Human CD45RA+ and CD45R0+ T cells exhibit similar CD3/T cell receptor-mediated transmembrane signaling capacities but differ in response to co-stimulatory signals

CD45RA⁺ cells have been described to be less responsive to CD3/T cell receptor (TcR)-mediated activation than CD45R0⁺ T cells. To analyze the underlying mechanism of the differential responses we compared CD3/TcR-triggered tyrosine phosphorylation in the two subsets and studied the role of co-stimulatory signals provided either by accessory cells or pharmacologic activation of protein kinase C by phorbol ester. Stimulation of purified CD45RA⁺ and CD45R0⁺ T cells with CD3/TcR antibodies induced similar patterns and intensities of tyrosine phosphorylation in the two subsets, but no proliferation. If accessory cells were used as the source of co-stimulatory signals, strong expression of the 55-kDa chain of the interleukin-2 (IL-2) receptor (CD25), significant IL-2 production and vigorous proliferation were observed in CD45R0⁺ cells, whereas CD45RA⁺ cells responded weakly. However, when CD3/TcR-mediated triggering was combined with activation of protein kinase C by phorbol ester, CD45RA⁺ cells responded strongly. These data indicate that the transmembrane signaling capacity of the T cell receptor expressed by CD45RA⁺ and CD45R0⁺ cells is similar and, therefore, is presumably not responsible for the differential reactivities of the two subsets. It is more likely that co-stimulatory signals determine whether CD3/TcR-initiated activation results in strong or weak responses.     

Comparison of lymphokine secretion and mRNA expression in the CD45RA+ and CD45RO+ subsets of human peripheral blood CD4+ and CD8+ lymphocytes

Flow cytometric analysis of human peripheral blood T lymphocytes demonstrated that the majority of the CD4⁺ cells were CD29⁺ or CD45RO⁺ “mature” cells while the CD8⁺ cells were primarily CD45RA⁺ “naive” cells. After an initial separation into CD4⁺ and CD8⁺ cells and a secondary separation into CD45 subsets, lymphokine secretion was assessed after phorbol 12-myristate 13-acetate and ionomycin or fixed anti-CD3 stimulation. Within the respective CD45 subsets, CD4⁺ cells produced more interleukin (IL)-2, IL-4, and IL-6; but the CD8⁺ cells secreted more interferon-γ and granulocyte/macrophage-colony-stimulating factor. Tumor necrosis factor-α secretion was similar in the matched CD45 subsets. Northern analysis revealed a parallel pattern of lymphokine mRNA expression in the four lymphocyte subsets. These results suggest that human CD8⁺ peripheral blood lymphocytes have a significant capacity to secrete lymphokines, and that the low lymphokine production observed in unseparated CD8⁺ cells reflects the higher percentage of less functional CD45RA⁺ cells.     

Cytomegalovirus infection induces the accumulation of short-lived, multifunctional CD4+CD45RA+CD27+ T cells: the potential involvement of interleukin-7 in this process

The relative roles that ageing and lifelong cytomegalovirus (CMV) infection have in shaping naive and memory CD4⁺ T-cell repertoires in healthy older people is unclear. Using multiple linear regression analysis we found that age itself is a stronger predictor than CMV seropositivity for the decrease in CD45RA⁺ CD27⁺ CD4⁺ T cells over time. In contrast, the increase in CD45RA⁻ CD27⁻ and CD45RA⁺ CD27⁻ CD4⁺ T cells is almost exclusively the result of CMV seropositivity, with age alone having no significant effect. Furthermore, the majority of the CD45RA⁻ CD27⁻ and CD45RA⁺ CD27⁻ CD4⁺ T cells in CMV-seropositive donors are specific for this virus. CD45RA⁺ CD27⁻ CD4⁺ T cells have significantly reduced CD28, interleukin-7 receptor α (IL-7Rα) and Bcl-2 expression, Akt (ser473) phosphorylation and reduced ability to survive after T-cell receptor activation compared with the other T-cell subsets in the same donors. Despite this, the CD45RA⁺ CD27⁻ subset is as multifunctional as the CD45RA⁻ CD27⁺ and CD45RA⁻ CD27⁻ CD4⁺ T-cell subsets, indicating that they are not an exhausted population. In addition, CD45RA⁺ CD27⁻ CD4⁺ T cells have cytotoxic potential as they express high levels of granzyme B and perforin. CD4⁺ memory T cells re-expressing CD45RA can be generated from the CD45RA⁻ CD27⁺ population by the addition of IL-7 and during this process these cells down-regulated expression of IL-7R and Bcl-2 and so resemble their counterparts in vivo. Finally we showed that the proportion of CD45RA⁺ CD27⁻ CD4⁺ T cells of multiple specificities was significantly higher in the bone marrow than the blood of the same individuals, suggesting that this may be a site where these cells are generated.     

