma (CM) has one of the highest response rates across cancers to immune checkpoint blockade (ICB) therapies, but mucosal melanoma (MM) responds poorly to ICB. MM patients have very few effective treatments options, making it critical to understand and improve anti-tumor immunity in these patients. MM is a rare subtype of melanoma, accounting for approximately 1% of all malignant melanoma diagnoses. MM occurs on mucosal surfaces such as nasal, vaginal, and anorectal, which provide a highly unique tumor microenvironment compared to CM arising in the skin. We compared the tumor immune microenvironment between CM and MM by multiplex immunofluorescence and discovered MM has decreased frequency of infiltrating immune cells. Using RNA sequencing, we investigated intra-tumoral differences in global gene expression between CM and MM that might explain the lack of immune cell infiltration and ICB response in MM. Compared to CM, MM had decreased expression of numerous innate immune genes in the RIG-I pathogen-sensing pathway. The RIG-I pathway produces an inflammatory response to destroy foreign pathogens, but is also important for ICB sensitivity and anti-tumor immunity in CM. Studies in CM demonstrate the RIG-I pathway enhances antigen presentation and promotes a favorable immune cell profile in the tumor microenvironment. Interestingly, the tumor microbiome, which is vastly different between CM and MM, has been shown to regulate the RIG-I pathway. We measured total fungal and bacterial load in MM and CM primary tumors and found increased levels of both bacteria and fungi in MM compared to CM. This raises the possibility that microbiome-mediated suppression of the RIG-I pathway underlies poor anti-tumor immunity and ICB response in MM, and that re-activation of this pathway may be a novel therapeutic strategy for overcoming ICB resistance in MM patients. Direct RIG-I agonists are still in clinical development, but FDA-approved hypomethylating agents, particularly 5’ aza-deoxycytidine (decitabine), can activate the RIG-I pathway and improve anti-tumor immunity by increasing the expression of RIG-I pathway activating genes, endogenous RNA retroviruses, tumor antigens, and natural killer (NK) cell ligands. We treated MM cell lines with decitabine and observed strong induction of RIG-I pathway genes, NK ligands, and tumor antigens. In conclusion, we have identified the loss of intra-tumoral RIG-I signaling as a potential microbiome-mediated mechanism underlying the poor tumor immune microenvironment and ICB response in MM. Combining decitabine, which is FDA-approved and now available orally, with ICB represents a novel treatment strategy for these difficult to treat MM patients that can be rapidly translated into the clinic. Citation Format: Morgan MacBeth, Richard Tobin, Robert Van Gulick, Martin D. McCarter, William A. Robinson, Kasey L. Couts. Loss of intra-tumoral RIG-I immune signaling is a potential microbiome-mediated mechanism underlying poor anti-tumor immunity and immunotherapy resistance in mucosal melanoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO048.

This study aimed to establish mechanistic links between the aging-associated changes in the functional status of mast cells and the altered responses of mesenteric tissue and mesenteric lymphatic vessels (MLVs) to acute inflammation. We used an in vivo model of acute peritoneal inflammation induced by lipopolysaccharide treatment of adult (9-month) and aged (24-month) F-344 rats. We analyzed contractility of isolated MLVs, mast cell activation, activation of nuclear factor-κB (NF-κB) without and with stabilization of mast cells by cromolyn or blockade of all types of histamine receptors and production of 27 major pro-inflammatory cytokines in adult and aged perilymphatic mesenteric tissues and blood. We found that the reactivity of aged contracting lymphatic vessels to LPS-induced acute inflammation was abolished and that activated mast cells trigger NF-κB signaling in the mesentery through release of histamine. The aging-associated basal activation of mesenteric mast cells limits acute inflammatory NF-κB activation in aged mesentery. We conclude that proper functioning of the mast cell/histamine/NF-κB axis is necessary for reactions of the lymphatic vessels to acute inflammatory stimuli as well as for interaction and trafficking of immune cells near and within the collecting lymphatics.

