Differential capacities of CD4+, CD8+, and CD4-CD8- T cell subsets to express IL-18 receptor and produce IFN-gamma in response to IL-18

IL-12 and IL-18 have the capacity to stimulate IFN-gamma production by T cells. Using a T cell clone, we reported that IL-18 responsiveness is generated only after exposure to IL-12. Here, we investigated the induction of IL-18 responsiveness in resting CD8+, CD4+, and CD4-CD8- T cells. Resting T cells respond to neither IL-12 nor IL-18. After stimulation with anti-CD3 plus anti-CD28 mAbs, CD8+, CD4+, and CD4-CD8- T cells expressed IL-12R, but not IL-18R, and produced IFN-gamma in response to IL-12. Cultures of T cells with anti-CD3/anti-CD28 in the presence of rIL-12 induced IL-18R expression and IL-18-stimulated IFN-gamma production, which reached higher levels than that induced by IL-12 stimulation. However, there was a substantial difference in the expression of IL-18R and IL-18-stimulated IFN-gamma production among T cell subsets. CD4+ cells expressed marginal levels of IL-18R and produced small amounts of IFN-gamma, whereas CD8+ cells expressed higher levels of IL-18R and produced more IFN-gamma than CD4+ cells. Moreover, CD4-CD8- cells expressed levels of IL-18R comparable to those for CD8+ cells but produced IFN-gamma one order higher than did CD8+ cells. These results indicate that the induction of IL-18R and IL-18 responsiveness by IL-12 represents a mechanism underlying enhanced IFN-gamma production by resting T cells, but the operation of this mechanism differs depending on the T cell subset stimulated.     

Identification of two distinct CD5- B cell subsets from human tonsils with different responses to CD40 monoclonal antibody

This study investigated the response of different CD5- B cell subsets to CD40 monoclonal antibody (mAb) in various combinations with interleukin (IL)-4 or rabbit anti-human mu chain antibody (a-mu-Ab). The different CD5- B cell subsets were isolated from tonsillar B cell suspensions depleted of CD5+ B cells and subsequently fractionated on Percoll density gradients. While resting CD5+ B cells proliferated and produced IgM molecules in response to a-mu-Ab, IL-4 and CD40 mAb as well as to Staphylococcus aureus Cowan strain I (SAC) and IL-2, resting CD5- B cells, which were co-purified in the same 60% Percoll fractions, consistently failed to respond. These cells were, however, activated by the stimuli employed, as demonstrated by their capacity to express the surface activation markers CD69, CD25 and CD71. Resting CD5+ B cells had the typical phenotype of mantle zone B cells (IgM+ IgD+ CD39+ CD38- CD10- CDw75dim), whereas resting CD5- B cells were CD38- CD39- CD10- CDw75 intermediate and expressed surface IgM but relatively little surface IgD and could not be classified as mantle zone or germinal center cells. The finding that purified germinal center cells (CD38+ CD10+ CD39- CDw75bright, IgG+) responded to CD40 mAb and IL-4 and also to SAC plus IL-2 further underlined the differences to resting CD5- B cells. However, some of the data collected suggest possible relationships between CD5- B cells and germinal center cells. The CD5- B cells isolated from the 50% Percoll fraction proliferated in response to a-mu-Ab, CD40 mAb and IL-4 as well as to SAC and IL-2. These cells had the same mantle zone B cell phenotype as the CD5+ B cells, but their capacity to respond to the stimuli in vitro was unrelated to a possible contamination with CD5+ B cells, as documented by the appropriate controls. Furthermore, upon exposure to SAC or phorbol esters, the large majority of CD5- B cells from the 50% Percoll fraction did not express surface CD5 and there was very little if any accumulation of CD5 mRNA. Finally, most of the cycling cells in the stimulated CD5- B cells did not express CD5. The CD5- B cells from the 50% Percoll fraction were comprised of a consistent proportion of cells that expressed surface activation markers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differentiation and stability of T helper 1 and 2 cells derived from naive human neonatal CD4+ T cells, analyzed at the single-cell level

