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.     

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.     

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.     

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.     

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.     

Changes in circulating lymphocyte subpopulations following administration of the leucocyte function-associated antigen-3 (LFA-3)/IgG1 fusion protein alefacept

Alefacept, a recombinant leucocyte function-associated antigen-3 (LFA-3)/IgG1 fusion protein approved for the treatment of psoriasis, is reported to reduce selectively the numbers of circulating CD4(+) CD45RO(+) and CD8(+) CD45RO(+) T cells, while sparing the naive cells. The purpose of the present study was to elucidate further the effect of alefacept on various circulating lymphocyte subsets. Sixteen patients, 12 with chronic plaque psoriasis and four with pustular psoriasis, received alefacept 7.5 mg once weekly for 12 weeks. Blood samples collected at study entry and after 12 weeks of treatment were analysed by four-colour flow cytometry. There were statistically significant reductions in the total number of conventional memory (CD45RA(-) CD27(+)) and effector (CD45RA(-) CD27(-) or CD45RA(+) CD27(-)) T cells, including CD4(+) and CD8(+) T cells expressing CD161 and CD8(+) T cells expressing cutaneous lymphocyte-associated antigen (CLA). Natural killer (NK) T cells were also reduced significantly, while no statistically significant changes were seen in NK cells and CD4(+) CD25(high) cells. The affected subpopulations were all characterized by a high expression of CD2. However, CD4(+) CD25(low), and CD4(+) CLA(+) cells, which also expressed relative high levels of CD2, were not reduced significantly. Our results suggest a heterogeneous effect of alefacept on the circulating memory T cell population, indicating that high expression of CD2 may not, by itself, be sufficient to explain the reduction in cell count for a specific subpopulation.     

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。     

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.     

Age-related modifications of the human alphabeta T cell repertoire due to different clonal expansions in the CD4+ and CD8+ subsets

We have studied the effects of a life-long antigen stimulation on the clonal heterogeneity of human peripheral T cell subsets, as defined by their CD45 isoform expression. CD4+ or CD8+ T cells were obtained from healthy donors ranging in age from 20 to 100 years, and sorted into CD45RA+ and CD45RO+ populations. A modified PCR-heteroduplex analysis was then used to directly compare the TCR Vbeta clonal make up of either compartment pair. We find that the CD4+ T cell repertoire remains largely polyclonal throughout life, since CD4+ expanded clones are rare and accumulate predominantly in the CD45RO+ compartment of exceptionally old donors (100 years old). In contrast, the CD8+ T cell subset contains expanded clones which are already detectable in young adults and become very frequent in 70- to 75-year-old donors in both CD45RA+ and CD45RO+ compartments analyzed. Interestingly, some expanded clones are detectable in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments of either CD4+ or CD8+ T cells. These results indicate that the age-dependent accumulation of expanded clones starts earlier and is more pronounced in the CD8+ than in the CD4+ T cell subset, reinforcing the concept that clonal expansion in the two subsets is controlled by substantially different mechanisms. Furthermore, whereas the finding of expanded CD45RO+ T cell clones is explained by antigen- driven proliferation, the detection of expanded clones in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments would support the hypothesis of reversion from the CD45RO+ to the CD45RA+ phenotype after antigen encounter.      

Selective recruitment of lymphocyte subsets to the inflamed appendix.

Total lymphocyte counts and the distribution of lymphocyte subsets were determined in peripheral venous blood and appendiceal mononuclear cells from 60 patients who underwent appendicectomy for the clinical diagnosis of appendicitis. A significant peripheral lymphopenia was observed in the 46 patients with histologically confirmed acute appendicitis which was accompanied by an increase in the appendiceal lymphocyte concentration. There was an even greater depletion of CD45RO+ (memory) T lymphocytes in peripheral blood and an increase in the inflamed appendix. Reciprocal changes were observed in the CD45RA+ (naive) T lymphocyte subset. These changes were reflected in the local arterial and venous CD45RA and CD45RO T lymphocyte subsets. Proliferation studies showed an expanded functional repertoire of T lymphocytes in the inflamed appendix. Selective recruitment of memory T lymphocytes from the peripheral blood to the inflamed appendix was demonstrated.     

Identical expression of CD45R isoforms by CD45RC+ 'revertant' memory and CD45RC+ naive CD4 T cells.

