Autocrine stimulation of IL-10 is critical to the enrichment of IL-10-producing CD40hiCD5+ regulatory B cells in vitro and in vivo

IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40^hiCD5⁺ B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40^hi is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10^−/−CD5⁺CD19⁺ B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40^hiCD5⁺ Breg cells in mice. However, the population of CD40^hiCD5⁺ B cells was minimal in IL-10^−/− mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40^hiCD5⁺ B cells and the autocrine effect of IL-10 is critical to the formation of CD40^hiCD5⁺ Breg cells. [BMB Reports 2015; 48(1): 54-59]     

Dendritic Cell-Specific Deletion of β-Catenin Results in Fewer Regulatory T-Cells without Exacerbating Autoimmune Collagen-Induced Arthritis

Dendritic cells (DCs) are professional antigen presenting cells that have the dual ability to stimulate immunity and maintain tolerance. However, the signalling pathways mediating tolerogenic DC function in vivo remain largely unknown. The β-catenin pathway has been suggested to promote a regulatory DC phenotype. The aim of this study was to unravel the role of β-catenin signalling to control DC function in the autoimmune collagen-induced arthritis model (CIA). Deletion of β-catenin specifically in DCs was achieved by crossing conditional knockout mice with a CD11c-Cre transgenic mouse line. Bone marrow-derived DCs (BMDCs) were generated and used to study the maturation profile of these cells in response to a TLR2 or TLR4 ligand stimulation. CIA was induced by intra-dermal immunization with 100 μg chicken type II collagen in complete Freund’s adjuvant on days 0 and 21. CIA incidence and severity was monitored macroscopically and by histology. The T cell profile as well as their cytokine production were analysed by flow cytometry. Lack of β-catenin specifically in DCs did not affect the spontaneous, TLR2- or TLR4-induced maturation and activation of BMDCs or their cytokine production. Moreover, no effect on the incidence and severity of CIA was observed in mice lacking β-catenin in CD11c⁺ cells. A decreased frequency of splenic CD3⁺CD8⁺ T cells and of regulatory T cells (Tregs) (CD4⁺CD25^highFoxP3⁺), but no changes in the frequency of splenic Th17 (CCR6⁺CXCR3^-CCR4⁺), Th2 (CCR6^-CXCR3^-CCR4⁺) and Th1 (CCR6^-CXCR3⁺CCR4^-) cells were observed in these mice under CIA condition. Furthermore, the expression of IL-17A, IL-17F, IL-22, IL-4 or IFNγ was also not affected. Our data indicate that ablation of β-catenin expression in DCs did not alter the course and severity of CIA. We conclude that although deletion of β-catenin resulted in a lower frequency of Tregs, this decrease was not sufficient to aggravate the onset and severity of CIA.     

Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4⁺ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-2^−/− DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-2^−/− DCs. Exogenous IL-2, added in excess of 500 U/ml to co-cultures with IL-2^−/− DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function.     

CD4+CD25?Nrp1+ T Cells Synergize with Rapamycin to Prevent Murine Cardiac Allorejection in Immunocompetent Recipients

Besides CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs), other immunosuppressive T cells also participated in the regulation of immune tolerance. Reportedly, neuropilin-1 (Nrp1) might be one of the molecules by which regulatory cells exert their suppressive effects. Indeed, CD4⁺CD25⁻Nrp1⁺ T cells exhibit potent suppressive function in autoimmune inflammatory responses. Here we investigated the specific role of CD4⁺CD25⁻Nrp1⁺ T cells in the setting of the transplant immune response. Through MLR assays, we found that CD4⁺CD25⁻Nrp1⁺ T cells suppressed the proliferation of naive CD4⁺CD25⁻ T cells activated by allogeneic antigen-stimulation. Adoptive transfer of CD4⁺CD25⁻Nrp1⁺ T cells synergized with rapamycin to induce long-term graft survival in fully MHC-mismatched murine heart transplantation, which was associated with decreased IFN-γ, IL-17 and increased IL-10, TGF-β, Foxp3 and Nrp1 expression in the grafts. Importantly, our data indicated that CD4⁺CD25⁻Nrp1⁺ T cell transfer augments the accumulation of Tregs in the recipient, and creates conditions that favored induction of hyporesponsiveness of the T effector cells. In conclusion, this translational study indicates the possible therapeutic potential of CD4⁺CD25⁻Nrp1⁺ T cells in preventing allorejection. CD4⁺Nrp1⁺ T cells might therefore be used in bulk as a population of immunosuppressive cells with more beneficial properties concerning ex vivo isolation as compared to Foxp3⁺ Tregs.     