CD45RB expression defines two interconvertible subsets of human CD4+ T cells with memory function

Reciprocal expression of CD45RA and CD45RO in human CD4⁺ T cells defines populations understood to be naive cells (CD45RA⁺CD45RO^?) and memory cells (CD45RA^?CD45RO⁺). We investigate two subsets of CD45RA^?CD45RO⁺ CD4⁺ human T cells which differ by fourfold in their expression of the CD45RB isoform; one is CD45RB^bright and the other is CD45RB^intermediate. In contrast, CD45RA⁺ naive cells are all CD45RB^bright. Both subsets of CD45RA^? cells proliferate in response to recall antigens so we designate them MEM 1 (CD45RO⁺RB^bright) and MEM 2 (CD45RO⁺RB^intermediate). CD45RA and CD45RB expression are regulated independently during in vitro activation of naive cells. When MEM 1 cells are activated they tend to down-regulate CD45RB expression, whereas activated MEM 2 cells tend to up-regulate CD45RB expression. Thus, in contrast to the stability of the CD45RA^?CD45RO⁺ phenotype, the MEM 1 and MEM 2 phenotypes are labile and may interconvert. MEM 1 and MEM 2 cells produced comparable amounts of interleukin(IL)?2, IL?4, and IL-5 though MEM 1 cells produced slightly more interferon(IFN)-γ (mean 1.7-fold more). MEM 1 cells consistently proliferated more (mean 2.3-fold more) than MEM 2 cells early during in vitro activation. Thus, differential expression of CD45RB within CD45RA^? cells defines two subsets that have similar properties except for somewhast greater IFN-γ production and proliferative responses by MEM 1 cells. Variability in CD45RB expression may represent a mechanism for fine-tuning the responsiveness of memory cells in vivo.     

In vitro responses of human CD45R0brightRA− and CD45R0−RAbright T cell subsets and their relationship to memory and naive T cells

The cellular basis of immunological memory, particularly with respect to T cells is not understood. In humans, monoclonal antibodies to CD45 have been used to identify memory (CD45R0) and naive (CD45RA) T cells. However, this identification has been called into question by various studies which suggest that high molecular weight CD45 isoforms may be re-expressed by previously activated cells. In the present study, using cultures which supported responses of naive T cells, we examined the responses of purified CD45R0^brightRA^? or CD45R0^?-RA^bright T cell subsets. The former subset was found to respond preferentially to recall antigens with minimal responses apparent to neo-(or non-recall)-antigens. The inverse pattern was found for CD45R0^?RA^bright T cells, which converted to CD45R0^brightRA^? after stimulation with a neo-antigen. Moreover, the two populations of T cells exhibited distinct response kinetics with a faster response evident from the CD45R0^brightRA^?T cells compared to the CD45R0^?RA^bright subset. The poor responses of CD45R0^?RA^bright T cells to recall antigens compared to neo-antigens suggests that this putative naive population is specifically depleted of reactive T cells following an encounter with antigen. We propose that T cell priming results in the stimulation of many CD45R0^?RA^bright T cells with various T cell receptor specificities from which memory T cells are selected for survival. If re-expression of higher molecular weight isoforms does occur in humans in vivo, our results suggest that R0 expression would be retained (CD45R0⁺RA⁺). Alternatively, if primed CD45R0^?RA^bright T cells exist, they are not prevalent in peripheral blood and thus may be sequestered within lymphoid tissues. Our data support the view that in human peripheral blood, CD45R0^bright and CD45RA^bright expression identify memory and naive CD4⁺ T cells, respectively.     

