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.     

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.     

Cationic amino acid transport in human T lymphocytes is markedly increased in the CD45RA, CD8+ population after activation.

Membrane transport of cationic amino acids is essential for cells which are actively metabolizing L-arginine or L-lysine. In human cells most of this transport occurs through y+, a transport system which is only now being characterized at the molecular level. We have previously shown that phytohaemagglutinin (PHA) stimulation of peripheral blood E rosette positive (T) lymphocytes specifically activated lysine transport through system y+, whereas Staphlyococcus aureus Cowan A (SAC) stimulation of the E rosette negative fraction did not. We have now analysed this effect in PHA-activated CD4, CD8, CD45RO and CD45RA T-cell subsets. Both PHA-activated CD4+ and CD8+ T cells have increased lysine transport through y+, and in seven out of eight experiments, more activity was seen in the CD8+ fraction. In contrast, marked differences in y+ activity were seen between the PHA-activated CD45RO and CD45RA subsets. Thus in six experiments y+ activity was markedly increased in the CD45RA (naive T cell) population but not in the CD45RO (memory) cells. In one further experiment the activated CD45RO, CD4- population (enriched for CD45RA+, CD8+) was studied and y+ activity was shown to be maximal in this cell subset. Transport of arginine is essential for nitric oxide synthesis. Our findings therefore suggest that activated CD45RA, CD8+ T cells are capable of nitric oxide production.     

CD8+ T-cell subsets defined by expression of CD45 isoforms differ in their capacity to produce IL-2, IFN-gamma and TNF-beta.

Expression of different isoforms of CD45, the leucocyte common antigen (LCA), on T-cell subsets has permitted distinctions between the functional activities of subpopulations within the major CD4+ T-cell subset. With respect to cytokine production, the expression on CD4+ cells of CD45RA, a high molecular weight isoform, defines a population which produces only interleukin-2 (IL-2) and tumour necrosis factor-beta (TNF-beta) in quantity, with peak production of IL-2 occurring after 24-48 hr stimulation, while the CD4+ population bearing high levels of CD45RO, a low molecular weight isoform, can produce a wide range of cytokines within 24 hr of activation. The literature is conflicting on the capacities for cytokine production of CD8+ subsets divided on the basis of either CD45RA or CD45RO expression. The aim of this study was to attempt to clarify this area by determining the amount and kinetics of production of IL-2, interferon-gamma (IFN-gamma) and TNF-beta in CD8+ cells separated on the basis of both CD45RA and CD45RO isoform expression. The results showed that CD8+ CD45RA- and CD8+ CD45RO+ T lymphocytes produce significantly more of all three cytokines than do CD8+ CD45RA+ or CD8+ CD45RO- T cells. The kinetics for IFN-gamma and TNF-beta production were similar for both subsets, while IL-2 production was delayed by approximately 3 hr in the CD8+ CD45RO- population as compared to the CD8+ CD45RO+ subset. It is suggested that some of the confusion over cytokine production by these CD8+ subsets may be attributable to different conditions for isolation causing pre-activation of positively selected populations. It is also suggested that while CD8+ CD45RA+ cells are shown to acquire CD45RO upon activation, as do CD4+ CD45RA+ cells, the results of the present study argue for a different relationship between CD8+ subsets separated on the basis of CD45 isoform expression than between the corresponding CD4+ subsets.     

Distribution of Alloreactivity Amongst the CD45 Isoforms of Circulating CD4 and CD8 T Lymphocytes

The expression of different isoforms of the CD45 surface molecule allows lymphocytes to be divided into two nonoverlapping categories, CD45RA and CD45RO. Previous studies of CD4 T cells have shown that responses to soluble antigens are present predominantly in the RO subset and to mitogens in the RA, alloreactivity being present in both subsets. Responses of CD8 T cells have not been investigated in such detail, nor have responses been compared to CD4 cells. Here we report the alloreactive responses of both CD45RA and RO subsets amongst both CD4 and CD8 T lymphocytes. Following isolation of CD4 and CD8 cells with immunomagnetic beads, CD45 subsets were separated by negative depletion using specific monoclonal antibodies. CD45RA populations were greater than 97% pure and CD45RO cells greater than 91%. One-way primary mixed lymphocyte reactions were established using the purified responder cells with irradiated allogeneic peripheral blood mononuclear cells as stimulators; experiments were all repeated at least three times. In assays of CD4⁺ RA and RO subsets, reactivity was present in both isoforms, being consistently, but not significantly, greater amongst the RO subset. With CD8⁺ T cells, reactivity was also present in both isoforms, but was significantly greater in the CD45RA subset, with mean proliferation 2.5–3-fold that of the CD45RO cells ( P  < 0.05).     