The development of CD4+ T helper (Th) type 1 and 2 cells is essential for the eradication of pathogens, but can also be responsible for various pathological disorders. Therefore, modulation of Th cell differentiation may have clinical utility in the treatment of human disease. Here, we show that interleukin (IL) 12 and IL-4 directly induce human neonatal CD4- T cells, activated via CD3 and CD28, to differentiate into Th1 and Th2 subsets. In contrast, IL-13, which shares many biological activities with IL-4, failed to induce T cell differentiation, consistent with the observation that human T cells do not express IL-13 receptors. Both the IL-12-induced Th1 subset and the IL-4-induced Th2 subset produce large quantities of IL-10, confirming that human IL-10 is not a typical human Th2 cytokine. Interestingly, IL-4-driven Th2 cell differentiation was completely prevented by an IL-4 mutant protein (IL-4.Y124D), indicating that this molecule acts as a strong IL-4 receptor antagonist. Analysis of single T cells producing interferon gamma or IL-4 revealed that induction of Th1 cell differentiation occurred rapidly and required only 4 d of priming of the neonatal CD4+ T cells in the presence of IL-12. The IL-12-induced Th1 cell phenotype was stable and was not significantly affected when repeatedly stimulated in the presence of recombinant IL-4. In contrast, the differentiation of Th2 cells occurred slowly and required not only 6 d of priming, but also additional restimulation of the primed CD4+ T cells in the presence of IL-4. Moreover, IL-4-induced Th2 cell phenotypes were not stable and could rapidly be reverted into a population predominantly containing Th0 and Th1 cells, after a single restimulation in the presence of IL-12. The observed differences in stability of IL-12- and IL-4-induced human Th1 and Th2 subsets, respectively, may have implications for cytokine-based therapies of chronic disease.     

Estrogen blocks early T cell development in the thymus

PROBLEM: Pregnancy and estrogen are known to suppress B lymphopoiesis as well as lead to thymic involution in the mouse. Additionally, estrogen deficiency by oophorectomy reportedly causes a selective increase in the B220+ B cells in the murine bone marrow. The purpose of this study was to determine if estrogens played a regulatory role in T cell development. METHODS: The first experimental group consisted of 5-6-week-old Balb/c mice that received subcutaneous pellets of placebo, estriol, estradiol, or progesterone. The thymus glands were examined 2-4 weeks after treatment. The second group consisted of 6-week-old Balb/c mice who underwent either bilateral oophorectomy or a sham procedure. Two weeks after the surgery, extensive phenotypic characterization of the thymus and spleen cells was performed by flow cytometry using monoclonal antibodies to surface markers of T cell subsets. RESULTS: Estrogen treatment causes a dramatic reduction of thymic size and cellularity. All defined T cell subsets of CD4 and CD8 were reduced, with a disproportionate loss of CD4+CD8+ double positive cells. Examination of the triple negative (CD3-CD4-CD8-) subset revealed a striking loss of TN developmental progression of the early precursor cells. Based on the expression of CD44 (pgp-1) and CD25 (IL-2R alpha) markers, the TN thymic compartment was composed almost entirely of the earliest population (CD44+, CD25-), with the remaining maturational stages (CD44+, CD25+; CD44-, CD25+; CD44-, CD25-) depleted. In contrast, all T cell developmental stages in the thymus were found to be in normal proportions in the oophorectomized mice, with no differences in the splenic T and B cell subsets. CONCLUSIONS: The study demonstrates that estrogen but not progesterone blocks T cell development in the thymus. However, contrary to our expectation, estrogen deprivation by oophorectomy does not enhance T cell development.     

