CD4+CD25+ regulatory cells in acquired MHC tolerance

Summary: Tolerance to self-antigens is an ongoing process that begins centrally during T-cell maturation in the thymus and continues throughout the cell's life in the periphery by a network of regulated restraints. Remaining self-reactive T-cells that escape intrathymic deletion may be silenced within the peripheral immune system by specialized regulatory CD4⁺ cells. By analogy, regulatory CD4⁺ cells that control immunity to "acquired self" should arise in circumstances where the immune system acquires tolerance to foreign MHC, such as the tolerance that develops following the exposure to foreign MHC antigens during the neonatal period. We have used this classic model of neonatal tolerance to examine the role of regulatory CD4⁺ cells in acquired tolerance to disparate class I and class II MHC. Adoptive transfer of unfractionated but not CD4⁺-depleted spleen cells from neonatal tolerant mice into SCID recipients inhibited skin graft rejection by immunocompetent CD8⁺ T cells. Using 5-bromo-2'-deoxyuridine incorporation, standard cytotoxic T-lymphocyte assays, short-term interferon-γ ELISPOT, and intracellular FACS analysis to study CD8⁺ T-cell effector function, we demonstrated that neonatal tolerant mice contain CD4⁺CD25⁺ cells that suppress the development of anti-donor CD8⁺ T-cell responses in vitro . We conclude that regulatory CD4⁺CD25⁺ cells initiate and/or maintain tolerance by preventing the development of CD8⁺ T-cell alloreactivity.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

The CD4+CD8+ and CD4+ Subsets of FOXP3+ Thymocytes Differ in their Response to Growth Factor Deprivation or Stimulation

The timing of thymic regulatory T (Treg) cell commitment remains unclear. Specifically, there is disagreement as to whether the CD4⁺CD8⁺ FOXP3⁺ thymocytes are precursors of mature CD4⁺ FOXP3⁺ Treg cells, or an independent Treg cell lineage. We reasoned that precursors should be more susceptible to apoptosis than mature Treg cells, and tested this by growth factor removal and anti-CD3 stimulation. Both treatments resulted in an increase of CD4⁺ FOXP3⁺ thymocytes, whereas the frequency of CD4⁺CD8⁺ FOXP3⁺ thymocytes decreased significantly. These changes were accompanied by an increase of annexin⁺ apoptotic cells. Both of these FOXP3⁺ subsets expressed higher levels of Bcl-2 and BIM than other thymocytes, and while in our setting expression of BIM seemed to predispose the cells to apoptosis, Bcl-2 had no apparent protective effect. These results indicate that CD4⁺CD8⁺ FOXP3⁺ thymocytes are more susceptible to apoptosis than mature CD4⁺ FOXP3⁺ Treg cells. This is consistent with the view that they are still immature and thus likely to represent a precursor population.     

Natural CD4+ CD25+ regulatory T cells. Their role in the control of superantigen responses

Summary: CD4 regulatory T cells have a major role in controlling the immune response to self and foreign antigens. Natural CD4⁺ CD25⁺ T cells are a major component of the regulatory subset. Their absence is associated with the development of autoimmune and inflammatory diseases and with abnormal peripheral T-cell homeostasis. Two main characteristics discriminate natural CD4⁺ CD25⁺ T cells from their CD4⁺ CD25⁻ counterparts, namely their cytokine production profile and their behavior during tolerance induction. Natural CD4⁺ CD25⁺ T cells produce interleukin (IL)-10, a cytokine that contributes to their regulatory role. They do not produce IL-2 and are dependent on exogenous IL-2 for proliferation in vitro and in vivo . Studies of their response to superantigen administration in vivo show that they are resistant to clonal deletion but can be tolerized by anergy. Their resistance to apoptosis may contribute to their continuous regulatory function, as it allows them to maintain permanent control over effector T cells.     

Expression of CD28 on CD8+ and CD4+ Lymphocytes During HIV Infection

CD28 interaction with B7 molecules, expressed on the membranes of antigen-presenting cells, costimulates cytokine production, T-cell proliferation and generation of cytotoxic lymphocytes. The expression of CD28 markers on CD4⁺ and CD8⁺ lymphocytes was studied in a group of subjects at various stages of HIV infection. A reduction in the percentage of CD28-bearing CD4⁺ and CD8⁺ cell subsets was observed during the asymptomatic stage of the disease. This reduction was more pronounced in AIDS than in non-AIDS patients. At the same time, an increase in the absolute CD8⁺CD28⁻ cell number (greater in stage A than in stage B and C subjects) was observed in HIV-infected patients. The finding of an altered pattern of CD28 expression on T cells might per se explain certain early defects in the cytokine pattern and in the immune response peculiar to HIV-infected patients.     

