Human herpesvirus 6 infection of CD4+ T-cell subsets

The immune system includes CD4+ regulatory T (T reg) cells that play a role in self-tolerance and demonstrate functional variations that govern immune responses. HHV-6 is an important immunosuppressive virus that completely replicates in vivo and in vitro in only CD4+ T cells. However, there have been no reports of the specific T-cell subpopulation that permits the replication of this virus. Here, we evaluated the infectivity of HHV-6 to specific T-cell populations such as CD4+CD25 high, which includes the majority of T reg cells, and CD4+CD25(-). These cells were isolated from peripheral blood and then expanded. The expanded cell fractions were then infected with the HHV-6 variant B strain, and the spreads of infected cells were evaluated by immunofluorescence. Viral growth was also quantified by real-time PCR. The effects of virus infection on cytokine production from these T-cell subsets were examined using ELISA. Our results revealed that both these fractions permitted complete HHV-6 replication. Virus infection enhanced the production of both Th1- and Th2-type cytokines from CD4+CD25(-) T cells; however, only Th2-type cytokine release was augmented from viral-infected CD4+CD25 high T cells. Further, while virusinfected CD4+CD25 high T cells shift their antiviral immunity toward Th2 dominance by producing IL-10, the role of virus-infected CD4+CD25(-) T cells remains obscure.     

Role of acid sphingomyelinase bioactivity in human CD4+ T-cell activation and immune responses

Acid sphingomyelinase (ASM), a lipid hydrolase enzyme, has the potential to modulate various cellular activation responses via the generation of ceramide and by interaction with cellular receptors. We have hypothesized that ASM modulates CD4⁺ T-cell receptor activation and impacts immune responses. We first observed interactions of ASM with the intracellular domains of both CD3 and CD28. ASM further mediates T-cell proliferation after anti-CD3/CD28 antibody stimulation and alters CD4⁺ T-cell activation signals by generating ceramide. We noted that various pharmacological inhibitors of ASM or knockdown of ASM using small hairpin RNA inhibit CD3/CD28-mediated CD4⁺ T-cell proliferation and activation. Furthermore, such blockade of ASM bioactivity by biochemical inhibitors and/or molecular-targeted knockdown of ASM broadly abrogate T-helper cell responses. In conclusion, we detail immune, pivotal roles of ASM in adaptive immune T-cell responses, and propose that these pathways might provide novel targets for the therapy of autoimmune and inflammatory diseases.Acid sphingomyelinase (ASM), a lipid hydrolase enzyme localized to lysosomes and cell membranes, converts sphingomyelin to ceramide,¹ an important lipid messenger mediating cell signaling.^{2, 3} Through the generation of ceramide, ASM appears to have an important role in regulating cell differentiation, proliferation, and apoptosis.^{1, 4} Abnormalities in ASM bioactivity result in multiple system disorders. As an example, patients with Niemann–Pick disease, who have mutations in the ASM gene, exhibit neurological symptoms at early age, and develop visceral organ abnormalities in later life.⁴ Patients with Niemann–Pick disease are at risk of infections,⁵ as can be modeled in ASM-deficient mice.^{6, 7} This phenotype has been attributed to phagocyte dysfunction.⁸ Recently, however, ASM function has also been described and noted in various other non-phagocytic immune cells, for example, regulating cytotoxic granule secretion by CD8⁺ T cells.⁹ASM has been reported to modulate T-cell receptor (TCR) signaling initiated by TNF,¹⁰ mediate CD28 signals,¹¹ and induce or rescue CD4⁺ T cells from apoptosis under certain circumstances.^{12, 13} By generating ceramide, ASM serves as a regulator of intracellular downstream signaling. However, the exact manner whereby ASM participates in TCR/CD3 or/and CD28 signaling remains controversial.^{10, 11, 14} Furthermore, the molecular mechanisms as to how ASM regulates CD4⁺ T-cell activation are still largely unexplored.Adaptive immune responses are important in the maintenance of human immune homeostasis. Imbalances in T-helper cell (Th) responses associated with aberrant CD4⁺ T-cell activation contribute to the development of inflammation as in human autoimmune diseases.^{15, 16} It remains unclear whether or how ASM might dictate Th responses during the progression of inflammatory diseases.In the present study, we confirm that ASM interacts with CD3 and CD28, and mediates intracellular signals that control CD4⁺ T-cell activation. ASM inhibition either by pharmacological inhibitors of ASM or knockdown of ASM results in decreased ceramide production. This leads to non-responsiveness of CD4⁺ T-cell to CD3/CD28 engagement, and causes globally diminished Th responses. These data suggest the pivotal role of ASM in CD3/CD28 intracellular signaling and adaptive immune responses, and also provide a potential target for the therapy of immune disease.     