Expression of the Fc receptor for IgG (Fc gamma RII/CDw32) by human circulating T and B lymphocytes

Tissue-specific isoforms of the human FcR for IgG Fc gamma RII (CDw32) have previously been described by using mAb. These mAb were shown to exhibit different patterns of reactivity with lymphocytes. Among human PBL, Fc gamma RII has been detected on B cells but not T cells when assessed by flow cytometry and microscopy with the use of mAb KB61 and 41H16. Although KB61 and 41H16 were found to react with B cells, the mAb IV.3, CIKM5, and 2E1 did not react with any PBL subset. In this study, we show that KB61 and 41H16 react strongly with the majority (93-96%) of B cells (CD20+), and weakly with a proportion (18-42%) of T cells (CD3+), including 10 to 14% of CD4+ and 27 to 69% of CD8+ cells. In addition, mRNA for Fc gamma RII was detected in purified CD3+CD8high+ lymphocytes by polymerase chain reaction. KB61 and 41H16 also reacted with a majority of CD3-CD16/CD56+ cells, and CD3-CD20- cells. These findings indicate that a subset of T cells and non-T/non-B cells express Fc gamma RII, and are of interest in the light of previous studies which postulate that human Fc gamma R+ cells and Fc gamma RII+ murine T cells suppress the B cell immune response.     

Differential expression of CD40 ligand on T cell subsets. Implications for different roles of CD45RA+ and CD45RO+ cells in IgE production

Physical contact between human T lymphocytes and B lymphocytes is required for the induction of IgE production. In the present study, we examined the abilities of CD45RA+ and CD45RO+ human T cell subsets to provide help for IgE production by human peripheral blood B cells in the presence of IL-4. Purified peripheral CD45RA+ T cells are much better inducers of IgE synthesis than are CD45RO+ T cells. Activation of CD45RA+ T cells, but not CD45RO+ T cells, via the TCR/CD3 complex is sufficient to confer the ability to provide IgE help, suggesting that an inducible T cell surface molecule plays an important role in this system. The CD40 ligand, an inducible T cell surface molecule, is expressed at higher levels on CD45RA+ T cells as compared with CD45RO+ T cells following CD3-stimulation. Blocking of the CD40-CD40 ligand interaction in vitro by the addition of a soluble form of B cell CD40 Ag completely blocks IgE production induced by CD45RA+ T cells. Finally, the in vitro conversion of CD45RA+ T cells to the CD45RO+ phenotype is accompanied by a loss in the ability of these cells to express the CD40 ligand in response to anti-CD3 stimulation as well as a loss in their ability to provide IgE help. These results suggest that both CD45 subsets may play significant and distinct roles in the induction of IgE production under physiologic conditions: CD45RO+ T cells provide IL-4 and the CD45RA+ subset provides the second signal via the CD40 ligand.     

Comparisons of endothelial cell G- and F-actin distribution in situ and in vitro

Precursor of T lymphocytes undergo proliferation and maturation under the influence of the thymic microenvironment. In our study, we have attempted to determine the distribution of human postnatal thymocytes in division according to their stage of differentiation. Our data show that about 11.5% of all thymic cells are in S/G2/M phases, and that a subset of the cortical and precortical subpopulations contains most of the dividing cells. Rate of cell division is maintained at high levels from the prethymocyte precursor along the successive stages of differentiation represented by CD1+CD3-CD4-CD8- and CD1+CD3-CD4+CD8- cells. The percentage of dividing cells is maximal in an intermediate subset of CD1+CD3-CD4+CD8-CD45RO+ cells defined by the distinct expression of class I HLAdim/high molecules, which could contain cells in transit from prethymocytes to double-positive cortical cells. The CD3- fraction of the double-positive cortical cells contains most of the dividing thymocytes, although the rate of division within this subset is much less than that of the precursor CD1+CD3-CD4+CD8- cells. In a linear scheme of differentiation, cell division stops at or near the point of initiation of CD3 expression. These results suggest that in human thymus cell expansion takes place before the initiation of the positive selection process. According to this view the stringency of the selection process would require the previous generation of a large number of precursors to permit the production of sufficient numbers of mature T cells.     