Objective: Cinobufotalin (CINO), a cardiotonic steroid (CTS) or bufadienolide, is extracted from the skin secretions of giant toads and is utilized in traditional Chinese medicine (Chan Su). CINO has been used as a cardiotonic, diuretic and a hemostatic agent. Recently, CINO has been shown to inhibit lung cancer cell function and has been implicated in several other disease processes. In this study, we pursued the potential anticancer application of CINO using the ovarian tumor cell line SK-OV-3. Study Design: We evaluated the effect of CINO on cultures of SK-OV-3. Cells were treated with 0.1, 0.5, 1, 5, and 10 μM CINO. Cell proliferation, migration, invasion, and viability were measured using commercially available kits. Cell cycle progression was evaluated by a Cell Cycle Phase Determination Kit. Apoptosis was evaluated by an Apoptotic Blebs Assay Kit; cell cycle arrest and apoptotic signaling were determined by fluorescence-activated cell sorting (FACS) analysis. Results: CINO at ≥ 0.5 μM inhibited SKOV-3 cell proliferation, migration, and invasion (p < 0.05). There was a higher (p < 0.05) percentage of S phase cells in groups treated with CINO at 0.5 μM. CINO at ≥ 0.5 μM down regulated expression of Proliferating Cell Nuclear Antigen (PCNA) and caused cell death. Conclusion: This data demonstrates that CINO impairs SK-OV-3 cell function via cell cycle arrest and apoptotic signaling, suggesting CINO be further investigated as a novel anti-ovarian cancer agent.

Class II invariant chain peptide (CLIP) expression has been demonstrated to play a pivotal role in the regulation of B cell function after nonspecific polyclonal expansion. Several studies have shown vitamin D3 helps regulate the immune response. We hypothesized that activated vitamin D3 suppresses CLIP expression on activated B-cells after nonspecific activation or priming of C57BL/6 mice with CpG. This study showed activated vitamin D3 actively reduced CLIP expression and decreased the number of CLIP+B-lymphocytes in a dose and formulation dependent fashion. Flow cytometry was used to analyze changes in mean fluorescent intensity (MFI) based on changes in concentration of CLIP on activated B-lymphocytes after treatment with the various formulations of vitamin D3. The human formulation of activated vitamin D (calcitriol) had the most dramatic reduction in CLIP density at an MFI of 257.3 [baseline of 701.1 (Pvalue = 0.01)]. Cholecalciferol and alfacalcidiol had no significant reduction in MFI at 667.7 and 743.0, respectively. Calcitriol seemed to best reduce CLIP overexpression in this ex vivo model. Bioactive vitamin D3 may be an effective compliment to other B cell suppression therapeutics to augment downregulation of nonspecific inflammation associated with many autoimmune disorders. Further study is necessary to confirm these findings.

Immunotherapy, such as anti-PD1, has improved the survival of patients with metastatic melanoma. However, predicting which patients will respond to immunotherapy remains a significant knowledge gap. In this study we analyzed pre-immunotherapy treated tumors from 52 patients with metastatic melanoma and monitored their response based on RECIST 1.1 criteria. The responders group contained 21 patients that had a complete or partial response, while the 31 non-responders had stable or progressive disease. Whole exome sequencing (WES) was used to identify biomarkers of anti-PD1 response from somatic mutations between the two groups. Variants in codons G34 and G41 in NFKBIE, a negative regulator of NFkB, were found exclusively in the responders. Mutations in NKBIE-related genes were also enriched in the responder group compared to the non-responders. Patients that harbored NFKBIE-related gene mutations also had a higher mutational burden, decreased tumor volume with treatment, and increased progression-free survival. RNA sequencing on a subset of tumor samples identified that CD83 was highly expressed in our responder group. Additionally, Gene Set Enrichment Analysis showed that the TNFalpha signaling via NFkB pathway was one of the top pathways with differential expression in responders vs. non-responders. In vitro NFkB activity assays indicated that the G34E variant caused loss-of-function of NFKBIE, and resulted in activation of NFkB signaling. Flow cytometry assays indicated that G34E variant was associated with upregulation of CD83 in human melanoma cell lines. These results suggest that NFkB activation and signaling in tumor cells contributes to a favorable anti-PD1 treatment response, and clinical screening to include aberrations in NFkB-related genes should be considered.

Background Immune checkpoint inhibitors have improved overall survival rates for many cancers, yet the majority of patients do not respond to treatment and succumb to disease progression. One tumor-related mechanism limiting the efficacy of immunotherapies in melanoma is the recruitment and expansion of myeloid-derived suppressor cells (MDSCs). Therefore, targeting MDSCs in combination with immunotherapies is an attractive strategy to improve response rates and effectiveness. Methods We tested this strategy by designing a randomized phase II clinical trial treating advanced melanoma patients with either Ipilimumab monotherapy or Ipilimumab plus all-trans retinoic acid (ATRA). Clinicaltrails.gov identifier ( NCT02403778 ). The frequency of circulating MDSCs and the activation of CD8(+) T cells was measured by flow cytometry. Expression of immunosuppressive genes was measured with quantitative real time-PCR. T cell suppressive functions were measured by mixed lymphocyte reaction. Results Here we show that in vitro treatment with ATRA decreases immunosuppressive function of MDSCs in mixed lymphocyte reactions. Additionally, ATRA reduces the expression of immunosuppressive genes including PD-L1, IL-10, and indoleamine 2,3‑dioxygenase by MDSCs. Furthermore, the addition of ATRA to standard of care Ipilimumab therapy appears safe, as ATRA did not increase the frequency of grade 3 or 4 adverse events. Finally, ATRA significantly decreased the frequency of circulating MDSCs compared to Ipilimumab treatment alone in advanced-stage melanoma patients. Conclusions These results illustrate the importance of MDSCs in immunotherapy resistance and provide evidence that targeting MDSCs in cancer patients may augment immunotherapeutic approaches.