Naive and memory CD4 T cells are frequently defined by exon-specific monoclonal antibodies (mAb) which stain (or not) high- or low-molecular-weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a 'memory' phenotype (CD45RA-, RB-, RC-, or CD45RO+) may revert ('revertants') and re-express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T-cell development. Furthermore, the picture may be incomplete since an exon-specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti-CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3-exon isoform ABC, the 2-exon isoforms AB and BC, and the null isoform O. Both immature CD45RC- (i.e. CD4+8- 'single positive' thymocytes, and peripheral Thy-1+ recent thymic emigrants) and mature CD45RC- 'antigen-experienced' CD4 T cells had message for single-exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen ('truly native') and CD45RC+ antigen-experienced revertant memory T cells. This observation has implications for understanding long-term immunological memory.     

Maternal and neonatal lymphocyte subpopulations at delivery and 3 days postpartum: increased coexpression of CD45 isoforms

PROBLEM: Huge physiologic changes during parturition involve immune cells. Alterations in maternal and neonatal lymphocytes postpartum might ascertain the subpopulations that are most affected and may possibly be of importance in the process. METHOD OF STUDY: Peripheral blood was taken from 20 healthy women at vaginal delivery and 3 days later, concomitantly with cord and peripheral blood from their newborns. Lymphocyte immunophenotyping was done by three-color flow-cytometry. RESULTS: Maternal T helper cells were decreased and natural killer (NK) cells were significantly increased during labor. Percentage of CD4(+) and percentage and absolute count of CD8(+) cells coexpressing CD45RA and CD45RO antigens were higher than 3 days later. In cord blood NK cells were considerably increased and more CD4(+) cells expressed CD45RO antigen. CONCLUSION: Coexpression of CD45RA and CD45RO molecules indicates activation of maternal CD4(+) and CD8(+) lymphocytes. NK cells increase suggests their possible association with parturition processes. Lymphocyte subsets in cord blood correspond to maternal subsets to some extent.     

Lymphocyte subpopulations from patients with primary antibody deficiency do not show increased telomere erosion

Telomere erosion and residual replicative capacity can be used as markers of the replicative history of somatic cells. We have investigated telomere length, in vitro replicative capacity and rate of telomere erosion in T and B lymphocyte populations from patients with primary antibody deficiency requiring immunoglobulin replacement therapy. We found no significant differences in telomere lengths of B cells, or of CD4+, CD8+, CD45RA+(naive) and CD45RO+(memory) T cell populations between patients and age matched controls. Overall, telomere length correlated inversely with age, and was reduced in memory (CD45RO+) as compared with naive (CD45RA+) T cells. In vitro long term (6 months) cell cultures showed no differences between patients and controls in the mitogen-stimulated replicative potential of T cell subpopulations (CD4+, CD8+, CD45RA+, CD45RO+), or in the rates of telomere erosion with cellular replication in these cell populations. The rate of telomere erosion per population doubling in CD45RA+cells, however, was greater than in CD45RO+cells in both patients and controls. These data suggest that premature immune exhaustion is unlikely to represent a long-term complication of primary antibody deficiency.     

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.     

Age dependency and mutual relations in T and B lymphocyte abnormalities in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is primary hypogammaglobulinaemia with an unknown aetiopathogenesis. Although various abnormalities of T and B cells have been described, their pathogenetic roles are unclear. We determined T and B lymphocyte subsets known to be abnormal in CVID in order to disclose possible relations between numerical abnormalities in those cells. Markers associated with B cell development (CD21, CD27, IgM, IgD) were determined on B lymphocytes (CD19+); T lymphocyte development (CD45RA, CD45RO, CD62L) and activation markers (CD25, CD27, CD28, CD29, CD38, CD57, HLA-DR) were determined on CD4+ and CD8+ T lymphocytes in 42 CVID patients and in 33 healthy controls. Abnormalities in CD4+ T lymphocyte activation markers (increase in CD29, HLA-DR, CD45RO, decrease in CD27, CD62L, CD45RA) were observed particularly in patients with a decreased number of memory (CD27+) and mature (CD21+) B cells (group Ia according to the Freiburg group's classification), while abnormalities observed in CD8+ cells (increase in CD27 and CD28 and decrease in HLA-DR, CD57 and CD38) did not depend upon grouping patients together according to B lymphocyte developmental subpopulations. We observed correlations between immature B cells (IgM+ CD21-) and expression of CD27, CD62L, CD45RA, CD45RO and HLA-DR on CD4+ T cells in CVID patients but not in the control group. The expression of CD27 and CD45RA on CD4+ T lymphocytes, such as the percentage of IgD+ CD27- and IgD+ CD27+ cells in B lymphocytes, showed age dependency to be more significant than in the control group. Our study demonstrates that T and B lymphocyte abnormalities in CVID are partially related to each other. Some of those abnormalities are not definite, but may evolve with age of the patient.     

