CD45 isoform phenotypes of human T cells: CD4(+)CD45RA(-)RO(+) memory T cells re-acquire CD45RA without losing CD45RO

We have studied the alterations in CD45R phenotypes of CD4(+)CD45RA(-)RO(+) T cells in recipients of T cell-depleted bone marrow grafts. These patients are convenient models because early after transplantation, their T cell compartment is repopulated through expansion of mature T cells and contains only cells with a memory phenotype. In addition, re-expression of CD45RA by former CD4(+)CD45RA(-) T cells can be accurately monitored in the pool of recipient T cells that, in the absence of recipient stem cells, can not be replenished with CD45RA(+) T cells through the thymic pathway. We found that CD4(+)CD45RA(-)RO(+) recipient T cells could re-express CD45RA but never reverted to a genuine CD4(+)CD45RA(+)RO(-) naive phenotype. Even 5 years after transplantation, they still co-expressed CD45RO. In addition, the level of CD45RA and CD45RC expression was lower ( approximately 35 %) than that of naive cells. In contrast, the level of CD45RB expression was comparable to that of naive cells. We conclude that CD4(+)CD45RA(-)RO(+) T cells may re-express CD45(high) isoforms but remain distinguishable from naive cells by their lower expression of CD45RA / RC and co-expression of CD45RO. Therefore, it is likely that the long-lived memory T cell will be found in the population expressing both low and high molecular CD45 isoforms.     

Definition of the HLA-A2 restricted peptides recognized by human CD8+ effector T cells by flow-assisted sorting of the CD8+ CD45RA+ CD28- T cell subpopulation

In response to antigenic stimulation, naive MHC-class I restricted and antigen-specific CD8+ CD45RA+ CD28+ T cells undergo clonal expansion, differentiate into CD8+ CD45RO+ memory T cells and convert to CD8+ CD45RA+ CD28- T cells displaying potent immune effector functions upon re-encounter with the nominal antigen. We show that the effector CD8+ CD45RA+ CD28- T cell subset is expanded in peripheral blood lymphocytes (PBL) from patients with human papilloma virus (HPV)+ cervical lesions as well as in PBL from patients with pulmonary tuberculosis. Flow-cytometric cell sorted CD8+ CD45RA+ CD28- and CD8+ CD45RA+ CD28- T cells were tested for recognition of HLA-A2 restricted peptides derived either from the human papillomavirus (HPV)16-E7 gene product, or from M. tuberculosis antigens. Mostly CD8+ CD45+ CD28- T cells define antigen/peptide-specific and MHC-restricted responses. These data were confirmed in PBL from patients with tuberculosis using HLA-A2 tetramer-complexes loaded with a peptide from the M. tuberculosis Ag85b antigen by flow cytometry. The sorting of this T cell subset enables to determine the fine specificity of CD8+ effector T cells without the need for in vitro manipulation.     

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.     

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells

Aside from an intermediate stage in thymic T-cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T-cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti-CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA-mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA-mediated effect. Autologous CD4+ and CD8+ T-cell co-cultures in the presence of PHA induced this CD4dimCD8bright T-cell expression by 33%, demonstrating a role for CD4 cells in the PHA-mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4- counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominantly T-cell receptor (TCR) alphabeta cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.     

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.     

Deficiency of suppressor-inducer (CD4+CD45RA+) T cells in autism

CD4+ cells are a heterogenous population of lymphocytes including at least two distinct subpopulations: CD45RA+ cells, inducers of suppressor T cells and CDw29+ cells, inducers of helper function for antibody production. To investigate the possibility that immune abnormalities in autism may involve abnormal distribution of these helper subpopulations, monoclonal antibodies were used in flow cytometric analysis to characterize peripheral blood lymphocytes of 36 subjects with autism. The autistic subjects as compared to a group of 35 healthy age-matched subjects had a significantly reduced number of lymphocytes, a decreased number of CD2+ T cells and reduced numbers of CD4+ and CD4+CD45RA+ lymphocytes. The numbers of B (CD20+) cells, suppressor T (CD8+) cells, inducers of helper function (CD4+CDw29+) and natural killer (CD56+) cells were not altered in the autistic subjects. Our results suggest that an alteration in the suppressor-inducer T-cell subset is associated with autism.     

