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.     

胰腺癌患者外周血中树突状细胞的分离、纯化与扩增

目的 探讨从胰腺癌患者外周血中分离、纯化与扩增树突状细胞（DC）的有效方法。方法 分别从健康人（10例，对照组）和胰腺癌患者（12例，实验组）外周血中分离出单核细胞，而后将实验组的单核细胞与GM－CSF及IL－4共同培养，用免疫荧光法和流式细胞仪检测培养前、后的DC数量及DC表面HLA－DR及B7－2的表达水平，并与对照组比较。结果 与对照组相比，实验组DC表面HLA－DR及B7－2表达水平较低（P＜0.01）；经GM－CSF及IL－4联合培养7天后，其单核细胞中的DC数较培养前明显增多（P＜0.01），且DC表面HLA－DR及B7－2表达水平较培养前明显增高（P＜0.01），与对照组相比则无明显差异（P＞0.05）。结论 GM－CSF与IL－4的联合应用能有效地从胰腺癌患者外周血中制备出大量具有功能活性的DC。     

Development of a Microarray for Studying Porcine Cytokine Production in Blood Mononuclear Cells and Intestinal Biopsies

A microarray for demonstration of a limited number of porcine cytokines was initiated. Polymerase chain reaction (PCR) products were synthesized for four house‐keeping genes, cyclophilin, β‐actin, hypoxanthine phosphoribosyltransferase (HPRT) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and the following cytokines: interleukin (IL)‐1β, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12 p35, IL‐12 p40, IL‐18, interferon (IFN)‐α, IFN‐β, IFN‐γ, tumour necrosis factor (TNF)‐α, macrophage inhibition factor (MIF) and granulocyte/macrophage colony‐stimulating factor (GM‐CSF). Cytokine production was induced by incubation of porcine peripheral blood mononuclear cells (PBMC) with Concanavalin A (ConA) or oligodeoxynucleotide (ODN) 2216. RNA was isolated after 6 or 24 h from stimulated cells or unstimulated control cells and from intestinal biopsies. Cytokine expression was analysed using a 3‐DNA Array 350^TM labelling kit from Genisphere. Data were normalized using external control genes and analysed with the genepix pro 5.0 software. All the cytokines could be induced in PBMC and expressed on the array and the cytokines IL‐6 and IFN‐α were also analysed at protein level. All but one cytokine were expressed in samples from intestinal biopsies. Densitometric analyses of PCR products of the house‐keeping genes were performed to validate the results from the microarray. Thus, this microarray will enable analyses of the cytokine profile during local and systemic infections in the pig.     

CD4+ CD25+ regulatory T cells suppressed the indirect xenogeneic immune response mediated by porcine epithelial cell pulsed dendritic cells

Nishimura T, Onda M, Takao S. CD4⁺ CD25⁺ regulatory T cells suppressed the indirect xenogeneic immune response mediated by porcine epithelial cell pulsed dendritic cells. Xenotransplantation 2010; 17: 313–323. © 2010 John Wiley & Sons A/S. Abstract: Background: CD4⁺ CD25⁺ regulatory T cells have been reported to suppress T cell‐mediated xenogeneic immune responses. Although the direct T cell response to xenogeneic cells is important, the indirect xenogeneic immune response mediated by dendritic cells (DCs) is also likely involved in rejection. We have generated an in vitro indirect immune reaction model and evaluated the effect of CD4⁺ CD25⁺ regulatory T cells on this system. Methods: Human DCs were generated from peripheral blood and cultured with X‐ray‐irradiated porcine kidney epithelial cells. Porcine cell‐pulsed DCs were mixed with autologous CD4⁺ T cells, CD4⁺ CD25^? T cells and/or CD4⁺ CD25⁺ T cells. After 7 days of culture, T cell proliferation was measured. Results: The co‐culture of human DCs and X‐ray‐irradiated porcine epithelial cells resulted in observable DC phagocytic activity within 2 days. These porcine cell–pulsed DCs stimulated CD4⁺ T cell proliferation much more potently than unpulsed DCs or porcine cells. This proliferation was blocked by CTLA4‐Ig or an anti‐HLA‐DR antibody. CD4⁺ CD25⁺ regulatory T cells also suppressed CD4⁺ CD25^? T cell proliferation in response to porcine cell‐pulsed DCs. Conclusions: An in vitro model of the indirect xenogeneic immune response was established. Porcine cell‐pulsed DCs stimulated CD4⁺ T cells, and CD4⁺ CD25⁺ regulatory T cells suppressed this response.     

