Generation of recombinant guinea pig antibody fragments to the human GABAC receptor

Regulatory T cells (Tregs) are of crucial importance to suppress graft versus host disease (GvHD) post allogeneic stem cell transplantation (SCT), but are also known to impair antitumor immunity. However, Treg longitudinal studies are rare and in this respect advanced flowcytometric approaches for Treg characterization are necessary. To investigate the relation of both the percentage and the absolute numbers of Tregs on GvHD or relapse we measured CD4(+)CD25(+/hi)CD127(lo/-) Tregs in 239 peripheral blood (PB) samples of 16 patients during the first two years post-SCT. A 10-color flowcytometric panel was established to evaluate Treg subpopulations and has been tested in ten healthy individuals. In patients we demonstrated a decrease in CD127 expression on T cells early post-SCT which increases during the first year. Moreover, Tregs reached higher absolute numbers in patients with GvHD≤grade I compared to those with GvHD grades II-IV. In contrast, the percentage of Tregs was significantly higher in patients with GvHD grades II-IV or disease relapse compared to those without GvHD. These patients fit into the range of healthy individuals where a median value of 7.5% and 6.4% of T helper cells were characterized as CD4(+)CD25(+/hi)CD127(lo/-) and CD4(+)CD25(+/hi) Tregs, respectively. Furthermore, Tregs could be further subdivided into 40% na?ve, 51% central memory and 9% effector memory Tregs. Our results showed for the first time a downregulation of CD127 expression on T cells including Tregs in patients early post-SCT. Additionally, new insights into the recovery of Tregs regarding GvHD and relapse were provided.     

Cell-surface IL-7 receptor expression facilitates the purification of FOXP3(+) regulatory T cells

FOXP3+ regulatory T (Treg) cells have crucial roles in maintaining self-tolerance and modulating adaptive immune responses. Functional studies of Treg cells have been hampered by a lack of suitable cell-surface markers that specifically enable their purification without contamination by non-regulatory CD25+ effector T cells. Two recent studies have demonstrated that downregulation of the interleukin-7 receptor (CD127) distinguishes Treg cells from activated T cells, facilitating both Treg-cell purification and their functional characterization in human diseases. CD127 uniquely enables the purification of FOXP3+ Treg cells and, potentially, also "adaptive" regulatory T-cell subsets from the CD4+CD25- T-cell population.     

Identification and monitoring of effector and regulatory T cells during experimental arthritis based on differential expression of CD25 and CD134

Major problems in the analysis of CD4+ effector cell and regulatory T cell (Treg) populations in an activated immune system are caused by the facts that both cell types can express CD25 and that the discriminatory marker forkhead box p3 can only be analyzed in nonviable (permeabilized) cells. Here, we show that CD134 (OX40) can be used as a discriminatory marker combined with CD25 to isolate and characterize viable CD4+ effector cells and Tregs. Before and during adjuvant arthritis in rats, coexpression of CD134 and CD25 identified activated Tregs consistently, as these T cells proliferated poorly to disease-associated antigens and were suppressive in vitro and in vivo. Depending on the time of isolation and location, CD4+ T cell populations expressing CD134 or CD25 contained effector/memory T cells. Analysis of the function, phenotype, and amount of the CD4+ T cell subsets in different lymph node stations revealed spatiotemporal differences in effector cell and Treg compartments during experimental arthritis.     

Distributional characteristics of CD25 and CD127 on CD4+ T cell subsets in chronic HCV infection

Attenuated CD4+ T-cell-mediated immune responses are involved in persistence of HCV infection, but the mechanism remains undefined. In this study, the proportions of CD4+ T cell subsets, naïve, central memory, effector memory and effector cells, along with CD25 (IL-2Rα) and CD127 (IL-7Rα) expression on different CD4+ T cell subsets, were measured by polychromatic flow cytometry in 24 chronic HCV-infected individuals and 21 healthy controls. A significant decrease in naïve CD4+ T cells and an increase of central memory and effector memory CD4+ T cells were found in HCV-infected patients compared with healthy controls. HCV-infected patients showed a lower level of CD127 expression in all CD4+ T cells subsets, especially in central memory and effector CD4+ T cells. In terms of total CD4+ T cells, an increase in CD25+ regular T cells (CD4+ CD25+ CD127lo) was found in HCV-infected patients. Interestingly, naïve CD4+ T cells showed increased CD25 expression, while effector memory and effector CD4+ T cells had lower CD25 expression. These data indicated that variations in different fractions of CD4+ T cells, including the phenotypic profile and expression level of CD25 and CD127, may be associated with low efficiency of immune response in chronic HCV infection. These results will strengthen the understanding of pathogenesis and dysfunction of CD4+ T cell immunity during long-term HCV persistence.     