CD4+CD25+ regulatory T cells: I. Phenotype and physiology

The immune system protects us against foreign pathogens. However, if fine discrimination between self and non-self is not carried out properly, immunological attacks against self may be launched leading to autoimmune diseases, estimated to afflict up to 5% of the population. During the last decade it has become increasingly clear that regulatory CD4⁺CD25⁺ T cells (T_reg cells) play an important role in the maintenance of immunological self-tolerance, and that this cell subset exerts its function by suppressing the proliferation or function of autoreactive T cells. Based on human and murine observations, this review presents a characterization of the phenotype and functions of the Treg cells in vitro and in vivo . An overview of the surface molecules associated with and the cytokines produced by the Treg cells is given and the origin, activation requirements and mode of action of the Treg cells are discussed. Finally, we address the possibility that Treg cells may play a central role in immune homeostasis, regulating not only autoimmune responses, but also immune responses toward foreign antigens.     

Characterization of T-Cell Receptor Vβ Repertoire in Ovarian Tumour-Reacting CD3+ CD8+ CD4− CTL Lines

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T-cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3⁺ CD8⁺ CD4⁻ cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3⁺ CD8⁺ CD4⁻ lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8⁺ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour-reactive CD3⁺ CD8⁺ CD4⁻ CTL from different individuals of known HLA types.     

CD8+CD28− T suppressor cells and the induction of antigen-specific, antigen-presenting cell-mediated suppression of Th reactivity

Summary: Human CD8⁺CD28⁻ suppressor T cells (Ts) are a subset of T cells generated in the course of in vitro and in vivo immunizations. Ts recognize MHC class I:peptide complexes and inhibit the reactivity of T helper cells (Th) with cognate antigen specificity. We have demonstrated for the first time that CD8⁺CD28⁻ Ts represent a unique subset of regulatory cells that induces the differentiation of tolerogenic antigen-presenting cells, initiating a suppressive loop which results in the induction and spreading of Th unresponsiveness.     

The role of B7 co-stimulation in activation and differentiation ofCD4+and CD8+T cells

Summary: The functional significance of B7 co-stimulation in T-cell activation was described first in the context of preventing the induction of anergy. The functions of this pathway are far more complex than initially appreciated in view of the existence of two B7 molecules which have specificities for both CD28 and CTLA-4, which serve to amplify and terminate T-cell responses respectively Mice lacking B7 co-stimulators and CD28 and CTLA-4 co-stimulatory receptors are helping to clarify the functions of this key immunoregulatory pathway. In this review we will focus on the role of B7 co-stimulation in the activation and differentiation of CD4⁺ helper cells and CD8⁺ cytotoxic cells. The contribution of B7 co-stimulation to CD⁺ responses depends upon the activation history of the T-cell and the strength of the T-cell antigen receptor signal. B7 co-stimulation contributes to in Cerleukin (IL)-2 production by both naive and previously activated CD4⁺ T cells. B7 co-stimulation is most critical for the differentiation of naive CD4+ T cells to IL-4 producers, but predominately influences IL-2 production by previously activated CD4+ cells. B7 co-stimulation is important in development of cytotoxic T cells through both effects on T-helper cells and by direct co-stimulation of CDS⁺ cells.     

The Therapeutic Effect of Vasoactive Intestinal Peptide on Experimental Arthritis is Associated with CD4+CD25+ T Regulatory Cells

Vasoactive intestinal peptide (VIP) has been found to act as a potent anti-inflammatory factor through regulating the production of both anti- and pro-inflammatory mediators and promoting Th2-type responses. In this study, we used Chicken collagen II-induced experimental arthritis (CIA) model in Wistar rats to investigate the potential effects of VIP on rheumatoid arthritis. Our results showed that in vivo treatment of CIA-induced rats with VIP had great protective benefit at both clinical and histological levels. Disease suppression was associated with the inhibition of T cells proliferation, shifting of the immune response toward a Th2-type response and expanded CD4⁺CD25⁺ Treg in the periphery, which inhibited autoreactive T cell activation/expansion. In conclusion, the study provides evidence that VIP had great protective effect on CIA through its inhibition actions on pathogenic T cells.     