Induction of porcine‐specific regulatory T cells with high specificity and expression of IL‐10 and TGF‐β1 using baboon‐derived tolerogenic dendritic cells

Background

Regulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo‐organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen‐specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non‐human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno‐specific Treg would benefit research on immune tolerance in xenotransplantation using this model system.

Method

Baboon tolerogenic dendritic cells (tolDC) were generated in 3 days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti‐inflammatory cytokines. After loading with porcine‐specific (PS) in vitro‐transcribed RNA (ivtRNA), tolDC were used to induce CD4⁺ T cells to become porcine‐specific Treg (PSTreg) in cocultures supplemented with IL‐2 and rapamycin for 10 days. Anti‐inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine‐specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity.

Results

TolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL‐10 and TGF‐β1, whereas IL‐12p40 and IFN‐γ were not expressed. PSTreg were successfully generated in cocultures of CD4⁺ T cells and PS ivtRNA‐loaded tolDC. They exhibited a CD3⁺ CD4⁺ CD25⁺ CD127^low/? CD45RA^low Foxp3⁺ phenotype and were characterized by high expression of IL‐10 and TGF‐β1 mRNA and protein. They showed upregulated expression of EBI3 and GARP mRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL‐10 and TGF‐β1 cytokine upon interaction with PSTeff and suppressing IFN‐γ expression on PSTeff.

Conclusion

In this study, a fast 3‐day method to generate baboon‐derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine‐antigen specificity and expression of IL‐10 and TGF‐β1. Porcine‐specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons.     

Intrarenal B Cell Cytokines Promote Transplant Fibrosis and Tubular Atrophy

Renal transplantation is the optimum treatment for end‐stage renal failure. B cells have been identified in chronic allograft damage (CAD) and associated with the development of tertiary lymphoid tissue within the human renal allograft. We performed renal transplantation in mice to model CAD and identified B cells forming tertiary lymphoid tissue with germinal centers. Intra‐allograft B220⁺ B cells comprised of IgM^high CD23^? B cells, IgM^lo CD23⁺ B cells, and IgM^lo CD23^? B cells with elevated expression of CD86. Depletion of B cells with anti‐CD20 was associated with an improvement in CAD but only when administered after transplantation and not before. Isolated intra‐allograft B cells were cultured and shown to synthesize multiple cytokines, the most abundant of these were GRO‐α (CXCL1), RANTES (CCL5), IL‐6 and MCP‐1 (CCL2). Tubular loss was observed with T cell accumulation within the allograft and development of interstitial fibrosis, whilst type III collagen deposition was observed in areas of F4/80⁺ macrophages and PDGFR‐β⁺ and transgelin⁺ fibroblasts, all of which were reduced by B cell depletion. We have shown that intra‐allograft B cells are key mediators of CAD. B cells possibly contribute to CAD by intra‐allograft secretion of cytokines and chemokines.

     

Thymosin-α1 modulates dendritic cell differentiation from human CD14+ monocytes

Introduction. Thymosin-α1 (Tα1) has established immunomodulatory effects on T cells, NK cells, and macrophages. The objective of this study was to determine the effect of Tα1 on DC differentiation, activation, and functional maturation. Methods. CD14-positive monocytes were purified from human peripheral blood mononuclear cells (PBMC) by MACS separator. Cells were treated with GM-CSF and IL-4 in the absence or presence of Tα1 and cell-surface markers were monitored using FACS analysis. Functional endocytosis of DCs was analyzed by antigen-uptake of FITC-conjugated Dextran beads. Ability of antigen presentation of matured DCs (mDCs) was monitored using an allogeneic mixed lymphocyte reaction assay (MLR) by [³H]-thymidine incorporation. Finally, the release of Th1 and Th2 polarizing cytokines by allogeneic T cells was analyzed by Bioplex cytokine assay. Results. Tα1 up regulated the iDC surface expression of CD11c^lo and CD11b^lo by 2- and 30-fold, respectively. iDC surface costimulatory molecules CD40, CD83, and CD86 were also up-regulated about 10% by Tα1 treatment. There was about 10% of reduced antigen uptake ability by Tα1-treated iDCs. During TNF-α-induced maturation, levels of MHC class II^lo and CD4^lo markers were up regulated by 3-fold in the presence of Tα1. Furthermore, Tα1-treated mDCs increased allogeneic CD3⁺ T cell proliferation by 2-fold as compared with non-Tα1-treated mDCs. However, there were no significant differences in Th1 or Th2 types of cytokines production by allogeneic T cells stimulated by either Tα1-treated or nontreated mDCs. Conclusions. Tα1 significantly enhances the differentiation and maturation of CD14⁺ monocytes derived iDCs. Furthermore, Tα1 decreases iDCs to endocytose foreign antigens and increases mDCs capacity for antigen presentation and stimulation of allogeneic T cell proliferation. These results suggest that Tα1 exerts its immunomodulatory effects on the immune system via direct interaction with DCs.     

