Effect of Surgically-Induced Weight Loss on Leukocyte Indicators of Chronic Inflammation in Morbid Obesity

Background: Recent evidence suggests that morbid obesity is a chronic inflammatory condition that may be associated with immune dysfunction.To test this hypothesis, we investigated several leukocyte cell surface markers of chronic inflammation and followed their response to surgically-induced weight loss. Methods: 26 patients having Roux-en-Y gastric bypass (RYGBP) for morbid obesity (BMI>40) were compared to 10 normal controls (BMI<25). Relative monocyte and neutrophil frequencies and expression of the activation antigens CD11b (adhesion molecule), CD16 (Fc receptor), and CD62L (Lselectin), were evaluated by flow cytometry preoperatively and at 1, 3, 6 and 12 months after RYGBP. Cases served as their own controls but were also compared to non-obese controls. The results were statistically analyzed using Student's t-test and ANOVA for parametric values and Mann-Whitney along with Kruskal-Wallis ANOVA for nonparametric values Results: The control group had mean age 37 ± 7.6 with mean 23 ± 2.5 and no comorbidities. The mean age of the sample group was 40.36 ± 13.7 with mean BMI 52 ± 8.2. The neutrophil and monocyte relative frequencies of CD11b (monocytes and neutrophils), and CD16 (neutrophils only) were comparable to controls at baseline and did not change significantly with weight loss throughout the study period. However, a significant reduction of CD62L (Lselectin) expression was noted in monocytes and neutrophils at baseline (neutrophils 103 vs 240 gmf, p<0.001) (monocytes104 vs 246 gmf, P<0.001) when compared to normal controls. Levels of L-selectin normalized by 6 months in both monocytes and neutrophils, and by 12 months had become abnormally elevated in monocytes (monocytes 391 gmf, P=0.007); in neutrophils, there was an upward trend that did not reach significance.The expression of the LPS receptor CD14 in the study group was elevated significantly compared to controls at baseline (1129 vs 719 gmf, P=0.004); this marker appeared to return to normal by 3 months. Monocyte CD14+/CD16+ subset percentage were also elevated significantly at baseline (14.3% vs 5.25%, P <0.001), declined throughout the time period but was still significant at 1 year (8.8%, P<0.001). Eosinophil percentages were elevated at baseline (3.3% obese vs 1.8% controls, P=0.003) and remained so throughout the time period. Conclusion: Deficiencies in the immune system of morbidly obese individuals include elevated levels of eosinophils, monocyte CD14, and monocyte CD14+/CD16+ subsets, with depression of monocyte and neutrophil CD62L. These abnormal levels reverse rapidly with surgically-induced weight loss. RYGBP is not only a weight loss operation but also appears to be an immune restorative procedure.     

Surgical influence on TH1/TH2 balance and monocyte surface antigen expression and its relation to infectious complications

Abstract Malignancy and operation for it cause several alterations in immune function that are considered to be concerned with the development of infectious complications. Forty-three patients who underwent curative surgery for gastrointestinal malignancies were entered into this study and were divided into two groups, those with and those without postoperative infection. Changes in the proportion of Th1/Th2 subsets in CD4⁺ T cells and the expression of human leukocyte antigen (HLA)-DR and CD16a molecules on monocytes were measured by flow cytometry before and after surgery. We performed intracellular cytokine stainings to exactly detect Th1/Th2 subsets. The proportions of interferon-γ-producing CD4⁺ T (Th1) cells in the preoperative state were almost equal in the two groups, and the proportion decreased on postoperative day (POD) 1 in both groups. On POD 7, the proportion of Th1 cells recovered to the preoperative level in the noninfection group, while the suppression was further reinforced in the infection group (26.8% versus 18.3%, p < 0.005). In contrast, the proportion of interleukin-4-producing CD4⁺ T (Th2) cells in the infection group (11.3%) was already suppressed in the preoperative state when compared with the noninfection group (17.3%, p < 0.005). Changes in HLA-DR and CD16a expression on monocytes were similar to the changes in the proportion of Th1 cells. These results indicate that the suppression of Th1 cell and monocyte functions during the early phase of the postoperative course was directly related to the occurrence of infectious complications and that several immunological impairments have already occurred in the preoperative state in cancer patients. Electronic Publication     

