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Summary: CD4 T cells contribute to protection against pathogens through numerous mechanisms. Incorporating the goal of memory CD4 T‐cell generation into vaccine strategies therefore offers a powerful approach to improve their efficacy, especially in situations where humoral responses alone cannot confer long‐term immunity. These threats include viruses such as influenza that mutate coat proteins to avoid neutralizing antibodies, but that are targeted by T cells that recognize more conserved protein epitopes shared by different strains. A major barrier in the design of such vaccines is that the mechanisms controlling the efficiency with which memory cells form remain incompletely understood. Here, we discuss recent insights into fate decisions controlling memory generation. We focus on the importance of three general cues: interleukin‐2, antigen and co‐stimulatory interactions. It is increasingly clear that these signals have a powerful influence on the capacity of CD4 T cells to form memory during two distinct phases of the immune response. First, through ‘programming’ that occurs during initial priming, and second, through ‘checkpoints’ that operate later during the effector stage. These findings indicate that novel vaccine strategies must seek to optimize cognate interactions, during which interleukin‐2‐, antigen‐ and co‐stimulation‐dependent signals are tightly linked, well beyond initial antigen encounter to induce robust memory CD4 T cells. [ABSTRACT FROM AUTHOR]

The p21-activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus-derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T-cell subsets including Foxp3+ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3+ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3+ Treg cells upon T-cell receptor and interleukin-2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3+ Treg cells. [ABSTRACT FROM AUTHOR]

Relatively little is known about regulatory T (Treg) cells and their functional responses in dogs. We have used the cross-reactive anti-mouse/rat Foxp3 antibody clone FJK-16s to identify a population of canine CD4 FOXP3 T cells in both the peripheral blood (PB) and popliteal lymph node (LN). FOXP3 cells in both PB and LN yielded positive staining with the newly developed anti-murine/human Helios antibody clone 22F6, consistent with the notion that they were naturally occurring Treg cells. Stimulation of mononuclear cells of LN origin with concanavalin A (Con A) in vitro yielded increased proportions and median fluorescence intensity of FOXP3 expression by both CD4 and CD8 T cells. Removal of the Con A and continued culture disclosed a CD4 FOXP3 population, distinct from the CD4 FOXP3 T cells; very few CD8 FOXP3 T cells were observed, though CD8 FOXP3 cells were present in equal abundance to CD4 FOXP3 cells. The CD4 FOXP3 T cells were thought to represent activated Treg cells, in contrast to the FOXP3 cells, which were thought to be a more heterogeneous population comprising predominantly activated conventional T cells. Co-staining with interferon-γ (IFN-γ) supported this notion, because the FOXP3 T cells were almost exclusively IFN-γ, whereas the FOXP3 cells expressed a more heterogeneous IFN-γ phenotype. Following activation of mononuclear cells with Con A and interleukin-2, the 5% of CD4 T cells showing the highest CD25 expression (CD4 CD25) were enriched in cells expressing FOXP3. These cells were anergic in vitro, in contrast to the 20% of CD4 T cells with the lowest CD25 expression (CD4 CD25), which proliferated readily. The CD4 CD25 FOXP3 T cells were able to suppress the proliferation of responder CD4 T cells in vitro, in contrast to the CD4 CD25 cells, which showed no regulatory properties. [ABSTRACT FROM AUTHOR]

The biological outcome of receptor-mediated signalling often depends on the duration of engagement. Because CD40 signalling is controlled by the regulated expression of its ligand, CD154, the mechanisms that regulate CD154 expression probably determine the strength and duration of CD40 signalling. Here, we demonstrate that CD154 expression on the surface of mouse CD4 T cells can be separated into an early phase, occurring between 0 and 24 hr after T-cell activation, and a later extended phase, occurring after 24 hr. The early phase of CD154 expression did not require costimulation and was probably influenced by the strength of T-cell receptor (TCR) signalling alone. However, later CD154 expression was highly dependent on costimulation through either CD28 or inducible costimulator (ICOS). Although CD28 signalling interleukin (IL)-2 secretion, ICOS not, suggesting that costimulation enhance CD154 expression independently of IL-2 production. In fact, anti-CD28 treatment could still induce late-phase CD154 on anti-CD3-stimulated CD4 T cells expressing a mutated form of CD28 that not lead to the induction of IL-2. However, this CD154 induction was somewhat weaker than that of wild-type CD28-expressing cells, suggesting that direct signalling and IL-2-mediated signalling co-operatively responsible for the levels of CD154 induced by CD28. Finally, we show that the second phase of CD154 expression negatively regulated B-cell terminal differentiation and antibody secretion. These results demonstrate that TCR signalling and costimulation each regulate different phases of CD154 expression and control the biological outcome of CD40 signalling on B cells. [ABSTRACT FROM AUTHOR]

