IL-4 synthesis byin vivo-primed memory CD4+ T Cells: II. Presence of IL-4 is not required for IL-4 synthesis in primed CD4+ T Cells

Previous studies have shown that the presence of IL-4 is required for the development of IL-4 synthesis in naive CD4⁺ T cells. The purpose of our current studies was to investigate the role of IL-4 in the development of IL-4 synthesis in primed memory T cells. We therefore examined CD4⁺ T cells taken from lymph nodes of BALB/c mice immunized with keyhole limpet hemocyanin (KLH) and restimulatedin vitro with KLH. Our results with such primed resting CD4⁺ T cells programmed to produce IL-4 indicated that the production of IL-4 did not require the presence of IL-4 (although the presence of IL-2 was absolutely necessary), and was only slightly limited by the presence of anti-IL-4 MAb. These results with resting memory T cells were not biased by the presence of activated T cells already producing substantial quantities of IL-4, since we demonstrated that high-density memory T cells could produce IL-4 in the absence of IL-4, and because T cells that actively produce IL-4 do not persistin vivo very long after antigen exposure. These results indicate that IL-4 synthesis in T cells committed to IL-4 production can indeed occur in the absence of IL-4 when culture conditions have been optimized and suggest that therapies with anti-IL-4 MAb or with soluble IL-4 receptors designed to control the development of IL-4 synthesis in memory T cells from individuals exhibiting excessive IL-4 synthesis will be unsuccessful. Therefore, other therapies, for example, utilizing IL-12, will be required to modulate the relatively fixed programs in memory T cells that direct the development of cytokine synthesis.     

CD4+ T cell-independent maintenance and expansion of memory CD8+ T cells derived from in vitro dendritic cell activation

CD4+ T cells are essential for the maintenance of CD8+ memory T (Tm) cells following acute infection, but the importance of CD4+ T cells for the maintenance and expansion of CD8+ Tm cells to non-infectious antigens remains mostly unknown. Here, we showed that ovalbumin (OVA)-specific CD8+ Tm cell precursors derived from in vitro stimulation of TCR transgenic OT I CD8+ T cells with OVA protein-pulsed bone marrow-derived dendritic cells (DCOVA) can give rise to functional CD8+ Tm cells after adoptively transferred into mice. These CD8+ Tm cells can be maintained and remain fully functional in CD4+ T cell-absent environments in vivo. Furthermore, CD4+ T cells are not essential for the expansion of these CD8+ Tm cells. Finally, these in vitro DCOVA-activated CD8+ Tm cells maintained in CD4-deficient mice are also able to confer fully protective immunity against a later challenge of OVA-expressing tumor cells. Collectively, these findings demonstrate that in contrast to acute infections, maintenance and expansion of CD8+ Tm cells after priming with OVA protein-pulsed dendritic cells are independent of CD4+ T cells.     

Chemokines,chemokine receptors and CD4+CD25+ regulatory T cells

The fields of regulatory T (Treg) cells and chemokines/chemokine receptors have progressed rapidly in the last few years. Treg cells, especially CD4⁺CD25⁺ Treg cells, play a critical role in maintaining self-tolerance and immune homeostasis. Chemokines and chemokine receptors are crucial for lymphoid development, homing and immunological regulation. This review will discuss the biological effects of chemokines and chemokine receptors on regulating the migration and development of CD4⁺CD25⁺ Treg cells, and the potential clinical implications of these findings when considering chemokine receptors as therapeutic targets.     