Hypoxia increases membranal and secreted HLA‐DR in endothelial cells,rendering them T‐cell activators

Transplantation involves preoperative ischemic periods that contribute to endothelial cell (EC) dysfunction and T‐cell activation, leading to graft rejection. As hypoxia is a major constituent of ischemia, we evaluated its effect on the ability of ECs to express HLA‐DR, which is required for presentation of antigens to T cells, and by itself serves as an important target for allogeneic T cells. Primary human umbilical vein ECs (HUVEC) and the human endothelial cell line EaHy926 were incubated in normoxia or hypoxia (PO₂ < 0.3%). Hypoxia increased the membranal expression (by 4–6 fold, P < 0.01) and secretion (by sixfold, P < 0.05) of HLA‐DR protein, without influencing the accumulation of its mRNA. Alternative splicing, attenuated trafficking, or shedding from the plasma membrane were not observed, but the lysosomal inhibitor bafilomycin A1 reduced HLA‐DR secretion. Hypoxia‐induced endothelial HLA‐DR elevated and diminished the secretion of IL‐2 and IL‐10, respectively, from co‐cultured allogeneic CD4⁺ T cells in a HLA‐DR‐dependent manner, as demonstrated by the use of monoclonal anti‐HLA‐DR. Our results indicate a yet not fully understood post‐translational mechanism(s), which elevate both membranal and soluble HLA‐DR expression. This elevation is involved in allogeneic T‐cell activation, highlighting the pivotal role of ECs in ischemia/hypoxia‐associated injury and graft rejection.     

Profiles of B-cell subsets in immunologically stable renal allograft recipients and end-stage renal disease patients

BackgroundPost-transplantation pharmacotherapies typically employ combinations of immunosuppressive agents that have been designed for targeted inhibition of T-cells and T-cell subsets. Studies of acute and chronic effects of clinically employed immunosuppressive agents on B-cells and B-cell subsets are significantly fewer in number and warrant further investigation. Accordingly, the goal of the present cross-sectional study is to functionally evaluate differences of B-cell subsets in patients with end-stage renal disease (ESRD) and immunologically stable renal transplant patients.Patients and methodsOf 103 patients who underwent renal transplantation, 73 patients were immunologically stable without rejection or infection. Among them, 34 patients were one-year post-transplantation, and 39 patients were five-year post-transplantation. The study also included 35 ESRD patients and 36 healthy volunteers. Flow cytometry identified B-cell subsets in the study groups.ResultsRenal allograft recipients had reduced percentages of total B-cells (CD19+) and regulatory B-cells (B_reg) (CD38^highCD27 + CD24+) compared with healthy controls. The percentage of transitional B-cells (IgM + CD38^highCD24^high) and marginal zone (MZ) B-cells (IgD-CD27+) was reduced in transplant recipients compared with patients with ESRD and healthy volunteers. The highest percentage of plasma cells (PCs) (CD38^highCD27 + CD24-) was in patients with ESRD. In five-year post-transplantation group, CD38^lowCD21- B-cells increased when compared with the other groups. Healthy volunteers and patients with ESRD had fewer unswitched memory (UM) B-cells (IgM + IgD + CD38^lowCD27+), and increased isotype switched memory (ISM) B-cells (IgM-IgD-CD38^lowCD27+). There was no difference in the percentage of naïve B-cells (IgD + CD27-) among diverse groups.ConclusionsThe percentages of the total, transitional, B_reg, PCs, MZ, and UM B-cell subsets in immunologically stable renal allograft recipients were significantly different from healthy controls. However, B-cell subsets in patients with ESRD were minimally different with immunologically stable renal allograft recipients.     