Blockade of Retinol Metabolism Protects T Cell-Induced Hepatitis by Increasing Migration of Regulatory T Cells

Retinols are metabolized into retinoic acids by alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (Raldh). However, their roles have yet to be clarified in hepatitis despite enriched retinols in hepatic stellate cells (HSCs). Therefore, we investigated the effects of retinols on Concanavalin A (Con A)-mediated hepatitis.Con A was injected into wild type (WT), Raldh1 knock-out (Raldh1^−/−), CCL2^−/− and CCR2^−/− mice. For migration study of regulatory T cells (Tregs), we used in vivo and ex vivo adoptive transfer systems. Blockade of retinol metabolism in mice given 4-methylpyrazole, an inhibitor of ADH, and ablated Raldh1 gene manifested increased migration of Tregs, eventually protected against Con A-mediated hepatitis by decreasing interferon-γ in T cells. Moreover, interferon-γ treatment increased the expression of ADH3 and Raldh1, but it suppressed that of CCL2 and IL-6 in HSCs. However, the expression of CCL2 and IL-6 was inversely increased upon the pharmacologic or genetic ablation of ADH3 and Raldh1 in HSCs. Indeed, IL-6 treatment increased CCR2 expression of Tregs. In migration assay, ablated CCR2 in Tregs showed reduced migration to HSCs. In adoptive transfer of Tregs in vivo and ex vivo, Raldh1-deficient mice showed more increased migration of Tregs than WT mice. Furthermore, inhibited retinol metabolism increased survival rate (75%) compared with that of the controls (25%) in Con A-induced hepatitis.These results suggest that blockade of retinol metabolism protects against acute liver injury by increased Treg migration, and it may represent a novel therapeutic strategy to control T cell-mediated acute hepatitis.     

CD4+FOXP3+ Regulatory T Cells Exhibit Impaired Ability to Suppress Effector T Cell Proliferation in Patients with Turner Syndrome

Objective

We investigated whether the frequency, phenotype, and suppressive function of CD4⁺FOXP3⁺ regulatory T cells (Tregs) are altered in young TS patients with the 45,X karyotype compared to age-matched controls.

Design and Methods

Peripheral blood mononuclear cells from young TS patients (n = 24, 17.4–35.9 years) and healthy controls (n = 16) were stained with various Treg markers to characterize their phenotypes. Based on the presence of thyroid autoimmunity, patients were categorized into TS (–) (n = 7) and TS (+) (n = 17). Tregs sorted for CD4⁺CD25^bright were co-cultured with autologous CD4⁺CD25⁻ target cells in the presence of anti-CD3 and -CD28 antibodies to assess their suppressive function.

Results

Despite a lower frequency of CD4⁺ T cells in the TS (-) and TS (+) patients (mean 30.8% and 31.7%, vs. 41.2%; P = 0.003 and P < 0.001, respectively), both groups exhibited a higher frequency of FOXP3⁺ Tregs among CD4⁺ T cells compared with controls (means 1.99% and 2.05%, vs. 1.33%; P = 0.029 and P = 0.004, respectively). There were no differences in the expression of CTLA-4 and the frequency of Tregs expressing CXCR3⁺, and CCR4⁺CCR6⁺ among the three groups. However, the ability of Tregs to suppress the in vitro proliferation of autologous CD4⁺CD25⁻ T cells was significantly impaired in the TS (–) and TS (+) patients compared to controls (P = 0.003 and P = 0.041). Meanwhile, both the TS (–) and TS (+) groups had lower frequencies of naïve cells (P = 0.001 for both) but higher frequencies of effector memory cells (P = 0.004 and P = 0.002) than did the healthy control group.