慢性乙型肝炎患者外周血CD45RA+、CD45RO+T淋巴细胞亚群的特点及临床意义

目的：探讨慢性乙型肝炎患者外周血CD4^＋CD45RA^＋、CD4^＋CD45RO^＋、CD8^＋CD45RA^＋和CD8^＋CD45RO^＋T淋巴细胞亚群的特点及其与肝病病情的关系。方法：采集46例轻中度慢性乙型肝炎患者、58例重度慢性乙型肝炎患者和30例健康人的外周抗凝血，应用流式细胞技术三色荧光分析法对其外周血中CD4^＋CD45RA^＋、CD4^＋CD45RO^＋、CD8^＋CD45RA^＋和CD8^＋CD45RO＋T淋巴细胞亚群进行检测。结果：轻中、重度慢性乙型肝炎患者与正常人相比，其外周血中CD4^＋、CD8^＋T细胞均无明显改变；CD8^＋CD45RA^＋T细胞均明显降低，CD8^＋CD45RO^＋T细胞均明显增高，而CD4^＋CD45RA^＋、CD4^＋CD45RO^＋T细胞均无明显改变；重度慢性乙型肝炎患者与轻中度慢性乙型肝炎患者相比，CD8^＋CD45RA^＋T细胞明显降低（P〈0.05），CD8^＋CD45RO^＋T细胞明显升高（P〈0.05）。结论：乙型肝炎慢性化过程中，CD8^＋CD45RO^＋T细胞起重要作用且与慢性乙型肝炎患者病情的进展呈正相关；检测CD4^＋CD45RA^＋、CD4^＋CD45RO^＋、CD8^＋CD45RA^＋和CD8^＋CD45RO^＋T淋巴细胞亚群比检测CD4^＋和CD8^＋T细胞亚群更能正确、充分、全面地了解慢性乙型肝炎的发病机制和预后，从而有效地指导临床治疗。     

Human CD4+ T cells expressing CD45RA acquire the lymphokine gene expression of CD45RO+ T-helper cells after activation in vitro.

CD4+ T cells were separated into subpopulations according to their expression of different isoforms of the CD45R molecule, i.e. CD45RA and CD45RO. The separated cells were activated with staphylococcal enterotoxin A (SEA) in the presence of formalin fixed Raji cells. Each set of cells was activated twice with a 6-day interval, and the lymphokine gene expression during the first 3 days after initiation of each stimulation was followed by use of polymerase chain reaction (PCR) technology. The lymphokine messenger RNA (mRNA) profiles were found to differ between the subsets, since after the first stimulation the CD45RA+ cells produced mRNA encoding interleukin-2 (IL-2) and IL-1 alpha, whereas the CD45RO+ cells transcribed genes for IL-1 alpha, IL-2, IL-4, IL-5 and interferon-gamma (IFN-gamma). After 6 days of SEA stimulation both populations were mainly CD45RO reactive, and when restimulated displayed the lymphokine mRNA profile restricted to this subset. These results indicate that the CD45RA subset is a precursor of the CD45RO and further strengthen the hypothesis that the former cell population represents naive whereas the latter subset represents memory T cells within the CD4 subset.     

Age-related increase in the fraction of CD27-CD4+ T cells and IL-4 production as a feature of CD4+ T cell differentiation in vivo.

The influence of ageing on phenotype and function of CD4+ T cells was studied by comparing young (19-28 years of age) and aged (75-84 years of age) donors that were selected using the SENIEUR protocol to exclude underlying disease. An age-related increase was observed in the relative number of memory cells, not only on the basis of a decreased CD45RA and increased CD45RO expression, but also on the basis of a decrease in the fraction of CD27+CD4+ T cells. Our observation that the absolute number of CD45RO+CD4+ T cells was increased, while absolute numbers of CD27-CD4+ T cells remained unchanged in aged donors, indicates that the latter subset does not merely reflect the size of the CD45RO+CD4+ T cell pool. The increased fraction of memory cells in the aged was functionally reflected in an increased IL-4 production and T cell proliferation, when cells were activated with the combination of anti-CD2 and anti-CD28, whereas IL-2 production was comparable between both groups. No differences were observed with respect to proliferative T cell responses or IL-2 production using plate-bound anti-CD3 or phytohaemagglutinin (PHA). The observation that IL-4 production correlated with the fraction of memory cells in young donors but not in aged donors suggests different functional characteristics of this subset in aged donors.     