Lymphocyte subsets and mitogen stimulation of blood lymphocytes in normal pregnancy

PROBLEM: In normal pregnancy the maternal immune system should be directed towards tolerance or suppression in order not to reject the partly foreign feto-placental unit. The aim of this investigation was to find hallmarks of systemic immunosuppression during normal pregnancy. METHODS: Five healthy primigravidae were examined during pregnancy and postpartum with flow cytometric analysis to define T and B lymphocyte subsets in peripheral blood. In addition, we studied the proliferative response of lymphocytes to mitogens or interleukin-2 (IL-2) alone or in combination with immunomodulating drugs or interleukin-4 (IL-4). The results were compared to healthy, non-pregnant women. RESULTS: During pregnancy and early puerperium we noted an immune balance in favour of suppression, as measured by increased numbers of T "helper/suppressor" (CD4+CD45RA+) and "suppressor"/effector T cells (CD8+S6F1-), and decreased numbers of T "helper/inducer" (CD4+CD29+), T "helper/memory" (CD4+CD45RO+), killer/effector T cells (CD8+S6F1+), and Natural Killer cells (CD56+), as well as decreased numbers of activated lymphocytes expressing IL-2 receptor (CD25+) and T cells expressing HLA-DR (HLA-DR+CD3+). During pregnancy, lymphocyte proliferation was impaired in autologous serum with concanavalin A (ConA), phytohemagglutinin (PHA), or IL-2. A difference in proliferative response to PHA or IL-2 between cultures with AB serum and autologous serum is suggestive of an immunosuppressor factor in serum during pregnancy. Indomethacin significantly increased lymphocyte proliferation in autologous serum with ConA, indicating PGE2 mediated suppressor activity during pregnancy. Chlorambucil and cimetidine modulated the proliferative response to ConA, indicating an alkylating agent sensitive and a histamine dependent suppressor activity during pregnancy. CONCLUSIONS: During normal pregnancy, a state of systemic suppression of the maternal immune system seems to be present.     

Functional diversity of the CD8(+) T-cell response to Epstein-Barr virus (EBV): implications for the pathogenesis of EBV-associated lymphoproliferative disorders

Epstein-Barr virus (EBV)-specific CD8(+) cytotoxic T cells are thought to be critical for the control of EBV, which persists in healthy individuals as a latent infection of B cells. However, recent observations have indicated that CD8(+) T-cell responses are not uniformly cytotoxic and that CD8(+) T cells may be subdivided into type 1 and type 2 subsets that parallel the classically described Th1 and Th2 subsets of CD4(+) T cells. Using two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, we have identified two distinct but overlapping subsets of EBV-specific CD8(+) T cells, the first of which expressed high levels of interferon gamma (IFNgamma), but little or no interleukin-4 (IL-4), whereas the second subset was IFNgamma+/IL-4(+) double-positive. A significant proportion of EBV-specific CD8(+) T cells also expressed IL-13. Subsequent analysis of a panel of 27 EBV-specific CD8(+) T-cell clones showed inverse relationships between EBV-specific cytotoxicity and secretion of IL-4, IL-10, and IFNgamma, respectively. IL-10 was not secreted by the 11 most strongly cytotoxic clones, suggesting that IL-10 secretion may provide a functional definition of an EBV-specific type 2 CD8(+) T-cell subset with reduced EBV-specific cytotoxicity. Finally, we have demonstrated that EBV-specific CD8(+) T cells that express type 2 cytokines possess the ability to activate resting B cells. EBV-specific CD8(+) T cells thus have the potential to reactivate latent EBV infection in vivo and may contribute to the development of EBV-associated lymphoproliferative disorders and lymphoma.     