Age-related accumulation of LFA-1high cells in a CD8+CD45RAhigh T cell population

The differential expression of CD45 isoforms has been suggested as a marker for stages of post-thymic T cell development, that is, CD45RA+CD45R0^? T cells and CD45RA^?CD45R0⁺ T cells are supposed to be virgin and memory cells respectively. Recently, several adhesion molecules have been shown to be up-regulated on the cell surface of memory T cells, and have been suggested to serve as a memory marker. In this study, we investigated the levels of LFA-1 expression on T cells in various subpopulations defined by CD45 isoform expression in donors of various ages. In CD4⁺ T cells, the proportion of LFA-1^high cells among CD45RA^highCD45R0-T cells remained low in all age groups and did not show significant accumulation with age. CD4⁺CD45RA^?CD45R0^highTcells expressed LFA-1 at a higher level than CD4⁺CD45RA^highCD45R0^? T cells. Thus, the currently prevailing view that CD45RA and CD45R0 can be markers for virgin and primed cells was consistent with LFA-1 expression in CD4⁺ T cell population. In CD8⁺ T cells, however, CD45RA^highCD45R0^? T cells consisted of two distinct subpopulations, LFA-1^low and LFA-1^high cells, whereas CD45RA^?CD45R0^high T cells were almost exclusively LFA-1^high When CD29 expression was examined in place of LFA-1 expression, similar results were obtained; CD45RA^high CD45R0^? T cells consisted of two distinct subpopulations, CD29-^{to low} and CD^29high cells, while CD45RA-CD45R0^high T cells were mostly CD29^high. The proportion of LFA-1^high cells in the CD8+CD45RA^high T cell subpopulation increased significantly as a function of age (r = 0.9, p < 0.001). It ranged from 8% in a 14-year-old donor to 94% in a 79-year-old donor. Furthermore, the proportion of CD8⁺CD45RA^highLFA-1^high cells in the CD8⁺ T cell population increased significantly as a function of age (r = 0.85, p < 0.001). On the other hand, the proportion of LEA-1^high cells in CD8⁺CD45RA^? T cell subpopulation exceeded 90% in most donors irrespective of age. These results indicate that the CD8⁺CD45RA^highCD45R0^? T cell subpopulation contains a considerable number of LFA-1^high cells and CD29^high cells, phenotypically similar to previously activated cells. Thus, in terms of LFA-1 and CD29 expressions, the simple scheme that CD45RA is a marker of virgin cells is not applicable to the CD8⁺ T cell population.     

Interleukin-13 is produced by activated human CD45RA+ and CD45R0+ T cells: modulation by interleukin-4 and interleukin-12

Interleukin (IL)-13 is a cytokine originally identified as a product of activated T cells. Little is known, however, about IL-13 production by human T cells and its modulation by other cytokines. Here, we show that IL-13 is produced by activated human CD4⁺ and CD8⁺ CD45R0⁺ memory T cells and CD4⁺ and CD8⁺ CD45RA⁺ naive T cells. In contrast, IL-4, which shares many biological activities with IL-13, is only produced by CD45R0⁺ T cells following activation. Analysis of intracellular cytokine production by single CD45RA⁺ and CD45R0⁺ T cells indicated that IL-13 continued to be produced for more than 24 h after stimulation, whereas IL-4 could not be detected after 24 h. These data were confirmed by measurement of specific mRNA and suggest that IL-13, unlike IL-4, but like interferon-γ (IFN-γ), is a cytokine with long-lasting kinetics. The majority of human CD45R0⁺ T cells produced IL-4 and IL-13 simultaneously. In contrast, IFN-γ protein was generally not co-expressed with IL-4 or IL-13. IL-4 added to primary cultures of highly purified peripheral blood T cells activated by the combination of anti-CD3+anti-CD28 mAb enhanced IL-13 production by CD45RA⁺ and to a lesser extent by CD45R0⁺ T cells. Under these conditions, however, IL-12 inhibited IL-13 production by CD45RA⁺ T cells and to a lesser extent by CD45R0⁺ T cells in a dose-dependent fashion. These inhibiting effects were not related to enhanced IFN-γ production induced by IL-12, since IFN-γ by itself did not affect IL-13 production. Collectively, our data indicate that IL-13 is produced by peripheral blood T cells which also produce IL-4, but not IFN-γ, and by naive CD45RA⁺ T cells which, in contrast, fail to produce IL-4. These observations, together with the long-lasting production of IL-13, suggest that IL-13 may have IL-4-like functions in situations where T cell-derived IL-4 is still absent or where its production has already been down-regulated.     