Cytomegalovirus-specific regulatory and effector T cells share TCR clonality--possible relation to repetitive CMV infections

Cytomegalovirus (CMV) infections have a major impact on morbidity and mortality of transplant patients. Among the complex antiviral T‐cell response, CMV‐IE‐1 antigen‐specific CD8+ cells are crucial for preventing CMV disease but do not protect from recurring/lasting CMV reactivation. Recently, we confirmed that adoptive transfer of autologous IE‐1/pp65‐specific T‐cell lines was able to combat severe CMV disease; however, the control of CMV infection was only temporary. We hypothesized that CMV‐induced regulatory T cells (iTreg) might be related to recurring/lasting CMV infection. In fact, kidney transplant patients with recurring CMV infections expressed enhanced suppression on CMV response. Analysis of in vitro expanded CD4+ epitope‐specific cells revealed that CMV‐specific CD4+CD25^high Treg cells functionally suppress CD25^low effector T cells (Teff) upon epitope‐specific reactivation. Their phenotype is similar to iTreg – CD39^high/Helios‐/IL‐2^low/IFNγ^high/IL‐10±/TGFß‐LAP±/FOXP3+ and methylated foxp3 locus. Remarkably, in vitro expanded CD4+CD25^high iTreg share the same dominant TCR‐Vβ‐CDR3 clones with functionally distinct CD4+CD25^low Teff. Moreover, the same clones were present in freshly isolated CD4+CD25^high and CD4+CD25^low T cells suggesting their in vivo generation. These findings directly demonstrate that Teff and iTreg can differentiate from one “mother” clone with specificity to the same viral epitope and indicate that peripheral iTreg generation is related to frequent antigen appearance.     

Human monocyte heterogeneity–a nephrological perspective

Monocytes are key components of the innate immune system and are circulating precursors of tissue macrophages. Phenotypically and functionally, monocytes are a heterogeneous leukocyte subset. Based on the expression of CD14 and CD16, three human monocyte subsets can be distinguished: CD14++CD16?, CD14++CD16+ and CD14(+)CD16+ monocytes. The latter two subsets are often summarized as CD16+ monocytes. As these CD16+ cells are expanded in inflammatory conditions including end-stage renal disease, they have traditionally been termed proinflammatory monocytes, which is in contrast to murine monocyte nomenclature. More, each dialysis session induces a transient CD16+ monocytopenia.. In end-stage renal disease, both higher predialytic counts of CD16+ monocytes, and dialysis-induced CD16+ monocyte kinetic are predictors of cardiovascular outcome. So far, the functional differences of monocyte subsets and their pathophysiological role are still insufficiently understood.     

Differential Expression of CD45 on Canine Microglial Cells

CD45, also called leucocyte common antigen is a transmembrane protein tyrosine phosphatase on the surface of nearly all white blood cells and has a functional role in signal transduction. In the brain, the expression of CD45 can be used to distinguish microglial cells with a characteristic phenotype of CD11b/c⁺ and CD45^low from other central nervous system (CNS) macrophages which show an expression of CD11b/c⁺ and CD45^high. In the course of pathological changes in the CNS, microglia in rodents is known to readily upregulate expression of various surface molecules, such as CD45. Understanding the mechanisms that regulate expression of surface molecules is essential to study the pathogenesis of CNS diseases. In the present study, the expression of CD45 on microglia of 42 dogs was examined ex vivo by means of flow cytometry. The dogs were classified in two groups according to the histopathological diagnosis in the CNS. All dogs without changes in the CNS (group I; n = 22) only showed low percentages of CD45⁺ microglial cells. In group II consisting of 20 dogs with different intracranial diseases varying results were obtained. Thirteen dogs showed a low percentage of CD45⁺ microglial cells whereas seven dogs exhibited high percentages of microglial cells expressing CD45. Evaluation of expression intensity in these seven dogs revealed two subpopulations of CD45⁺ microglial cells: a large subpopulation with CD45^low and a small subpopulation with CD45^high. The expression intensity of CD45^high was comparable with that of canine monocytes. It was attempted to correlate these findings to age of the animals, underlying disease, duration of clinical signs, medical treatment, occurrence of seizure activity and the expression of other surface molecules. It appeared that dogs with high percentages of CD45⁺ suffered from long‐lasting CNS disease with seizures. In future studies, the reason and consequences for upregulated CD45 in long‐lasting CNS diseases has to be further evaluated.     