Induction and maintenance of self tolerance: the role of CD4+CD25+ regulatory T cells

The immune system responds vigorously to invading pathogens (non-self, foreign), while remaining unresponsive (tolerant) to the body's own components and circulating constituents (self). This indifference to self components is a result of finely orchestrated events of thymic negative selection (central tolerance) of developing T cells that are autoaggressive combined with those operative in the periphery (peripheral tolerance) to control the activity of potentially autoreactive T cells that escaped thymic tolerance. Recently, autoimmune regulator expressed in the thymus has been identified as a critical mediator of central tolerance towards tissue-specific antigens. In the periphery, a variety of regulatory T cells are involved in effecting tolerance. There is immense interest and excitement about the newly identified subset of CD4(+)CD25(+) T cells. This is a unique subset of CD4(+) T cells that bear CD25 (IL-2Ralpha chain) on the cell surface in the na?ve state and express FoxP3 as a unique marker. These cells suppress the activity of autoreactive effector T cells primarily via cell-cell contact. The deficiency and/or altered function of CD4(+)CD25(+) T cells is associated with autoimmunity. Mice deficient in FoxP3 (scurfy mice) bear an autoimmune phenotype, and human males with mutations in the corresponding gene express the phenotype of wide-spread autoimmunity, the immune dysregulation, polyendocrinopathy and enteropathy, and X-linked syndrome. In vitro expansion of antigen-specific CD4(+)CD25(+) T cells and their adoptive transfer into patients suffering from autoimmunity is emerging as a promising new therapeutic approach for these debilitating disorders.     

Toll-like receptors and immune regulation: their direct and indirect modulation on regulatory CD4+ CD25+ T cells

Regulatory CD4(+) CD25(+) T (Treg) cells with the ability to suppress host immune responses against self- or non-self antigens play important roles in the processes of autoimmunity, transplant rejection, infectious diseases and cancers. The proper regulation of CD4(+) CD25(+) Treg cells is thus critical for optimal immune responses. Toll-like receptor (TLR)-mediated recognition of specific structures of invading pathogens initiates innate as well as adaptive immune responses via antigen-presenting cells (APCs). Interestingly, new evidence suggests that TLR signalling may directly or indirectly regulate the immunosuppressive function of CD4(+) CD25(+) Treg cells in immune responses. TLR signalling may shift the balance between CD4(+) T-helper cells and Treg cells, and subsequently influence the outcome of the immune response. This immunomodulation pathway may therefore have potential applications in the treatment of graft rejection, autoimmune diseases, infection diseases and cancers.     

The number and function of circulating dendritic cells may limit effector memory CD4+ T-cell responses in HIV patients responding to antiretroviral therapy

Some HIV patients who previously experienced severe immunodeficiency retain low pathogen-specific T-cell responses despite a virological response to antiretroviral therapy (ART). To identify correlates with dysfunction in accessory cell populations, HIV patients were stratified into groups maintaining high or low CD4⁺ T-cell IFN-γ responses to cytomegalovirus (CMV) over 4–8 years on ART. Myeloid dendritic cells (mDC), plasmacytoid (p) DC, M-DC8⁺ cells and monocytes were enumerated and mRNA of cytokines and activation molecules were quantitated in purified subpopulations. Proportions of pDC were lower (p = 0.043) and mDC were higher (p = 0.043) in low responders. TRAIL receptor 2 (DR5) mRNA levels in pDC (p = 0.0008) and mDC (p = 0.0062) were lower in high responders compared to controls. Levels of IL-15 mRNA were higher in mDC from high responders (p = 0.015) and levels of IL-10 mRNA were higher in M-DC8⁺ cells from low responders (p = 0.036). Hence CMV-specific CD4⁺ T-cell IFN-γ responses may be affected by numbers and function of circulating DC.     