MHC class I-selected CD4-CD8-TCR-alpha beta+ T cells are a potential source of IL-4 during primary immune response

Differentiation of naive CD4+ lymphocytes into either Th1 or Th2 cells is influenced by the cytokine present during initial Ag priming. IL-4 is the critical element in the induction of Th2 response; however, its origin during a primary immune response is not well defined. In the present study, we characterized a novel potential source of IL-4, the class I-selected CD4-CD8-TCR-alpha beta+ T cells. In a first set of experiments, we demonstrated that CD4-CD8-TCR-alpha beta+ thymocytes produce a large amount of IL-4 after in vitro anti-CD3 stimulation. This phenomenon was not observed in class I-deficient mice, demonstrating that among these cells, the class I-selected subset was predominantly responsible for IL-4 production. Further studies focused on the in vivo IL-4-producing capacity of peripheral CD4-CD8-TCR-alpha beta+ T cells. To this end, a single injection of anti-CD3 mAb, which promptly induces IL-4 mRNA expression, was used. Peripheral CD4-CD8-TCR-alpha beta+ T cells express high levels of IL-4 mRNA in response to in vivo anti-CD3 challenge. Furthermore, analysis performed in mice lacking MHC class I or class II molecules demonstrates that both the class I-selected subset of CD4-CD8-TCR+ and CD4+ peripheral T lymphocytes are the major IL-4 producers after in vivo anti-CD3 stimulation. These findings suggest that class I-selected CD4-CD8-TCR-alpha beta+ and CD4+ T cell populations are important sources of IL-4 probably implicated in the development of specific Th2 immune responses.     

Ecologic studies of rodent reservoirs: their relevance for human health

To gain insight into the immunomodulatory effects of vitamin E (VE), immune cell population analyses were conducted using thymus and spleen from male broilers fed diets with various levels of VE supplementation (0, 17, 46, and 87 mg dl-alpha-tocopherol acetate/kg of feed). At 2 and 7 wk of age, the percentages of B cells, macrophages, and T cell subsets, delineated by the expression of CD4, CD8, and T cell receptor (TCR) isotype, in thymus and spleen were determined by flow cytometry. The percentages of thymic and splenic B cells and macrophages from 2- and 7-wk-old chickens, as well as the percentage of thymic T cells in 2-wk-old chickens, were unaffected by VE treatment. However, 7-wk-old broilers maintained on 87 mg VE/kg feed had a higher percentage of CD4+CD8- thymocytes, a higher CD4+CD8- to CD4-CD8+ thymocyte ratio, and a lower percentage of CD4+CD8+ thymocytes than chickens receiving no dietary VE supplementation. The VE-induced increase in the percentage of CD4+CD8- thymocytes was due to an increase in the TCR2+CD4+CD8- thymocyte subset, whereas the decrease in the percentage of CD4+CD8+ thymocytes involved all TCR defined T cell subsets. In the spleen, the percentage of CD4+CD8- T cells was lower in 2-wk-old chickens and higher in 7-wk-old chickens maintained on 87 mg/kg feed than in chickens receiving no dietary VE supplementation. The decrease in CD4+CD8- splenocytes at 2 wk of age was due to a decline in the percentage of TCR2+CD4+CD8- splenocytes, whereas the increase in CD4+CD8- splenocytes in 7-wk-old chicks was due to an increase in the percentages of all TCR defined CD4+CD8- T cell subsets. These data support an immunomodulatory effect of VE on CD4+CD8- T cells.     

Improving tumour targeting and decreasing normal tissue uptake by optimizing the stoichiometry of a two-step biotinylated-antibody/streptavidin-based targeting strategy: studies in a nude mouse xenograft model