Expression of naive/memory (CD45RA/CD45RO) markers by peripheral blood CD4+ and CD8+ T cells in children with asthma

Introduction: The role of CD4⁺ T cells in the immunopathogenesis of asthma is well documented. Little is known about the role of CD8⁺ T cells. The aim of this study was to assess peripheral blood subsets of CD4⁺ and CD8⁺ T cells expressing naive/memory markers (CD45RA⁺/RO⁺) and the activation marker (CD25⁺) in children with allergic asthma. Materials and Methods: Peripheral blood mononuclear cells were isolated from children with allergic asthma and healthy children. T cell subsets were analyzed by flow cytometry for the expressions of CD45RA, CD45RO, and CD25. In this study, some differences in the memory compartment of peripheral blood T cells between asthmatic children and healthy controls were detected. Results: The absolute number of CD8⁺ T cells expressing CD45RO was significantly elevated and the percentages of CD3⁺ T cells expressing activation marker CD25 and of CD4⁺ T cells expressing memory marker CD45RO were significantly lower in children with asthma compared with controls. No correlation was found between severity of asthma and peripheral blood lymphocyte subsets. Conclusions: There were some differences in the memory compartment of peripheral blood T cells between asthmatic children and healthy controls. The increase in the number of CD8⁺ T cells expressing the memory marker (CD45RO) in children with allergic asthma may indicate that CD8⁺ T cells play a role in the pathogenesis of asthma.     

Phenotypic changes associated with activation of CD45RA+ and CD45RO+ T cells.

Resting CD45RO+, mature/memory, T cells are phenotypically distinct from intermediate CD45RO+/CD45RA+ and CD45RA+, immature/virgin, T cells, and are characterized by high levels of expression of a number of adhesion molecules, such as CD2, CD18, CD58 and CD29. The kinetics of up-regulation of molecules, like CD25 and CD54 associated with activation, were similar in both subsets and suggested that their high level expression was associated with later events rather than initial recognition and signal transduction. CD45RA+ T cells, unlike CD45RO+ T cells, were unable to proliferate in response to mitogenic combinations of CD2 monoclonal antibodies (mAb), although in combination with submitogenic doses of PMA both up-regulation of cell-surface molecules and proliferation occurred. In addition, recruitment of CD45RA+ T cells by CD2 mAb-activated CD45RO+ T cells can occur.     

CD45RA expression on HCMV-specific effector memory CD8+ T cells is associated with the duration and intensity of HCMV replication after transplantation

In this cross-sectional study on 42 solid organ transplant recipients, the association of kinetics of human cytomegalovirus (HCMV) replication and EMRA HCMV-specific CD8+ T cells was investigated. Correlation was observed between the duration of HCMV replication after transplantation and CD45RA+CD27− (r = 0.609; p = 0.004), CD45RA+ CD28− (r = 0.579; p = 0.008) or CD45RA+CCR7− (r = 0.488; p = 0.029) HCMV-specific CD8+ T cells percentages. In the multivariate regression analyses, CD45RA+CD27−, CD45RA+CD28− or CD45RA+CCR7− HCMV-specific CD8+ T cells percentages increased 5.58% (p = 0.001), 5.35% (p = 0.001) or 4.49% (p = 0.012), respectively, with every 10-day increase in the duration of HCMV replication. Moreover, CD45RA+CD27− or CD45RA+CD28− frequencies increased 4.16% (p = 0.024) or 3.58% (p = 0.049), respectively, with every unity increase in log₁₀ genomes/mL. These observations support the major association between the frequency of EMRA HCMV-specific CD8+ T cells and the duration of post-transplant HCMV replication episodes in solid organ transplantation recipients.     

CD4+ CD45RA+ and CD4+ CD45RO+ T cells differ in their TCR-associated signaling responses.

Peripheral CD4+ T cells can be divided into two different functional populations based on the expression of distinct isoforms of the surface molecule CD45. We have investigated the differences in the proximal signaling induced by anti-CD3 monoclonal antibody in purified populations of "naive" CD45RA+ and "memory" CD45RO+ human CD4+ T cells. Expression of cell surface CD3, CD4 and CD28 was comparable between RA+ and RO+ cells. However, TCR-directed stimulation in the form of anti-CD3 produced markedly different patterns of intracellular signaling. Greater inositol triphosphate generation occurred in naive cells and the rise in intracellular free calcium was also substantially greater in naive than in memory cells. Cells with the naive phenotype were considerably more active in TCR-dependent tyrosine phosphorylation, both at an overall level and specifically in terms of TCR-zeta and ZAP-70 phosphorylation. Despite these differences in phosphorylation, the amounts of TCR-zeta, ZAP-70 and Ick were equivalent between the two subsets. These findings suggest that the TCR-dependent signaling is differentially regulated in naive and memory CD4+ T cells. This may be due to differences in the way that the two isoforms of the CD45 phosphatase regulate the activity of proximal kinases in the TCR signaling pathway, and could be an important means by which the unique functions of differentiated T cell populations are maintained.     