Phenotypic characterization of regulatory CD4+CD25+ T cells in rats

CD25 has become widely used as a marker for a subset of regulatory CD4(+) T cells present in the thymus and periphery of mice, rats and humans. However, CD25 is also expressed on conventionally activated T cells that are not regulatory and not all peripheral regulatory T cells express CD25. The identification of a stable and unique marker for regulatory T cells would therefore be valuable. This study provides a detailed account of the phenotype of CD4(+)CD25(+) regulatory T cells in rats. In the thymus, CD4(+)CD8(-)CD25(+) cells were found to have a more mature phenotype than the corresponding CD4(+)CD8(-)CD25(-) cells with respect to expression of Thy1 (CD90), CD53 and CD44, suggesting that CD25 expression, and perhaps commitment to regulatory function, might be a late event in thymocyte development. CD4(+)CD25(+) cells in both the thymus and periphery were found to have enriched and heterogeneous expression of activation markers such as OX40 (CD134) and OX48 (an antibody determined in this study to be specific for CD86). CD4(+)CD25(+) T cells were also found to have enriched expression of CD80, at both the mRNA and protein level. However, functional studies in vitro and in vivo showed that neither OX40 or CD86 were useful markers for the further subdivision of regulatory T cells. Our studies indicate that, at present, CD25 remains the most useful marker to enrich for regulatory CD4(+) T cells in rats and no further subdivision of the regulatory component of CD4(+)CD25(-)CD45RC(low) T cells has yet been achieved.     

Inhibition of accelerated rejection mediated by alloreactive CD4+ memory T cells and prolonged allograft survival by arsenic trioxide

The aim of this study was to evaluate and determine the potential mechanisms of As₂O₃ in accelerated rejection mediated by alloreactive CD4⁺ memory T cells. Vascularized heterotopic cardiac transplantation from C57BL/6 mice to nude mice (pre-transferred CD4⁺ memory T cells) was performed on Day 0, and As₂O₃ was administered to recipient mice from Day 0 to 10. As a result, As₂O₃ could reduce the proliferation of allo-primed CD4⁺ memory T cells in vitro in MLR and the baseline rate of proliferation was restored by the addition of exogenous IL-2. In vivo, compared with the control[+] group, the mean survival time of cardiac allografts in the As₂O₃ group was prolonged from 5.8?±?0.7 to 14.2?±?2.5 days. Five days after transplantation, the relative gene expression of IL-2, IFN-γ and Foxp3 was reduced in the grafts by As₂O₃ treatment, but the expression of IL-10 and TGF-β was increased. Correspondingly, the proportions of CD4⁺ T cells, CD4⁺ memory T cells and regulatory T cells (Tregs), both in recipient spleens and lymph nodes, were lowered. These results indicate the potential of As₂O₃ as a novel immunosuppressant targeting CD4⁺ memory T cells.     

CD8+ T cells and not CD4+ T cells are hyporesponsive to CD28- and CD40L-mediated activation in HIV-infected subjects

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4⁺ or CD8⁺ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8⁺ and CD4⁺ T cells from HIV-infected and HIV⁻ subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8⁺ T cells from patients proliferated consistently less than controls, while responses from CD4⁺ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4⁺ and CD8⁺ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-γ) in CD4⁺ T cells. Compared with controls, CD4⁺ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-γ, IL-4 and IL-5, while CD8⁺ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4⁺ T cells from HIV⁺ subjects to various costimulatory pathways are relatively intact, whereas CD8⁺ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8⁺ T cell failure might contribute to viral and neoplastic complications of HIV infection.     

CD4+CD25+ T regulatory cells inhibit cytotoxic activity of T CD8+ and NK lymphocytes in the direct cell-to-cell interaction

There are reports suggesting an influence of CD4(+)CD25(+) T regulatory cells (Treg) on cytotoxic lymphocytes. The aim of the study was to evaluate such an influence. Cytotoxic activity was examined in the cultures of peripheral blood mononuclear cells (PBMC) as well as in the cultures of separate T CD8(+) or NK cells mixed with Treg and other subpopulations of PBMC. We found that the production of IFNgamma, perforin and cytotoxic activity of T CD8(+) or NK cells were decreased in the presence of Treg, however, the percentage of conjugates formed by cytotoxic cells with target cells during cytotoxic reaction was decreased only in the cultures of T CD8(+) cells. Inhibition of the cytotoxic reactions in the presence of Treg cells was found to be associated with the generation of conglomerates formed by CD4(+)CD25(+) and the cytotoxic cells, as observed under the fluorescence microscope. Treg produced IL10 when mixed with the cytotoxic lymphocytes, however, an addition of anti-IL10 mAb into the cultures did not affect the results. It is concluded that Treg were able to inhibit both T CD8+ and NK lymphocyte cytotoxic activities in a direct cell-to-cell interaction. Treg decreased the number of T CD8+ cells attached to the target cells, while the mechanism underlying a decrease in NK cytotoxicity remained unclear.     

