CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

CD38 identifies a hypo-proliferative IL-13-secreting CD4+ T-cell subset that does not fit into existing naive and memory phenotype paradigms

CD38 is commonly regarded as an activation marker for human T cells. Herein, we show that CD38 expression identifies a hypo-proliferative CD4(+) T-cell subset that, following TCR stimulation, retains expression of naive cell surface markers including CD45RA, CD62L and CCR7. Hypo-proliferation was mediated by reduced CD25 up-regulation upon TCR stimulation compared to CD4(+) CD38(-) cells and lack of responsiveness to exogenous IL-2. Instead, CD4(+) CD38(+) T cells expressed CD127, and hypo-proliferation was reversed by addition of IL-7, further associated with increased STAT5 phosphorylation. This phenotype was exacerbated by addition of an agonistic CD38-binding antibody, suggesting that signaling through CD38 promotes this cell profile. Activated CD4(+) CD38(+) cells had a bias towards IL-13 secretion, but not other Th2 cytokines such as IL-4 or IL-5. In comparison, the CD4(+) CD38(-) cells had a clear bias towards secretion of Th1-associated cytokines IFN-γ and TNF. The existence of such CD4(+) CD38(+) T cells may play an important role in pathologies such as asthma, which are associated with IL-13, but not IL-4 and IL-5. Coupled with responsiveness to IL-7 but not IL-2, and the involvement of CD38 ligation, our results highlight a unique T-cell subpopulation that does not fit into existing naive and memory cell paradigms.     

Differential effect of interleukin 1 on naive and memory CD4+ T cells.

Freshly derived murine CD4+ T cells are divided into naive and memory cells based on the expression of CD45 isoforms. Cross-linking the T cell receptor CD3 complex either by plastic-bound anti-CD3 antibodies or the antibody presented on non-lymphoid Fc gamma receptor type II-positive Chinese hamster ovary cells in absence of competent antigen-presenting cells fails to activate naive cells to either secrete cytokines or to proliferate. In contrast, memory cells secrete their characteristic cytokines [interleukin (IL) 2, IL4, and interferon-gamma] and show significant proliferation to this stimulus. IL 1 however, is required for their optimal clonal expansion. Differential expression of IL 1 receptor mRNA in memory cells also correlate with their responsiveness to IL 1. Thus, these data reveal a basic difference in the requirements for activation of naive and memory CD4+ T cells.     

Minimal activation of memory CD8+ T cell by tissue-derived dendritic cells favors the stimulation of naive CD8+ T cells

Of the many dendritic cell (DC) subsets, DCs expressing the monomorphic coreceptor CD8 alpha-chain (CD8alpha) are localized permanently in lymphoid organs, whereas 'tissue-derived DCs' remain in nonlymphoid tissues until they 'capture' antigen and then move to local lymph nodes. Here we show that after lung infection, both naive and memory CD8+ 'killer' T cells responded to influenza virus antigens presented by lymph node-resident CD8alpha+ DCs, but only naive cells responded to antigens presented by lung-derived DCs. This difference provides a mechanism for priming naive T cell responses in conditions in which robust memory predominates. Our findings have implications for immunity to pathogens that can mutate their T cell epitopes, such as influenza virus and human immunodeficiency virus, and challenge the long-held view that memory T cells have less-stringent requirements for activation than naive T cells have.     

Differences in responsiveness to CD3 stimulation between naive and memory CD4+ T cells cannot be overcome by CD28 costimulation

Activation of naive CD4⁺ T cells is essential for the induction of primary immune responses. However, this subset is less responsive to signaling via T cell receptor/CD3 (TcR/CD3) complex than memory CD4⁺ cells. For mitogenic activation of T cells, in addition to triggering of the TcR/CD3 complex, costimulatory signals are required that can be generated by surface structures present on the antigen-presenting cells. We investigated here whether differences in responsiveness to TcR/CD3 stimulation of naive and memory cells can be overcome by the costimulatory pathway B7/CD28. Using a B7-dependent system we show that even in the presence of optimal CD28 costimulation, CD4⁺ naive cells still have more stringent TcR/CD3 activation requirements than memory cells. Furthermore, titration of the B7 signal revealed that for activation of naive CD4⁺ cells a higher level of cross-linking of CD28 molecules is required than for memory cells. Thus, our results show that at least two signals are required for activation of both CD4⁺ memory and naive cells, but that for activation of naive cells higher cross-linking of both CD3 and CD28 molecules is necessary.     