CD14(++)CD16+ monocytes but not total monocyte numbers predict cardiovascular events in dialysis patients

Migration of monocytes into the vessel wall contributes to the onset and progression of atherosclerosis. Because monocytes are a heterogeneous population, we determined potential associations between monocyte subsets and cardiovascular events in a prospective cohort of 94 dialysis patients followed for 35 months. The incidence of cardiovascular events and death measured by Kaplan-Meier plots and flow cytometric analysis of monocyte subsets showed that total leukocyte and monocyte numbers failed to predict event-free survival. Among monocyte subsets, a high CD14(++)CD16(+) monocyte number was associated with higher rates of cardiovascular events and death. In a multivariate proportional hazards model adjusted for classical cardiovascular risk factors, patients with CD14(++)CD16(+) monocyte numbers in the top quartile were at higher risk of cardiovascular events and death compared to patients in the lowest quartile. Our study suggests that the number of CD14(++)CD16(+) monocytes was independently associated with cardiovascular events and death in a high-risk population of dialysis patients.     

Peripheral blood monocyte activation during coronary artery bypass grafting with or without cardiopulmonary bypass

Objective. The aim of this prospective, randomized study was to investigate the impact of coronary artery bypass grafting (CABG) on peripheral monocytes and to evaluate the additional effect of cardiopulmonary bypass (CPB).

Design. Twenty patients admitted for elective CABG were randomized to either on-pump (ONCAB, n=9) or off-pump (OFFCAB, n=11) surgery and blood samples were drawn before, during and 24 h after the operation. The total number of monocytes and the proportion of the more mature CD16⁺/CD14⁺ monocytes were measured. Expression of activation markers (CD11b, CD35 and CD62L) and oxidative burst were determined using flow cytometry on both resting and in vitro stimulated cells. Serum concentrations of soluble CD14 and monocytes/macrophage chemotactic protein 1 (MCP-1) were analysed.

Results. During surgery there was a selective decrease in the proportion of CD16⁺/CD14⁺ monocytes compared to total monocytes. These had returned to preoperative values 24 h after surgery while the total number of monocytes had increased more than 100%. Intracellular production of oxygen free radical H₂O₂ was increased in the ONCAB group during surgery compared to OFFCAB. Monocyte expression and in vitro mobilization of complement receptors, CD11b and CD35, were similar in both study groups during and after surgery as was the expression of CD62L. Serum levels of MCP-1 decreased during surgery as did soluble CD14, both with increased levels again the day after surgery.

Conclusion. It is concluded that the circulating monocyte population is activated during and as a consequence of CABG. There were few apparent additional effects of CPB found in this study. In this setting the inflammation caused by the surgery procedure per se probably surpasses the impact of the CPB on circulating blood monocytes.     

Monocyte function-associated antigen expression during and after pediatric cardiac surgery

OBJECTIVE: Systemic inflammatory response syndrome and infectious complications are major causes of morbidity and mortality after cardiopulmonary bypass. Recent work in adult patients suggests that the balance between proinflammatory and anti-inflammatory mediators is important. We hypothesized that the expression of different function-related receptors on circulating monocytes might reflect the net response of the inflammatory reaction. METHODS: We performed a prospective and observational study in a tertiary pediatric cardiac center in a population of children (n = 40) undergoing elective cardiac surgery. Expression of receptors on the surface of monocytes was assessed before, during, and after surgical intervention. RESULTS: Early monocyte activation was demonstrated by changes of the expression of the chemokine receptor CCR2, which was inversely correlated with plasma levels of monocyte chemotactic protein 1 (rho = -0.54, P = .002). High levels of monocyte chemotactic protein 1 were found in children with high expression of the adhesion receptor CD11b/CD18 on circulating monocytes. The intensity of human leukocyte antigen DR expression rapidly decreased in all children after the onset of cardiopulmonary bypass ( P < .001). Low human leukocyte antigen DR expression was correlated with increased plasma levels of interleukin 10 postoperatively. Children who had signs of bacterial pneumonia postoperatively had lower levels of human leukocyte antigen DR expression before surgical intervention (relative risk, 13.3; P = .007). CONCLUSIONS: The expression of monocyte function-related receptors is altered after cardiac surgery. Early activation of monocytes by monocyte chemotactic protein 1 possibly released from the heart is followed by an anti-inflammatory response with suppression of monocyte human leukocyte antigen DR expression. The increased risk of bacterial infection after pediatric cardiac surgery can be anticipated by surveillance of monocyte function before surgical intervention.     