Conclusions

The Tregs of the TS patients could not efficiently suppress the proliferation of autologous effector T cells, despite their increased frequency in peripheral CD4⁺ T cells.     

HIV-1 Vpr Accelerates Viral Replication during Acute Infection by Exploitation of Proliferating CD4+ T Cells In Vivo

The precise role of viral protein R (Vpr), an HIV-1-encoded protein, during HIV-1 infection and its contribution to the development of AIDS remain unclear. Previous reports have shown that Vpr has the ability to cause G₂ cell cycle arrest and apoptosis in HIV-1-infected cells in vitro. In addition, vpr is highly conserved in transmitted/founder HIV-1s and in all primate lentiviruses, which are evolutionarily related to HIV-1. Although these findings suggest an important role of Vpr in HIV-1 pathogenesis, its direct evidence in vivo has not been shown. Here, by using a human hematopoietic stem cell-transplanted humanized mouse model, we demonstrated that Vpr causes G₂ cell cycle arrest and apoptosis predominantly in proliferating CCR5⁺ CD4⁺ T cells, which mainly consist of regulatory CD4⁺ T cells (Tregs), resulting in Treg depletion and enhanced virus production during acute infection. The Vpr-dependent enhancement of virus replication and Treg depletion is observed in CCR5-tropic but not CXCR4-tropic HIV-1-infected mice, suggesting that these effects are dependent on the coreceptor usage by HIV-1. Immune activation was observed in CCR5-tropic wild-type but not in vpr-deficient HIV-1-infected humanized mice. When humanized mice were treated with denileukin diftitox (DD), to deplete Tregs, DD-treated humanized mice showed massive activation/proliferation of memory T cells compared to the untreated group. This activation/proliferation enhanced CCR5 expression in memory CD4⁺ T cells and rendered them more susceptible to CCR5-tropic wild-type HIV-1 infection than to vpr-deficient virus. Taken together, these results suggest that Vpr takes advantage of proliferating CCR5⁺ CD4⁺ T cells for enhancing viremia of CCR5-tropic HIV-1. Because Tregs exist in a higher cycling state than other T cell subsets, Tregs appear to be more vulnerable to exploitation by Vpr during acute HIV-1 infection.     

Human Regulatory T Cells of G-CSF Mobilized Allogeneic Stem Cell Donors Qualify for Clinical Application

Recent clinical studies demonstrate the high potency of regulatory T cells (Tregs) to control graft-versus-host disease in hematopoietic stem cell transplantation (SCT). However, the adoptive transfer of Tregs is limited by their low frequency in unstimulated donors and considerable concerns that G-CSF induced SC mobilization might have negative effects on the stability and function of Tregs. The isolation of Tregs from the G-CSF mobilized SC grafts would extend this novel strategy for tolerance induction to the unrelated setting and simplify global clinical application. We characterized CD4⁺CD25^highCD127⁻ Tregs from SC donors before and after G-CSF mobilization for their phenotype, function, and stability. After G-CSF application the Treg cell yield increased significantly. Donor Tregs retained their cytokine profile, phenotypic characteristics and in vitro expansion capacity after SC mobilization. Most importantly, in vivo G-CSF stimulated Tregs remained highly suppressive on the proliferation of effector T cells, also after in vitro expansion, and displayed a stable phenotype in epigenetic studies. The surface expression of CXCR3 is transiently reduced. However, donor-derived Tregs maintain their migratory properties after G-CSF stimulation. Therefore, the adoptive transfer of Tregs from G-CSF mobilized SC donors seems to be a feasible and safe strategy for clinical application in allogeneic SCT.     