In the mouse the maturation stage of the peripheral CD4+ CD45RA+ subset is different from that of the CD8+ CD45RA+ subset.

In vitro studies have suggested that the presence of CD45RA on subsets of CD4 and CD8 cells defines naive T cells and that, in response to antigen, CD45RA+ cells become CD45RA- along a differentiation pathway. To test the hypothesis that CD45RA+ cells are naive cells which have just left the thymus, young mice were thymectomized. This would be predicted to lead to a fall in the size of the peripheral pool of CD45RA+ T cells. However, the changes in the size of this pool would also be dependent on the life-span and self renewal capacity of the CD45RA+ T cells in the periphery. Therefore, to test the contribution of the thymus to the peripheral CD45RA+ pool, the percentage of CD45RA+ cells among spleen lymphocyte subsets was studied from 10 days up to 2 years of age in thymectomized and control mice. We also studied the expression of the memory marker CD44 on the CD45RA subsets of CD4 and CD8 cells, as well as the effect of in vitro activation on expression of CD45RA. Our results show that CD8+ CD45RA+ cells are mainly CD44- and their maintenance is dependent on the presence of the thymus. In contrast, the majority of CD4+ CD45RA+ are CD44+ and are not affected by thymectomy. This indicates that the maturation stage of CD8+ CD45RA+ cells is different from that of CD4+ CD45RA+ cells.     

Antigen-independent adhesion of CD45RA (naive) and CD45RO (memory) CD4 T cells to B cells.

We found that naive (CD45RA+) CD4 T cells have a lower capacity of adhesion to Epstein-Barr virus (EBV) immortalized B cells than memory (CD45RO+) CD4 T cells, as judged by conjugate formation. This would appear to be due to differences in the expression of adhesion molecules [lymphocyte function-associated antigen (LFA)-1, CD2]. However, kinetic studies showed that the degree of adhesion of naive T cells to B cells was stable over 60 min while that of memory T cells, like that of unseparated CD4 T cells, was characterized by a rapid formation and rapid dissociation of conjugates. This could be explained by a difference in the sensitivity of naive and memory CD4 T cells to down-regulation of antigen-independent adhesion by CD4-MHC class II interaction. Indeed, memory T cells also adhered stably to MHC class II(-) B cells. The adhesion of memory T cells, but not naive T cells, to MHC class II(+) B cells was sensitive to inhibition by OKT4a an anti-CD4 antibody, human immunodeficiency (HIV) gp160 (env) protein and a 12-mer peptide encompassing the 35-46 sequence of the HLA, DR beta 1 domain and previously shown to inhibit activation of HLA class II-restricted CD4 T cell responses. Since MHC class II expression did not influence the degree of conjugate formation by naive or memory CD4 T cells with B cells, CD4-MHC class II interaction does not appear to be involved in binding itself, but may down-regulate the adhesion of memory but not naive CD4 T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human CD4+CD45R0+ and CD4+CD45RA+ T cells synergize in response to alloantigens.

Alloantigens, unlike recall antigens, activate both CD45RA+ (naive) and CD45R0+ (memory) CD4+ cells to the same extent. These T cell subsets may therefore interact with each other in response to alloantigens on transplanted grafts. We have investigated if the ability of activated CD4+CD45RA+ and CD4+CD45R0+ T cells to produce and respond to interleukin 2 (IL2) and IL4 may be involved in this interaction. After activation, both subsets up-regulate their IL2 receptor (IL2R) and IL4R expression, yet IL4 substantially enhanced the proliferation of the CD4+CD45RA+ but not of the CD4+CD45R0+ T cell subset, while IL2 increased the proliferation of CD4+CD45R0+ but not of the CD4+CD45RA+ T cells. Significantly, the CD4+CD45RA+ T cells synthesized two- to threefold more mRNA for IL2 than the CD4+CD45R0+ subset, while the CD4+CD45R0+ T cells synthesized mRNA for IL4 and interferon-gamma exclusively. The addition of IL2 to alloactivated CD4+CD45R0+ T cells further up-regulated their production of all three lymphokine mRNA; in contrast, IL4 induced an increase in mRNA for IL2 in only the alloactivated CD4+CD45RA+ subset. The reciprocity in the ability of both these CD4+ T cells to synthesize and respond to IL2 and IL4 may provide a rationale for the regulation of lymphokine interactions in vivo. Furthermore, the synergy between these subsets in response to alloantigens, which was directly quantitated by co-culturing CD4+CD45RA+ and CD4+CD45R0+ cells together prior to activation, may potentiate the alloreactivity against transplanted grafts in vivo.     