In Vivo Mobilization and Functional Characterization of Nonhuman Primate Monocytic Myeloid‐Derived Suppressor Cells

Increasing evidence from small animal models shows that myeloid‐derived suppressor cells (MDSCs) can play a crucial role in inhibiting allograft rejection and promoting transplant tolerance. We identified CD3^?CD20^?HLA‐DR^?CD14⁺CD33⁺CD11b⁺ cells in peripheral blood of healthy rhesus macaques. These putative monocytic MDSCs constituted 2.1% ± 1.7% of lin^?HLA‐DR^? peripheral blood mononuclear cells. Administration of granulocyte‐macrophage colony‐stimulating factor (CSF) and granulocyte CSF increased their incidence to 5.3% ± 3.4%. The total number of MDSCs that could be flow sorted from a single whole rhesus leukapheresis product was 38 ± 13 × 10⁶ (n = 10 monkeys). Freshly isolated or cryopreserved MDSCs from mobilized monkeys incorporated in cultures of anti‐CD3– and anti‐CD28–stimulated autologous T cells markedly suppressed CD4⁺ and CD8⁺ T cell proliferation and cytokine secretion (interferon γ, IL‐17A). Moreover, these MDSCs enhanced CD4⁺CD25^hiFoxp3⁺ regulatory T cell (Treg) expansion while inhibiting proliferation of activated memory T cells and increasing Treg relative to effector and terminally differentiated memory T cells. Inhibition of arginase‐1, but not inducible nitric oxide synthase activity, partially reversed the inhibitory effect of the MDSCs on CD8⁺ T cell proliferation. Consequently, functional MDSCs can be isolated from nonhuman primates for prospective use as therapeutic cellular vaccines in transplantation.

     

Phase I/II trial of subcutaneous interleukin-2, granulocyte-macrophage colony-stimulating factor and interferon-α in patients with metastatic renal cell carcinoma

Study Type – Therapy (individual cohort) Level of Evidence 2b

OBJECTIVE

? To determine, in a phase I/II trial, the maximum tolerated dose (MTD), clinical activity and safety of concurrent subcutaneous (s.c.) interleukin‐2 (IL‐2), interferon‐α2b (IFN‐α) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF).

PATIENTS AND METHODS

? Patients with metastatic renal cell carcinoma (RCC) received on a 3+3 trial design escalating doses of s.c. GM‐CSF, IL‐2 and IFN‐α. ? Dose‐limiting toxicities (DLTs) during the first 6‐week cycle were used to determine the MTD. ? A phase II trial was then initiated to determine clinical activity.

RESULTS

? A total of sixty patients were enrolled in the study (phase I = 31; phase II = 29). ? Two DLTs were observed (G3 nausea/vomiting and fatigue) and the MTD was determined to be GM‐CSF 5.0 µg/kg/day, IL‐2 9.0 mIU/m²/day and IFN‐α 5.0 mU/m²/day. ? Patients received a median (range) of four (one to 11) cycles of therapy. G3 adverse events were reported in 10 of 31 (32%) patients. ? The overall response rate was 20% (one complete response and 11 partial responses), including patients who were rendered free of disease with surgery. ? The median progression‐free survival and overall survival were 6.0 and 23.4 months, respectively.

CONCLUSIONS

? Immunotherapy with concurrent s.c. GM‐CSF, IL‐2 and IFN‐α is generally well tolerated. ? The overall response rate observed with this combination continues to show the efficacy of immunotherapy in a selected group of metastatic RCC patients.     