Control of NK cell functions by CD4+CD25+ regulatory T cells

Regulatory T cells (Treg) are key players in the maintenance of peripheral tolerance. As a result of suppressive effects on CD4+ and CD8+ effector T cells, Treg control the adaptive immune system and prevent autoimmunity. In addition, they inhibit B lymphocytes, dendritic cells, and monocytes/macrophages. It is interesting that several recent papers show that CD4+CD25+ Treg are also able to inhibit NK cells. Thus, Treg exert their control on immune responses from the onset (triggering of innate immune cells) to the effector phase of adaptive immunity (B and T cell-mediated responses). That Treg inhibit NK cells suggests that their uncontrolled activation might break self-tolerance and induce "innate" autoimmune pathology. Conversely, Treg-mediated suppression of NK cell functions might have negative effects, as these cells are important in defense against infections and cancer. It is conceivable that Treg might dampen efficient activation of NK cells in these diseases.     

Peripheral CD4 loss of regulatory T cells is associated with persistent viraemia in chronic HIV infection

Chronic HIV infection is associated with T cell abnormalities and altered effector function. Regulatory T cells (Treg) are CD4+ T cells that play a critical role in regulating the immune system. The impact of regulatory T cells on HIV infection and disease progression may be highly significant. We hypothesize that chronic antigenic stimulation from a persistent, high viraemic state may promote a population of Treg that contributes to HIV-associated immune dysfunction. We evaluated the pattern of Treg in chronically infected, HIV-positive individuals over a course of 6 months. Treg are depleted at a distinct rate from that of absolute CD4 cells and loss of Treg is slower in the presence of viral suppression. In vitro depletion of CD25+ CD4+ cells resulted in increased Gag-specific CD4 and CD8 responses. A significant correlation between ex vivo measurement of Treg and Gag-specific CD4 T cell responses was observed (r=-0 x 41, P=0 x 018) with a trend observed with Gag-specific CD8 T cell responses (P=0 x 07). The impact of HIV infection on the Treg population directly complicates the measured effect of Treg on the immune dysfunction although our data support the important role of Treg on modulating the effector T cell response in chronic infection.     

Characterization of mouse CD4 T cell subsets defined by expression of KLRG1

The mouse killer cell lectin-like receptor G1 (KLRG1) is an inhibitory receptor known to be expressed on a subset of NK cells and antigen-experienced CD8 T cells. Here, we have characterized expression of KLRG1 on CD4+ T cells from normal mice. While a polyclonal TCR repertoire suggests thymic origin of KLRG1+ CD4+ cells, KLRG1 expression was found to be restricted to peripheral CD4+ T cells. Based on phenotypic analyses, a minority of KLRG1+ CD4+ cells are effector/memory cells with a proliferative history. The majority of KLRG1+ CD4+ cells are, however, bona fide Treg cells that depend on IL-2 and/or CD28 and express both FoxP3 and high levels of intracellular CD152. KLRG1-expressing Treg are contained within the CD38+ subset but are only partially overlapping with the CD25+ CD4+ Treg subset. In functional assays, KLRG1+ CD4+ cells were anergic to TCR stimulation with respect to proliferation, and sorted KLRG1+ CD25+ CD4+ cells were equal or superior to KLRG1+ CD25- CD4+ cells, which were more potent than KLRG1- CD25+ CD4+ cells in suppressing responder cell proliferation. Together, our results demonstrate that KLRG1 expression defines novel and distinctive subsets of senescent effector/memory and potent regulatory CD4+ T cells.     