Antiviral CD4 and CD8 T-cell memory: differences in the size of the response and activation requirements

Following acute lymphocytic choriomeningitis virus (LCMV) infection, there is a potent antiviral CD8 T-cell response that eliminates the infection. This initial CD8 T-cell response is followed by a period of memory during which elevated numbers of virus-specific CD8 T cells remain in the mouse. CD4 T cells are also activated after LCMV infection, but relatively less is known about the magnitude and duration of the CD4 response. In this study, we used intracellular staining for interferon-gamma to measure both CD4 and CD8 responses in the same mice at the single cell level. After LCMV infection, there was an increase in the number of activated CD4 T cells and an associated increase in the number of virus-specific CD4 T cells. At the peak of this expansion phase, the frequency of virus-specific CD4 T cells was 1 in 20 (0.5-1.0 x 10(6) per spleen). Like the CD8 response, long-term CD4 memory could be found up to a year after the infection with frequencies of approximately 1 in 260 (0.5-1.5 x 10(5) per spleen). However, the magnitude of virus-specific CD8 T cells was greater than virus-specific CD4 T cells during all phases of the immune response (expansion, death, and memory). At day 8, there were 20- to 35-fold more virus-specific CD8 T cells than CD4 T cells. This initial difference in cell number lasted into the memory phase as there remained a ten- to 20-fold difference in the CD8 and CD4 responses. These results highlight the importance of the expansion phase in determining the size of the memory T-cell pool. In addition to the difference in the magnitude, the activation requirements of CD8 and CD4 T-cell responses were different: CD8 T responses were not affected by blockade of CD40-CD40 ligand interaction whereas CD4 responses were reduced 90%. So while there is long-term memory in both the CD8 and CD4 compartments, the rules regulating the activation of CD8 and CD4 T cells and the overall magnitude of the responses are different.     

Maturation- and differentiation-dependent responsiveness of human CD4+ T helper subsets.

The human CD45R0+ (memory) CD4+ T cell population can be subdivided into a large (82%) CD27+ and a small (18%) CD27- subset. Within the CD45R0+CD27- subset, cells accumulate that have been persistently stimulated by Ag in vivo. As an apparent consequence, TLC with a differentiated functional phenotype, producing either high levels of IFN-gamma (Th1-like), high levels of IL-4 (Th2-like) or high amounts of both these cytokines (here referred to as Thx) can primarily be generated from the CD27- memory CD4+ T cell subset. In this study we examined the requirements for induction of proliferation of distinct CD4+CD45R0+ Th subsets. Immobilized CD3 mAb induced proliferation with comparable magnitude and kinetics in all types of TLC. However, interference with intracellular signaling pathways in this activation system, resulted in a strong inhibition of proliferation in TLC derived from CD27+ cells whereas, in contrast, TLC from CD27- cells were relatively resistant to elevation of [cAMP]i, inhibition of protein kinase C activation and the immunosuppressive effects of cyclosporin A. Stimulation with CD3 mAb in soluble form resulted in Il-4 secretion by Th2-like and Thx-type TLC but did not induce IFN-gamma or Il-2 secretion in any Th subset. Interestingly, Th2-like cells but not Thx-type cells were able to use endogenously produced Il-4 for proliferation. These data demonstrate a differential sensitivity of CD45R0+CD4+ Th subsets for immune activation and suppression, which correlated with their maturation stage, as reflected by CD27 membrane expression, as well as with their effector phenotype.     