The Modulation of Endoplasmic Reticulum Stress by Chemical Chaperone Upregulates Immune Negative Cytokine IL-35 in Apolipoprotein E-Deficient Mice

Interleukin (IL)-35 is a newly identified immune negative molecule which is secreted by CD4⁺Foxp3⁺ T regulatory cells (Tregs) and contributes to their suppressive capacity. Early data have shown that IL-35 inhibits development of several autoimmune diseases. However, the role of IL-35 in atherosclerosis, a lipid-driven chronic inflammatory disease in arterial wall, remains to be investigated. Here, we found that IL-35 was involved in atherosclerosis in apolipoprotein E-deficient (ApoE^−/−) mice. ApoE^−/− mice with established atherosclerotic lesion displayed a lower level of IL-35 compared to age-matched wild type C57BL/6 mice without plaque. However, IL-35 expression increased significantly in ApoE^−/− mice with attenuated plaque. More importantly, we found that modulation of ER stress treated by chemical chaperone, 4-Phenyl butyric acid (PBA) in vivo, mainly upregulated immune negative regulating molecule IL-35, as well as IL-10 and Foxp3, accompanied by increased Tregs. However, no obvious impact on pro-inflammatory molecules such as TNF-α, IFN-γ, IL-17 and IL-23 was observed, which provides new insight into the benefit of ER stress recovery from attenuated plaque. Our results suggest that IL-35 might have a potential value for atherosclerotic therapy.     

Peripheral regulatory cells immunophenotyping in Primary Sj?gren's Syndrome: a cross-sectional study

Introduction

IL-10--producing B cells, Foxp3-expressing T cells (Tregs) and the IDO-expressing dendritic cells (pDC) are able to modulate inflammatory processes, to induce immunological tolerance and, in turn, to inhibit the pathogenesis of autoimmune disease.The aim of the study was to characterize and to enumerate peripheral IL-10--producing B cells, Tregs and pDCregs in primary Sjögren''s Syndrome (pSS) patients in regard of their clinical and serologic activity.

Methods

Fifty pSS patients and 25 healthy individuals were included in the study. CD19⁺--expressing peripheral B lymphocytes were purified by positive selection. CD19⁺/CD24^hi/CD38^hi/IL-10--producing B cells, CD4⁺/CD25^hi/Foxp3⁺ and CD8⁺/CD28^-/Foxp3⁺ Tregs, as well as CCR6⁺/CD123⁺/IDO⁺ DCs, were quantitated by flow cytometry.

Results

Immature/transitional circulating IgA⁺ IL-10--producing B cells had higher levels in pSS patients versus control group, whereas CD19⁺/CD38^hi/IgG⁺/IL-10⁺ cells had lower percentage versus control. Indeed CD19⁺/CD24^hi/CD38^hi/CD5⁺/IL-10⁺, CD19⁺/CD24^hi/CD38^hi/CD10⁺/IL-10⁺, CD19⁺/CD24^hi/CD38^hi/CD20⁺/IL-10⁺, CD19⁺/CD24^hi/CD38^hi/CD27^-/IL-10⁺, and CD19⁺/CD24^hi/CD38^hi/CXCR7⁺/IL-10⁺ cells had higher frequency in clinical inactive pSS patients when compared with control group. Remarkably, only percentages of CD19⁺/CD24^hi/CD38^hi/CD10⁺/IL-10⁺ and CD19⁺/CD24^hi/CD38^hi/CD27^-/IL-10⁺ subsets were increased in pSS serologic inactive versus control group (P < 0.05). The percentage of IDO-expressing pDC cells was higher in pSS patients regardless of their clinical or serologic activity. There were no statistically significant differences in the percentage of CD4⁺/CD25^hi/Foxp3⁺ Tregs between patient groups versus controls. Nonetheless, a decrease in the frequency of CD8⁺/CD28^-/Foxp3⁺ Tregs was found in inactive pSS patients versus controls (P < 0.05).

Conclusions

The findings of this exploratory study show that clinical inactive pSS patients have an increased frequency of IL-10--producing B cells and IDO-expressing pDC cells.     