Impact of Human Mutant TGFβ1/Fc Protein on Memory and Regulatory T Cell Homeostasis Following Lymphodepletion in Nonhuman Primates

Transforming growth factor β1 (TGFβ1) plays a key role in T cell homeostasis and peripheral tolerance. We evaluated the influence of a novel human mutant TGFβ1/Fc (human IgG4 Fc) fusion protein on memory CD4⁺ and CD8⁺ T cell (Tmem) responses in vitro and their recovery following antithymocyte globulin (ATG)–mediated lymphodepletion in monkeys. TGFβ1/Fc induced Smad2/3 protein phosphorylation in rhesus and human peripheral blood mononuclear cells and augmented the suppressive effect of rapamycin on rhesus Tmem proliferation after either alloactivation or anti‐CD3/CD28 stimulation. In combination with IL‐2, the incidence of CD4⁺CD25^hiFoxp3^hi regulatory T cells (Treg) and Treg:Th17 ratios were increased. In lymphodepleted monkeys, whole blood trough levels of infused TGFβ1/Fc were maintained between 2 and 7 μg/mL for 35 days. Following ATG administration, total T cell numbers were reduced markedly. In those given TGFβ1/Fc infusion, CD8⁺ T cell recovery to predepletion levels was delayed compared to controls. Additionally, numbers of CD4⁺CD25^hiCD127^lo Treg increased at 4–6 weeks after depletion but subsequently declined to predepletion levels by 12 weeks. In all monkeys, CD4⁺CD25^hiFoxp3^hi Treg/CD4⁺IL‐17⁺ cell ratios were reduced, particularly after stopping TGFβ1/Fc infusion. Thus, human TGFβ1/Fc infusion may delay Tmem recovery following lymphodepletion in nonhuman primates. Combined (low‐dose) IL‐2 infusion may be required to improve the Treg:Th17 ratio following lymphodepletion.     

Diltiazem induces regulatory T cells in vitro by modulating human dendritic cell maturation

Diltiazem is a calcium channel antagonist that has been commonly associated with currently used immunosuppressants to prevent acute graft rejection in humans. In this study, we examined the possibility that diltiazem may affect human dendritic cell (DC) functions in response to lipopolysaccharide (LPS) stimulation and may induce the generation of DC with the capacity to generate CD4⁺ regulatory T cells (Tregs). Blood monocytes were cultured in the presence of diltiazem at the beginning of their differentiation process into DC. Monocyte‐derived DCs were stimulated with LPS, and DCs differentiated in the presence of diltiazem showed a decreased interleukin (IL)‐12 production and an enhanced IL‐10 production. When cultured with CD4⁺CD45RA⁺ they were able to enhance the CD4⁺Foxp3⁺ T‐cell population and to induce slowly proliferating T cells, which showed a significant increase of transforming growth factor (TGF)‐β production. These T cells suppress proliferation of activated autologous T cells, and we show that this effect is attributable to soluble factors, primarily to TGF‐β. Blockade of TGF‐β by specific monoclonal antibodies reversed this inhibitory effect. Herein, we provide new evidence that diltiazem‐conditioned monocyte‐derived DC induce T cells which acquire a regulatory phenotype and activity similar to those described for Tregs.     