Alterations in levels of CD28-/CD8+ suppressor cell precursor and CD45RO+/CD4+ memory T lymphocytes in the peripheral blood of multiple sclerosis patients.

A comprehensive peripheral blood immunophenotype analysis of 16 multiple sclerosis (MS) patients was performed by three-color flow cytometric analysis, and the results were compared with those for age-matched healthy controls. The cell subsets quantified included T cells (CD3+), B cells (CD19+), NK cells (CD56+), CD4+ and CD8+ T cells, cytotoxic (CD28+) and suppressor precursor (CD28-) CD8+ T cells, CD45RA+ and CD45RO+ T cells (CD4+ and CD8+), and CD5+ T and B cells. Analysis of MS patients' peripheral blood revealed essentially normal levels of total T, B, and NK cells. In agreement with results obtained by other investigators, it was found that MS patients had an increased CD4/CD8 ratio, primarily due to a decrease in CD8+ T cells. MS patients were found to have a significantly decreased level of suppressor precursor (CD28-) CD8+ T cells compared with that of controls but to have normal levels of cytotoxic (CD28+) CD8+ T cells. These data indicate that MS patients do not have a general decrease in CD8+ T cells but that they have a specific decrease in the suppressor precursor subset only and normal levels of cytotoxic CD8+ T cells. MS patients also had a significant increase in memory (CD45RO+) CD4+ T cells and displayed a trend towards a decrease in naive (CD45RA+) T cells in the peripheral blood.     

Inhibitory effect of IL-4 on the sepharose-CD3-induced proliferation of the CD4CD45RO human T cell subset

CD45R monoclonal antibodies are able to distinguish two different subsets of the CD4 human T cells. This phenotypic split is accompanied by functional diversity. In this report we have analyzed the capabilities of CD45R subsets of CD4 human T cells to use interleukin 2 (IL-2) and IL-4 as growth factors. We have found that both cell subsets are able to proliferate after stimulation with Sepharose-CD3 in the presence of externally added IL-2 or IL-4. However, the response to IL-4 of CD4CD45RO cells was comparatively lower than the response of CD4CD45RA cells. Both cell subsets showed a good response to Sepharose-CD3 plus adherent cells (AC), but when IL-4 was present in the culture only the CD4CD45RA cells showed an enhancement in the Sepharose-CD3-induced proliferation, while proliferation of the CD4CD45RO T cell subset was inhibited. Similar effects were seen, however, in the response to CD4CD45RA or CD4CD45RO cells to Sepharose-CD3 plus IL-2. Although the precise mechanism of the inhibitory effect of IL-4 is not known, the results obtained suggest that IL-4 could interfere in some way with the signalling of IL-2 to the proliferation of the CD4CD45RO T cell subset.     

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.     

Lymphocyte diversity in a 9-year-old boy with idiopathic CD4+ T cell lymphocytopenia

Since CD4+ lymphocytopenia can be caused by disturbed thymic T-cell maturation, we investigated the T-cell subsets of a 9-year-old boy fulfilling the diagnostic criteria for CD4+ lymphocytopenia in a follow-up period of 4 years. We found (I) reduced CD45RA expression, (II) enhanced CD45RO expression and (III) a significant increase in gamma delta TCR-bearing T cells. An accelerated apoptosis (11%) was observed in the CD45RO+, but not CD45RA+ subset. These findings provide evidence that a disturbed thymic T-cell maturation process might play a role in the pathogenesis of CD4+ lymphocytopenia.     

Locomotor responses of human CD45 lymphocyte subsets: preferential locomotion of CD45RO+ lymphocytes in response to attractants and mitogens.