Antigen-independent adhesion of CD4 CD45RA T cells from cord blood

Antigen-independent adhesion of resting adult CD4⁺ CD45RO⁺ T cells to B lymphocytes has been shown to be transient and can be down-regulated by CD4 major histocompatibility complex (MHC) class II molecule interactions. Conversely, adhesion of adult CD4+ CD45RA+ subpopulation to B cells is not regulated by ligands of CD4. We have investigated the regulation of adhesion of cord blood CD45RA⁺ CD4⁺ T lymphocytes. In contrast to adult CD45RA⁺ CD4⁺ T cells, cord blood CD45RA⁺ CD4⁺ T cells were strongly sensitive to the down-regulation of adhesion mediated by the CD4-HLA class II interaction, since adhesion to MHC class II(⁺) B cells was transient and inhibited by an anti-CD4 antibody. In addition, human immunodeficiency virus gpl60, synthetic gpl06-derived peptides encompassing a CD4 binding site inhibited conjugate formation between cord blood CD45RA⁺ CD4+ T cells and B cells. Following activation of the cord blood CD4 T cells by an anti-CD3 antibody, a conversion from a transient to a stable adhesion pattern of cord blood CD4 T cells to B cells occurred in 2 days. The reversal to a transient adhesion occurred at day 8 following anti-CD3 activation in correlation with a complete shift to a CD45RO phenotype of the cord blood CD4 T cells. These data suggest that CD4 T cell adhesion can be developmentally regulated.     

Rapid re-expression of CD45RC on rat CD4 T cells in vitro correlates with a change in function

Rat CD4⁺ T cells are divided phenotypically by the anti-CD45RC monoclonal antibody OX22 into subsets with contrasting functions. Stimulation of T cells in vitro is known to induce a change in isoform from CD45RC⁺ to CD45RC^?. We have investigated the in vitro conditions which promote a switch in isoform in the opposite direction. We observed that a majority of CD45RC^? CD4 T cells (> 90%) spontaneously re-expressed CD45RC during the first 1-3 days of culture in both the presence and absence of alloantigen. The T cells remained CD45RC⁺ when cultured for 7 days in serum-free growth medium. However, alloantigen-activated lymphocytes, expressing the interleukin-2 receptor (IL-2R), down-regulated CD45RC by day 4 and remained CD45RC^? during the course of the experiment. Using mixtures of allotype-marked CD45RC⁺ and CD45RC^?T cells, it was demonstrated that each subset showed comparable survival, IL-2R expression and time courses of activation in response to alloantigen. The repertoire of neither subset was, therefore, deficient in terms of allorecognition. The rapid re-expression of CD45RC in culture was accompanied by a change in function: CD45RC⁺ “converts”, obtained by overnight culture of CD45RC^? T cells, induced significantly higher graft-versus-host responses. Thus, the transition in culture from CD45RC^? to CD45RC⁺ reflects a major functional reprogramming of the cell and not a trivial modulation of a surface antigen.     

CD38+ CD45RBlow CD4+ T cells: a population of T cells with immune regulatory activities in vitro

An antibody reactive with CD38 revealed both phenotypic and functional heterogeneity amongst CD45RB^low cells. Functional analysis of the CD38⁺ and CD38⁻ fractions showed that the latter contained T cells which responded to recall antigens and produced high levels of cytokine in response to polyclonal stimulation. In contrast, the CD38⁺ population failed to proliferate or to produce detectable levels of cytokines. Despite appearing unresponsive, the CD38⁺ population significantly inhibited anti-CD3-induced proliferation and cytokine secretion by the reciprocal CD38⁻ population. Immune suppression required stimulation through the TCR and was dependent on a physical interaction between regulatory and responding CD4⁺ populations. It did not involve killing of the responding T cells or secretion of IL-10 or TGF-β. Despite some similarities there is no direct correlation between the in vitro suppression characteristic of the CD38⁺ CD45RB^low subset and in vivo suppression which has been shown to be mediated by unseparated CD45RB^low CD4⁺ T cells. However, these results demonstrate that two functionally distinct subsets of T cells reside within the antigen-exposed or CD45RB^low CD4⁺ T cell population and are thus generated in vivo: (1) conventional memory T cells which proliferate and secrete cytokines in response to activation and (2) a population of regulatory T cells which inhibit T cell activation in vitro. Antibodies reactive with CD38 may provide a useful tool with which to study the role of these T cell subsets in the induction and regulation of the immune response.     