Age-related changes in mature CD4+ T cells: cell cycle analysis

T cell proliferative responses decrease with age, but the mechanisms responsible are unknown. We examined the impact of age on memory and naive CD4(+) T cell entry and progression through the cell cycle using acridine orange to identify cell cycle stage. For both subsets, fewer stimulated cells from old donors were able to enter and progress through the first cell cycle, with an increased number of cells arrested in G(0) and fewer cells in post G(0) phases. The number of dead cells as assessed by sub-G(0) DNA was also significantly greater in the old group. CD4(+) T cells from old mice also exhibited a significant reduction in clonal history as assessed by CFSE staining. This was associated with a significant decline in cyclin D2 mRNA and protein. We propose that decreases in cyclin D2 are at least partially responsible for the proliferative decline found in aged CD4(+) T cells.     

The complexity of the CD4 T-cell responses: old and new T-cell subsets

The T-cell compartment of the immune system reacts to an enormous variety of antigens, including self antigens, due to its a wide repertoire of T-cell clones. Self-reactive T cells undergo a negative selection process resulting in apoptosis of T cells with high affinity for self-peptides. Self-reactive T cells escaped to negative selection are then controlled by natural T regulatory (Treg) cells. Regulation also controls excessive effector T-cell responses. Three types of effector T cells are recognized: T helper 1 (Th1) cells, which protect against intracellular bacteria; Th2 cells, which play a role against parasites; Th17 cells, which would face extracellular bacteria, but also are involved in autoimmunity. Effector T-cell polarization is determined by the complex interaction of antigen-presenting cells with naive T cells and involves a multitude of factors, including the dominant cytokine environment, costimulatory molecules, type and load of antigen presented and signaling cascades. The decision for the immune response to go in a certain direction is based not onto one signal alone, rather onto many different elements acting synergistically, antagonistically and through feedback loops leading to activation of Th1, Th2, or Th17 responses. Both Th1 and Th2 can be suppressed by adaptive Treg cells through contact-dependent mechanisms and/or cytokines.     

Distinct mechanisms of CD4+ and CD8+ T-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4(+) and CD8(+) T cells. Infection of primary T-cell cultures with ELI6 induced CD4(+) T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4(+) and CD8(+) T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4(+) T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8(+) T cells was triggered by a soluble factor(s) secreted by CD4(+) T cells. HIV-1 virions activated CD4(+) and CD8(+) T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25(+)HLA-DR(+) T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4(+) and CD8(+) T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.     

Functional uncoupling of T-cell receptor engagement and Lck activation in anergic human thymic CD4+ T cells

Human thymic CD1a-CD4+ T cells in the final stage of thymic maturation are susceptible to anergy induced by a superantigen, toxic shock syndrome toxin-1 (TSST-1). Thymic CD4+ T-cell blasts, established by stimulating human thymic CD1a-CD4+ T cells with TSST-1 in vitro, produce a low level of interleukin-2 after restimulation with TSST-1, whereas TSST-1-induced adult peripheral blood (APB) CD4+ T-cell blasts produce high levels of interleukin-2. The extent of tyrosine phosphorylation of the T-cell receptor zeta chain induced after restimulation with TSST-1 was 2-4-fold higher in APB CD4+ T-cell blasts than in thymic CD4+ T-cell blasts. The tyrosine kinase activity of Lck was low in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, the Lck kinase activity increased in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Surprisingly, Lck was highly tyrosine-phosphorylated in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, it was markedly dephosphorylated in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Lck from APB CD4+ T-cell blasts bound the peptide containing the phosphotyrosine at the negative regulatory site of Lck-505 indicating that the site of dephosphorylation in TSST-1-activated T-cell blasts is Tyr-505. Confocal microscopy demonstrated that colocalization of Lck and CD45 was induced after restimulation with TSST-1 in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Further, remarkable accumulation of Lck in the membrane raft was observed in restimulated APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. These data indicate that interaction between Lck and CD45 is suppressed physically in thymic CD4+ T-cell blasts and plays a critical role in sustaining an anergic state.     