Expression of CD45 isoforms by fresh and activated human gamma delta T lymphocytes and natural killer cells.

Naive and primed alpha beta T cells can be distinguished on the basis of their differential expression of CD45RA and CD45RO, respectively. The present study indicates that these CD45-isoforms also identify naive and primed maturational stages of gamma delta T cells and natural killer (NK) cells. In peripheral blood, all V gamma 9-V delta 2 gamma delta T cells reportedly express CD45RO whereas all V delta gamma delta T cells lack CD45RO. Here, we show that these CD45RO- V delta gamma delta T cells all express CD45RA and the CD45RO+ V.9-V delta 2 gamma delta cells lack expression of CD45RA. The V delta T cells acquired CD45RO expression and lost part of their surface CD45RA, following in vitro activation with phytohaemagglutinin or IL-2. Also the CD3-CD16+ NK cells in peripheral blood that are uniformly CD45RA+ CD45RO- completely converted to the CD45RA-CD45RO+ phenotype upon in vitro activation. Moreover, all cloned V.9-V delta 2 and V delta 1 T cells and NK cells express CD45RO and lack expression of CD45RA. Our results strongly suggest that CD45RA and CD45RO are genuine markers for naive and primed lymphocytes that represent distinct differentiation lineages.     

Lymphocyte membrane antigen expression and intracellular cytokine patterns in an asymptomatic patient with persistently high serum levels of IgE.

BACKGROUND: Patients completely asymptomatic with extremely high levels of IgE have rarely been reported. One such case, in which the immunophenotype pattern of lymphocyte subsets and their cytokine profile were investigated, is described here. OBJECTIVE: To assess whether the cytokine production was consistent with a T helper 2-type immune response, as suggested by theories regarding the functional polarization of helper and cytotoxic T cells in hyper-IgE conditions. METHODS: An asymptomatic 79-year-old man presented with persistent high levels of serum IgE and sporadic hypereosinophilia without any evidence of an underlying pathologic condition. We investigated the immunophenotype of circulating lymphocytes, the expression/release of CD30 (a member of the tumor necrosis factor receptor family preferentially associated with T helper 2-type immune responses) and the intracellular patterns of interferon-gamma (IFN-gamma), Interleukin-2 (IL-2), IL-4, IL-5, and IL-10 production by T cell subsets, as evaluated by single-cell flow-cytometric analysis. RESULTS: The majority of lymphocytes displayed the membrane immunophenotype of NK cells. Both CD4+ and CD8+ T cells were reduced and expressed the "memory" (CD4+/CD45RO+) and the "naive" (CD8+/CD45RA+) phenotypes, respectively. Among CD4+ T cells, CD30 expression was increased in the resting condition and was further inducible following stimulation with mitogenic anti-CD3. Interleukin-4, IL-2, and IL-10 production by CD4+ T cells was increased, whereas IFN-gamma was reduced as compared with normals. CONCLUSIONS: The data suggest that a polarization of CD4+ T cells towards a T helper 0/2-type cytokine pattern occurred in this patient in spite of CD4+ cell reduction and NK cell expansion.     

Rapid conversion of naive to effector T cell function counteracts diminished primary human newborn T cell responses.