Age-dependent modifications of Type 1 and Type 2 cytokines within virgin and memory CD4+ T cells in humans

Several alterations in immune function and a concomitant progressive increase in pro-inflammatory status are the major characteristics of ageing process. Cytokines play a key role during ageing acting both in regulatory communication among cells and in effector activity during an immune response. The impact of age on intracellular Type 1 (IFN-gamma and TNF-alpha) and Type 2 (IL-4) cytokines, after stimulation with PMA/ionomycin, was determined in three CD4+ T subsets, i.e. CD95- CD28+ (virgin), CD95+ CD28+ (activated/memory), and CD95+ CD28- (effector/memory) from 47 subjects aged between 21 and 99 years. The percentage of IFN-gamma positive cells significantly decreased in virgin CD4+ subset both in old and nonagenarian subjects, as well as in activated/memory T cells from old in comparison with young subjects. The percentage of TNF-alpha positive cells significantly decreased in activated/memory CD4+ subset from old people. Regarding Type 2 cytokines, IL-4 positive cells significantly increased in activated/memory CD4+ subset from nonagenarians. On the whole our data indicate that: (1) different Type 1 and Type 2 cytokine-positive CD4+ T subsets are differently affected by ageing process; (2) activated/memory T cells appear to be the most affected subset; (3) a shift towards an increased role of Type 2 cytokines and a diminished role of Type 1 cytokines emerges with ageing.     

Decreased 4-1BB expression on CD4+CD25 high regulatory T cells in peripheral blood of patients with multiple sclerosis

As a tumour necrosis factor receptor superfamily member, 4-1BB (CD137) is preferentially expressed in CD4+CD25+ regulatory T cells (Tregs) and has been suggested to play an important role in regulating the generation or function of Tregs. Recent studies of human Tregs have shown that blood CD4+CD25(high) T cells were much closer to Tregs in terms of their functionality. Furthermore, CD4+CD25(high) Tregs have been found to have a decreased effector function in patients with multiple sclerosis (MS). In this study, we examined the expression of 4-1BB and soluble 4-1BB (s4-1BB) protein levels in the peripheral blood of MS patients. Compared with healthy controls, MS patients had decreased 4-1BB expression in their CD4+C25(high) Tregs and increased plasma s4-1BB protein levels. Moreover, the plasma s4-1BB levels of MS patients were shown to be inversely correlated with the 4-1BB surface expression of CD4+CD25(high) Tregs. The down-regulated 4-1BB expression on CD4+CD25(high) Tregs of MS patients may be involved in the impaired immunoactivity of these Tregs. The elevated s4-1BB levels may, at least in part, function as a self-regulatory attempt to inhibit antigen-driven proliferation of Tregs or their immunosuppressive activity.     