CD4+CD25+ regulatory T cells: II. Origin, disease models and clinical aspects

Autoimmune diseases afflict approximately 5% of the population and reflect a failure in the immune system to discriminate between self and non-self resulting in the breakdown of self-tolerance. Regulatory CD4⁺CD25⁺ T cells (T_reg cells) have been shown to play an important role in the maintenance of immune homeostasis and self-tolerance by counteracting the development and effector functions of potentially autoreactive T cells. We have in the previous APMIS review described the phenotype and physiology of T_reg cells. The present overview deals with the thymic origin of T_reg cells and their role in disease models such as autoimmune gastritis and inflammatory bowel disease. Finally, we will consider some aspects of the therapeutic potential of T_reg cells.     

The Allogeneic but not Syngeneic Dendritic Cells Effectively Generated Regulatory T Cells from Total Cd4+ Population without Exogenous Cytokines

Dendritic cells (DC) play a critical role in both the expansion of natural regulatory T cells (nTreg) and conversion of induced Treg (iTreg) from their precursors. In the present study, we evaluated the potential of DC to generate Treg from total CD4⁺ population which contains both nTreg and the precursors, and found that allogeneic (allo-DC) but not syngeneic DC (syn-DC) could effectively generated Foxp3⁺ Treg from total CD4⁺ population in the absence of exogenous cytokines. Compared with freshly purified CD4⁺ T cells, allo-DC-stimulated CD4⁺ T cells showed increased percentage of CD4⁺CD25⁺Foxp3⁺ Treg by 5–7-folds while syn-DC-stimulated CD4⁺ T cells did not. Furthermore, we demonstrated that the significant amounts of endogenous IL-2 and TGF-β, at least partially, contributed to the expansion of nTreg and conversion of iTreg in this cocultural system, respectively. Importantly, similar to nTreg, these allo-DC-generated Treg were capable of suppressing T cell response in vitro . Thus, our research provides a novel and efficient strategy for generation of Treg from total CD4⁺ population.     

T Helper-Independent Activation of Human CD8+ Cells: the Role of CD28 Costimulation

The concept that activation of MHC class I-restricted CD8⁺ cells entirely depends on help from MHC class II-restricted CD4⁺ T cells has recently been supplemented with an alternative model in which CD8⁺ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8⁺ T cells in two different in vitro models. Freshly isolated CD8⁺ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting FcγR⁺ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8⁺ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8⁺ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8⁺ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8⁺ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance.     

Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance

Summary: There is accumulating evidence that T-cell-mediated dominant control of self-reactive T-cells contributes to the maintenance of immunologic self-tolerance and its alteration can cause autoimmune disease. Efforts to delineate such a regulatory T-cell population have revealed that CD25⁺ cells in the CD4⁺ population in normal naive animals bear the ability to prevent autoimmune disease in vivo and, upon antigenic stimulation, suppress the activation/proliferation of other T cells in vitro . The CD25⁺ CD4⁺ regulatory T cells, which are naturally anergic and suppressive, appear to be produced by the normal thymus as a functionally distinct subpopulation of T cells. They play critical roles not only in preventing autoimmunity but also in controlling tumor immunity and transplantation tolerance.     

Suppression of allergic inflammation by allergen-DNA-modified dendritic cells depends on the induction of Foxp3+ Regulatory T cells

CD4⁺CD25⁺Foxp3⁺Regulatory T cells (Tregs) play important roles in regulating allergic inflammation. To analyse if allergen-DNA-modified dendritic cells (DC) can suppress allergic responses and what roles Treg cells play in DC-based allergen-specific immunotherapy. Immature DC were transfected with retrovirus encoding Der p2 DNA, and administered to mice that sensitized and challenged with Der p2 protein. After Treg cells were depleted with anti-CD25 mAb, mice were re-challenged to observe the airway inflammation, and Treg cells in spleen CD4⁺ T cells. And responses of spleen CD4⁺ T cells to Der p2 were determined. Co-culture of naïve CD4⁺ T cells with allergen-modified DC induced Foxp3+ Tregs. Sensitized and challenged mice developed allergic airway inflammation and Th2 responses, and decreased Foxp3⁺ Tregs. Treatment with allergen-modified-DC suppressed airway inflammation and Th2 responses, and increased IL-10 and IFN-γ production and Foxp3⁺ Tregs significantly; and eliminated the responses of CD4⁺ T cells to allergen. Administration of anit-CD25 mAb eliminated all the effects of modified-DC except for the increasing of IFN-γ. Allergen-modified DC can induce immune tolerance to allergens and reverse the established Th2 responses induced by allergen, with dependence on the induction of Foxp3⁺ Tregs.     