T cell receptor beta chain (TCR-Vβ) repertoire of circulating CD4+ CD25−, CD4+ CD25low and CD4+ CD25high T cells in patients with long-term renal allograft survival

The mechanisms underlying maintenance of renal allografts in humans under minimal or conventional immunosuppression are poorly understood. There is evidence that CD4⁺ CD25⁺ regulatory T cells and clonal deletion, among other mechanisms of tolerance, could play a key role in clinical allograft survival. Twenty‐four TCR‐Vβ families were assessed in CD4⁺ CD25^?, CD4⁺ CD25^low and CD4⁺ CD25^high T cells from patients with long‐term renal allograft survival (LTS), patients exhibiting chronic rejection (ChrRx), patients on dialysis (Dial) and healthy controls (HC) by flow cytometry. LTS patients presented a higher variability in their TCR‐Vβ repertoire, such decreased percentage of Vβ2⁺, Vβ8a⁺ and Vβ13⁺ in CD4⁺ CD25^low and ^high compared with CD4⁺ CD25^? subset and increased Vβ4 and Vβ7 families in CD4⁺ CD25^high T cells exclusively. Additionally, LTS patients, particularly those that were not receiving calcineurin inhibitors (CNI), had increased percentages of CD4⁺ CD25^high T cells when compared with Dial (P < 0.05) and ChrRx (P < 0.05) patients. Our results suggest that a differential expression of particular TCR‐Vβ families and high levels of circulating CD4⁺ CD25^high T cells in long‐term surviving renal transplant patients could contribute to an active and specific state of immunologic suppression. However, the increase in this T cell subset with regulatory phenotype can be affected by CNI.     

Hyperlipidemia Alters Regulatory T Cell Function and Promotes Resistance to Tolerance Induction Through Costimulatory Molecule Blockade

Recent work from our laboratory has shown that hyperlipidemia promotes accelerated rejection of vascularized cardiac allografts in mice by inducing anti‐donor Th17 reactivity and production of IL‐17. Here, we show that hyperlipidemia also affects FoxP3⁺ regulatory T cells (Tregs). Hyperlipidemia promotes the development of Tregs that express low levels of CD25. Hyperlipidemia also promotes a decrease in central Tregs and an increase in effector Tregs that appears to account for the increase in the frequency of CD25^low Tregs. Alterations in Treg subsets also appear to lead to alterations in Treg function. The ability of FoxP3⁺, CD25^high_, CD4⁺ Tregs from hyperlipidemic mice to inhibit proliferation of effector T cells stimulated with anti‐CD3 and CD28 was reduced when compared with Tregs from control mice. Regulatory T cells isolated from hyperlipidemic recipients exhibit increased activation of Akt, and a reduction in Bim levels that permits the expansion of FoxP3⁺CD25^lowCD4⁺ T cells. Hyperlipidemic mice were also resistant to tolerance induction using costimulatory molecule blockade consisting of anti‐CD154 and CTLA4Ig, a strategy that requires Tregs. Together, our data suggest that hyperlipidemia profoundly affects Treg subsets and function as well as the ability to induce tolerance.     

Myeloid‐derived suppressor cells increase and inhibit donor‐reactive T cell responses to graft intestinal epithelium in intestinal transplant patients

Recent advances in immunosuppressive regimens have decreased acute cellular rejection (ACR) rates and improved intestinal and multivisceral transplant (ITx) recipient survival. We investigated the role of myeloid‐derived suppressor cells (MDSCs) in ITx. We identified MDSCs as CD33⁺CD11b⁺ lineage(CD3/CD56/CD19)^?HLA‐DR^?/low cells with 3 subsets, CD14^?CD15^? (e‐MDSCs), CD14⁺CD15^? (M‐MDSCs), and CD14^?CD15⁺ (PMN‐MDSCs), in peripheral blood mononuclear cells (PBMCs) and mononuclear cells in the grafted intestinal mucosa. Total MDSC numbers increased in PBMCs after ITx; among MDSC subsets, M‐MDSC numbers were maintained at a high level after 2 months post ITx. The MDSC numbers decreased in ITx recipients with ACR. MDSC numbers were positively correlated with serum interleukin (IL)‐6 levels and the glucocorticoid administration index. IL‐6 and methylprednisolone enhanced the differentiation of bone marrow cells to MDSCs in vitro. M‐MDSCs and e‐MDSCs expressed CCR1, ‐2, and ‐3; e‐MDSCs and PMN‐MDSCs expressed CXCR2; and intestinal grafts expressed the corresponding chemokine ligands after ITx. Of note, the percentage of MDSCs among intestinal mucosal CD45⁺ cells increased after ITx. A novel in vitro assay demonstrated that MDSCs suppressed donor‐reactive T cell–mediated destruction of donor intestinal epithelial organoids. Taken together, our results suggest that MDSCs accumulate in the recipient PBMCs and the grafted intestinal mucosa in ITx, and may regulate ACR.     