Engineering of regulatory T cells by means of mRNA electroporation in a GMP-compliant manner

Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance. This unique capacity of Tregs, in combination with proof-of-principle in preclinical studies, highlights the potential clinical use of Tregs for the treatment of autoimmunity and transplant rejection. Although proven to be safe and well tolerated in the first clinical trials, only modest clinical results were observed. In this regard, it has been hypothesized that current challenges lie in the development of antigen-specific Tregs.Here, we present an innovative, good manufacturing practices (GMP)-compliant manufacturing protocol for Tregs applicable in a clinical-grade setting, allowing efficient and safe redirection of Treg specificity. First, a soluble polymer conjugated with antibodies to CD3 and CD28 and high amounts of exogenous IL-2 for in vitro Treg expansion resulted in a >70-fold and 185-fold increase of a pure population of CD4⁺CD127^?CD25^hi Tregs and CD4⁺CD127^?CD25⁺CD45RA⁺ Tregs, respectively. Next, as a proof-of-principle, expanded Tregs were engineered by means of TCR-encoding mRNA electroporation to generate antigen-specific Tregs. This resulted in an expression of the newly introduced TCR in up to 85% of Tregs. Moreover, we did not observe a negative effect on the phenotype of Tregs, as demonstrated by the expression of FOXP3, Helios, CTLA-4 and CCR4, nor on the TSDR methylation status. Importantly, mRNA-engineered Tregs were still able to induce in vitro suppression of effector T cells and produced anti-inflammatory, but not pro-inflammatory, cytokines when activated.In conclusion, our findings demonstrate that high numbers of stable and functional Tregs can be obtained with high purity and successfully engineered for gain of function, in a GMP-compliant manner. We envisage that this clinical-grade protocol will provide solid basis for future clinical application of mRNA-engineered Tregs.     

IL-27 Receptor Signalling Restricts the Formation of Pathogenic,Terminally Differentiated Th1 Cells during Malaria Infection by Repressing IL-12 Dependent Signals

The IL-27R, WSX-1, is required to limit IFN-γ production by effector CD4⁺ T cells in a number of different inflammatory conditions but the molecular basis of WSX-1-mediated regulation of Th1 responses in vivo during infection has not been investigated in detail. In this study we demonstrate that WSX-1 signalling suppresses the development of pathogenic, terminally differentiated (KLRG-1⁺) Th1 cells during malaria infection and establishes a restrictive threshold to constrain the emergent Th1 response. Importantly, we show that WSX-1 regulates cell-intrinsic responsiveness to IL-12 and IL-2, but the fate of the effector CD4⁺ T cell pool during malaria infection is controlled primarily through IL-12 dependent signals. Finally, we show that WSX-1 regulates Th1 cell terminal differentiation during malaria infection through IL-10 and Foxp3 independent mechanisms; the kinetics and magnitude of the Th1 response, and the degree of Th1 cell terminal differentiation, were comparable in WT, IL-10R1^−/− and IL-10^−/− mice and the numbers and phenotype of Foxp3⁺ cells were largely unaltered in WSX-1^−/− mice during infection. As expected, depletion of Foxp3⁺ cells did not enhance Th1 cell polarisation or terminal differentiation during malaria infection. Our results significantly expand our understanding of how IL-27 regulates Th1 responses in vivo during inflammatory conditions and establishes WSX-1 as a critical and non-redundant regulator of the emergent Th1 effector response during malaria infection.     

CD4+ T Cells Expressing Latency-Associated Peptide and Foxp3 Are an Activated Subgroup of Regulatory T Cells Enriched in Patients with Colorectal Cancer