Lyme disease is a multisystem infection transmitted by tick vectors with an incidence of up to 300,000 individuals/yr in the United States. The primary treatments are oral or i.v. antibiotics. Despite treatment, some individuals do not recover and have prolonged symptoms affecting multiple organs, including the nervous system and connective tissues. Inflammatory arthritis is a common symptom associated with Lyme pathology. In the past decades, γδ T cells have emerged as candidates that contribute to the transition from innate to adaptive responses. These cells are also differentially regulated within the synovia of patients affected by RLA. Here, we review and discuss potential cellular mechanisms involving γδ T cells and DCs in RLA. TLR signaling and antigen processing and presentation will be the key concepts that we review in aid of understanding the impact of γδ T cells in RLA.

Excessive innate immune system activation and inflammation during pregnancy can lead to organ injury and dysfunction and preeclampsia (PE); however, the molecular mechanisms involved are unknown. We tested the hypothesis that Toll-like receptor (TLR) activation induces major histocompatibility complex (MHC) class II invariant chain peptide (CLIP) expression on immune cells, makes them pro-inflammatory, and are necessary to cause PE-like features in mice. Treatment with VG1177, a competitive antagonist peptide for CLIP in the groove of MHC class II, was able to both prevent and treat PE-like features in mice. We then determined that γ–δ T cells are critical for the development of PE-like features in mice since γ–δ T-cell knockout mice, like CLIP deficient mice, are resistant to developing PE-like features. Placentas from women with PE exhibit significantly increased levels of γ–δ T cells. These preclinical data demonstrate that CLIP expression and activated γ–δ T cells are responsible for the development of immunologic PE-like features and that temporarily antagonizing CLIP and/or γ–δ T cells may be a therapeutic strategy for PE.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that represent a formidable obstacle to the successful treatment of cancer. Patients with high frequencies of MDSCs have significantly decreased progression-free survival (PFS) and overall survival (OS). Whereas there is experimental evidence that the reduction of the number and/or suppressive function of MDSCs in mice improves the efficacy of anti-cancer therapies, there is notably less evidence for this therapeutic strategy in human clinical trials. Here, we discuss currently available data concerning MDSCs from human clinical trials and explore the evidence that targeting MDSCs may improve the efficacy of cancer therapies.

Immune checkpoint blockade (ICB) treatments, such as anti-PD1, improves survival in patients with metastatic melanoma. Despite durable response rates, 40-50% of patients show no benefit. Implicated in acquired resistance is the downregulation of antigen presentation, via dysfunction of the interferon-receptor pathway. However, little is known about intrinsic response mechanisms to ICB. The NF-κB-pathway is important for immune response as a key transcription factor for several antigen presentation proteins and cytokine/chemokine production. This study explored NF-κB signaling in patients with favorable, or unfavorable responses to anti-PD1 therapy. Fifty-two patients with metastatic melanoma were grouped into complete or partial responders (CR/PR; n = 6 and 15, respectively), and stable or progressive disease (SD/PD; n = 16 and 15, respectively). Consistent with other studies, tumor mutational burden assessed by whole exome sequencing (WES) was higher in the CR/PR group (p = 0.03). WES analysis also showed an enrichment of variants in NF-κB-pathway related genes within the CR/PR group (14/21, 67%) compared with SD/PD patients (6/31, 19%), p = 0.001. Notably, the hotspot variants G34E/R and G41E in NFKBIE, a negative regulator of NF-κB, were found exclusively in patients with complete or partial response to ICB (5/21, 24%). RNA-seq analysis of 19 tumors showed that the TNFα/NF-κB signaling pathway is upregulated in the CR/PR group, and highly expressed genes included CD83, HLA-DQB, and NFATC1. Therefore we hypothesized that when activated, particularly within the melanoma cells, this pathway contributes to an anti-tumor immune response. Using HEK293 cells, we demonstrated NFKBIEG34E increased NF-κB activity compared with the wildtype, upon TNFα stimulation. Consistently, flow cytometry also show an increase of targets of NF-κB transcription such as CD83, MHC class II, and PDL1. Similarly, the melanoma cell line A375, which harbors the NFKBIEG34E mutation, showed increases in protein expression when stimulated with TNFα and INFγ. Furthermore, progression free survival increased in patients that had tumors with an NF-κB-pathway variant compared with patients that were pathway wildtype (p = 0.03). Patients harboring tumors with NF-κB-pathway variants also had a greater response by RECIST criteria compared with wildtype. Taken together, our results suggest that an increase of pro-immunogenic NF-κB signaling in the tumor may contribute to the favorable response to ICB, highlighting a role for CD83 in the tumor microenvironment that merits further examination. Citation Format: Keith Wells, Jennifer Hintzsche, Carol M. Amato, Richard Tobin, Victoria Vorwald, Martin McCarter, Yiqun Shellman, Aik Choon Tan, William Robinson. Investigating the role of NF- κB signaling and immune checkpoint blockade therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5002.