外科术后危重患者单核细胞HLA-DR表达的临床研究

目的研究外科术后危重患者外周血CD14^＋单核细胞HLA—DR的表达及动态变化与其预后的关系。方法应用流式细胞术检测并比较30例外科手术后呼吸机脱机困难而转入ICU的危重患者和28名同期体检健康者的外周血CD14^＋单核细胞HLA—DR的表达率。动态观察入ICU第1、4、7天单核细胞HLA-DR的表达率的变化,并记录APACHEⅡ评分、全身性感染相关性器官功能衰竭评分（SOFA）及28d的短期预后。结果与健康志愿者相比,外科术后危重患者外周血CD14^＋单核细胞HLA—DR的表达率降低（P〈0．01）。人ICU第1天的HLA—DR表达率与SOFA、APACHEⅡ评分及预后无相关性。第7天HLA—DR表达率较第1天提高的患者其28d的短期预后较好（P〈0．01）。结论外科术后危重患者外周血CD14^＋单核细胞HLA—DR的表达率较正常人降低;入ICU当天的单核细胞HLA—DR的表达率不能提示预后,而在治疗过程中,动态地观察外周血CD14^＋单核细胞HLA—DR的表达率才能对患者的预后作出正确的评估。     

Impact of Basiliximab on regulatory T‐cells early after kidney transplantation: down‐regulation of CD25 by receptor modulation

Monoclonal anti‐CD25‐antibodies are successfully applied in organ transplantation to reduce the incidence of acute graft rejection. However, targeting the CD25 molecule might not only affect activated T‐cells but also regulatory T‐cells (T_regs) constitutively expressing the CD4⁺CD25⁺CD127^lowFoxP3⁺ phenotype. In this study, we investigated the influence of the anti‐CD25‐antibody Basiliximab on the frequency of T_regs early after kidney transplantation comparing individuals receiving/not receiving induction therapy (n = 14 and n = 7). Following Basiliximab administration, a distinct loss of CD4⁺CD25^high T‐cells was observed lasting for at least 6 weeks. This was not accompanied by a disappearance of the entire CD4⁺CD25⁺FoxP3⁺ T_regs but rather a decreased expression density of CD25 on the latter. In addition, a transient rise in CD4⁺CD25^?FoxP3⁺ T‐cells was found which expressed the CD127^low phenotype. Thus, a phenotypic shift of T_regs from the CD25⁺ to the CD25^? compartment was suggested. This was supported by in vitro findings showing that the disappearance of CD4⁺CD25^high cells in the presence of Basiliximab was due to down‐regulation of CD25 expression meanwhile the suppressive function of these cells was maintained. In conclusion, Basiliximab therapy directly affects CD4⁺CD25⁺CD127^lowFoxP3⁺ T_regs but does not seem to be associated with functional consequences. Thus, it is unlikely that Basiliximab treatment negatively influences strategies involving T_regs to promote tolerance after organ transplantation.     