CD8alpha+beta(low) effector T cells in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by the loss of self-tolerance to nuclear antigens. Aberrant T-cell function plays a central role in lupus pathogenesis. We and others previously demonstrated that peripheral TCRαβ⁺CD3⁺ T cells express CD8β either at a high (CD8β^high) or low density (CD8β^low), thereby defining two functionally distinct subsets. CD8β^low T cells express predominantly CD8αα and less CD8αβ as a coreceptor, display a differentiated phenotype and exert effector function. CD8β^high T cells appear to be the precursors expressing predominantly the heterodimeric efficient CD8αβ coreceptor, exhibiting a naïve phenotype and high proliferative capacity. In the present study, the distribution and functional properties of CD8β^high and CD8β^low T cells of SLE patients were compared ( n  = 20) with those of healthy subjects ( n  = 16). It was found that expansion of CD8β^low T-cell subset correlated with disease activity indicating chronic antigenic stimulation leading to a major lack of naïve CD8β^high precursor T cells in SLE. Functional characteristics of CD8β^low T cells including production of cytokines and cytotoxic granules were not significantly different between patients with SLE and healthy individuals. We speculate that unbalanced CD8β^high/CD8β^low T-cell relation reflects a skewed homeostasis within the CD8⁺ T-cell compartment towards fully differentiated effector T cells possibly due to persistent antigen stimulation in SLE.     

T cell control of staphylococcal enterotoxin B (SEB) lethal sensitivity in mice: CD4+ CD45RB(bright)/CD4+ CD45RB(dim) balance defines susceptibility to SEB toxicity

Radiation chimeras, generated by transplantation of SCID bone marrow into C3H/HeJ mice, show lethal susceptibility to staphylococcal enterotoxin B (SEB), thus constituting a valid murine model for SEB shock. This SEB sensitivity is due to the ability of the irradiated host to restore residual T cell populations, since the SCID donor bone marrow is unable to generate T cells. SCID bone marrow transplanted into irradiated nude mice does not generate SEB-sensitive chimeras, as a consequence of the inability of the recipient nude mice to develop mature T cells. Thymectomy of normal recipient mice prior to bone marrow transplantation does not affect the development of susceptibility to SEB, suggesting that postthymic, residual T cells of the host probably mediate this SEB sensitivity. In vivo depletion experiments show that CD4+ T cells are required for the SEB-triggered shock, while CD8+ cells suppress it. A further examination of the T helper subpopulations in the SEB-sensitive mice reveals a prevalence of CD4+ CD45RB(dim) cells over CD4+ CD45RB(bright) cells. This T helper balance was statistically significant when correlated with SEB-induced mortality. Our model provides a possible explanation for the SEB resistance of normal mice: they have a prevalence of CD4+ CD45RB(dim) over CD4+ CD45RB(bright) cells.     

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     

CD45RO+ memory T cells but not CD45RA+ naive T cells can be efficiently activated by remote co-stimulation with B7

Co-stimulatory signals are absolutely required for T cell activationafter TCR–MHC-peptide interaction. The most importantco-stimulatory signal known so far is mediated by the interactionof CD28 on T cells with B7 on APC. Here we demonstrate thatthe co-stimulatory signal from the B7 molecule does not necessarilyhave to come from the same cell which presents antigen. Titrationcurves obtained by limiting the amount of anti-CD3 mAb suggeststhat the same amount of TCR–CD3 cross-linking is requiredfor full T cell activation whether B7 is present on the sameor on another cell, but that the kinetics of T cell activationis slower when B7 is present on a separate cell from the primarysignal. Finally and most importantly we also show that CD45RO⁺memory T cells, but not CD45RA⁺ naive T cells, can be efficientlyactivated when B7 is expressed on bystander cells. These findingsimply that co-stimulatory activation requirements of B7 aremore stringent for naive than for memory T cells, which couldbe an important mechanism involved in the maintenance of self-tolerance.