Objective: Reduced numbers of regulatory T (T reg ) cells have been observed in visceral adipose tissue of obese mice and humans. However, it is unknown whether human obesity affects circulating Treg cells and whether their number is associated with markers of systemic inflammation or glucose intolerance. Design and Methods: Peripheral blood mononuclear cells were isolated from venous blood of obese (BMI ! 27 kg/m 2 ; n ¼ 30) and nonobese (BMI ! 27 kg/m 2 ; n ¼ 13) individuals and analyzed using flow cytometry for the expression of CD4, CD25, and Foxp3. Results: Reduced circulating T reg -cell numbers were detected in obese compared with nonobese study participants (P ¼ 0.038). Circulating CD4 þ CD25 þ CD127 À Foxp3 T reg cells inversely correlated with body weight (P ¼ 0.009), BMI (P ¼ 0.004) and plasma leptin levels (P ¼ 0.004) and were reduced in subjects with hsCRP ! 3.0 mg/L (P ¼ 0.034) or HbA1c ! 5.5% (P < 0.005). Receiver operating characteristic curve analysis revealed a cutoff of circulating Treg cells < 1.06% to be predictive for hsCRP levels ! 3.0 mg/L, and logistic regression showed that the risk of having hsCRP levels ! 3.0 mg/L was increased 9.6-fold (P ¼ 0.008), if T reg cells were below this threshold. The T reg cutoff for HbA1c levels ! 5.5% was 0.73%, and this cutoff also predicted an increased risk of having elevated levels of both hsCRP and HbA1c, if only obese subjects were examined. Conclusion: Our findings thus reveal an association between circulating T reg cells and measures of adiposity, inflammation, and glucose intolerance. Although further prospective studies are needed, we present data suggesting that the determination of T reg cells might be useful to identify obese subjects at increased risk of developing cardiovascular and/or metabolic complications.

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BackgroundBreast and colon cancer rank second and third, respectively, in world-wide prevalence of malignancies and present a large unmet medical need. The correlation between lymphocyte infiltration into the tumor microenvironment and efficacy of anti-cancer immunotherapies has been established. Therefore, relevant and cost-saving pre-clinical models are needed for developing new treatment approaches to predominant human tumor types. HuCD34NCG mice facilitate studying human immune responses in vivo elicited by experimental therapeutic antibodies. We characterized growth kinetics and human immune responses to checkpoint blockade in human breast and colon tumor-bearing HuCD34NCG mice. Aging, non tumor-bearing HuCD34NCG mice were also monitored for indicators of spontaneous hematopoietic cancer formation.MethodsHSC engraftment was quality controlled prior to inoculating HuCD34NCG mice with either colon adenocarcinoma (COLO 205) or triple negative breast cancer (MDA-MB-436) cells (both purchased from American Type Culture Collection, Manassas, VA). Mice were randomized into treatment groups based on tumor size, and checkpoint inhibitor antibodies were dosed twice weekly (anti-human PD-1, BioXcell clone: RMP1-14 or Keytruda; anti-human CTLA-4, BioXcell clone: BN13; and combination therapy). Body weights, general health status and survival were monitored. Peripheral blood (PB) and selected tissues were analyzed for the presence and composition of human immune cells by acoustic focusing flow cytometry. Non tumor-bearing aged HuCD34NCG mice (27 weeks post-engraftment) were sampled biweekly over ten weeks for lymphoma immunophenotyping.ResultsBoth tumor-bearing models showed significant anti-hPD-1 and anti-hCTLA-4 responses, but combination therapy only enhanced growth reduction significantly in MDA-MB-436 tumors. Flow cytometric analysis identified viable human leukocytes in tumor and spleen at study termination. These tumor-infiltrating lymphocytes (TIL) and splenocytes from surviving COLO 205 and MDA-MB-436 mice consisted of a total T-cell phenotype (CD3+) with proliferating (Ki67+), CD4+, CD8+ and Treg subsets. Additionally, myeloid cells (CD11b+, CD11c+) and M1/M2 macrophages were detected within these infiltrates. Splenic and tumor-infiltrating T-cells readily secreted human cytokines (IFN-γ, IL-2, TNF-α) and granzyme B upon ex vivo activation exhibiting polyfunctional and cytotoxic capabilities in all treatment groups. Baseline murine and human cytokine levels were distinguished in plasma from aging, non tumor-bearing HuCD34NCGs. Their phenotypes also showed no conclusive indicators of abnormal blood cells developing or graft failure.ConclusionsBreast and colon tumor cell-line derived models were established in HuCD34NCG mice. Standard checkpoint inhibitor treatment promoted human T-cell infiltration into tumor microenvironments inhibiting growth. These results demonstrate that HuCD34NCG are a robust and relevant host for various human cell xenotransplants to advance preclinical immuno-oncology drug development.Ethics ApprovalAnimal studies were executed in compliance with local Charles River IACUC guidelines, IACUC number I-033.

The aim of this study was to elucidate functional and molecular effects of mycophenolic acid (MPA) on non-lymphatic, kidney epithelial cells treated with transforming growth factor (TGF). MPA effects were studied using HK2 cells incubated with EGF and TGF. The reversibility of these effects was verified using guanosine and 8-aminoguanosine. The following assays were applied: cell proliferation, viability, collagen matrix contraction, scratch wound closure, spindle index, FACS with anti-CD29 and anti-CD326, promoter demethylation of RAS protein activator like 1 (RASAL1), as well as gene expression of RASAL1, integrin 1ß (ITGB1) (CD29) and epithelial cell adhesion molecule (EpCam) (CD326). Cell proliferation was inhibited by increasing concentrations of MPA, whereas neither apoptosis nor cytotoxicity was detected. Stimulation with EGF and/or TGF led to a significant collagen matrix contraction that was successfully inhibited by MPA. In addition, scratch wound closure was inhibited by incubation with TGF alone or with EGF. Under the same conditions, cell morphology (spindle shape) and molecular phenotype (ITGB1(High)EpCam(Low)/ITGB1(Low)EpCam(High)) were both significantly changed, suggesting an epithelial to mesenchymal transformation. Cell morphology and motility, as well as molecular phenotype, were reversible after MPA treatment with TGF transformation in both presence/absence of EGF, thereby suggesting a correlation with the previously described antifibrotic effects of MPA. Dysregulation of TGF signal transduction appears to be related to progression of fibrosis. A TGF-transformed kidney epithelial cell line derived from human proximal tubules was used to study whether the immunosuppressive drug: MPA possesses any functional or molecular antifibrotic effects. Functional and morphological in vitro changes induced by both the TGF and epithelial-growth-factor were reversible by treatment with MPA. An inhibitory effect of MPA on the TGF pathway appears to be responsible for the previously described antifibrotic effects of the MPA in the COL4A3-deficient mouse model of renal fibrosis.