e21016 Background: Currently there is a very limited data reporting response of adrenal metastases to immunotherapy in patients with metastatic malignant melanoma. Certain sites in the body may serve as sanctuary sites due to tumor microenvironment. Lower response rates and shorter PFS are reported in malignant melanoma with liver metastases while higher objective response rates are seen with lung metastases. We hypothesized that adrenal glands are sanctuary sites for melanoma metastases and thus would have poor response to immunotherapy due to steroid production in the adrenals making microenvironment less optimum for the anti-tumor effects of immunotherapy. Methods: A retrospective single-institution chart review of all malignant melanoma with metastasis to adrenal gland treated at University Colorado Hospital from 2008 till 2018, with age ≥ 18 yrs of age who received at least one dose of immunotherapy. Immune-related response criteria (irRC) was used to assess the response to immunotherapy on the imaging. Kaplan Meier estimate, and Chi square test were used for statistical analysis. Results: Sixty-five patients met the inclusion criteria. Mean age of 61 yrs. with range from 25 to 90 yrs. 71% were males and 95% were Caucasians. Median duration of follow up was 51.3 months with range of 1.5-212 months. Forty-three percent were cutaneous, 35% unknown primary, 11% each with mucosal and ocular melanoma. Based on the category of malignant melanoma, 60% were metastatic, 12% nodular, 8% choroidal, 8% superficial spreading, 6% acral lentiginous and 6% other. BRAF mutations were present in 38%, wild type in 54% and unknown in 8%. NRAS was mutated in 18% and wild type in 17% and unknown in 65% of the patients. Comparison of response between adrenal metastasis and non-adrenal metastasis, showed that 71% adrenal metastases had progressive disease compared to 54% of non-adrenal metastases. The overall response rate (ORR) to immunotherapy was 15% in adrenal metastases compared to 20% of non-adrenal metastases. Disease control rate (DCR) was 29% in adrenal metastases compared to 46% non-adrenal metastases with p value = 0.015. Conclusions: The adrenal glands appear to be a sanctuary site for metastases in patients with malignant melanoma receiving immunotherapy. The reasons behind this are unclear but may be related to the microenvironment and local corticosteroid production suppressing the immune response. Preliminary data examining cellular responses in nine surgically removed adrenal metastases support this hypothesis and will be presented.

