Alloantibodies produced by recipient B cells and plasma cells pose a significant risk for rejection after solid organ transplantation, including intestinal transplantation (ITx). Dynamic turnover of recipient B cells and plasma cells in intestinal allografts and their roles in mediating rejection have not been investigated. We analyzed the chimerism, phenotype and clonotypes of B cells and plasma cells in the allograft and peripheral blood of ITx recipients using multicolor flow cytometry and high-throughput BCR sequencing. Serial analyses of ileal biopsies demonstrated eventual replacement of lamina propria donor B cells by the recipient, over periods of >1 year in some patients (Fig.1). Recipient B cell replacement rates were often faster than those of T cells. In patients with donor T cell macrochimerism (>4%) in blood, replacement of donor cells by recipient cells tended to occur slowly, over a period of several months. In contrast, in patients without T cell macrochimerism and with early rejection, recipient B cells and T cells rapidly replaced those of the donor in the graft. High levels (>10%) of donor B cell chimerism in blood were only observed in multivisceral transplant (MVTx) recipients and peak donor B cell blood chimerism was generally higher than that of donor T cells within the same patient. Phenotypic analyses of lamina propria B cells on sequential biopsies of a 2-year-old recipient of an allograft from a 6-month-old donor revealed the early presence of naïve donor and recipient B cells in the graft that over time were largely replaced by both class-switched and non-switched memory B cells of donor and recipient origin. Recipient-derived surface IgG+ and IgA+ B cells appeared in the long-term graft of a patient who had had DSA but not in a DSA- patient. Even when recipient B cells had almost fully replaced donor-derived B cells, donor-derived plasma cells (CD138+CD38+) persisted long-term (POD>900), demonstrating the existence of long-lived plasma cells in human intestine. The presence of recipient plasma cells in the graft may correlate with rejection, as they were only detected during rejection episodes. (Fig.2). IgH V region sequencing on sorted recipient B cells from ileal specimens of a MVTx patient on Days 29, 32, 43, 57, 78, 105 and 141 post-Tx revealed clonal overlap, especially at later time points. The fraction of recipient B cells co-expressing CD45RB and CD69 in the ileal biopsies increased with time in 3 out of 3 patients. (Fig.3) CD45RB+CD69+ B cells may belong to a resident memory pool that contributes to tissue-based immunity (Weisel et al, in preparation). In summary, the appearance of recipient plasma cells and IgG+/IgA+ B cells in the graft correlates with rejection and DSA development, respectively. We hypothesize that graft-repopulating recipient B cell clones may expand and acquire a B resident memory phenotype, promoting organ-specific alloimmunity. The study was funded by the National Institute of Allergy and Infectious Diseases grant P01 AI106697. Research reported in this study was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056.

Translating studies on T cell function and modulation from mouse models to humans requires extrapolating in vivo results on mouse T cell responses in lymphoid organs (spleen and lymph nodes [LN]) to human peripheral blood T cells. However, our understanding of T cell responses in human lymphoid sites and their relation to peripheral blood remains sparse. In this study, we used a unique human tissue resource to study human T cells in different anatomical compartments within individual donors and identify a subset of memory CD8+ T cells in LN, which maintain a distinct differentiation and functional profile compared with memory CD8+ T cells in blood, spleen, bone marrow, and lungs. Whole-transcriptome and high-dimensional cytometry by time-of-flight profiling reveals that LN memory CD8+ T cells express signatures of quiescence and self-renewal compared with corresponding populations in blood, spleen, bone marrow, and lung. LN memory T cells exhibit a distinct transcriptional signature, including expression of stem cell–associated transcription factors TCF-1 and LEF-1, T follicular helper cell markers CXCR5 and CXCR4, and reduced expression of effector molecules. LN memory T cells display high homology to a subset of mouse CD8+ T cells identified in chronic infection models that respond to checkpoint blockade immunotherapy. Functionally, human LN memory T cells exhibit increased proliferation to TCR-mediated stimulation and maintain higher TCR clonal diversity compared with memory T cells from blood and other sites. These findings establish human LN as reservoirs for memory T cells with high capacities for expansion and diverse recognition and important targets for immunotherapies.