Expression in vivo of CD45RA,CD45RB and CD44 on T cell receptor-transgenic CD8+ T cells following immunization

We used mice transgenic for a major histocompatibility complex class I-restricted T cell receptor to study the changes of phenotype in vivo which follow priming by antigen of CD8 T cells. We show that following priming with peptide, CD44 on CD8 T cells is up-regulated. The change of phenotype was relatively stable, as primed CD8 cells isolated from thymectomized mice 6 weeks after priming still expressed increased levels of CD44. CD8 T cells in these mice are still responsive to peptide and could represent long-lived primed cells. No downregulation in vivo of the CD45RA or CD45RB isoforms was found, indicating that there is a differential regulation of the expression of CD44 and CD45RB by activated CD8 transgenic T cells. These results contradict earlier studies in vitro which showed that CD8 T cells which have been primed earlier belong to the CD45RA^? or CD45RB^? subset.     

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.     

Membrane CD45R isoform exchange on CD4 T cells is rapid,frequent and dynamic in vivo

CD4 T cells bearing high (240–190 kDa) and low (180 kDa) molecular mass isoforms of the leukocyte common antigen CD45 define functionally distinct subsets which have been equated with naive and memory T cells. In the rat, CD4 T cells expressing a high molecular mass isoform [identified by monoclonal antibody MRC-OX22 (anti-CD45RC)] exchange this for the 180 kDa molecule (CD45RC^?) when stimulated by antigen. Here we show, by transferring mature allotype-marked CD45RC^? CD4 T cells (depleted of immature Thy-1⁺ CD45RC^? recent thymic emigrants) into normal euthymic recipients, that many T cells re-express the high molecular mass isoform in less than 6 h. By 24 h, 30–60% of CD45RC^? CD4 T cells became CD45RC⁺; within a week the entire cohort appeared to exchange the low for the high molecular mass isoform. Isoform exchange was dynamic and many CD4 T cells returned once again to the CD45RC^? state. CD45RC^? CD4 T cells declined in number more rapidly than the CD45RC⁺ subset after transfer. The results suggest that CD45R isoforms distinguish between resting T cells (CD45RC⁺) and those which have encountered antigen in the recent past. CD45R isoforms would appear to be unsuitable markers of naive and memory T cells.     

3H11, a unique cell surface molecule involved in the function of the CD45RA+subset of CD4+ cells

We have developed a mAb anti-3H11 by immunizing mice with aT cell line derived from the Callithrix Jacchus (common marmoset).anti-3H11 is reactive with 48% of unfractlonated T cells, 62%of CD4⁺ cells and 39% of CD8⁺ cells. Among CD4 cells, anti-3H11preferentially reacts with the CD45RA⁺ T cell subset. The majorityof helper activity for pokeweed mitogen (PWM)-driven B cellIgG synthesis and T cell response to recall antigen such astetanus toxold was found within the 3H11-CD4 cell population,whereas anti-3H11⁺CD4⁺ cells provided poor helper function forPWM-driven B cell IgG synthesis and were more responsive toconcanavalln A and autologous mixed lymphocyte reaction. Biochemicalcharacterization showed that anti-3H11 precipitated a singleprotein band with a relative molecular weight of 32,000 from¹²⁵l-surface labeled cell lysate. Biochemical, phenotyplc andfunctional studies revealed that the 3H11 molecule appearedto be different from previously established molecules on theT cell surface. Interestingly, addition of anti-3H11 to thecombination of CD4 and B cells in the presence of CDS cellsbut not to the combination of CD4 and B cells resulted in enhancementof the suppression of PWM-driven B cell IgG synthesis. Moreover,anti-3H11 had a co-mitogenic effect on T cells via the CD2 andCD3 pathways, and this co-mitogenic activity is restricted tothe CD45RA⁺ T cells. Taken together, our results show that the3H11 molecule is a novel antigen which may play an importantrole in the activation and function of the CD45RA⁺ subset ofT cells.     