T regulatory cells are critical for the maintenance,anamnestic expansion and protection elicited by vaccine‐induced CD8 T cells

T regulatory (Treg) cells are critical for preventing autoimmunity and suppressing immune responses during cancer and chronic infection. However, the role of Treg cells in the generation of vaccine‐induced immune memory remains ill‐defined. Using the mouse model of lymphocytic choriomeningitis virus (LCMV) infection, we demonstrate that transient absence of Treg cells during effector to memory CD8 T‐cell transition results in a permanent impairment in the maintenance, function and recall capacity of CD8 T cells. Memory CD8 T cells in mice that were transiently depleted of Treg cells exhibited defective up‐regulation of memory markers with a significant decrease in polyfunctionality. However, Treg‐depleted mice showed no significant change in CD4 T‐cell responses, and antibody levels relative to control. Altogether, this study evaluates the role of Treg cells in the formation of immune memory and demonstrates an important role for Treg cells in promoting memory CD8 T‐cell differentiation and vaccine‐induced immune protection against intracellular pathogens.     

Rapid assessment of in vitro expanded human regulatory T cell function

Human regulatory T cells (Treg) are able to actively suppress autoreactive immune responses. Phenotypically, they are broadly characterized as CD4+, CD25+, CD127(lo/?) and FoxP3+. CD45RA can be used to further differentiate the population into na?ve (CD45RA(+)) and induced (CD45RA?) Treg. The functional potential of Treg is routinely determined by assessing their ability to suppress T cell function in 3-5day proliferation assays. Since Treg are being explored for therapeutic use, a short-term functional assay could serve as a valuable tool for evaluating the potency of Treg. Therefore, an assay designed to measure Treg suppression of activation marker expression by responder T cells in 7 to 20h has been examined in this report. Using flow cytometry, expression of CD69 and CD154 on T cells, in response to stimulation with CD3/CD28 beads, was used as a measure of activation in the assay. Treg from healthy volunteers were sorted as CD4+CD25+CD127(lo/?)CD45RA+ cells with a BD FACSAria? II. The highly purified Treg were then expanded in vitro and their function was assessed in short term activation marker suppression assays using autologous PBMC as responder cells. The data suggest that this short term suppression assay could be a reliable surrogate for assessing Treg functional potential.     

Bim mediates apoptosis of CD127(lo) effector T cells and limits T cell memory

Following an acute T cell response, most activated effector cells die, while some survive and become memory cells. The pro-apoptotic Bcl-2 family member, Bcl-2 interacting mediator of death (Bim) is critical for eliminating most effector T cells, while expression of CD127 (IL-7Ralpha) has been proposed to mark effector cells destined to become memory cells. Here, we examined the effects of Bim on the death of effector T cells in relationship to CD127 expression and on development of T cell memory following lymphocytic choriomeningitis virus (LCMV) infection. We found that large numbers of CD127(lo) LCMV-specific CD4(+) and CD8(+) T cells were lost in wild-type mice, but were spared in Bim(-/-) mice. Further, while the numbers of CD127(hi) T cells declined only slightly during contraction of the response in wild-type mice, they increased significantly in Bim(-/-) mice due to re-expression of CD127 on CD127(lo) T cells that had avoided apoptosis. Functional memory T cells were significantly increased in Bim(-/-) mice; however, they underwent a slow attrition due to decreased proliferative renewal. Taken together, these data suggest that the absence of Bim-mediated death of LCMV-specific CD4(+) and CD8(+) T cells in vivo can increase T cell memory, but other homeostatic mechanisms control the long-term maintenance of memory cells.     