Peripheral T-cell lymphomas (PTCLs) are often diagnosed after demonstration of T-lineage-related antigen expression on neoplastic lymphocytes by paraffin immunoperoxidase (PIP). However, complete T-cell subset analysis for helper, suppressor/cytotoxic, alphabeta, and gammadelta phenotypes has not been examined by PIP. Therefore, PIP was performed for CD4, CD8, T-cell intracellular antigen (TIA)-1, and betaF1 expression in 31 PTCLs previously studied for CD4 and CD8 by flow cytometry. The CD4 and CD8 results from both methods were compared. All betaF1- PTCLs were studied for T-cell receptor (TCR)gammadelta by PIP. PIP showed 71% correlation with the 21 PTCLs that had distinct CD4+ CD8- or CD4- CD8+ phenotypes by flow cytometry, with 64% and 90% sensitivity for CD4 and CD8 expression, respectively. Tumor cells in four of six PTCLs that had no clear CD4 or CD8 predominance or coexpression of these antigens by flow cytometry were shown to be CD4+ CD8- or CD4- CD8+ by PIP. Twelve (39%) PTCLs demonstrated a cytotoxic (TIA-1+) phenotype by PIP, including eight CD4- CD8+, one CD4+ CD8- and three CD4- CD8- cases. Of 30 immunoreactive PTCLs, 26 (87%) were alphabeta (betaF1+) by PIP. Both large cell cases among four betaF1- PTCLs were TCRgammadelta+ by PIP, including one gammadelta+ case confirmed by flow cytometry. We conclude that CD4 and CD8 T-cell subsets can be assigned for most PTCLs by PIP, with CD4 showing moderate and CD8 showing strong correlation with flow cytometric results. PIP can also define CD4 or CD8 expression on tumor cells in the PTCLs in which flow cytometry produces inconclusive results. Cytotoxic PTCLs can be identified easily with TIA-1, which can also distinguish cytotoxic from "suppressor" CD8+ PTCLs. Most PTCLs are derived from alphabeta T-cells, however some large cell gammadelta PTCLs may be identified by PIP.     

Differential effects of type I IFNs on the growth of WC1- CD8+ gamma delta T cells and WC1+ CD8- gamma delta T cells in vitro

Type I IFNs have a broad array of immunoregulatory functions that include up-regulation of type 1 immune responses through enhancing differentiation and activation of CD8+ T cells and CD4+ Th1 cells. Ovine trophoblast IFN-tau is a recently described type I IFN with the potential for therapeutic use, based on its potent antiviral activity yet low toxicity. Studies were designed to determine the immunoregulatory effects of IFN-tau on Ag-stimulated T cells, and a novel effect of type I IFNs on gammadelta T cells was observed. In cultures of parasite Ag-stimulated bovine T cells that contained a mixture of alphabeta and gammadelta T cells, both IFN-tau and IFN-alpha suppressed the expansion of WC1+ CD2- CD6- CD8- gammadelta T cells, yet stimulated the growth of WC1- CD2+ CD6+ CD8+ gammadelta T cells and CD8+ alphabeta T cells. The CD8+ gammadelta T cell subset expressed high levels of the IL-2R alpha-chain. Furthermore, we showed that type I IFN enhanced IL-2 production by these Ag-stimulated T cell lines. In short term cultures of PBMC, IL-2 stimulated an expansion of WC1- CD6+ CD8+ gammadelta T cells, which was significantly increased by IFN-tau, even though IFN-tau alone did not support cell survival. These studies demonstrate for the first time that type I IFNs differentially modulate the proliferation of different subsets of gammadelta T cells, which appears to act in part via IL-2.     

Human natural killer cell development from bone marrow progenitors: analysis of phenotype, cytotoxicity and growth

We have developed a culture system allowing for generation of NK cells from human CD34+ bone marrow progenitors. The appearance of NK cells expressing CD56+, CD3- phenotype and large granular lymphocyte morphology was observed after 2-3 weeks of culture with IL-2. The NK cell appearance coincided with development of lytic activity. NK cells generated in bone marrow cultures proliferated actively (expansion index ranged from 2- to 200-fold). The phenotype of NK cells generated from CD34+ bone marrow deviated from peripheral blood NK cells in that a lower percentage of the former cells expressed CD16, CD2, CD7, and CD8 antigens. NK cells were also generated from CD34+ populations depleted of the CD34+, CD33+ subset indicating that myeloid-committed progenitors are not required for NK cell development. The dose of IL-2 was not important for generation of NK cells; however, only high doses of IL-2 supported development of optimal NK cell cytotoxic potential. Addition of TNF-alpha facilitated IL-2-dependent NK cell generation. These data showed that NK cells can develop from early bone marrow progenitors and that this system may be instrumental in studies on NK cell lineage and differentiation.     