The CD45RO+ population of lymphocytes from human blood contains a higher proportion of locomotor cells than the CD45RA+ population. Direct from blood there were few locomotor lymphocytes (< 15%), but, among these, a higher proportion of CD45RO+ than of CD45RA+ cells responded to the chemotactic stimuli, foetal calf serum (FCS) and interleukin-2 (IL-2) in polarization assays. Likewise, after overnight culture, a higher proportion of CD45RO+ cells responded to IL-8. Culture for 24-72 hr in activators such as anti-CD3, purified protein derivative (PPD), phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) or in an allogeneic mixed leucocyte reaction (AMLR) increased the proportion of locomotor lymphocytes to 20-60%, and the CD45RO+ subset showed proportionately more polarized cells than the CD45RA+ subset after culture with all the above activators. Preferential migration of CD45RO+ cells into collagen gels was also seen after culture in antigenic stimuli (PPD or AMLR) but not with polyclonal activators (alpha CD3 or Con A). Double labelling showed that, within the CD4+ and CD8+ subsets, antigen-stimulated CD45RO+ T cells invaded collagen gels in higher proportions than CD45RA+ T cells. Clustering of lymphocytes with accessory cells is an essential prerequisite for locomotion and, after culture in alpha CD3, CD45RO+ lymphocytes were found preferentially in clusters with monocytes. In all of the above populations, CD45RO+ lymphocytes were larger in size. These findings suggest that, not only selective adhesion to vascular endothelium as reported earlier, but also selective locomotion recruits CD45RO+ lymphocytes into sites of inflammation.     

Parallel pattern of expression of CD43 and of LFA-1 on the CD45RA+ (naive) and CD45RO+ (memory) subsets of human CD4+ and CD8+ cells. Correlation with the aggregative response of the cells to CD43 monoclonal antibodies.

Human peripheral blood T cells form large aggregates when cultured in the presence of antibodies to the highly sialylated protein CD43. About 25% of the cells in such cultures do not aggregate, however, although virtually all T cells express CD43. To find out if these cells constitute a distinct subpopulation of T cells, we analysed the expression of CD43 and lymphocyte function-associated antigen-1 (LFA-1) and examined the aggregation induced by CD43 monoclonal antibodies (mAb) in CD4+ and CD8+ cells and in their CD45RA+ (naive) and CD45RO+ (memory) subsets, respectively. We found that CD43-stimulated CD8+ cells aggregated more rapidly and formed larger aggregates than CD4+ cells. Furthermore, whereas CD8+CD45RO+ cells formed compact clusters after some hours of incubation, a majority (about 75%) of the CD4+CD45RA+ cells remained as singles even after overnight culture. Flow cytometry analysis showed that the patterns of expression of CD43 and of LFA-1 on the different subsets were strikingly parallel to each other. Thus, CD4+ and CD8+ memory (CD45RO+) T cells expressed higher levels of CD43 than the corresponding naive cells, suggesting that increased levels of CD43 expression are, like LFA-1 expression, a marker of primed or recently activated cells. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 125I-labelled subsets showed that CD8+ cells expressed about twice as much CD43 as CD4+ cells. In a particular donor where the mean size of the cells in the four subsets was close to equal, CD4+ memory cells showed a 1.4-fold and CD8+ memory cells a twofold increase in CD43 compared to their corresponding naive populations. The propensity of memory T cells to extravasate may be facilitated by high expression of CD43.     

Intraepithelial TcR alpha/beta+ lymphocytes express CD45RO more often than the TcR gamma/delta+ counterparts in coeliac disease.

Expression of CD45RO on intraepithelial lymphocytes (IEL) bearing the T-cell receptor (TcR) alpha/beta or gamma/delta was studied in situ by three-colour immunofluorescence on jejunal tissue sections from 21 patients with coeliac disease and eight controls. CD45RA-TcR alpha/beta+ IEL expressed CD45RO significantly more often (75%) than the preferentially expanded TcR gamma/delta+ counterpart (59%). Triple staining for CD3, CD4/8 and CD45RA or CD45RB revealed that all CD3 + 4 - 8 - IEL (taken to be TcR gamma/delta+) expressed CD45RB and none were CD45RA. CD45RO positivity was of the same magnitude (66%) on the predominating monoclonal antibody delta TCS1-reactive fraction of TcR gamma/delta+ cells as on the remainder of the TcR gamma/delta+ subset. These results suggest that gluten exposition in patients with coeliac disease leads to accumulation of CD45RA-, putative antigen-primed memory cells of both TcR phenotypes. The less marked CD45RO expression within the preferentially expanded TcR gamma/delta+ subset of IEL may be of particular biological interest.     