TGF-beta1 production by CD4+ CD25+ regulatory T cells is not essential for suppression of intestinal inflammation

Naturally occurring CD4+ CD25+ regulatory T cells (Treg) are potent suppressors of CD4+ and CD8+ T cell responses in vitro and inhibit several organ-specific autoimmune diseases. While most in vitro studies suggest that CD4+ CD25+ Treg cells adopt a cytokine-independent but cell contact-dependent mode of T cell regulation, their precise mechanism of suppression in vivo remains largely unknown. Here we examine the functional contribution of Treg cell-derived TGF-beta1 and effector T cell responsiveness to TGF-beta in CD4+ CD25+ T cell-mediated suppression of inflammatory bowel disease (IBD). We show that CD4+ CD25+ Treg cells from either TGF-beta1+/+ or neonatal TGF-beta1-/- mice can suppress the incidence and severity of IBD as well as colonic IFN-gamma mRNA expression induced by WT CD4+ CD25- effector T cells. Furthermore, TGF-beta-resistant Smad3-/- CD4+ CD25+ Treg cells are equivalent to WT Treg cells in their capacity to suppress disease induced by either WT or Smad3-/- CD4+ CD25- effector T cells. Finally, anti-TGF-beta treatment exacerbates the colitogenic potential of CD4+ CD25- effector T cells in the absence of CD4+ CD25+ Treg cells. Together, these data demonstrate that in certain situations CD4+ CD25+ T cells are able to suppress intestinal inflammation by a mechanism not requiring Treg cell-derived TGF-beta1 or effector T cell/Treg cell responsiveness to TGF-beta via Smad3.     

CD127 expression and regulation are altered in the memory CD8 T cells of HIV-infected patients--reversal by highly active anti-retroviral therapy (HAART)

HIV infection activates abnormally the immune system and the chronic phase is accompanied by marked alterations in the CD8 compartment. The expression of CD127 (IL-7R alpha chain) by memory CD8 T lymphocytes in HIV-infected patients is analysed and reported. The memory CD8 T cell subset was characterized by expression of CD45RA and CD27 markers, and CD127 cell surface expression was measured ex vivo by four-colour flow cytometry. HIV infection was associated with a fall in the proportion of CD127(+) cells among memory CD8 lymphocytes that resulted in a higher CD127(-) CD45RA(-)CD27(+) CD8 T cell count in HIV-infected patients. Diminished CD127 cell surface expression [mean fluorescence intensity (MFI)] by positive cells was also observed in this subset. The data suggest that these defects were reversed by highly active anti-retroviral therapy (HAART). The regulation of CD127 expression was also studied in vitro. Down-regulation of CD127 by interleukin (IL)-7 was observed in memory CD8 lymphocytes from healthy donors and HAART patients. Expression of CD127 by memory CD8 lymphocytes cultured in the absence of IL-7 confirmed that IL-7R regulation is altered in viraemic patients. Under the same experimental conditions, memory CD8 lymphocytes from HAART patients were shown to express CD127 at levels comparable to cells from healthy individuals. Altered CD127 cell surface expression and defective CD127 regulation in the memory CD8 T lymphocytes of HIV-infected patients are potential mechanisms by which these cells may be impeded in their physiological response to endogenous IL-7 stimulatory signals. Our data suggest that these defects are reversed during the immune reconstitution that follows HAART.     

In vivo depletion of CD4+CD25+ regulatory T cells in cats

To establish a characterized model of regulatory T cell (Treg) depletion in the cat we assessed the kinetics of depletion and rebound in peripheral and central lymphoid compartments after treatment with anti-CD25 antibody as determined by cell surface markers and FOXP3 mRNA expression. An 82% decrease in circulating CD4+CD25+ Tregs was observed by day 11 after treatment. CD4+CD25+ cells were also reduced in the thymus (69%), secondary lymphoid tissues (66%), and gut (67%). Although CD4+CD25+ cells rebound by day 35 post-treatment, FOXP3 levels remain depressed suggesting anti-CD25 antibody treatment has a sustainable diminutive effect on the Treg population. To determine whether CD25+ Treg depletion strategies also deplete activated CD25+ effector cells, cats were immunized with feline immunodeficiency virus (FIV) p24-GST recombinant protein, allowing them to develop a measurable memory response, prior to depletion with anti-CD25 antibody. Anti-FIV p24-GST effector cell activity in peripheral blood after depletion was sustained as determined by antigen-specific T cell proliferation and humoral responses against FIV p24-GST with an ELISA for antigen-specific feline IgG. Furthermore, development of an anti-mouse response in Treg-depleted cats was similar to control levels indicating the retained capacity to respond to a novel antigen. We conclude that despite alterations in CD25+ cell levels during depletion, the feline immune system remains functional. We demonstrate here a model for the study of disease pathogenesis in the context of reduced numbers of immunosuppressive CD4+CD25+ Tregs throughout the feline immune system.     