Signalling through CD4 by human immunodeficiency virus (HIV)-1 envelope glycoprotein (gpl20) and/or anti-CD4 antibodies can promote T-cell activation and anergy. Interleukin (IL)-16 is a competence growth factor for CD4+ T cells that can induce a G0 to G1 cell cycle transition but cannot induce cell division. The receptor of this cytokine is thought to be the CD4 molecule, although the binding epitope of IL-16 differs from that of HIV. We have demonstrated that both HIV-1/gp120 and IL-16 induced CD4+ T-cell dysfunction, as indicated by suppression of mitogen-induced IL-2 production. Two anti-CD4 antibodies with different binding sites on CD4 also showed an inhibitory effect on IL-2 production. These results indicate that promotion of CD4+ T-cell anergy via the CD4 molecule does not depend on the binding sites of the CD4 ligands. [ABSTRACT FROM AUTHOR]

A critical role for protein kinase C (PKC) in signal transduction events has been well established. Moreover, studies of regulation in PKC levels suggest participation in mediating long-term cellular functions. Protein kinase C-β (PKC-β) has been reported to be involved in interleukin-2 (IL-2) synthesis in T lymphocytes. In this study, the role of PKC-β in intracellular accumulation of IL-2 was investigated using specific inhibitors. Preincubation with two different PKC inhibitors, one specific for classical isotypes (α and βI) Go6976, and one which inhibits both classical and non- classical isotypes, GF109203X, caused a complete block in cytoplasmic IL-2 accumulation when naive CD4 T cells were stimulated in the presence of CD2+CD28+phorbol myristate acetate (PMA). In contrast, preincubation with up to 1000 ng/ml of cyclosporin A (CsA) resulted in a reduction in the intracellular IL-2 detected, as observed by a decrease in the proportion of positive cells as well as a fall in the mean fluorescence intensity (MFI). CsA did not influence PKC-β translocation. Flow cytometric assessments of PKC-β and its isoforms βI and βII correlated with Western blotting analysis and these results were further supported by the use of PKC-β-positive (HUT 78) and -negative (BW5147) T-cell lines. Using the specific inhibitors, Go6976 and GF109203X, the findings in this study suggest that activation and translocation of PKC-β is critical for accumulation of intracellular IL-2. The influence of CsA in reducing but not blocking IL-2 synthesis is discussed. PMA-induced down-regulation of the CD4 antigen was observed in the presence of Go6976 and but not GF109203X, suggesting regulation by non-classical PKC isoforms. [ABSTRACT FROM AUTHOR]

The CD31 antigen (PECAM-1) has been reported to be a stable marker for a human CD4 T-cell subpopulation unable to produce interleukin-4 (IL-4). We show here that CD31 expression is not stable inasmuch as CD4 T-cell lines and clones derived from cell-sorted neonatal CD31+ cells lose CD31 upon repetitive cycles of stimulation and IL-2 expansion. Moreover, various cytokines (IL-1α, IL-4, IL-6, transforming growth factor-β) fail to reinduce CD31 on CD31- clones. Whereas all CD31+ CD4 T cells rapidly express high levels of the CD45RO antigen and downregulate the L-selectin antigen after priming, CD31 disappears more slowly because only part of the cells lose CD31 expression upon each cycle of stimulation. Loss of CD31 reflects a functional maturation of CD45RO+ cells since, in a system which favours the development of Th2 effectors, IL-4 is produced by CD31- but not CD31+ effector T cells, whereas interferon-), is produced by both types of cells. However, CD31 is not a Th1 marker since it is not expressed on several Th1 antigen-specific clones. We conclude that CD31 is a maturation marker expressed on the great majority of naive CD45RO- CD4 T cells and on a subset of CD45RO+ CD4 T cells that are at an intermediate stage of maturation. [ABSTRACT FROM AUTHOR]

The article presents a study which assayed the precursors of cytotoxic T cells and T-helper cells after injection of tumor cells. The author proposes that the interleukin-2 and interleukin-4 secreting T cells found in the lymph nodes of tumor recipients are homologues of Th0 cells. The results suggest that mice with immune deviation associated with the anterior chamber do not develop memory CD4+ T cells.