Characterization of baboon NK cells and their xenogeneic activity

Kennett SB, Porter CM, Horvath‐Arcidiacono JA, Bloom ET. Characterization of baboon NK cells and their xenogeneic activity. Xenotransplantation 2010; 17: 288–299. © 2010 John Wiley & Sons A/S. Abstract: Background: Baboons are commonly used as models for transplantation and preclinical testing of various types of therapeutic agents. For proper assessment of information gathered from these models, differences between the baboon and human immune systems need to be characterized. Natural killer (NK) cells are the first line of defense against many infectious agents and cancer and are important mediators of transplantation rejection reactions, particularly during xenotransplantation. In this study, we examined baboon NK cell function and developed methods for purifying and expanding these cells. Methods: Baboon NK cells were analyzed using a combination of extracellular and intracellular cell staining, cell sorting, interleukin (IL)‐2 mediated stimulation and expansion, and 4 h cytotoxicity assays with human and pig target cell lines. Results: Baboon peripheral blood mononuclear cell (PBMC) exert very low but detectable cytolytic activity against both human (K562) and pig (PAEC, J2) target cells, and this activity is enhanced within 4 h of treatment with IL‐2. Like human NK cells, many baboon PBMC express the lytic enzymes granzyme A, granzyme B, and perforin. Based on these markers, we identified a subpopulation of CD3^? baboon lymphocytes that are CD8^dim and CD16^bright that likely represents the baboon NK cells. These cells also are characterized by expression of the natural cytotoxicity receptor NKp46. Baboon CD3^?NKp46⁺ cells purified by flow cytometric cell sorting have high cytolytic capacity that can be further enhanced by IL‐2 stimulation. These baboon NK cells can be expanded in vitro and retain extremely high cytolytic capacity. While fresh baboon lymphocytes express very little CD56, the expanded baboon NK cells are predominantly CD56⁺; approximately 10% of the expanded NK cells are CD56^dim, and the remainder are CD56^bright. Conclusions: Baboon NK cells that are IL‐2 responsive can be identified on the basis of a CD3^?NKp46⁺CD8^dimCD16^+/? or CD3^?CD8^dimCD16^bright phenotype and can be isolated and expanded in culture. These results may allow for a more accurate representation of the human innate immune system in baboon models and more accurate analyses of the role of the baboon innate immune system cells in preclinical models.     

Clinical and immunomodulatory effects of bevacizumab and low‐dose interleukin‐2 in patients with metastatic renal cell carcinoma: results from a phase II trial

Study Type – Therapy (case series) Level of Evidence 4 What’s known on the subject? and What does the study add? As single agents, both IL‐2 and bevacizumab have demonstrated clinical activity in advanced RCC. This study demonstrated the feasibility of the combination, its safety and unfortunately the lack of enhanced activity if one compares the combination with existing data of bevacizumab alone.

OBJECTIVE

Low‐dose interleukin‐2 (IL‐2) is a historical treatment for metastatic renal cell carcinoma (mRCC). Increased vascular endothelial growth factor (VEGF) levels inhibit dendritic cell (DC) differentiation and augment production of immunosuppressive regulatory T (Treg) cells. Bevacizumab is an antibody that binds to VEGF, has activity in mRCC and may augment the anti‐tumour immune effects of IL‐2. To determine the clinical and immunomodulatory effects of this combination, a prospective, phase II trial of bevacizumab plus low‐dose IL‐2 was conducted.

PATIENTS AND METHODS

Patients with untreated mRCC received bevacizumab (10 mg/kg i.v. every 2 weeks) and IL‐2 (125 000 units/kg/day subcutaneously from Monday to Friday for 6 consecutive weeks followed by a 2‐week rest period). Endpoints included progression‐free survival, Response Evaluation Criteria in Solid Tumors‐defined objective response rate, immunomodulatory effects and safety.

RESULTS

Between January 2005 and September 2007, twenty‐six patients with untreated mRCC were enrolled. The median progression‐free survival was 9.6 months (95% CI, 4.1–16.9 months) The objective response rate was 15% and an additional 38% of patients had tumour burden reduction of <30%. Grade 3 constitutional adverse events (fatigue, fever/chills) and neutropenia were observed in 42% and 12% of patients, respectively. Peripheral blood CD1c⁺ myeloid and CD303⁺ plasmacytoid DC increased during treatment as did IL‐8 levels and CD4⁺ CD25⁺ FoxP3⁺ Treg cells. No changes in T helper type 1/2‐associated cytokines were observed.

CONCLUSION

Bevacizumab plus low‐dose IL‐2 has modest clinical activity in mRCC. Toxicity was largely IL‐2 related without enhancement of bevacizumab‐related toxicity. Biological data indicate inhibition of VEGF levels and increase of immunosuppressive Treg cells without an effect on DC activation.     