Background: Recruitment and expansion of immunosuppressive myeloid cells present a significant barrier to the successful treatment of melanoma. We aimed to identify tumor-secreted factors that altered the frequency of MDSCs and correlated with clinical outcomes in advanced melanoma patients. We hypothesized that production of IL-6 and IL-8 by melanoma tumors would lead to expansion and accumulation of MDSCs and correlate with long-term clinical outcomes. Methods: Expression of IL-6 and IL-8 in melanoma tumors as well as the plasma concentration of IL-6 and IL-8 were measured and compared with the frequency of circulating MDSCs in a total of 52 stage IV melanoma patients. These measures were correlated with tumor burden, BRAF status, lactic acid dehydrogenase (LDH) levels and with clinical outcomes. Samples were collected beginning in January 2011 and clinical follow-up was collected through January 2018. Results: The plasma concentration of both IL-6 and IL-8 correlated with tumor burden (p < 0.0001 and p= 0.0122, respectively). Patients with high plasma concentrations of either IL-6 (40%) or IL-8 (63%) had worse median overall survival (MOS) (7.5 months, p < 0.0001 and 9.2 months, p = 0.0015, respectively) compared to patients with low concentrations, where MOS was not reached. Patients with high plasma concentrations of both IL-6 and IL-8 (35%) had even worse MOS (6.7 months) than patients with low levels of both cytokines (MOS not reached, 31%) p < 0.0001. We observed that patients with lower circulating concentrations and lower tumoral expression of IL-6 and IL-8 showed decreased frequencies of circulating MDSCs. Furthermore, patients with low frequencies of MDSCs (MOS not reached) had better overall survival than patients with high frequencies of these cells (MOS 11.9 months) p = 0.0224. Multivariable analysis showed that when adjusting for variables BRAF status and LDH in the model, the hazard for the event of death for subjects with both high IL-6 and IL-8 is 4.46 times (95% CI, 1.4, 14.2) that of the hazard for the subjects with low IL-6 and IL-8 (p = 0.01). Additionally, the hazard of death for subjects with higher total MDSCs is 3.3 times (95% CI, 1.2, 9.0) that for subjects with low total MDSCs. Conclusions: The durability of the nearly 7-year follow-up time for these advanced melanoma patients further strengthens the implied importance of IL-6 and IL-8 in the accumulation of MDSC’s in melanoma patients. These data provide clues for potential therapeutic targets in advanced melanoma patients. Citation Format: Richard P. Tobin, Kimberly R. Jordan, Dana Davis, Victoria M. Vorwald, Kasey Couts, Dexiang Gao, Derek E Smith, William A Robinson, Virginia Borges, Martin D McCarter. Tumor-produced IL-6 and IL-8 are associated with MDSC accumulation and correlate with long-term clinical outcomes in melanoma patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A117.

Objective: Inflammation is necessary for successfulpregnancy; however, excessive inflammation plays acentral role in the development of the pregnancy-specifichypertensive disorder preeclampsia. Numerous anti-inflammatory cytokines are decreased in women withpreeclampsia but the role of individual cytokines in bloodpressure regulation during pregnancy is unknown.Therefore, we examined whether the lack of the potentanti-inflammatory cytokine interleukin-4 (IL-4) would besufficient to elicit a preeclampsia-like syndrome in mice,and when coupled with immune system activation thatthese symptoms would be further augmented.Methods: Measures of splenic immune cells, placentalinflammation, blood pressure, endothelial function, andurinary protein excretion were performed in pregnant IL-4-deficient mice as well as in pregnant IL-4-deficient micetreated with the Toll-like receptor 3 agonistpolyinosinic:polycytidylic (poly I:C).Results: Pregnant IL-4-deficient mice exhibited alteredsplenic immune cell subsets, increased levels of pro-inflammatory cytokines, placental inflammation, mildhypertension, endothelial dysfunction, and proteinuriacompared to pregnant control mice. Compared topregnant control mice treated with poly I:C which exhibitpreeclampsia-like symptoms, poly I:C-treated pregnant IL-4-deficient mice exhibited a further increase in pro-inflammatory cytokine levels, which was associated withaugmented SBP and endothelial dysfunction.Conclusion: Collectively, these data show that theabsence of IL-4 is sufficient to induce mild preeclampsia-like symptoms in mice due to excessive inflammation.Thus, the anti-inflammatory effects of IL-4 are important inpreventing hypertension during pregnancy.Keywords: anti-inflammatory, cytokines, endothelialfunction, pregnancy, pro-inflammatory, SBPAbbreviations: ACh, acetylcholine; DAMPs, danger-associated molecular patterns; ICAM-1, intracellularadhesion molecule-1; IFNg, interferon g; IL, interleukin;PAMPs, pathogen-associated molecular patterns; polyI???C, polyinosinic???polycytidylic; SNP, sodiumnitroprusside; TGFb1, transforming growth factor b1; Th, Thelper type; TLR, Toll-like receptor; TNFa, tumor necrosisfactor a

Hemorrhagic shock (HS)-induced microvascular hyperpermeability poses a serious challenge in the management of trauma patients. Microvascular hyperpermeability occurs mainly because of the disruption of endothelial cell adherens junctions, where the "intrinsic" apoptotic signaling plays a regulatory role. The purpose of this study was to understand the role of the "extrinsic" apoptotic signaling molecules, particularly Fas-Fas ligand interaction in microvascular endothelial barrier integrity. Rat lung microvascular endothelial cells (RLMECs) were exposed to HS serum in the presence or absence of the Fas ligand inhibitor, FasFc. The effect of HS serum on Fas receptor and Fas ligand expression on RLMECs was determined by flow cytometry. Endothelial cell permeability was determined by monolayer permeability assay and the barrier integrity by β-catenin immunofluorescence. Mitochondrial reactive oxygen species formation was determined using dihydrorhodamine 123 probe by fluorescent microscopy. Mitochondrial transmembrane potential was studied by fluorescent microscopy as well as flow cytometry. Caspase 3 enzyme activity was assayed fluorometrically. Rat lung microvascular endothelial cells exposed to HS serum showed increase in Fas receptor and Fas ligand expression levels. FasFc treatment showed protection against HS serum-induced disruption of the adherens junctions and monolayer hyperpermeability (P < 0.05) in the endothelial cells. Pretreatment with FasFc also decreased HS serum-induced increase in mitochondrial reactive oxygen species formation, restored HS serum-induced drop in mitochondrial transmembrane potential, and reduced HS serum-induced caspase 3 activity in RLMECs. These findings open new avenues for drug development to manage HS-induced microvascular hyperpermeability by targeting the Fas-Fas ligand-mediated pathway.