Importance of B cell co-stimulation in CD4(+) T cell differentiation: X-linked agammaglobulinaemia, a human model

We were interested in the question of whether the congenital lack of B cells actually had any influence on the development of the T cell compartment in patients with agammaglobulinaemia. Sixteen patients with X‐linked agammaglobulinaemia (XLA) due to mutations in Btk, nine patients affected by common variable immune deficiency (CVID) with <2% of peripheral B cells and 20 healthy volunteers were enrolled. The T cell phenotype was determined with FACSCalibur and CellQuest Pro software. Mann–Whitney two‐tailed analysis was used for statistical analysis. The CD4 T cell memory compartment was reduced in patients with XLA of all ages. This T cell subset encompasses both CD4⁺CD45RO⁺ and CD4⁺CD45RO⁺CXCR5⁺ cells and both subsets were decreased significantly when compared to healthy controls: P = 0·001 and P < 0·0001, respectively. This observation was confirmed in patients with CVID who had <2% B cells, suggesting that not the lack of Bruton's tyrosine kinase but the lack of B cells is most probably the cause of the impaired CD4 T cell maturation. We postulate that this defect is a correlate of the observed paucity of germinal centres in XLA. Our results support the importance of the interplay between B and T cells in the germinal centre for the activation of CD4 T cells in humans.     

CD8+ and CD45RA+ human peripheral blood lymphocytes are potent sources of macrophage inflammatory protein 1α, interleukin-8 and RANTES

The chemokines macrophage inflammatory protein 1α (MIP 1α), interleukin-8 (IL-8) and RANTES are potent regulators of leukocyte trafficking. Examination of chemokine secretion by human peripheral blood lymphocytes after stimulation with anti-CD3 or phorbol 12, 13 myristate acetate and ionomycin showed CD8⁺ cells were the dominant source of MIP 1α and RANTES. Although production of MIP 1α and IL-8 were similar in pharmacologically stimulated CD4⁺ CD45RA⁺, CD4⁺ CD45RO⁺, and CD8⁺ CD45RA⁺ cells, the largest amounts of MIP 1α and RANTES were secreted by CD8⁺ CD45RO⁺ lymphocytes. A parallel pattern of prolonged chemokine mRNA expression for at least 18 h after activation was observed in the T cell subsets. These results confirm that human T lymphocytes have a unique capacity for secretion of these three chemokines. In addition, CD8⁺ cells have an unrecognized role in recruiting cells to sites of inflammation, and adult human CD45RA⁺ cells have a physiologically significant secretory capacity.     

CD4+CD45RA+T cells from adults respond to recall antigens after CD28 ligation

The leukocyte common antigen isoforms CD45RA and CD45RO havelong been used to discriminate human naive and memory T cellsrespectively. This model was largely based on the observationthat CD45RO⁺ T cells respond preferentially to and show a higherfrequency of precursors specific for recall antigens. However,CD45RA⁺ T cells have more stringent requirements for stimulationand standard in vitro assays may favour CD45RO⁺ cells in thisrespect. We tested the hypothesis that CD45RAf T cells respondpoorly to in vitro stimulation with recall antigens becauseof inadequate stimulation rather than a lack of precursors.Limiting dilution analyses (LDA) for tetanus toxoid (lT)-specificT cells were performed in the presence or absence of exogenousantLCD28 antibody. Addition of antLCD28 yielded no proliferationin the absence of specific antigen. The precursor frequencyfor lT in the CD4⁺ CD45RO⁺ population was –1:4000, whilethe frequency of CD4⁺ CD45RA⁺ T cells specific for lT was 4-to >>20-fold lower. Addition of anti-CD28 antibody didnot significantly alter the apparent precursor frequency forCD45RA⁺ cells but yielded an enhancement of the value for CD45RA⁺cells by 3- to >>5-fold. No enhancement of antigen-specificproliferation by antLCD28 was observed with CD45RA⁺ T cellsderived from cord blood, although phytohemagglutinin responsesof these cells were amplified by CD28 antibody. These resultsindicate that conventional LDA underestimate the true precursorfrequency of antigen-specific cells within the adult CD45RA⁺population and support the possibility that a small number ofcells revert from a primed (CD45RO⁺) to an unprimed (CD45RA⁺)state. The majority of memory T cells, however, appear to residein the CD45RO⁺ population     