CD4+CD29+T cells are blamed for the persistent inflammatory response in ulcerative colitis

Ulcerative colitis (UC) is a chronic gastrointestinal disorder eliciting occurrence of colorectal cancer, the third most common human malignancy. The diagnosis of UC is based on clinical symptoms combined with typical findings on endoscopy, radiology, and ultimately pathology. We investigated the variation trend of CD4⁺CD29⁺T cells together with MPO, VCAM-1 in different periods of rat UC model and UC patients. We also evaluated the relationship between CD4⁺CD29⁺T cells and disease severity. UC model was induced by administering DNCB liquid and acetate solution. We found upregulated expression of CD4⁺CD29⁺T cells in both peripheral blood and colon from rats, and a similar trend for MPO and VCAM-1 in colon (P < 0.05); the expression was especially enhanced in UC rats at two weeks after the model was established (P < 0.01). Such upregulation was also indicated in active and remission UC patients as compared to the healthy and enteritis groups (P < 0.05), with the highest expression level detected in the active UC patients (P < 0.01). Pearson correlation analysis showed a positive correlation of CD4⁺CD29⁺T cells in rat and human peripheral blood with DAI score (r_rat = 0.712, r_human = 0.677, P < 0.01), and MPO in colon (r_rat = 0.514, r_human = 0.682, P < 0.05). These results suggest that CD4⁺CD29⁺T cells may act as major effector cell subsets in persistent inflammatory responses for UC and that infiltration into colon inflammation may be induced by the combination of VCAM-1 and CD29.     

Low-dose antigen-experienced CD4+ T cells display reduced clonal expansion but facilitate an effective memory pool in response to secondary exposure

The strength and duration of an antigenic signal at the time of initial stimulation were assumed to affect the development and response of effectors and memory cells to secondary stimulation with the same antigen. To test this assumption, we used T-cell receptor (TCR)-transgenic CD4⁺ T cells that were stimulated in vitro with various antigen doses. The primary effector CD4⁺ T cells generated in response to low-dose antigen in vitro exhibited reduced clonal expansion upon secondary antigenic exposure after adoptive transfer to hosts. However, the magnitude of their contraction was much smaller than both those generated by high-dose antigen stimulation and by naïve CD4⁺ T cells, resulting in higher numbers of antigen-specific CD4⁺ T cells remaining until the memory stage. Moreover, secondary effectors and memory cells developed by secondary antigen exposure were not functionally impaired. In hosts given the low-dose antigen-experienced CD4⁺ T cells, we also observed accelerated recall responses upon injection of antigen-bearing antigen-presenting cells. These results suggest that primary TCR stimulation is important for developing optimal effectors during initial antigen exposure to confer long-lasting memory CD4⁺ T cells in response to secondary exposure.     

Human CD4+ effector T lymphocytes generated upon TCR engagement with self-peptides respond defectively to IL-7 in their transition to memory cells

The peripheral repertoire of CD4⁺ T lymphocytes contains autoreactive cells that remain tolerant through several mechanisms. However, nonspecific CD4⁺ T cells can be activated in physiological conditions as in the course of an ongoing immune response, and their outcome is not yet fully understood. Here, we investigate the fate of human naive CD4⁺ lymphocytes activated by dendritic cells (DCs) presenting endogenous self-peptides in comparison with lymphocytes involved in alloresponses. We generated memory cells (Tmem) from primary effectors activated with mature autologous DCs plus interleukin (IL)-2 (Tm_auto), simulating the circumstances of an active immune response, or allogeneic DCs (Tm_allo). Tmem were generated from effector cells that were rested in the absence of antigenic stimuli, with or without IL-7. Tmem were less activated than effectors (demonstrated by CD25 downregulation) particularly with IL-7, suggesting that this cytokine may favour the transition to quiescence. Tm_auto and Tm_allo showed an effector memory phenotype, and responded similarly to polyclonal and antigen-specific stimuli. Biochemically, IL-7-treated Tm_allo were closely related to conventional memory lymphocytes based on Erk-1/2 activation, whereas Tm_auto were more similar to effectors. Autologous effectors exhibited lower responses to IL-7 than allogeneic cells, which were reflected in their reduced proliferation and higher cell death. This was not related to IL-7 receptor expression but rather to signalling deficiencies, according to STAT5 activation These results suggest that ineffective responses to IL-7 could impair the transition to memory cells of naive CD4⁺ T lymphocytes recognizing self-peptides in the setting of strong costimulation.     