Placental microRNA (miRNA) expression is known to be dysregulated during preeclampsia (PE), a hypertensive pregnancy disorder but the role it plays in the pathogenesis of PE is currently unknown. We have previously demonstrated that placental miR-155 expression is upregulated (P-PIC: 3.15 fold, p

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells that are increased in the peripheral blood of cancer patients and limit productive immune responses against tumors. Immunosuppressive MDSCs are well characterized in murine splenic tissue and are found at higher frequencies in spleens of tumor-bearing mice. However, no studies have yet analyzed these cells in parallel human spleens. We hypothesized that MDSCs would be increased in the spleens of human cancer patients, similar to tumor-bearing mice. We compared the frequency and function of MDSC subsets in dissociated human spleen from 16 patients with benign pancreatic cysts and 26 patients with a variety of cancers. We found that total MDSCs (Linneg CD11bpos CD33pos HLA-DRneg), granulocytic MDSCs (additional markers CD14neg CD15pos), and monocytic MDSCs (CD14pos CD15neg) were identified in human spleen. The monocytic subset was the most prominent in both spleen and peripheral blood and the granulocytic subset was expanded in the spleen relative to matched peripheral blood samples. Importantly, the frequency of CD15pos MDSCs in the spleen was increased in patients with cancer compared to patients with benign pancreatic cysts and was associated with a significantly increased risk of death and decreased overall survival. Finally, MDSCs isolated from the spleen suppressed T cell responses, demonstrating for the first time the functional capacity of human splenic MDSCs. These data suggest that the human spleen is a potential source of large quantities of cells with immunosuppressive function for future characterization and in-depth studies of human MDSCs.

TLR stimulation results in polyclonal B cell activation, ectopic CLIP expression, release of T cell-derived TNFα, and reversal with high affinity peptide treatment. Infectious pathogens produce compounds called Toll ligands that activate TLRs on lymphocytes. Acute activation triggered by certain TLRs appears to “jump start” the innate immune response, characterized by the release of inflammatory cytokines and cellular expansion. In some individuals, there is a failure to control acute inflammation, resulting in postinfectious, chronic inflammation. Susceptibility to chronic inflammation is strongly associated with an individualˈs MHC genes. Recent clinical trials for several autoimmune diseases characterized by chronic inflammation suggest that B lymphocyte depletion therapies dampen chronic immune activation. However, currently, there is no known mechanism that accounts for the correlation among TLR activation, MHC genetics, and a pathological role for B-lymphocytes. Our hypothesis is that TLR-activated B cells (B cells that have been polyclonally activated in the absence of antigen-specific signals) are not controlled properly by T cell-dependent B cell death, thereby causing B cell-dependent chronic inflammation. Here, we show that treatment with Toll ligands results in polyclonal B cell activation accompanied by ectopic expression of CLIP. Furthermore, by adoptively transferring purified CLIP+ B cells in syngeneic animals, we find that CLIP+ B cells induce production of TNF-α by host T cells. Finally, we demonstrate that CLIP-targeted peptide competition results in the death of polyclonally activated CLIP+ B cells.