Class II HLA Epitope Matching—A Strategy to Minimize De Novo Donor‐Specific Antibody Development and Improve Outcomes

De novo donor‐specific antibody (dnDSA) develops in 15–25% of renal transplant recipients within 5 years of transplantation and is associated with 40% lower graft survival at 10 years. HLA epitope matching is a novel strategy that may minimize dnDSA development. HLAMatchmaker software was used to characterize epitope mismatches at 395 potential HLA‐DR/DQ/DP conformational epitopes for 286 donor–recipient pairs. Epitope specificities were assigned using single antigen HLA bead analysis and correlated with known monoclonal alloantibody epitope targets. Locus‐specific epitope mismatches were more numerous in patients who developed HLA‐DR dnDSA alone (21.4 vs. 13.2, p < 0.02) or HLA‐DQ dnDSA alone (27.5 vs. 17.3, p < 0.001). An optimal threshold for epitope mismatches (10 for HLA‐DR, 17 for HLA‐DQ) was defined that was associated with minimal development of Class II dnDSA. Applying these thresholds, zero and 2.7% of patients developed dnDSA against HLA‐DR and HLA‐DQ, respectively, after a median of 6.9 years. Epitope specificity analysis revealed that 3 HLA‐DR and 3 HLA‐DQ epitopes were independent multivariate predictors of Class II dnDSA. HLA‐DR and DQ epitope matching outperforms traditional low‐resolution antigen‐based matching and has the potential to minimize the risk of de novo Class II DSA development, thereby improving long‐term graft outcome.     

Ex Vivo Expansion of Human Tregs by Rabbit ATG Is Dependent on Intact STAT3‐Signaling in CD4+ T Cells and Requires the Presence of Monocytes

The addition of low, nondepleting doses of rabbit antithymocyte globulin (ATG) to human peripheral blood mononuclear cells has been shown to expand functional CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) in vitro. This report is the first to elucidate the exact cellular mechanisms of ATG‐mediated Treg expansion. CD4⁺ T cells require monocytes, but not other antigen presenting cell subsets, to be present in coculture to expand Tregs. However, T cells do not require direct cell–cell contact with monocytes, suggesting the importance of soluble factors. Moreover, ATG initially “reprograms” CD4⁺ T cells, but not monocytes, and induces STAT3 and STAT5 signaling in CD4⁺ cells. These reprogrammed CD4⁺ T cells subsequently secrete GM‐CSF and IL‐10 only in case of intact STAT3 signaling, which in turn promote the generation of tolerogenic CD14⁺CD11c⁺ dendritic cells characterized by enhanced IL‐10 and decreased IL‐12 production. Treg expansion following ATG treatment is accompanied by enhanced gene expression of both GM‐CSF and Bcl‐2, but not TGF‐β, in peripheral blood mononuclear cells. These results demonstrate that ex vivo expansion of human Tregs by ATG is due to its ability to reprogram CD4⁺ T cells in a STAT3‐dependent but TGF‐β‐independent manner, leading to the generation of monocyte‐derived dendritic cells with a tolerogenic cytokine profile.     

Differential Ly6C Expression after Renal Ischemia-Reperfusion Identifies Unique Macrophage Populations

Macrophages are a heterogeneous cell type implicated in injury, repair, and fibrosis after AKI, but the macrophage population associated with each phase is unclear. In this study, we used a renal bilateral ischemia-reperfusion injury mouse model to identify unique monocyte/macrophage populations by differential expression of Ly6C in CD11b⁺ cells and to define the function of these cells in the pathophysiology of disease on the basis of microarray gene signatures and reduction strategies. Macrophage populations were isolated from kidney homogenates by fluorescence-activated cell sorting for whole genome microarray analysis. The CD11b⁺/Ly6C^high population associated with the onset of renal injury and increase in proinflammatory cytokines, whereas the CD11b⁺/Ly6C^intermediate population peaked during kidney repair. The CD11b⁺/Ly6C^low population emerged with developing renal fibrosis. Principal component and hierarchical cluster analyses identified gene signatures unique to each population. The CD11b⁺/Ly6C^intermediate population had a distinct phenotype of wound healing, confirmed by results of studies inhibiting the macrophage colony-stimulating factor 1 receptor,whereas the CD11b⁺/Ly6C^low population had a profibrotic phenotype. All populations, including the CD11b⁺/Ly6C^high population, carried differential inflammatory signatures. The expression of M2-specific markers was detected in both the CD11b⁺/Ly6C^intermediate and CD11b⁺/Ly6C^low populations, suggesting these in vivo populations do not fit into the traditional classifications defined by in vitro systems. Results of this study in a renal ischemia-reperfusion injury model allow phenotype and function to be assigned to CD11b⁺/Ly6C⁺ monocyte/macrophage populations in the pathophysiology of disease after AKI.