Latency-associated peptide (LAP) - expressing regulatory T cells (Tregs) are important for immunological self-tolerance and immune homeostasis. In order to investigate the role of LAP in human CD4⁺Foxp3⁺ Tregs, we designed a cross-sectional study that involved 42 colorectal cancer (CRC) patients. The phenotypes, cytokine-release patterns, and suppressive ability of Tregs isolated from peripheral blood and tumor tissues were analyzed. We found that the population of LAP-positive CD4⁺Foxp3⁺ Tregs significantly increased in peripheral blood and cancer tissues of CRC patients as compared to that in the peripheral blood and tissues of healthy subjects. Both LAP⁺ and LAP⁻ Tregs had a similar effector/memory phenotype. However, LAP⁺ Tregs expressed more effector molecules, including tumor necrosis factor receptor II, granzyme B, perforin, Ki67, and CCR5, than their LAP⁻ negative counterparts. The in vitro immunosuppressive activity of LAP⁺ Tregs, exerted via a transforming growth factor-β–mediated mechanism, was more potent than that of LAP⁻ Tregs. Furthermore, the enrichment of LAP⁺ Treg population in peripheral blood mononuclear cells (PBMCs) of CRC patients correlated with cancer metastases. In conclusion, we found that LAP⁺ Foxp3⁺ CD4⁺ Treg cells represented an activated subgroup of Tregs having more potent regulatory activity in CRC patients. The increased frequency of LAP⁺ Tregs in PBMCs of CRC patients suggests their potential role in controlling immune response to cancer and presents LAP as a marker of tumor-specific Tregs in CRC patients.     

Functional Modulation of Regulatory T Cells by IL-2

The suppressive function of regulatory T cells (Tregs) is critical to the maintenance of immune homeostasis in vivo and yet, the specific identification of Tregs by phenotypic markers is not perfect. Tregs were originally identified in the CD4⁺CD25⁺ fraction of T cells, but FoxP3 expression was later included as an additional marker of Tregs as FoxP3 expression was identified as being critical to the development and function of these cells. Intracellular expression of FoxP3 makes it difficult in using to isolate live and not permeabilized cells for functional assays. As such CD4⁺CD25⁺ fraction is still frequently used for functional assays of Tregs. Although, the CD4⁺CD25⁺ fraction substantially overlaps with the FoxP3⁺ fraction, the minor mismatch between CD4⁺CD25⁺ and FoxP3⁺ fractions may confound the functional characteristics of Tregs. In this study, we isolated CD4⁺FoxP3⁺ as well as CD4⁺CD25⁺ fractions from Foxp3 knock-in mice, and compared their proliferative and suppressive activity in the presence or absence of various concentrations of IL-2. Our results showed comparable patterns of proliferative and suppressive responses for both fractions, except that contrary to the CD4⁺CD25⁺ fraction the FoxP3⁺ fraction did not proliferate in an autocrine fashion even in response to a strong stimulation. In presence of exogenous IL-2, both CD4⁺CD25⁺ and CD4⁺FoxP3⁺ fractions were more sensitive than the CD4⁺CD25^- responder cells in proliferative responsiveness. In addition, a low dose IL-2 enhanced whereas a high dose abrogated the suppressive activities of the CD4⁺CD25⁺ and CD4⁺FoxP3⁺ fractions. These results may provide an additional understanding of the characteristics of the various fractions of isolated Tregs based on phenotype and function and the role of varying levels of exogenous IL-2 on the suppressive activity of these cells.     

Overexpression of IL-10 in C2D Macrophages Promotes a Macrophage Phenotypic Switch in Adipose Tissue Environments

Adipose tissue macrophages are a heterogeneous collection of classically activated (M1) and alternatively activated (M2) macrophages. Interleukin 10 (IL-10) is an anti-inflammatory cytokine, secreted by a variety of cell types including M2 macrophages. We generated a macrophage cell line stably overexpressing IL-10 (C2D-IL10) and analyzed the C2D-IL10 cells for several macrophage markers after exposure to adipocytes compared to C2D cells transfected with an empty vector (C2D-vector). C2D-IL10 macrophage cells expressed more CD206 when co-cultured with adipocytes than C2D-vector cells; while the co-cultured cell mixture also expressed higher levels of Il4, Il10, Il1β and Tnf. Since regular C2D cells traffic to adipose tissue after adoptive transfer, we explored the impact of constitutive IL-10 expression on C2D-IL10 macrophages in adipose tissue in vivo. Adipose tissue-isolated C2D-IL10 cells increased the percentage of CD206⁺, CD301⁺, CD11c⁻CD206⁺ (M2) and CD11c⁺CD206⁺ (M1b) on their cell surface, compared to isolated C2D-vector cells. These data suggest that the expression of IL-10 remains stable, alters the C2D-IL10 macrophage cell surface phenotype and may play a role in regulating macrophage interactions with the adipose tissue.     