BACKGROUND AND SIGNIFICANCE: Glioblastoma Multiforme (GBM) is a deadly and highly aggressive brain tumor with grim prognosis. At present none of the treatment strategies extend the life of most patients beyond a year. A contribution from the immune system in the control and/or growth of tumors is currently under intense investigation. However, the strategies GBM cells use to hide from the immune system are poorly understood. EXPERIMENTAL APPROACH: To address this issue, we compared the tumor graft acceptance of the mouse GBM 261 neurosphere (mouse glioma cell line/derived from a tumor from a C57Bl6 strain background) into C57Bl6 mice with three genetic modifications (CD74, invariant chain deficient mice; gamma delta knockout mice; and control, wild type C57Bl6 mice). Neurospheres were made from 261 cells by plating 10^5 cells/mL in DMEM/F12 media supplemented with 20 ng/mL EGF and 10 ng/mL bFGF, 0.002% heparin and B27 supplement without retinol. Cells started showing neurosphere morphology at around 6 days. The 261 neurospheres were injected stereotactically in the caudate nucleus and mice were observed for 4-6 weeks after neurosphere injection. RESULTS: The data showed that the tumor acceptance in C57Bl6 mice was higher than acceptance in either CD74 deficient or Gamma Delta TCR deficient mice. In addition, C57Bl6 mice showed rapid tumor growth with robust neurological deficit as early as 4 weeks. No neurological deficit was observed in invariant chain deficient mice or gamma delta knockout mice. CONCLUSIONS: These data suggest a contribution from CD74 as well as gamma delta T cells in tumor graft acceptance. Further experiments are ongoing to determine the potential role of CD74 and/or gamma delta T cells in the progression of GBM.

Lipopolysaccharide (LPS) is a cell wall component of gram-negative bacteria that can activate toll-like receptor 4, which in turn, activates the innate immune system. Chronic immune system activation is linked to blunted afferent arteriolar autoregulatory behavior and kidney injury. MHC Class II-associated invariant peptide (CLIP) provides a critical step in antigen processing, presentation, and adaptive immune system activation. Accordingly, we postulated that treatment with a competitive CLIP antagonist (CAP) during chronic low-dose LPS exposure would preserve afferent arteriolar autoregulatory behavior. Rats were implanted with osmotic minipumps (day 0) for infusion of LPS (0.01mg/kg/day) or saline (0.9% NaCl; 0.5μl/hr) for 8 days and then the kidneys were harvested for juxtamedullary nephron studies. Four groups (n=6/group) were studied: Control, LPS, LPS + CAP and Sham + CAP. Both LPS + CAP and Sham + CAP groups were treated with CAP (3mg/kg/day; i.p.) on days 1-7. Autoregulatory behavior was assessed in these groups by increasing perfusion pressure in 15 mmHg increments from 65 to 170 mmHg. Starting baseline diameters were similar across the control (15.2 ± 1.2 μm), LPS (14.1 ± 1.0 μm), LPS + CAP (15.1 ± 1.0), and Sham + CAP (15.7 ± 0.6) groups. When perfusion pressure was increased from 65 to 170 mmHg, control and sham + CAP afferent arteriolar diameter decreased significantly by 26 ± 4% and 25 ± 2% (P

Preeclampsia (PE), a hypertensive disorder of pregnancy, is associated with vascular endothelial dysfunction and excessive immunity and inflammation. However, it is unclear which innate immune cells propagate the pro-inflammatory state. Gamma-delta T (gdT) cells can secrete tolerogenic anti-inflammatory cytokines or cytotoxic pro-inflammatory cytokines depending on their activation status. gdT cells from women with PE produce significantly more IFNg and perforin and are less susceptible to apoptosis than gdT cells from normal pregnant women. We hypothesized that Toll-like receptor (TLR) activation in gdT cells induces inflammation and causes PE-like features in mice and that gdT cell KO mice would be resistant to developing TLR-induced PE-like features. Activation of splenocytes isolated from day 14 normal pregnant mice with the TLR3 agonist poly I:C or the TLR7 agonist R837 for 24 hours significantly increased gdT cells as well as IFNg and TNFa production. We have reported that poly I:C or R837 treatment of normal pregnant mice elicits a pregnancy-dependent PE-like syndrome by inducing a pro-inflammatory immune response. Pregnant poly I:C-treated and R837-treated mice had significantly increased splenic levels of gdT cells and plasma levels of IFNg and TNFa compared to pregnant vehicle-treated mice. Pregnant gdT cell KO mice treated with poly I:C or R837 did not develop hypertension or endothelial dysfunction. These data demonstrate that gdT cells mediate the TLR-induced PE-like features in mice and depletion of gdT cells may reduce the severity of PE in women.