Memory functions and death proneness in three CD4+CD45RO+ human T cell subsets

We propose a classification of human CD4(+)CD45RO(+) memory T cells into three new subsets based on cell surface expression levels of CD43. The first subset consists of cells whose CD43 expression is relatively high; this subset also contains the highest proportion of recall Ag-reactive precursors, and its constituent cells respond far more strongly than cells in either of the other subsets to immobilized CD3 Ab in addition to secreting substantially more IFN-gamma and IL-4. Cells of the second subset express similar levels of CD43 to naive cells, and they also respond weakly to TCR-mediated stimuli as judged by either their ability to proliferate or capacity for cytokine production. The third subsets consists of cells whose CD43 expression levels are clearly down-regulated; its cells appear to be anergic to TCR-mediated stimuli, and when examined ex vivo many of them appear to be undergoing either spontaneous apoptosis via a caspase-independent pathway or Fas-mediated apoptosis via a caspase-dependent pathway, even in the resting state. An analysis of telomere lengths revealed that the typical telomere of a cell in the second subset was significantly longer than the typical telomere in the first or third subset. Taken together, these results appear to indicate that CD4(+)CD45RO(+) T cells fall into three functionally differing subsets, one being a subset of cells with fully matured memory phenotype, a second being a less mature subset of cells that retain longer telomeres and whose memory functionality is marginal, and a third consisting of anergic cells that give every appearance of being death-prone and/or in the process of dying.     

Establishment and functional characterization of human herpesvirus 6-specific CD4+ human T-cell clones.

In order to clarify the protective immune responses against a newly identified herpesvirus, human herpesvirus 6 (HHV-6), we established HHV-6-specific human T-cell clones and examined their functional properties. Five CD3+CD4+CD8- T-cell clones, which proliferated in response to stimulation with two different strains of HHV-6 in the presence of autologous antigen-presenting cells but not with herpes simplex virus type 1 or human cytomegalovirus, were established from peripheral blood lymphocytes of a healthy individual. The proliferative response of all T-cell clones to HHV-6 antigen was inhibited by addition of anti-HLA-DR monoclonal antibody, indicating that these clones were human leukocyte antigen (HLA) class II DR restricted. Of the five clones, two lysed HHV-6-infected autologous lymphoblasts, but not HHV-6-infected allogeneic cells or natural killer-sensitive K562 cells (group 1); one showed cytotoxicity against HHV-6-infected autologous lymphoblasts as well as HHV-6-infected allogeneic cells and K562 cells (group 2); and the remaining two showed no cytotoxic activity (group 3). The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR monoclonal antibody to the culture, whereas this monoclonal antibody had no effect on the cytotoxicity of group 2 and did not induce the cytotoxicity of group 3. Perforin, which is one of the mediators of cytotoxicity, was abundantly expressed in group 1 and 2 clones. Moreover, all groups of clones produced gamma interferon after culture with antigen-presenting cells followed by HHV-6 antigen stimulation. These results suggest that HHV-6-specific CD4+ T cells have heterogeneous functions.     

Allogeneic differences in the dependence on CD4+ T-cell help for virus-specific CD8+ T-cell differentiation