Requirement of CD4+ Lymphocytes in IL-2-Stimulated NK Cell Proliferation

Growth requirements of human natural killer cells in IL-2-supplemented cultures were studied, NK cell proliferation was monitored by the MAC (morphology antibody chromosomes) technique and subset specific cell cycle analysis, which both enable direct determination of cell growth in specific lymphocyte subsets among heterogeneous lymphocyte populations. Our results show that even in the presence of saturating concentrations of IL-2, the proliferative capacity of purified CD16⁺ cells is quite low, but can be stimulated in a dose dependent manner by CD4⁺ cells. CD4⁺ cells could partially be replaced by IL-4 but not by various other commercially available cytokines. These results provide further evidence of the requirement of accessory stimuli in NK cell proliferation, and support the interpretation that NK cells have a direct regulatory role in specific T cell responses.     

CD4+ T-regulatory cells: toward therapy for human diseases

T-regulatory cells (Tregs) have a fundamental role in the establishment and maintenance of peripheral tolerance. There is now compelling evidence that deficits in the numbers and/or function of different types of Tregs can lead to autoimmunity, allergy, and graft rejection, whereas an over-abundance of Tregs can inhibit anti-tumor and anti-pathogen immunity. Experimental models in mice have demonstrated that manipulating the numbers and/or function of Tregs can decrease pathology in a wide range of contexts, including transplantation, autoimmunity, and cancer, and it is widely assumed that similar approaches will be possible in humans. Research into how Tregs can be manipulated therapeutically in humans is most advanced for two main types of CD4⁺ Tregs: forkhead box protein 3 (FOXP3)⁺ Tregs and interleukin-10-producing type 1 Tregs (Tr1 cells). The aim of this review is to highlight current information on the characteristics of human FOXP3⁺ Tregs and Tr1 cells that make them an attractive therapeutic target. We discuss the progress and limitations that must be overcome to develop methods to enhance Tregs in vivo , expand or induce them in vitro for adoptive transfer, and/or inhibit their function in vivo . Although many technical and theoretical challenges remain, the next decade will see the first clinical trials testing whether Treg-based therapies are effective in humans.     

Anti CD5 Sustains the Proliferative Response of IgM-Activated Human CD5+B Cells

The CD5 molecule is expressed by most T cells but it is present on a minor B cell subset. Whilst several studies have provided information on the physiological role of T cell CD5, the functional role of CD5 on B lymphocytes remains unclear. To address this question, tonsillar CD5⁺ B cells were sorted by dual-colour fluorescence and FACS. Sorted cells were stimulated with polyclonal anti-IgM antibodies (Ab), and monoclonal (MoAb) F(ab')₂ fragments of anti-CD5. Proliferative responses were evaluated by enumeration of Ki-67 positive cells using quantitative flow cytometry. Co-stimulation with anti-CD5 MoAb for 3 days did not affect the anti-IgM and IL2-induced proliferation of CD5⁺ B cells. This was seen under conditions where the anti-CD5 was soluble, adsorbed to the microwells or cross-linked by anti-mouse antibodies. Fewer CD25⁺ cells were detected, however, in the presence of anti-CD5. In contrast, the proliferative response of CD5⁺ B cells prestimulated for 3 days with IL-2 and anti-IgM, was sustained in a further 3-day culture period when anti-CD5 was added. It is concluded that CD5 occupancy might provide an additional signal to activated CD5⁺ B cells favouring their proliferation and differentiation into autoantibody secreting cells.     

Restricted and Individual Usage of T-cell Receptor β-Gene Variables in Nickel-Induced CD4+ and CD8+ Cells

Nickel allergy is manifested as contact allergic eczema elicited by delayed-type hypersensitivity, the reaction being mediated by T lymphocytes. We examined the T-cell receptor (TCR) β-chain variable gene segment (Vβ) use of nickel-induced CD4⁺ and CD8⁺ T cells in the peripheral blood of nickel-sensitive and non-sensitized subjects. The results show that each patient had an individual Vβ repertoire overexpressed, these being in CD4⁺ cells Vβ10 and Vβ13 (in subject A); Vβ1, Vβ2, Vβ13 and Vβ21 (subject B); Vβ1 and Vβ10 (subject C); Vβ9 and Vβ19 (subject D). Thus, no single Vβ gene dominated in a majority of the CD4⁺ samples. The Vβ genes overexpressed in patient CD8⁺ nickel-induced T cells were Vβ1 (in subject A), Vβ1 (subject B), Vβ1 and Vβ2 (subject C) and Vβ7 (subject D), domination of Vβ1 being seen in most of the CD8⁺ samples (75%). No specific overexpression of any Vβ genes in the nickel-allergic subjects was found in comparison with the non-sensitized subjects. In conclusion, an individual pattern of restricted Vβ genes was induced with nickel in CD4⁺ and CD8⁺ T cells in each nickel allergy patient.