Allogeneic cord blood regulatory T cells can resolve lung inflammation

Background aimsCD4⁺CD25⁺CD127^lo regulatory T cells (Tregs) are responsible for maintaining immune homeostasis. Tregs can be rendered defective and deficient as a result of the immune imbalance seen in lung injury, and such dysfunction can play a major role in continued tissue inflammation. The authors hypothesized that adoptive therapy with healthy allogeneic umbilical cord blood (UCB)-derived Tregs may be able to resolve inflammation.ResultsEx vivo-expanded UCB Tregs exhibited a unique phenotype with co-expression of CD45RA⁺CD45RO⁺ >80% and lung homing markers, including CD49d. UCB Tregs did not turn pathogenic when exposed to IL-6. Co-culture with increasing doses of dexamethasone led to a synergistic increase in UCB Treg-induced apoptosis of conventional T cells (Tcons), which translated into significantly higher suppression of proliferating Tcons, especially at a lower Treg:Tcon ratio. Multiple injections of UCB Tregs led to their preferential accumulation in lung tissue in an immune injury xenogenic model. A significant decrease in lung resident cytotoxic CD8⁺ T cells (P = 0.0218) correlated with a sustained decrease in their systemic distribution compared with controls (P < 0.0001) (n = 7 per arm) as well as a decrease in circulating human soluble CD40 ligand level (P = 0.031). Tissue architecture was preserved in the treatment arm, and a significant decrease in CD3⁺ and CD8⁺ burden was evident in immunohistochemistry analysis.ConclusionsUCB Treg adoptive therapy is a promising therapeutic strategy for treatment of lung injury.     

Differences in CD44 Surface Expression Levels and Function Discriminates IL-17 and IFN-γ Producing Helper T Cells

CD44 is a prominent activation marker which distinguishes memory and effector T cells from their naïve counterparts. It also plays a role in early T cell signaling events as it is bound to the lymphocyte-specific protein kinase and thereby enhances T cell receptor signalling. Here, we investigated whether IFN-γ and IL-17 producing T helper cells differ in their CD44 expression and their dependence of CD44 for differentiation. Stimulation of CD4⁺ T cells with allogeneic dendritic cells resulted in the formation of three distinguishable populations: CD44⁺, CD44⁺⁺ and CD44⁺⁺⁺. In vitro and in vivo generated allo-reactive IL-17 producing T helper cells were mainly CD44⁺⁺⁺ as compared to IFN-γ⁺ T helper cells, which were CD44⁺⁺. This effect was enhanced under polarizing conditions. T helper 17 polarization led to a shift towards the CD44⁺⁺⁺ population, whereas T helper 1 polarization diminished this population. Furthermore, blocking CD44 decreased IL-17 secretion, while IFN-γ was barely affected. Titration experiments revealed that low T cell receptor and CD28 stimulation supported T helper 17 rather than T helper 1 development. Under these conditions CD44 could act as a co-stimulatory molecule and replace CD28. Indeed, rested CD44⁺⁺⁺CD4⁺ T cells contained already more total and especially phosphorylated zeta-chain-associated protein kinase 70 as compared to CD44⁺⁺ cells. Our results support the notion, that CD44 enhances T cell receptor signaling strength by delivering lymphocyte-specific protein kinase, which is required for induction of IL-17 producing T helper cells.     

