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2. AbstractBackgroundCardiac allograft vasculopathy (CAV) is a major cause of late-graft failure and mortality following heart transplantation. The mechanisms underlying vascular remodeling are poorly understood. A major immune risk factor associated with the development of CAV is the presence of donor-specific antibodies (DSA) that induce chronic endothelial cell (EC) injury, leukocyte recruitment and inflammation resulting in thickening of arterial intima. Here, we molecularly characterized innate and adaptive immune cells present in the arteries of rejected cardiac allografts with DSA and identified protein and transcriptomic signatures distinguishing early and late CAV lesions.MethodsArterial areas of interest (AOIs) from CAV+DSA+ rejected cardiac allografts (N=3; 2 females, 1 male) were subjected to GeoMx digital spatial profiling (DSP). AOIs were scored on the level of CAV progression/neointimal thickening (22 AOIs total; 11 high and 11 low neointima) and were subjected to whole transcriptome and protein profiling.ResultsAOIs with low neointima significantly increased markers for activated inflammatory infiltrates, transcripts of EC activation and gene modules involved in activating metalloproteinases and TP53 regulation of caspases. Inflammatory and apoptotic protein markers significantly correlated with inflammatory modules in AOIs with low neointima. AOIs with high neointima increased TGFβ-regulated transcripts and modules enriched for platelet activation/aggregation. Proteins encoding SMCs and growth factors/survival correlated with modules enriched for proliferation/repair in AOIs with high neointima. Key transcripts in promoting proliferation, migration, and EndoMT were significantly associated with increasing neointima scores.ConclusionOur results reveal new protein and transcriptomic signatures associated with CAV progression. Lesions exhibiting inflammatory profiles appear to be early lesions that transition to later proliferative/pro-fibrotic phenotype CAV lesions. These findings should form the foundation for the identification of improved biomarkers to guide CAV treatment.

Sex differences in glioblastoma (GBM) incidence and outcome are well recognized, and emerging evidence suggests that these extend to genetic/epigenetic and cellular differences, including immune responses. However, the mechanisms driving immunological sex differences are not fully understood. Using GBM models, we demonstrate that T cells play a critical role in driving GBM sex differences. Male mice exhibited accelerated tumor growth, with decreased T cell infiltration and increased T cell exhaustion. Furthermore, a higher frequency of progenitor exhausted T cells was found in males, with improved responsiveness to anti-PD1 treatment. Bone marrow chimera and adoptive transfer models indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, which was further corroborated by in vitro exhaustion assays. Moreover, increased T cell exhaustion was observed in male GBM patients. These findings demonstrate sex-specific pre-determined behavior of T cells is critical in inducing sex differences in GBM progression and immunotherapy response.Statement of significanceImmunotherapies in GBM patients have been unsuccessful due to a variety of factors including the highly immunosuppressive tumor microenvironment in GBM. This study demonstrates that sex-specific T cell behaviors are predominantly intrinsically regulated, further suggesting sex-specific approaches can be leveraged to potentially improve therapeutic efficacy of immunotherapy in GBM.

Lymphocyte activation gene-3 (LAG3) is a coinhibitory receptor expressed by a wide range of immune cells. While LAG3 is predominantly studied in T cells, its functions in humoral immune responses remain poorly characterized. The goal of this study was to test the role of recipient LAG3 in a mouse model of renal allograft rejection. Compared to WT animals, naïve B6.LAG3-/-mice have increased splenic cellularity and higher frequencies of CD44himemory T cells, CXCR5hifollicular T cells, and B220+CD138+plasma cells yet do not develop spontaneous autoimmunity. Furthermore, naïve B6.LAG3-/-(H-2Db) mice have increased frequencies of memory T cells recognizing H-2Dd, H-2Ds, H-2Dqand H-2Dkalloantigens. In addition, naïve non-transplanted B6.LAG3-/-mice have elevated levels of serum IgG reactive to allogenic class I and class II MHC molecules. Whereas 4/4 B6.WT recipients spontaneously accept fully MHC-mismatched C3H (H-2k) renal allografts for longer than 60 d, recipient LAG3 deficiency led to rapid allograft rejection and elevated serum creatinine levels. Graft histology at rejection revealed minimal T cell infiltration, diffuse C4d staining, atrophic peritubular capillaries, endothelial swelling and edema characteristic of antibody mediated rejection (AMR). Compared to WT, LAG3-/-recipients had elevated frequencies of anti-donor IFNγ producing T cells and increased levels of anti-donor MHC antibodies. Recipient CD8+T cell depletion did not alter the rejection kinetics in LAG3-/-recipients, while B cell depletion significantly extended C3H kidney allograft survival. Conditional knockout mice were used to determine the importance of LAG3 expression by T and B lymphocytes. The absence of LAG3 on either T or B lymphocytes had no impact on graft rejection with both groups accepting kidney allografts for longer than 30 days, suggesting that LAG3 expression on either T or B cells is sufficient to regulate immune responses to kidney allografts. These results suggest that LAG3 is a regulator of both T and B cell responses to kidney allografts and is an attractive potential therapeutic target for AMR prevention and treatment.