The reduced incidence of graft versus host disease following the use of human cord blood as a source of stem cells for bone marrow reconstitution challenges our understanding of the immunocompetence of newborn T cells. Newborn CD4+ T cells express mainly the CD45RA phenotype and have been considered to respond comparably to adult CD4+ T cells exhibiting the CD45RA phenotype. We compared the in vitro kinetics of phenotypic conversion of newborn and adult CD4+CD45RA+ T cells to CD4+CD45RO+ T cells. The cytokine profile and B cell helper activity of the converted CD4+CD45RO+ T cell population were also determined. Newborn CD4+CD45RA+ T cells were converted to CD4+CD45RO+ with significantly faster time kinetics than adult CD4+CD45RA+ T cells, following either phytohaemagglutinin (PHA) or anti-CD2 activation. Freshly purified newborn naive T cells did not produce IL-2, IL-4 or interferon-gamma (IFN-gamma) following stimulation, whereas adult naive T cells secreted IL-2 and adult-derived CD4+CD45RO+ T cells secreted all three cytokines under the same stimulatory conditions. However, newborn and adult CD4+CD45RA+ T cells, following primary stimulation and maturation in vitro, acquired the ability to secrete a Th1-type cytokine profile of IL-2 and IFN-gamma after secondary stimulation. Newborn CD4+ naive T cells that acquired the CD45RO phenotype in vitro also gained B cell helper activity equivalent to that of adult in vitro matured CD4+ naive T cells. These findings suggest that newborn and adult CD4+CD45RA+ T cell subsets are differentially responsive to various stimuli. They show that newborn CD4+CD45RA+ naive T cells can transform more quickly than their adult counterparts into functionally equivalent CD4+CD45RO+ T cells, a process that may be important to counteract the immature immune environment which exists in the newborn.     

Loss of activation-induced CD45RO with maintenance of CD45RA expression during prolonged culture of T cells and NK cells.

The results of the present study show that activation-induced changes in CD45RA and CD45RO expression on T cells and natural killer (NK) cells are not unidirectional for all cells during a 5-week culture period. T cells and NK cells were generated from a resting subpopulation of peripheral blood mononuclear cells (PBMC) defined by sedimentation at Percoll high buoyant densities (p greater than 1.0640 g/ml) and unresponsiveness to IL-2. T cells were activated by a combination of PHA, sheep erythrocytes and IL-2-conditioned medium (IL-2-CM), and NK cells were activated by co-culture with gamma-irradiated malignant melanoma (MM-170) cells and IL-2-CM. Both T-cell and NK-cell cultures were maintained by subculture in IL-2-CM. NK cells and the CD45R(Abright)RO(dim/neg) subpopulation of T cells gained CD45RO following activation and this was accompanied by a two-fold decrease in CD45RA expression. In different cultures, CD45RO expression was not stable on 28-80% of T cells and 10-55% of NK cells. Cells with decreased CD45RO expression showed increased expression of CD45RA. Instability of CD45RO expression on cultured T cells and NK cells occurred at a time following the period of rapid cell growth when the cells were entering a quiescent phase. Both the CD4+ and CD8+ T-cell subpopulation showed similar changes in CD45 isoform expression. In contrast to the results obtained with the CD45R(Abright)RO(dim/neg) resting T cells, the CD45RO(bright)RA(dim/neg) subpopulation of resting T cells when activated and cultured under identical conditions retained CD45RO expression and remained CD45RAdim/neg. Thus a significant proportion of resting CD45R(Abright)RO(dim/neg) T cells is not related in a differentiation sequence to resting CD45RObrightRAdim/neg T cells, and therefore resting CD45RAbrightROdim/neg T cells and resting NK cells may be heterogeneous with respect to their activation history.     

Recent thymic emigrants and subsets of naive and memory T cells in the circulation of patients with head and neck cancer

Apoptosis of circulating CD8+ T lymphocytes is a frequent finding in patients with cancer. T-cell output by the thymus or antigen-driven expansion of circulating T cells could compensate for apoptosis and thus normalize their homeostasis. We studied the frequency of recent thymic emigrants (RTE) identified by T-cell receptor excision circles (TREC) and of naive and memory T-cell subsets in peripheral blood samples obtained from 39 patients with head and neck cancer (HNC) and 33 age-matched controls (NC). TREC numbers were determined by real-time quantitative PCR, and CD8+CD45RO-CD27+ or CD4+CD45RO-CD27+ T-cell subsets were quantified by flow cytometry. Age-associated decreases in TREC numbers and proportions of naive CD8+ and CD4+ T-cell subsets were significantly greater in cancer patients than NC. In contrast, the memory compartment was expanded, with increased proportions of CD4+CD45RO+ but not CD8+CD45RO+ T cells, in cancer patients vs. NC. These alterations did not normalize in patients who were NED. The data suggest that lower thymic output combined with rapid turnover of naive CD8+ T cells account for altered lymphocyte homeostasis in HNC patients. The defect persists long after curative treatments and may contribute to immune cell dysregulation in patients with cancer.