CD4⁺ T-cell help enables antiviral CD8⁺ T cells to differentiate into fully competent memory cells and sustains CD8⁺ T-cell-mediated immunity during persistent virus infection. We recently reported that mice of C57BL/6 and C3H strains differ in their dependence on CD28 and CD40L costimulation for long-term control of infection by polyoma virus, a persistent mouse pathogen. In this study, we asked whether mice of these inbred strains also vary in their requirement for CD4⁺ T-cell help for generating and maintaining polyoma virus-specific CD8⁺ T cells. CD4⁺ T-cell-depleted C57BL/6 mice mounted a robust antiviral CD8⁺ T-cell response during acute infection, whereas unhelped CD8⁺ T-cell effectors in C3H mice were functionally impaired during acute infection and failed to expand upon antigenic challenge during persistent infection. Using (C57BL/6 × C3H)F₁ mice, we found that the dispensability for CD4⁺ T-cell help for the H-2^b-restricted polyoma virus-specific CD8⁺ T-cell response during acute infection extends to the H-2^k-restricted antiviral CD8⁺ T cells. Our findings demonstrate that dependence on CD4⁺ T-cell help for antiviral CD8⁺ T-cell effector differentiation can vary among allogeneic strains of inbred mice.     

The cytomegalovirus-specific CD4+ T-cell response expands with age and markedly alters the CD4+ T-cell repertoire

Immune function in the elderly is associated with a number of phenotypic and functional abnormalities, and this phenomenon of immune senescence is associated with increased susceptibility to infection. The immune response to pathogens frequently declines with age, but the CD8(+) T-cell response to cytomegalovirus (CMV) is unusual, as it demonstrates a significant expansion over time. Here we have documented the CD4(+) T-cell immune response to CMV in healthy donors of different ages. The magnitude of the CMV-specific CD4(+) T-cell immune response increases from a mean of 2.2% of the CD4(+) T-cell pool in donors below 50 years of age to 4.7% in donors aged over 65 years. In addition, CMV-specific CD4(+) T cells in elderly donors demonstrate decreased production of interleukin-2 and less dependence on costimulation. CMV seropositivity is associated with marked changes in the phenotype of the overall CD4(+) T-cell repertoire in healthy aged donors, including an increase in CD57(+) expression and a decrease in CD28 and CD27 expression, a phenotypic profile characteristic of immune senescence. This memory inflation of CMV-specific CD4(+) T cells contributes to evidence that CMV infection may be damaging to immune function in elderly individuals.     

Requirement of cognate CD4+ T-cell recognition for the regulation of allospecific CTL by human CD4+ CD127- CD25+ FOXP3+ cells generated in MLR

Although immunoregulation of alloreactive human CTLs has been described, the direct influence of CD4(+) Tregs on CD8(+) cytotoxicity and the interactive mechanisms have not been well clarified. Therefore, human CD4(+)CD127(-)CD25(+)FOXP3(+) Tregs were generated in MLR, immunoselected and their allospecific regulatory functions and associated mechanisms were then tested using modified (51)Chromium release assays (Micro-CML), MLRs and CFSE-based multi-fluorochrome flow cytometry proliferation assays. It was observed that increased numbers of CD4(+)CD127(-)CD25(+)FOXP3(+) cells were generated after a 7 day MLR. After immunoselection for CD4(+)CD127(-)CD25(+) cells, they were designated as MLR-Tregs. When added as third component modulators, MLR-Tregs inhibited the alloreactive proliferation of autologous PBMC in a concentration dependent manner. The inhibition was quasi-antigen specific, in that the inhibition was non-specific at higher MLR-Treg modulator doses, but non-specificity disappeared with lower numbers at which specific inhibition was still significant. When tested in micro-CML assays CTL inhibition occurred with PBMC and purified CD8(+) responders. However, antigen specificity of CTL inhibition was observed only with unpurified PBMC responders and not with purified CD8(+) responders or even with CD8(+) responders plus Non-T "APC". However, allospecificity of CTL regulation was restored when autologous purified CD4(+) T cells were added to the CD8(+) responders. Proliferation of CD8(+) cells was suppressed by MLR-Tregs in the presence or absence of IL-2. Inhibition by MLR-Tregs was mediated through down-regulation of intracellular perforin, granzyme B and membrane-bound CD25 molecules on the responding CD8(+) cells. Therefore, it was concluded that human CD4(+)CD127(-)CD25(+)FOXP3(+) MLR-Tregs down-regulate alloreactive cytotoxic responses. Regulatory allospecificity, however, requires the presence of cognate responding CD4(+) T cells. CD8(+) CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25(+) activation epitopes.     