How do Regulatory T Cells Work?

CD4⁺ T cells are commonly divided into regulatory T (Treg) cells and conventional T helper (Th) cells. Th cells control adaptive immunity against pathogens and cancer by activating other effector immune cells. Treg cells are defined as CD4⁺ T cells in charge of suppressing potentially deleterious activities of Th cells. This review briefly summarizes the current knowledge in the Treg field and defines some key questions that remain to be answered. Suggested functions for Treg cells include: prevention of autoimmune diseases by maintaining self-tolerance; suppression of allergy, asthma and pathogen-induced immunopathology; feto-maternal tolerance; and oral tolerance. Identification of Treg cells remains problematic, because accumulating evidence suggests that all the presently-used Treg markers (CD25, CTLA-4, GITR, LAG-3, CD127 and Foxp3) represent general T-cell activation markers, rather than being truly Treg-specific. Treg-cell activation is antigen-specific, which implies that suppressive activities of Treg cells are antigen-dependent. It has been proposed that Treg cells would be self-reactive, but extensive TCR repertoire analysis suggests that self-reactivity may be the exception rather than the rule. The classification of Treg cells as a separate lineage remains controversial because the ability to suppress is not an exclusive Treg property. Suppressive activities attributed to Treg cells may in reality, at least in some experimental settings, be exerted by conventional Th cell subsets, such as Th1, Th2, Th17 and T follicular (Tfh) cells. Recent reports have also demonstrated that Foxp3⁺ Treg cells may differentiate in vivo into conventional effector Th cells, with or without concomitant downregulation of Foxp3.     

CD4+Ki67+ lymphocytes in HIV-infected patients are effector T cells accumulated in the G1 phase of the cell cycle

Entry into the cell cycle represents a fundamental step before generating an effector immune response. The relationship between the cell cycle and antigen-driven T cell response remains, however, poorly understood in virus infection, including HIV. We have identified a critical fraction of CD4+CD45RO+ memory T lymphocytes that express the Ki67 antigen in chronically HIV-infected patients. A high accumulation of Ki67 protein is detected in CD4+CD45RO+Ki67+ cells that are in the G1 phase of the cell cycle (92 ? 5 %) but not in S and G2 + M phases, in contrast to normal individuals. Of these cells, 20 - 60 % are spontaneously producing IL-2 and IFN-, unlike CD4+CD45RO+ that do not express Ki67. In addition, HIV-p24 antigen is detectable only in a small fraction CD4+Ki67+ cells. In conclusion, CD4+Ki67+ lymphocytes are circulating effector cells accumulated in the G1 phase of the cell cycle and may be involved in the immune surveillance during HIV infection.     

CD52 is a novel costimulatory molecule for induction of CD4+ regulatory T cells

We previously reported that 4C8 monoclonal antibody (mAb) provides a costimulatory signal to human CD4+ T cells and consequently induces regulatory T (Treg) cells, which are hypo-responsive and suppress the polyclonal response of bystander CD4+ cells in a contact-dependent manner. In this study, we identified the antigen of 4C8 mAb as CD52. Costimulation with Campath-1H, a humanized anti-CD52 mAb, also induced Treg cells. Anti-CD52-induced Treg cells suppressed the proliferation of both CD4+ and CD8+ T cells provided with polyclonal or allogeneic stimulation. When Treg cells were induced from Staphylococcal enterotoxin B (SEB) treated cells, they suppressed the response to SEB more efficiently than that to another superantigen, SEA. Furthermore, anti-CD52-induced Treg cells could be expanded by culture with IL-2 followed by CD52-costimulation, and co-injection of expanded Treg cells suppressed lethal xenogeneic graft versus host disease (GvHD) reactions in SCID mice caused by human peripheral blood mononuclear cells (PBMCs).