Hodgkin's reed-sternberg cell line (KM-H2) promotes a bidirectional differentiation of CD4+CD25+Foxp3+ T cells and CD4+ cytotoxic T lymphocytes from CD4+ naive T cells

A recent report revealed that a large population of Hodgkin's lymphoma-infiltrating lymphocytes (HLILs) consisted of regulatory T cells. In this study, we cocultured CD4+ naive T cells with KM-H2, which was established as a Hodgkin's Reed-Sternberg cell line, to clarify their ability to induce CD25+ Forkhead box P3+ (Foxp3+) T cells. The characteristic analyses of T cells cocultured with KM-H2 revealed the presence of CD4+CD25+ T cells. They expressed CTLA-4, glucocorticoid-induced TNFR family-related gene, and Foxp3 and could produce large amounts of IL-10. Conversely, KM-H2 also generated CD4+ CTLs, which expressed Granzyme B and T cell intracellular antigen-1 in addition to Foxp3+ T cells. They exhibit a strong cytotoxic effect against the parental KM-H2. In conclusion, KM-H2 promotes a bidirectional differentiation of CD4+ naive T cells toward Foxp3+ T cells and CD4+ CTLs. In addition to KM-H2, several cell lines that exhibit the APC function were able to generate Foxp3+ T cells and CD4+ CTLs. Conversely, the APC nonfunctioning cell lines examined did not induce both types of cells. Our findings suggest that the APC function of tumor cells is essential for the differentiation of CD4+ naive T cells into CD25+Foxp3+ T cells and CD4+ CTLs and at least partly explains the predominance of CD25+Foxp3+ T cells in HLILs and their contribution to a better prognosis. Therefore, in APC-functioning tumors, including classical Hodgkin lymphomas, which generate Foxp3+ T cells and CD4+ CTLs, these T cell repertories play a beneficial role synergistically in disease stability.     

Identical expression of CD45R isoforms by CD45RC+ 'revertant' memory and CD45RC+ naive CD4 T cells.

Naive and memory CD4 T cells are frequently defined by exon-specific monoclonal antibodies (mAb) which stain (or not) high- or low-molecular-weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a 'memory' phenotype (CD45RA-, RB-, RC-, or CD45RO+) may revert ('revertants') and re-express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T-cell development. Furthermore, the picture may be incomplete since an exon-specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti-CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3-exon isoform ABC, the 2-exon isoforms AB and BC, and the null isoform O. Both immature CD45RC- (i.e. CD4+8- 'single positive' thymocytes, and peripheral Thy-1+ recent thymic emigrants) and mature CD45RC- 'antigen-experienced' CD4 T cells had message for single-exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen ('truly native') and CD45RC+ antigen-experienced revertant memory T cells. This observation has implications for understanding long-term immunological memory.     

Towards a cellular definition of CD8+ T-cell memory: the role of CD4+ T-cell help in CD8+ T-cell responses

Whereas the definition of B-cell memory is based on well-known cellular properties and differentiation steps, the process of T-cell memory generation was, until recently, less well understood. A series of recent reports, however, have drastically modified our notion of CD8(+) memory T cells. They show that, in addition to division, the generation of efficient memory cells requires a previously unknown differentiation process. As a whole, the generation of CD8(+) memory T cells appears to mimic the generation of memory B cells. Both processes depend on the help of CD4(+) T cells, they are irreversible, they have the same mechanism, and they occur progressively during the late expansion phase of the primary immune response.     

Co-activation of naive CD4+ T cells and bone marrow-derived mast cells results in the development of Th2 cells

Activation of nalve dense CD4⁺ T cells by plate-bound anti-CD3antibodies favors the development of T_h1 cells which, upon re-stimulation,produce significant amounts of INF- but no IL-4. However, co-activationof such naive T cells in the presence of IgE [anti-dlnitrophenyl(DNP)]-loaded bone marrow-derived mast cells (BMMC) on platescoated with anti-CD3 antibodies and DNP-BSA led to the developmentof IL-4-produclng T_h2 cells. The same result could be observedif irradiated (800 rad) BMMC were applied as co-stimulators.Moreover, BMMC could be replaced by the supernatant of IgE-activatedBMMC suggesting that a soluble mediator, presumably IL-4, wasresponsible for this effect. This assumption was substantiatedusing neutralizing anti-IL-4 antibodies which abolished theBMMC-medlated T_h2 development in all cases. Addition of IL-12,a cytokine that was shown to antagonize the T_h2-promoting effectof IL-4 in vivo, could not inhibit the development of IL-4-producingT cells, but gave rise to a T cell population which producedrelatively high amounts of IL-4 and IFN-. Since BMMC representthe in vitro equivalent of mucosai mast cells these data suggestthat IgE-activated mucosai mast cells can bias an emerging Tcell dependent Immune response towards a T_h2 dominated reactionby the initial production of IL-4.     