The effect of Gal expression on pig cells on the human T-cell xenoresponse

Wilhite T, Ezzelarab C, Hara H, Long C, Ayares D, Cooper DKC, Ezzelarab M. The effect of Gal expression on pig cells on the human T cell xenoresponse. Xenotransplantation 2012; 19: 56–63. © 2012 John Wiley & Sons A/S. Abstract: Background: Lack of Gal expression on pig cells is associated with a reduced primate humoral immune response as well as a reduction in cytokine production by human cells in vitro. We investigated whether lack of Gal expression is associated with reduced human T‐cell response in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy humans and naïve baboons. Human CD4⁺ and CD8⁺ T cells were isolated. Porcine aortic endothelial cells (pAECs) were isolated from wild‐type (WT) and α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs. WT pAECs were treated with α‐galactosidase, reducing Gal expression. Swine leukocyte antigen (SLA) class I and II expression on pAECs was measured, as was T‐cell proliferation and cytokine production in response to pAECs. Results: Reduced Gal expression on WT pAECs after α‐galactosidase treatment was associated with reduced human PBMC proliferation (P < 0.005). SLA class I and II expression on WT and GTKO pAECs was comparable. Human CD4⁺ and CD8⁺ T‐cell proliferation was less against GTKO pAECs before (P < 0.001) and after (P < 0.01 and P < 0.05, respectively) activation. Human and baboon PBMC proliferation was less against GTKO pAECs before (P < 0.05) and after (P < 0.01 and P < 0.05, respectively) activation. Human PBMCs produced a comparable cytokine/chemokine response to WT and GTKO pAECs. However, there was less production of IFN‐γ/TNF‐α by CD4⁺ and IFN‐γ/granzyme B/IP‐10 by CD8⁺ T cells in response to GTKO pAECs. Conclusions: The absence of Gal on pig cells is associated with reduced human T‐cell proliferation (and possibly selected cytokine production). Adaptive primate T‐cell responses are likely to be reduced in GTKO xenograft recipients.     

Antigen‐dependent interactions between regulatory B cells and T cells at the T:B border inhibit subsequent T cell interactions with DCs

IL‐10+ regulatory B cells (Bregs) inhibit immune responses in various settings. While Bregs appear to inhibit inflammatory cytokine expression by CD4+ T cells and innate immune cells, their reported impact on CD8+ T cells is contradictory. Moreover, it remains unclear which effects of Bregs are direct versus indirect. Finally, the subanatomical localization of Breg suppressive function and the nature of their intercellular interactions remain unknown. Using novel tamoxifen‐inducible B cell–specific IL‐10 knockout mice, we found that Bregs inhibit CD8+ T cell proliferation and inhibit inflammatory cytokine expression by both CD4+ and CD8+ T cells. Sort‐purified Bregs from IL‐10‐reporter mice were adoptively transferred into wild‐type hosts and examined by live‐cell imaging. Bregs localized to the T:B border, specifically entered the T cell zone, and made more frequent and longer contacts with both CD4+ and CD8+ T cells than did non‐Bregs. These Breg:T cell interactions were antigen‐specific and reduced subsequent T:DC contacts. Thus, Bregs inhibit T cells through direct cognate interactions that subsequently reduce DC:T cell interactions.     

CD4+CD28null T cells are not alloreactive unless stimulated by interleukin‐15

Proinflammatory, cytotoxic CD4⁺CD28^null T cells can be substantially expanded in patients with end‐stage renal disease. These cells have been associated with the risk for rejection, but their alloreactive potential is unknown. CD4⁺CD28^null T cells were stimulated with HLA‐mismatched antigen presenting cells in the absence/presence of exogenous cytokines. Alloreactive potential was evaluated based on proliferation, degranulation, cytotoxicity, and cytokine production. Further, their suppressive capacity was assessed by measuring inhibition of proliferating alloreactive CD28⁺ T cells. CD4⁺CD28^null T cells contained alloreactive (CD137⁺) T cells but did not proliferate in response to allogeneic stimulation, unless interleukin (IL)‐15 was added. However, they could proliferate on stimulation with cytomegalovirus antigen without exogenous cytokines. IL‐15 increased the frequency of proliferating alloreactive CD4⁺CD28^null T cells to 30.5% without inducing CD28 expression (P < .05). After allogeneic stimulation together with IL‐15 and IL‐21, frequency of degranulating CD107a⁺CD4⁺CD28^null T cells increased significantly from 0.6% to 5.8% (P < .001). Granzyme B and perforin positivity remained similar, but production of interferon‐γ and tumor necrosis factor‐α increased by the combination of IL‐15 and IL‐21 (P < .001 and P < .05, respectively). Finally, CD4⁺CD28^null T cells did not show significant suppression. Thus, CD4⁺CD28^null T cells represent a population with absent alloreactivity unless IL‐15 is present.