PURPOSE To evaluate the accuracy of leg-to-leg bioelectrical impedance analysis (BIA) in assessing fat-free mass (FFM) using the TBF-300WA in comparison with hydrostatic weighing (HW) and skinfolds (SK) in high-school (HS) wrestlers in a hydrated state. METHODS Body composition was determined by BIA, HW, and three-site SK in 129 HS wrestlers (mean +/- SD, age: 15.5 +/- 1.3, height 1.70 +/- 0.08 m, body mass 65.6 +/- 13.1 kg). For all methods, body density (Db) was converted to percent body fat (%BF) using the Brozek equation. Hydration state was quantified by evaluating urine specific gravity. RESULTS There were no significant differences for estimated FFM between BIA (56.9 +/- 8.4 kg) and HW (56.2 +/- 9.9 kg) or between SK (56.1 +/- 8.9 kg) and HW. The standard errors of estimate for FFM with HW as the reference method were 3.64 kg for BIA and 1.97 kg for SK. Significant correlations were found for FFM between HW and BIA (r = 0.93, P < 0.001), and between HW and SK (r = 0.98, P < 0.001). The FFM difference between BIA and HW was significantly correlated with the FFM average of the two methods (r = -0.39, P < 0.001). The FFM difference between SK and HW was also significantly correlated with the FFM average (r = -0.44, P < 0.001). CONCLUSIONS This study demonstrates that SK should be considered as the preferred field method of estimating the FFM of HS wrestlers because of its greater individual precision than the leg-to-leg BIA method. Although the BIA is an attractive assessment tool and easy to use, it should be recommended as an alternative to the SK method only when trained skinfold technicians are not available.

125 Background: Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of immature immunosuppressive myeloid cells that are expanded in tumor bearing hosts. Melanoma patients with high frequencies of MDSCs have decreased overall survival and an increased risk of death and disease progression, as well as impaired responses to ipilimumab. Targeting MDSCs to decease their frequency or suppressive activity may provide an effective means to improve the efficacy of anti-cancer therapies. All-trans retinoic acid (ATRA) is a vitamin A derivative currently used to treat APL that may target MDSCs. ATRA differentiates immature myeloid cells into macrophages, dendritic cells, or granulocytes. ATRA has been tested as an MDSC targeting agent in two completed caner clinical trials and is being investigated in several other open and completed trials. Methods: We purified myeloid cells from LRS chambers collected from normal donors. The purified cells were treated with GM-CSF+IL-6, GM-CSF+IL-4, or melanoma conditioned media with or without ATRA. After 5 days of incubation, PCR was used to determine the expression of immunoregulatory genes. Additionally, we used a MLR to determine the effect of ATRA on MDSC’s T cell suppressive function. Results: Here we report that in vitro ATRA treatment decreases the expression of ARG1, NOX1, INOS, and PD-L1. Further, we report that ATRA treatment improved T cell proliferation. Conclusions: Controlling MDSC suppressive function and accumulation may provide a mechanism to improve the efficacy of many current cancer therapies. Here we show that ATRA reduces the expression of the immunosuppressive genes ARG1, NOX1, INOS, and PD-L1 in MDSCs. Additionally, we show that treatment of MDSCs with ATRA decreases MDSC’s ability to suppress T cell proliferation. These results indicate that ATRA may improve the efficacy of conventional and immnunotherapeutic treatments.

Infections can exacerbate heart failure and contribute to heart transplant rejection. We hypothesize that cardiac myosin binding protein C (MyBPC3), a thick filament protein, contributes to protection of the heart during an immune response to infection. We tested our hypothesis by comparing wild type (WT) vs. MyBPC3(-/-) mice for their immunologic response to non-pathogenic doses of Eschericia coli lipopolysaccharide (LPS). LPS caused increased hypertrophy, worsening of diastolic dysfunction, and increased apoptosis in MyBP3(-/-) hearts. We then performed flow cytometric analyses on immune cells isolated from the myocardium, the spleen, and the bone marrow. Prior to LPS challenge, MyBPC3(-/-) hearts have relatively fewer myeloid cells characterized by CD11b+GR1High in comparison to WT hearts. LPS challenge dramatically increased myeloid cell population in WT, but not in MyBPC3(-/-) hearts. Thus, myeloid population demonstrated inverse relationship with cardiac dysfunction and apoptosis. LPS increased B cells(CD19+) infiltration into MyBPC3(-/-) hearts only, suggesting that increases in B cells correlate with increased cardiac myocyte cell death. In spleen, LPS increased total number of immune cells of all types (CD45+) only in WT. MyBPC3(-/-) showed depressed myeloid cell populations in spleen and bone marrow. Thus, loss of MyBPC3 may have contributed to overall decreased myeloid population. These results suggest that CD11b+GR1High cells are myeloid derived suppressor cells (MDSC) which protect cardiac myocytes during an immune response, and B cells are associated with cardiac myocyte cell death. Furthermore, MyBPC3 may be necessary to recruit MDSCs to maintain protection of the heart during infection. Finally, these results imply that modulating recruitment of MDSCs and preservation of MyBPC3 may provide the basis for new treatments to prevent infection-associated exacerbation of heart failure and heart transplant rejection.