The CD7− T cell subset represents the majority of IL-5-secreting cells within CD4+CD45RA− T cells

Absence of CD7 is a stable phenotype in a subset of normal human T cells. Most circulating CD7⁻ T cells express the CD4⁺CD45RO⁺CD45RA⁻ memory phenotype. We analysed CD4⁺CD45RA⁻ peripheral blood lymphocytes that were separated into CD7⁺ and CD7⁻ for their in vitro cytokine secretion in response to different stimuli. The CD4⁺CD7⁻ subpopulation was found to secrete significantly higher levels of IL-5 compared with the CD4⁺CD7⁺ subset upon stimulation with ionomycin/phorbol myristate acetate (PMA) plus anti-CD28 MoAbs. In contrast to IL-5 secretion, IL-4 and interferon-gamma (IFN-γ) secretion was not significantly different in CD7⁺ and CD7⁻ T cells upon stimulation in vitro. The data indicate that the CD4⁺CD7⁻ T cell represents the majority of IL-5-secreting cells within the population of CD4⁺CD45RA⁻ memory T cells. Since CD4⁺CD7⁻ T cells were found to be enriched in various skin lesions associated with eosinophilic infiltration, the results of our study support the hypothesis that skin-infiltrating CD7⁻ T cells are one of the major sources of IL-5 responsible for the development of eosinophilic inflammation in certain skin diseases.     

Hyper-reactivity of mouse CD45RA − T cells

Mouse CD4 T cells have been partitioned into CD45RA and CD45RA^? subpopulations by means ot the monoclonal antibody 14.8. The CD45RA^? subpopulation proliferated more actively and generated more interleukin-4 (IL-4) in response to stimulation with anti-CD3 antibody and phytohemaglutinin, and more IL-2 in response to anti-CD3. This subpopulation is therefore hyper-reactive to these polyclonal stimulators, but does not show the bias towards T helper type 2 activity that has been found in studies with other related CD45 isoforms. No evidence of suppression was obtained by comparing proliferation of CD45RA^? cells in the presence and absence of CD45RA cells. Thus mouse CD4 T cells behave in these respects similarly to those of man, as is evident in a brief review of the quiescence-activation-quiescence cycle in the two species.     

Role of CD4+CD45RA+ T cells in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse

The non-obese diabetic (NOD) mouse spontaneously develops aT cell-mediated autoimmune disease, sharing many features withhuman insulin-dependent diabetes mellltus (IDDM), leading toinsulin-secreting ß cell destruction. The role ofCD4⁺ T cells has been evidenced at two levels. First, CD4⁺ Tcells from diabetic animals are required to transfer diabetesto non-diabetic recipients in conjunction with CD8⁺ effectorT cells. Second, suppressive CD4⁺ T cells have been characterizedin non-diabetic NOD mice. T cells with different functions canthus share the CD4⁺ phenotype. Since CD4⁺ T cells can be dividedinto at least two subgroups on the basis of CD45 isoform expression,we evaluated the distribution of CD4⁺ T cells expressing theCD45RA isoform on NOD mouse thymocytes and peripheral T cells.The percentage of CD45RA⁺ cells was dramatically increased amongthe most mature CD3^bright thymocytes and among CD4⁺ T cellsin lymph nodes of the NOD mouse as compared with control strains.This increase was related to the development of insulitls. Interestingly,the CD45RA isoform was expressed on most CD4⁺ T cells invadingthe islets. In vivo treatment with an antl-CD45RA mAb preventedthe development of insulitls and spontaneous diabetes in femaleanimals but not the transfer of diabetes by T cells collectedfrom diabetic NOD donors. These results indicate that anti-CD45RAmAb is only effective if given before the full commitment ofeffector T cells to the destruction of islet ß cells.ThusCD4⁺CD45RA⁺ T cells play a key role in early activation stepsof anti-islet immunity.