Age-related changes in naive and memory CD4+ T cells in healthy human children

Subsets of CD4+ T cells originally identified functionally as suppressor-inducer and helper-inducer populations have recently been reinterpreted as naive and memory maturational states. The subsets can be identified by the surface expression of CD45R and CDw29, respectively. Using two-color flow cytometric analysis, we measured these CD4+ T cell subsets in two samples of cord blood and in 26 healthy children between the ages of 1 and 19 years. As has been reported by others, we observed that the majority of CD4+ T cells in cord blood consist predominantly of the CD45R+ subset. With aging we could demonstrate a gradual acquisition of CDw29+, CD4+ T cells and a concomitant gradual decrease in the percentage of CD45R+, CD4+ T cells. These age-related changes are consistent with the concept of naive (CD45R+) and memory (CDw29+) subsets. Further, because of the dynamic changes, their utilization as prognostic indicators in immunologic disease states cannot be applied to children in the same manner as adults.     

Aurintricarboxylic acid promotes the conversion of naive CD4+CD25- T cells into Foxp3-expressing regulatory T cells

Naive peripheral CD4(+)CD25(-) T cells can be converted into Foxp3-expressing regulatory T cells under appropriate stimulation conditions. Considering that continuous exposure to antigens is one of the prerequisites for the differentiation and maintenance of Treg cells, we investigated whether preventing activation-induced cell death while providing continuous TCR stimulation could promote the expression of Foxp3 in murine naive CD4(+) T cells. Among the several anti-apoptotic agents tested, aurintricarboxylic acid (ATA) was found to induce the in vitro conversion of naive CD4(+) T cells into Foxp3(+) Treg cells with suppressive activity. Neutralizing studies with an antibody against transforming growth factor (TGF)-β revealed that ATA requires the presence of TGF-β to induce Foxp3 expression in naive CD4(+)CD25(-) T cells. Although ATA itself did not activate the Smad signaling pathway, it down-regulated the extracellular signal-regulated kinase and mammalian target of rapamycin signaling cascade in activated T cells. Lastly, combined exposure to ATA and TGF-β had a synergistic effect on the rate of induction and maintenance of Foxp3 expression. These results indicate that ATA could be exploited to efficiently prepare inducible regulatory T cells in vitro and may aid in more precisely identifying the specific signaling pathways that drive Foxp3 expression in T cells.     

CTLA-4 x Ig converts naive CD4+CD25- T cells into CD4+CD25+ regulatory T cells

CTLA-4 x Ig was originally designed as an immunosuppressive agent capable of interfering with the co-stimulation of T cells. In the present study, we demonstrate that CTLA-4 x Ig, in combination with TCR ligation, has the additional capacity to convert naive CD4+CD25- T cells into Foxp3+ regulatory T (T(reg)) cells, as well as to expand their numbers. The CD4+CD25+Foxp3+ T(reg) generated by CTLA-4 x Ig treatment in vitro potently suppress effector T cells. Extending this in vivo, we show that systemic administration of CTLA-4 x Ig increases the percentage of CD4+CD25(hi)Foxp3+ cells within mixed lymphocyte reaction-induced murine lymph nodes. Significantly, the in vitro conversion of naive CD4+CD25- T cells into T(reg) cells is antigen-presenting cell (APC) dependent. This finding, together with the further observation that this conversion can also be driven in vitro by an antibody that engages B7-2 ligand, suggests that CTLA-4 x Ig-driven T(reg) induction may be predicated upon active CTLA-4 x Ig to B7-2 signaling within APC, which elicits from them T(reg)-inducing potential. These findings extend CTLA-4 x Ig's functional repertoire, and at the same time, reinforce the concept that T cell anergy and active suppression are not entirely distinct processes and may be linked by some common molecular triggers.     

CD4-mediated functional activation of human CD4+CD25+ regulatory T cells

Naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells (CD25(+) Tregs) constitute a specialized population of T cells that is essential for the maintenance of peripheral self-tolerance. The immune regulatory function of CD25(+) Tregs depends upon their activation. We found that anti-CD4 antibodies activate the suppressive function of human CD25(+) Tregs in a dose-dependent manner. We demonstrate that CD4-activated CD25(+) Tregs suppress the proliferation of CD4(+) and CD8(+) T cells, their IL-2 and IFN-gamma production as well as the capacity of CD8(+) T cells to re-express CD25. By contrast, anti-CD4 stimulation did not induce suppressive activity in conventional CD4(+) T cells. These results identify CD4 as a trigger for the suppressive function of CD25(+) Tregs and suggest a possible CD4-mediated exploitation of these cells.     