Differential age-change in the numbers of CD4+CD45RA+ and CD4+CD29+ T cell subsets in human peripheral blood.

Peripheral blood mononuclear cells were obtained from people ranging in age from newborn to 102 years old and analyzed by dual color flow cytometer in terms of number and percentage of various subsets of T cells, B cells and natural killer cells (CD3, 4, 5, 8, 11b, 19, 20, 21, 25, 29, 45RA and 56). Numbers of T cells (CD3+ or CD5+ cells) significantly declined at the 3rd decade as compared with those of younger people, stayed at a relatively constant level between the 3rd and the 7th decade and gradually declined thereafter. In T cell subsets, both CD4 and CD8 positive positive cells decreased with age, but a decrease was more pronounced in the latter, showing an age-related increase of CD4/CD8 ratio. The most interesting finding was a contrasting age-change in two subsets of CD4+ T cells; i.e. a subset of suppressor inducer T cells (CD4+CD45RA+ naive cells) decreased with age, while a subset of helper inducer T cells (CD4+CD29+ memory cells) increased with age. CD20+ B cells also decreased with age in a manner similar to that observed in T cells. Natural killer cells (CD56) showed an increase in numbers with age. The relationship between these changes in various subsets of peripheral blood leukocytes and the age-related decline in immune functions has been discussed.     

CD45 isoform expression during T cell development in the thymus.

Various isoforms of leukocyte common antigen, or CD45, are expressed differentially on T cells at different stages of development and activation. We report studies on CD45 isoform expression on various subsets of human T cells using two- and three-color flow cytometry and cell depletion. Bone marrow cells that were depleted of CD3+ and HLA-DR+ cells were CD45RA-RO-. The earliest CD3-CD4-CD8-CD19- thymocytes were CD45RO- with 20%-30% CD45RA+ cells. The most prominent population of CD4+CD8+ double-positive thymocytes were CD45RA-RO+. Even the CD4+CD8+ blasts were greater than 90% CD45RO+. About 80% of single-positive thymocytes (CD4+CD8- or CD4-CD8+) were also CD45RO+. Only 4.3% of CD4+ and 18% of CD8+ single-positive thymocytes were CD45RA+. In contrast, cord blood T cells which represent the stage that immediately follows single-positive thymocytes, contained 90% CD45RA+ cells. Thus, in terms of CD45 isoform expression, single-positive thymocytes are more like double-positive cells than cord blood T cells. These results suggest the following sequence of CD45 isoform switching during T cell development: CD45RA-RO- or RA+RO- (double-negative thymocytes)----RA-RO+ (double-positive and most single-positive thymocytes)----RA+RO- (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.     

Deficiency of the expression of CD45RA isoform of CD45 common leukocyte antigen in CD4+ T lymphocytes in children with infantile cholestasis

The immunological background of the pathological changes that appear in infantile cholestasis (infections, inflammatory process in the liver) is largely unknown. With the use of double color flow cytometry, we assessed the distribution of functionally different lymphocyte subpopulations in the peripheral blood of 29 infants with extra and intra-hepatic cholestasis (12 and 17 patients, respectively), aged from 1 to 8.6 months. Control group consisted of 15 age-matched, healthy infants. We examined: (1) the expression of CD3, CD4, CD8, CD19 lymphocyte surface receptors; and (2) the distribution of lymphocyte subsets with distinctive surface Ag characteristics of 'naive' (CD45RA+) and 'memory' (CD45RO+) cells in both CD4+ and CD8+ cell populations. The surface markers expression was evaluated in terms of percentage of positive cells and receptor density. The following changes in the expression of lymphocyte surface markers are described: (1) a decrease in the percentage of total CD3+, CD4+ cells but normal percentage of CD8+ cells and elevated proportion of CD19+ B cells; (2) a reduction of the proportion of 'naive' CD4+ lymphocytes but normal percentage of 'naive' CD8+ as well as 'memory' CD4+ and CD8+ cell subsets; (3) a decrease in density of CD3, CD4+, CD8 receptors, and D45RA isoform in a subset of 'naive' CD4+ cells. We conclude that deficiency of 'naive' CD4+ T cell subset which possess important effector and immunoregulatory functions, and low expression of certain lymphocyte receptors known to be engaged in T cell activation, possibly reflect a defect of cell mediated immunity that may account for viral and bacterial infections, often observed in infants with cholestasis.     