The requirements for activation of an antigen specific T-cell clone via the CD3 complex.

We have studied the requirements for the activation of the haemagglutinin peptide specific cloned T-cell HA1.7 by anti-CD3 antibodies and the mechanism of modulation of the CD3 complex. Cross-linking of the complex either by a soluble antibody second layer or an immobilized matrix of antibody is necessary for induction of a proliferative response. This proliferative response is totally dependent on exogenously added IL-2. Modulation of the cross-linked CD3 complex is by patching, capping and internalization of the antigen and is not essential for the proliferative response.     

Multiple specificities in the murine CD4+ and CD8+ T-cell response to dengue virus.

The target epitopes, serotype specificity, and cytolytic function of dengue virus-specific T cells may influence their theoretical roles in protection against secondary infection as well as the immunopathogenesis of dengue hemorrhagic fever. To study these factors in an experimental system, we isolated dengue virus-specific CD4+ and CD8+ T-cell clones from dengue-2 virus-immunized BALB/c mice. The T-cell response to dengue virus in this mouse strain was heterogeneous; we identified at least five different CD4+ phenotypes and six different CD8+ phenotypes. Individual T-cell clones recognized epitopes on the dengue virus pre-M, E, NSl/NS2A, and NS3 proteins and were restricted by the I-Ad, I-Ed, Ld, and Kd antigens. Both serotype-specific and serotype-cross-reactive clones were isolated in the CD4+ and CD8+ subsets; among CD8+ clones, those that recognized the dengue virus structural proteins were serotype specific whereas those that recognized the nonstructural proteins were serotype cross-reactive. All of the CD8+ and one of five CD4+ clones lysed dengue virus-infected target cells. Using synthetic peptides, we identified an Ld-restricted epitope on the E protein (residues 331 to 339, SPCKIPFEI) and a Kd-restricted epitope on the NS3 protein (residues 296 to 310, ARGYISTRVEM GEAA). These data parallel previous findings of studies using human dengue virus-specific T-cell clones. This experimental mouse system may be useful for studying the role of the virus serotype and HLA haplotype on T-cell responses after primary dengue virus infection.     

CD4+CD45RA+ and CD4+CD45RA- T cell subsets in man maintain distinct function and CD45RA expression persists on a subpopulation of CD45RA+ cells after activation with Con A.

We have previously shown that Con A-induced suppressor T cells belong to the CD45RA+ subset. After unseparated T cells are activated with Con A, CD45RA expression increases to a maximum (Day 2), and then decreases significantly, but does not disappear entirely (Day 9), while CD29 expression increases steadily. In the present study, we examined the fate of these cell surface molecules on isolated CD4+CD45RA+ and CD4+CD45RA- cells following activation with Con A, and their relationship to the regulatory functions of these subsets. After activation of CD4+CD45RA+ cells with Con A, CD45RO and CD29 antigen expression rapidly increases (greater than 90%). While CD45RA expression is downregulated, approximately 40% of the cells continue to express low-density CD45RA in a stable fashion through Day 21. Despite these phenotypic changes, cells originally CD45RA+ continue to suppress IgG synthesis and provide only minimal B cell help. Furthermore, when cells originally CD45RA+ were sorted on the basis of continued presence, or loss of CD45RA antigen 14 days after activation, both populations demonstrated potent suppression and minimal help. In contrast, after activation with Con A, CD4+CD45A- cells maintain stable phenotype and provide significant help and minimal suppression. Immunoprecipitation of the CD45RA antigen from Day 14 activated CD4+CD45RA+ cells confirms the continued presence of the 205-kDa isoform, but reveals a significant decrease in the 220-kDa isoform. These results suggest that after activation with Con A, cells originally CD45RA+ remain functionally distinct from cells originally CD45RA-, and that CD45RA antigen persists on a subpopulation of CD45RA+ cells after activation with Con A.