Using anti-Tac and anti-Mik-β1 monoclonal antibodies to α and β subunits of the interleukin-2 receptor (IL-2R), respectively, a marked difference in expression of IL-2R subunits on blood CD4+ and CD8+ T cells was demonstrated between adults and newborns. In the adult blood, reciprocal expression of IL-2Rα and IL-2Rβ was observed in CD4+ and CD8+ T cells. Some CD4+ T cells expressing IL-2Rα were often detected, but IL=2Rβ+ CD4+ cells were very few. On the other hand, CD8+ T cells expressed significant IL-2Rβ but little IL-2Rα. In marked contrast to adult individuals, both CD4+ and CD8+ T cells from the newborns, which seemed to consist mainly of naive populations, showed only negligible expression of IL=2R subunits, it was found that IL-2R subunits appeared to be preferentially expressed on CD4 + and CD8+ T cells with memory phenotypes in the adult blood. Isolated memory (CD45RO+) CD4+ and CD8+ T cells, unlike naive (CD45RO-) ones, were able to proliferate in response to exogenous IL-2 as well as the recall antigen. The present results suggest that IL-2R subunits expressed on circulating T-cell subsets may play an important role in memory T-cell function. [ABSTRACT FROM AUTHOR]

Functionally distinct subsets within the T-helper (CD4+) cell population have been described in man, rat and mouse. We have shown previously that the CEM+45R- human lymphocytes are producers of IL-2 within 24 hr of polyclonal stimulation and are the major interferon (IFN) producers. The mAbs 4B4 (CDw29) and Leu 8 (p80) are here used together with Leu-18 (CD45R) to characterize the subpopulations of the CD4+ human lymphocytes in more detail. The majority of the CD45R+ cells were CDw29- and the majority of the CDw29+ cells were CD45R-. Most of the CD45R+ cells (78%) were also p80+. A significant number of cells (12%) were CDw29-CD45R-. The predominant subpopulations were defined to be CDw29- CD45R+p80+ (31%), CDw29+ CE45R-p80-(24%) and CDW29+CD45R-p80+(18%). Within the CD4+CD45R- subpopulation different subsets vary in their capacities to produce IFN and to produce IL-2 within 24 hr after activation. CD4+CDw29-CD45R-p80- T lymphocytes produced the largest quantities of IFN and IL-2. [ABSTRACT FROM AUTHOR]

Clones derived from highly purified human peripheral blood Leu l9+ cells in the presence of phytohaemagglutinin (PHA) and interleukin-2 (IL-2) expressed cytotoxic activity against natural killer (NK)-resistant as well as NK-sensitive targets. All 66 clones analysed had a germ line configuration of T-cell receptor (TCR) β genes and 38/40 also had unrearranged TCR γ genes. The two exceptions were both CD3+ clones, but these did not have a cytotoxic repertoire noticeably different from CD3- clones without TCR γ gene rearrangements. Clones were also obtained from highly purified CD4-8- cells, most of which were also cytotoxic for NK-resistant and NK-sensitive targets. About 90% of these clones were CD3+ but only around 50% remained negative for CD4 and CD8 while a significant number (127%) were positive for both CD4 and CDL All clones analysed had rearranged TCR γ genes and most had also rearranged TCR β genes, including 20/25 of the clones which were CD3+4-8- . Many of the clones showed two rearrangements of TCR β genes, and 3/4 CD3- clones had rearranged TCR β as well as TCR γ genes. There was no correlation between cytotoxic activity and TCR gene status or phenotype of these CD4-8- derived clones, except that clones which were Leu 19+ tended to have higher cytotoxic activity against NK-sensitive and NK- resistant targets than Leu 19-clones. The results strongly indicate that TCR β and γ gene products are not involved in the cytotoxicity mediated by these clones. They also suggest that some CD4-8 cells may be capable of limited differentiation in vitro. [ABSTRACT FROM AUTHOR]

Summary We examined the proliferative and cytokine-producing activities of CD4+ T cells from young mice of the senescence-accelerated mouse strain SAMP1, which had shown markedly low T-dependent antibody-producing responses. When splenic T cells were cultured with concanavalin A (Con A), the percentage of CD4+ cells decreased earlier in SAMP1 than in C3H/He mice. At 40 hr of culture, the percentage of BrdU-labelled proliferating CD4+ cells increased strongly in C3H/He, but only slightly in SAMP1. When purified CD4+ T cells were cultured with Con A, the percentage of 5-bromo-2′-deoxyuridine (BrdU)-labelled cells peaked at around 48 hr of culture in both strains, but decreased significantly at 64 hr in SAMP1. The production of interleukin (IL)-2 but not IL-4 or interferon-γ (IFN-γ) was significantly lower in SAMP1 than in C3H/He at 48 hr of culture. IL-2 production was also markedly low in SAMP1, even under the stimulation of anti-CD3 with anti-CD28 antibodies. The frequency of cells producing IL-2 was significantly lower in SAMP1 than in C3H/He at 6–24 hr of culture with Con A. The percentage of annexin-positive and propidium iodide (PI)-negative apoptotic cells was significantly higher in SAMP1 than in C3H/He at 96 hr of culture. Exogenous IL-2 prevented the decrease in BrdU-labelled cells and the increase in apoptotic cells in the SAMP1 cell culture. These results indicate that SAMP1 CD4+ T cells cannot produce IL-2 at levels sufficient to support cell proliferation and survival. This may account for the weak T-dependent antibody response in SAMP1 mice. [ABSTRACT FROM AUTHOR]