Differential infiltration by CD45RO and CD45RA subsets of T cells associated with human heart allograft rejection.

Subsets of T cells express different isoforms of the leukocyte common antigen CD45; those expressing the glycoprotein 220 isoform (CD45RA) have been characterized as naive in their response to antigens, and those expressing the glycoprotein 180 isoform (CD45RO) as memory T cells. The association between the rejection status of human cardiac allograft recipients and the relative infiltration of the CD45 subsets of both CD8+ and CD4+ T cells was examined using two-color immunohistological labeling techniques on 33 heart transplant biopsies, categorized by routine histological and clinical criteria as mild (requiring no treatment) or moderate (requiring antirejection therapy) rejection. Double-labeling was performed using pairs of monoclonal antibodies to define the following populations: CD4+ CD45RA+, CD4+ CD45RO+, CD8+ CD45RA+, and CD8+-CD45RO+. The number of cells per high-power field (HPF) for each of these cell subsets was counted in every biopsy. In cases with mild rejection, infiltration was predominant for CD4+ CD45RA+ cells (median = 5.0 cells/HPF) relative to CD4+ CD45RO+ (3.12 cells/HPF), CD8+ CD45RA+ (2.14 cells/HPF), and especially CD8+ CD45RO+ (1.22 cells/HPF) populations. In cases with moderate rejection, all four subpopulations increased but were essentially equivalent in intensity, such that in comparison to cases with mild rejection, the smallest increase was seen for CD4+ CD45RA+ cells (6.67 cells/HPF, P < 0.09) and the greatest for CD8+ CD45RO+ cells (7.00 cells/HPF, P < 0.002). A majority of CD8 cells expressed CD45RA in 14 of 16 (88%) cases of mild rejection compared to only 2 of 17 cases of moderate rejection. Moreover, the ratio of CD45RO+ to CD45RA+ cells in each biopsy was higher in moderate versus mild rejection for both CD4 (median ratios = 1.13 versus 0.68, respectively; P < 0.008) and CD8 (1.43 versus 0.58, respectively; P < 0.005) subsets. A majority of T cells expressed CD45RO in cases of moderate rejection (11 of 14 or 79%), compared to only 1 of 13 (8%) cases of mild rejection. These findings indicate that during generally self-limited mild acute cardiac allograft rejection there is a predominance of naive CD45RA+ T cells, especially of the CD4 phenotype, whereas during moderate rejection there is a significant shift toward activated CD45RO+ T cells, especially in the CD8 population.     

Abnormalities in the T and NK lymphocyte phenotype in patients with Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by spontaneous chromosomal instability with predisposition to immunodeficiency and cancer. In order to assess the cellular basis of the compromised immune response of NBS patients, the distribution of functionally distinct lymphocyte subsets in peripheral blood was evaluated by means of double-colour flow cytometry. The study involved the 36 lymphopenic patients with a total lymphocyte count < or =1500 microl (group A) and seven patients (group B) having the absolute lymphocyte count comparable with the age-matched controls (> or =3000 microl). Regardless of the total lymphocyte count the NBS patients showed: (1) profound deficiency of CD4+ and CD3/CD8+ T cell subsets and up to fourfold increase in natural killer (NK) cells, almost lack of naive CD4+ T cells expressing CD45RA isoform, unchanged percentage of naive CD8+ cell subset (CD8/CD45RA+) but bearing the CD8 receptor of low density (CD8low); (2) normal expression of CD45RA isoform in the CD56+ lymphocyte subset, profound decrease in alpha beta but up to threefold increase in gamma delta-T cell-receptor (TCR)-positive T cells; (3) shift towards the memory phenotype in both CD4+ and CD8+ lymphocyte subpopulations expressing CD45RO isoform (over-expression of CD45RO in terms of both the fluorescence intensity for CD45RO isoform and the number of positive cells); and (4) an increase in fluorescence intensity for the CD45RA isoform in NK cells population. These results indicate either a failure in T cell regeneration in the thymic pathway (deficiency of naive CD4+ cells) and/or more dominant contribution of non-thymic pathways in lymphocyte renewal reflected by an increase in the population of CD4+ and CD8+ memory cells, gamma delta-TCR positive T as well as NK cell subsets.