Introduction: Calcineurin (CN), a protein phosphatase activated by Ca2+-calmodulin, is involved in activation of NFAT. CN inhibitors such as cyclosporine A (CsA) and FK506 are potent inhibitors of T-cell responses and are commonly used to prevent graft rejection. Consistently, CNAβ-deficient mice exhibit defective thymocyte maturation in young mice. However, T cells undergo spontaneous activation in older Cnab-/- mice, and succumb to B-cell lymphomas. Methods: Splenocytes are cultured in the presence of anti-CD3/CD28 beads, TGFβ1 and IL-2 for 3 days. Cytokines in culture supernatants are determined by ELISA, CBA and MSD methods. Cells are stained with CD4, CD25, CD62L and intracellular FOXP3. Results: We observed that FOXP3+ Treg cells are decreased while mature activated T cells (CD62L-CD44+) are increased in KO mice compared to control mice. While TGFβ1 induced the expression of FOXP3 in WT cultures, it failed to induce FOXP3 in KO cultures. Cytokine profiles revealed that CNAβ is required to inhibit IL-4, IL-6, IL-10, IL-17 and IFNγ by TGFβ1. IHC analyses of lymphomas revealed that the lymphomas are of B cell origin and they do not express FOXP1, a marker for activated B cells (ABCs). Discussion: These data show that CNAβ is important for the development and homeostasis of mature T cells and Treg cells. The data also suggest that in the absence of CNAβ, a defective T-cell regulation results in activation of T cells, production of inflammatory cytokines and cause cancer.

Abstract Introduction: The TGFB pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Tgfb1−/−Rag2−/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer. Methods: To determine the function of TGFβ1 on colonic bacterial composition we used GEM models, Tgfb1−/−Rag2−/− and Tgfb1+/+Rag2−/−. The cecal microflora of 10 Tgfb1−/−Rag2−/− and 10 Tgfb1+/+Rag2−/− mice were isolated and serially diluted onto brucella (BRU), bacteroides bile esculin (BBE), and laked kanomycin-vancomycin (LKV) media under anaerobic conditions. Colony forming units (CFUs) were enumerated. Individual colony types were then streaked onto trypitcase soy agar with 5% sheep blood and grown anaerobically and aerobically. The bacteria were then gram-stained and biotyped utilizing a Dade Berhing MicroScan instrument. To further examine the bacterial composition we designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on fecal DNA. Results: The Dade Berhing Instrument showed that Tgfb1−/−Rag2−/− mice had a 4 fold increase in bacterial load and a 28 fold increase in Bacteroides species when compared with Tgfb1+/+Rag2−/− mice. The qRT-PCR results showed an increase in Bacteroides fragilis and Bacteroides distasonis and a significant decrease in Bacteroides thetaiotaomicron in the Tgfb1−/−Rag2−/− mice. These data suggest that loss of TGFβ1 alters the colonic microflora. Previous studies illustrated the importance of bacterial nutrient sources on bacterial composition. To examine if TGFβ1 is altering nutrient availability in the colon a previous micro-array was analyzed for candidate genes associated with glycoprotein metabolism. This showed changes in fucose metabolizing enzymes with the loss of TGFβ1. Conclusion: These findings suggest that TGFβ1 plays a role in bacterial load maintenance, possibly by altering available nutrient sources and when disrupted, can cause abnormalities in pathobionts (commensal bacterial with pathogenic potential) which could then lead to increased inflammation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1957.

Tgfb1 knockout mice develop severe autoimmune inflammatory lesions and die within 3 wks after birth. Since the calcineurin inhibitor FK506 inhibits the hyperresponsiveness of Tgfb1−/− thymocytes, and since calcineurin Aβ‐deficient (Cnab−/−)T cells are hyporesponsive to stimulation, we have generated Tgfb1−/− Cnab−/− mice (double knockout mice) to address whether CnAβ deficiency prevents T‐cell activation and inflammation in Tgfb1−/− mice. Here we show that in Tgfb1−/− Cnab−/− mice inflammation is reduced relative to that in Tgfb1−/− mice. Both CD4+ and CD8+ T cells in double knockout (DKO) mice are activated. To our surprise, we have found that T cells undergo spontaneous activation in Cnab KO mice. Further analysis of Cnab KO mice revealed that Foxp3 expression is down regulated and total Treg cell numbers are reduced suggesting that CnAβ could regulate T‐cell activation through generation of Treg cells. These data also suggest a dual role for CnAβ in T‐cell activation and regulation.Grant support: This study was supported by NIH AI067903 and CA84291 to TD, and by a grant from Shriners of North America to GFB.