Amplified B lymphocyte CD40 signaling drives regulatory B10 cell expansion in mice

Background

Aberrant CD40 ligand (CD154) expression occurs on both T cells and B cells in human lupus patients, which is suggested to enhance B cell CD40 signaling and play a role in disease pathogenesis. Transgenic mice expressing CD154 by their B cells (CD154^TG) have an expanded spleen B cell pool and produce autoantibodies (autoAbs). CD22 deficient (CD22^−/−) mice also produce autoAbs, and importantly, their B cells are hyper-proliferative following CD40 stimulation ex vivo. Combining these 2 genetic alterations in CD154^TGCD22^−/− mice was thereby predicted to intensify CD40 signaling and autoimmune disease due to autoreactive B cell expansion and/or activation.

Methodology/Principal Findings

CD154^TGCD22^−/− mice were assessed for their humoral immune responses and for changes in their endogenous lymphocyte subsets. Remarkably, CD154^TGCD22^−/− mice were not autoimmune, but instead generated minimal IgG responses against both self and foreign antigens. This paucity in IgG isotype switching occurred despite an expanded spleen B cell pool, higher serum IgM levels, and augmented ex vivo B cell proliferation. Impaired IgG responses in CD154^TGCD22^−/− mice were explained by a 16-fold expansion of functional, mature IL-10-competent regulatory spleen B cells (B10 cells: 26.7×10⁶±6 in CD154^TGCD22^−/− mice; 1.7×10⁶±0.4 in wild type mice, p<0.01), and an 11-fold expansion of B10 cells combined with their ex vivo-matured progenitors (B10+B10pro cells: 66×10⁶±3 in CD154^TGCD22^−/− mice; 6.1×10⁶±2 in wild type mice, p<0.01) that represented 39% of all spleen B cells.

Conclusions/Significance

These results demonstrate for the first time that the IL-10-producing B10 B cell subset has the capacity to suppress IgG humoral immune responses against both foreign and self antigens. Thereby, therapeutic agents that drive regulatory B10 cell expansion in vivo may inhibit pathogenic IgG autoAb production in humans.     

Relationships between IL-17+ Subsets,Tregs and pDCs That Distinguish among SIV Infected Elite Controllers,Low, Medium and High Viral Load Rhesus Macaques

Comprehensive studies of the frequencies and absolute numbers of the various cell lineages that synthesize IL-17 in the blood and corresponding gastrointestinal (GI) tissues, their correlation with CD4⁺ Tregs, CD8⁺ Tregs, total and IFN-α synthesizing plasmacytoid dendritic cells (pDC) relative to plasma viral load in SIV infection has been lacking. The unique availability of SIV infected rhesus macaques (RM) classified as Elite Controllers (EC), and those with Low, Intermediate and High Viral Loads (HVL) provided a unique opportunity to address this issue. Results of these studies showed that EC demonstrated a remarkable ability to reverse changes that are induced acutely by SIV in the various cell lineages. Highlights of the differences between EC and HVL RM within Gastro-intestinal tissues (GIT) was the maintenance and/or increases in the levels of IL-17 synthesizing CD4, CD8, and NK cells and pDCs associated with slight decreases in the levels of CD4⁺ Tregs and IFN-α synthesizing pDCs in EC as compared with decreases in the levels of IL-17 synthesizing CD4, CD8 and NK cells associated with increases in pDCs and IFN-α synthesizing pDCs in HVL monkeys. A previously underappreciated role for CD8⁺ Tregs was also noted with a moderate increase in ECs but further increases of CD8⁺ Tregs with increasing VL in viremic monkeys. Positive correlations between plasma VL and decreases in the levels of Th17, Tc17, NK-17, CD4⁺ Tregs and increases in the levels of CD8⁺ Tregs, total and IFN-α synthesizing pDCs were also noted. This study also identified 2 additional IL-17⁺ subsets in GIT as CD3^−/CD8⁺/NKG2a⁻ and CD3⁺/CD8⁺/NKG2a⁺ subsets. Studies also suggest a limited role for IFN-α synthesizing pDCs in chronic immune activation despite persistent up-regulation of ISGs. Finally, elevated persistent innate immune responses appear associated with poor prognosis. These findings provide an initial foundation for markers important to follow for vaccine design.