CD4+CD25+ T regulatory cells inhibit cytotoxic activity of CTL and NK cells in humans-impact of immunosenescence

We hypothesized that advanced age and medical conditions had an impact on the accumulation of CD4+CD25+ T regulatory cells (Treg), which in turn could deteriorate cytotoxic activity of CD8+ T and NK cells. Volunteers were divided according to the Senieur Protocol into healthy young and elderly and non-healthy young and elderly subjects. The numbers of Treg cells in peripheral blood, their influence on CD8+ T and NK cells and production of IL2 as well as apoptosis intensity of Treg cells were measured. The number of Treg cells was higher in both elderly groups than in respective young ones. Compared to healthy subjects, those with medical conditions were revealed to have higher numbers of Treg cells. In addition, the highest accumulation of Treg cells in non-healthy elderly could be a result of their resistance to undergo apoptosis. The frequency of Treg cells correlated inversely with the activity of autologous cytotoxic cells in PBMC and production of IL2 by autologous CD4+CD25- Th cells. Thus, these parameters were the most highly decreased in non-healthy subjects, notably in the elderly. However, these parameters improved in the cultures of pure sorted cells. The only subset capable of decreasing them to the levels noted in PBMC when added back was Treg cells, which proved the link between the number of Treg cells, cytotoxic activity and production of IL2. Concluding, we found that Treg accumulated as a result of ageing and/or medical conditions were capable of decreasing cytotoxic activity of CD8+ T and NK cells and production of IL2.     

Interleukin-15 selectively expands CD57+ CD28- CD4+ T cells, which are increased in active rheumatoid arthritis

Proinflammatory cytokines as well as CD4(+) T cells play critical roles in the pathogenesis of rheumatoid arthritis (RA). Recently, an increase of CD57(+) or CD28(-)CD4(+) T cells was demonstrated in RA, although the mechanism of the increase of these T cells is unclear. In this study, we first examined the relationship between CD57(+)CD4(+) T cells and CD28(-)CD4(+) T cells and found CD57(+)CD28(-)CD4(+) T cells, but neither CD57(+)CD28(+) nor CD57(-)CD28(+) cells, expanded in the peripheral blood of active RA. In vitro experiments revealed that CD57(+)CD28(-)CD4(+) T cells selectively expanded in response to IL-15. Furthermore IL-15-stimulated CD57(+)CD28(-)CD4(+) T cells induced TNF-alpha production from monocytes. These results suggest that CD57(+)CD28(-)CD4(+) T cells are involved in the pathogenesis of RA by responding to IL-15.     

Natural regulatory T cells: number and function are normal in the majority of patients with lupus nephritis

CD4(+) CD25(+) regulatory T cells have been shown to be a vital component of the mechanisms that prevent autoreactivity in mice and also in humans. Previous studies have examined CD4(+) CD25(hi) regulatory T cell frequency and function in patients with systemic lupus erythematosus (SLE) with mixed results. We investigated frequency, phenotype and function in 21 patients with SLE and six with inactive disease. We found no reduction in frequency of the CD25(hi) subset, although active disease was associated with an increased proportion of CD4(+) CD25(+) T cells. When examining function, in the majority of individuals suppression was comparable with controls, although cells isolated from one patient with active disease failed to suppress proliferation. On testing the effect of CD25(hi) depletion on the responses of whole peripheral blood mononuclear cells to nucleosomes we found that, where a response was detectable from patients, depletion augmented interferon-gamma secretion, demonstrating intact suppression of responses implicated in the pathogenesis of SLE. Our results did not confirm an association of failure in CD4(+) CD25(hi) regulatory T cell function or a reduction in their frequency with active disease. Instead, perturbations in the CD4(+) CD25(hi) regulatory T cell population may play a role in disease in only a minority of the patients afflicted by the diverse syndromes of SLE.