Immune memory in CD4+ CD45RA+ T cells.

This study addresses the question of whether human peripheral CD4+ CD45RA+ T cells possess antigen-specific immune memory. CD4+ CD45RA+ T cells were isolated by a combination of positive and negative selection. Putative CD4+ CD45RA+ cells expressed CD45RA (98.9%) and contained < 0.1% CD4+ CD45RO+ and < 0.5% CD4+ CD45RA+ CD45RO+ cells. Putative CD45RO+ cells expressed CD45RO (90%) and contained 9% CD45RA+ CD45RO+ and < 0.1% CD4+ CD45RA+ cells. The responder frequency of Dermatophagoides pteronyssinus-stimulated CD4+ CD45RA+ and CD4+ CD45RO+ T cells was determined in two atopic donors and found to be 1:11,314 and 1:8031 for CD4+ CD45RA+ and 1:1463 and 1:1408 for CD4+ CD45RO+ T cells. The responder frequencies of CD4+ CD45RA+ and CD4+ CD45RO+ T cells from two non-atopic, but exposed, donors were 1:78031 and 1:176,903 for CD4+ CD45RA+ and 1:9136 and 1:13,136 for CD4+ CD45RO+ T cells. T cells specific for D. pteronyssinus were cloned at limiting dilution following 10 days of bulk culture with D. pteronyssinus antigen. Sixty-eight clones were obtained from CD4+ CD45RO+ and 24 from CD4+ CD45RA+ T cells. All clones were CD3+ CD4+ CD45RO+ and proliferated in response to D. pteronyssinus antigens. Of 40 clones tested, none responded to Tubercule bacillus purified protein derivative (PPD). No difference was seen in the pattern of interleukin-4 (IL-4) or interferon-gamma (IFN-gamma) producing clones derived from CD4+ CD45RA+ and CD4+ CD45RO+ precursors, although freshly isolated and polyclonally activated CD4+ CD45RA+ T cells produced 20-30-fold lower levels of IL-4 and IFN-gamma than their CD4+ CD45RO+ counterparts. Sixty per cent of the clones used the same pool of V beta genes. These data support the hypothesis that immune memory resides in CD4+ CD45RA+ as well as CD4+ CD45RO+ T cells during the chronic immune response to inhaled antigen.     

Differential effects of interleukin-12 on the development of naive mouse CD4+ T cells

The influence of interleukin (IL)-12 and IL-4 on the differentiation of naive CD4⁺ T cells was studied in an accessory cell-free in vitro system. Dense CD4⁺ T cells were purified from unimmunized mice and activated using immobilized anti-CD3 monoclonal antibodies (mAb) in the presence of IL-4, IL-12, or a combination of both cytokines, and restimulated after 6 days by re-exposure to anti-CD3-coated culture wells. T cells initially activated in the presence of IL-4 produced substantial amounts of IL-4 and trace amounts of interferon (IFN)-γ after restimulation at day 6 with plate-bound anti-CD3 mAb. By contrast, T cells primed in the presence of IL-12 produced high levels of IFN-γ and only minimal amounts of IL-4, thus indicating that IL-12 and IL-4 by acting directly on stimulated naive CD4⁺ T cells support the development of T_H1 and T_H2 cells, respectively. When naive CD4⁺ T cells were stimulated in the presence of IL-12 together with IL-4 in comparable concentrations, the effect of IL-12 on T_H1 differentiation was largely inhibited by IL-4. On the other hand, IL-12 exerted no inhibitory effect on IL-4-induced T_H2 differentiation but rather enhanced the production of IL-4 after restimulation of the respective T cells. Decreasing amounts of IL-4 in combination with a high level of IL-12 led to an increasing production of IFN-γ by the emerging T cells and, simultaneously, to a relatively high production of IL-4. These data were confirmed by time-course experiments which revealed that the delayed addition of IL-4 to IL-12-primed T cell cultures resulted in a gradual restoration of IFN-γ production whereas in parallel the secretion of IL-4 was not reduced over a wide period of delay (6–72 h). These results, therefore, demonstrate that (a) IL-4 dominates the effect of IL-12, (b) IL-12 promotes the development of T_H1 cells; however, in the presence of IL-12 and relatively high levels of IL-4 also the development of T_H2-like cells is slightly but significantly enhanced by IL-12, and (c) high amounts of IL-12 in combination with relatively low levels of IL-4 give rise to a T cell population that upon rechallenge exhibited a cytokine profile resembling that of T_H0 cells.