Impact of social confrontation on rat CD4 T cells bearing different CD45R isoforms

The impact of social defeat on lymphocyte subpopulations and T helper subsets was investigated in Long Evans rats. CD4 T helper cell subsets with distinct functional properties and different cytokine profiles can be distinguished by using the mAbs OX-22 (anti-CD45RC) and OX-7 (anti-CD90, Thy1.1). Male intruders were exposed for 2, 6, or 48 h to aggressive resident pairs. All intruders were attacked upon introduction and were defeated as indicated by frequent display of full submissive postures. After 2 and 48 h of confrontation, drastic but differential effects on blood leukocyte numbers, CD4 and CD8a cells, and CD4 subsets were evident. However, after 6 h of confrontation most lymphocyte subset numbers corresponded to baseline levels. Focusing on CD4 subsets after 2 h of confrontation, we demonstrated that only the number of the CD45RC-CD90(-) subset declines, whereas neither the number of the CD45RC+CD90(-) subset nor the number of the CD45RC-CD90(+) subset (recent thymic emigrants) was influenced. Con A stimulation of sorted subsets identified the CD45RC-CD90(-) as a poor producer of IFN-gamma. The data clearly demonstrate that social factors might differentially influence not only T cell subsets but also T helper cell subsets with distinct cytokine profiles in a possibly time-dependent manner. Such a stress-induced shift toward a CD45RC+CD90(-)-dominated milieu may have important consequences in interpreting results obtained from mitogenic stimulation of blood lymphocytes and cytokine production profiles measured after such a stimulation. In addition, a shift toward a CD45RC+CD90(-) dominance may modify the type and magnitude of immune response, at least temporarily.     

Analysis of T-cell activation after bronchial allergen challenge in patients with atopic asthma

BACKGROUND: T helper cells are a heterogeneous group of cells that have phenotypic and functional differences. Activated T helper cells have been found in peripheral blood after allergen challenge of subjects with atopic asthma, but the phenotypes of specific T helper subpopulation involved remains to be identified. OBJECTIVE: To characterize the T cell activation markers that may be regulated by allergens, we analyzed peripheral blood lymphocytes obtained before and after allergen challenge from subjects with atopic asthma. METHODS: We analyzed the distribution of the cell surface activation markers, interleukin 2 receptor (IL-2R) and major histocompatibility complex class II antigens (MHC II) among T helper subpopulations classified as naive (CD45RA) or memory (CD45RO) phenotypes. Nine adult subjects with atopic asthma underwent bronchoprovacative allergen inhalation and isocapnic cold air hyperventilation (ISH) challenge followed by serial spirometry. Peripheral blood mononuclear cells (PBMC) were isolated at baseline and 2 and 24 hours after challenge. Four-color flow cytometry was used to analyze the expression and distribution in vivo of IL-2R and MHC II activation markers on naive and memory T cell subsets after challenge. RESULTS: At 2 and 24 hours after allergen challenge, there was a significant increase in the CD45RO+IL-2R+ T helper cells compared with baseline (mean +/- SE, baseline, 12.5% +/- 1% versus 2 hours, 18.1% +/- 1% and 24 hours, 17.8% +/- 2%, p < 0.025). MHC II expression was not significantly increased after challenge on naive and memory T helper cells and coexpression of IL-2R and MHC II was only found in a small proportion of CD45RO+ T helper cells (2.7% +/- 1%). No changes of IL-2R or MHC II expression on T helper subsets were observed after ISH challenge in the same patients. We also found that 31% to 46% of T helper cells coexpress CD45RA and CD45RO simultaneously, and upregulation of IL-2-R and MHC II expression occurs only on those T helper cells that express CD45RO. CONCLUSIONS: We have found that T helper cells express both CD45RA and CD45RO isoforms, which suggests the existence of a transitional phenotype among naive and memory T helper cells in peripheral blood. In subjects with atopic asthma, our in vivo analysis characterizes two populations of activated memory T helper cells based on the expression of IL-2R or MHC II surface molecules after allergen challenge.