Quantitative analysis of CD4+ T cell function in the course of human immunodeficiency virus infection. Gradual decline of both naive and memory alloreactive T cells.

Early in human immunodeficiency virus (HIV) infection CD4+ and CD8+ T cells are qualitatively affected. Loss of responses to recall antigen precedes impaired responses to allogeneic MHC and mitogens. The selective quantitative loss of memory T cells in early infection, only partially explains the observed defects. We investigated whether functional loss of T cells is preferentially observed for memory T cells or whether both naive and memory T cell subsets are affected in the course of HIV infection. We studied the proliferative response of CD4+ T cells from HIV-infected individuals to alloantigens, to which normally both naive and memory T cells respond, by limiting dilution analysis. The decreased proliferative response to alloantigens in HIV-infected individuals was associated with a decreased precursor frequency of alloreactive cells. The frequency was decreased in both the CD45RA+ (naive) and the CD45RO+ (memory) subset of CD4+ T cells. Analysis of four individuals in the course of HIV infection revealed similar kinetics of the decline in function in both subsets. Although initially T cell defects may be accounted for by the selective quantitative loss of memory cells, in later stages of HIV infection the function of both CD45RA+ and CD45RO+ cells is affected.     

Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1.

Many ligands of adhesion molecules mediate costimulation of T cell activation. The generality of this emerging concept is best determined by using model systems which exploit physiologically relevant ligands. We developed such an "antigen-specific" model system for stimulation of resting CD4+ human T cells using the following purified ligands: (a) major histocompatibility complex class II plus the superantigen Staphylococcus enterotoxin A, to engage the T cell receptor (TCR); (b) adhesion proteins vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and endothelial leukocyte adhesion molecule 1 (ELAM-1), to provide potential cell surface costimulatory signals; and (c) recombinant interleukin 1 beta (rIL-1 beta)/rIL-6 as costimulatory cytokines. In this biochemically defined system, we find that resting CD4+ T cells require costimulation in order to respond to TCR engagement. This costimulation can be provided by VCAM-1 or ICAM-1; however adhesion alone is not sufficient since ELAM-1 mediates adhesion but not costimulation. The cytokines IL-1 beta and IL-6 by themselves cannot mediate costimulation, but augment the adhesion ligand-mediated costimulation. Direct comparison with the model of TCR/CD3 engagement by CD3 monoclonal antibody demonstrated comparable costimulatory requirements in both systems, thereby authenticating the commonly used CD3 model. The costimulation mediated by the activation-dependent interaction of the VLA-4 and LFA-1 integrins with their respective ligands VCAM-1 and ICAM-1 leads to increased IL-2R alpha (CD25) expression and proliferation in both CD45RA+ CD4+ and CD45RO+ CD4+ T cells. The integrins also regulate the secretion of IL-2, IL-4, and granulocyte/macrophage colony-stimulating factor. In contrast the activation-independent adhesion of CD4+ T cell to ELAM-1 molecules does not lead to T cell stimulation as measured by proliferation, IL-2R alpha expression, or cytokine release. These findings imply that adhesion per se is not sufficient for costimulation, but rather that the costimulation conferred by the VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions reflects specialized accessory functions of these integrin pathways. The new finding that VLA-4/VCAM-1 mediates costimulation adds significance to observations that VCAM-1 is expressed on a unique set of potential antigen-presenting cells in vivo.     

Interleukin 2 induces the expression of CD45RO and the memory phenotype by CD45RA+ peripheral blood lymphocytes

The CD45RA and CD45RO isoforms of the leukocyte common antigen identify functionally distinct "naive" and "memory" T cell subsets. While antigenic and mitogenic stimuli are known to initiate transition from the naive to memory state, little is known about the role of cytokines in this process. This report demonstrates that in vitro exposure of purified CD45RA+/CD45RO- peripheral blood lymphocytes (PBL) to interleukin 2 (IL-2) promotes their conversion to the CD45RA-/CD45RO+ phenotype. Conversion to CD45RO occurs for both the CD3+ and CD3-/CD56+ lymphocyte subsets, but occurs more rapidly, and at lower IL-2 concentrations, in the CD3-/CD56+ population. Expression of CD45RO was observed only in response to IL-2 and was not observed during long-term culture in IL-4, IL-6, or IL-7. We also examined the effect of IL-2 on the expression of adhesion molecules by T cells. The expression of CD2, CD11a, and CDw29 increased, and expression of Leu-8 (LAM-1) decreased, on cultured CD45RA+/CD45RO- cells after they converted to expression of CD45RO. In contrast, lymphocytes that remained CD45RA+/CD45RO- after 10 d in culture exhibited no change from their baseline adhesion molecule profile. Finally, to test the role of endogenous IL-2 during T cell activation we stimulated CD45RA+/CD45RO- PBL with immobilized anti-CD3 in the presence of neutralizing anti-IL-2 antibody and/or cyclosporin A. Both agents significantly reduced the expression of CD45RO and the effect of cyclosporin A was reversed by exogenous IL-2. We conclude that IL-2 promotes CD45RA+ cells to express the memory phenotype and is a mediator of CD45RO expression after stimulation of the T cell receptor/CD3 complex.     

Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10

Interleukin-12 (IL-12) induces differentiation of T helper 1 (Th1) cells, primarily through its ability to prime T cells for high interferon-gamma (IFN-gamma) production. We now report that the presence of IL-12 during the first several days of in vitro clonal expansion in limiting dilution cultures of polyclonally stimulated human peripheral blood CD4+ and CD8+ T cells also induces stable priming for high IL-10 production. This effect was demonstrated with T cells from both healthy donors and HIV+ patients. Priming for IL-4 production, which requires IL-4, was maximum in cultures containing both IL-12 and IL-4. IL-4 modestly inhibited the IL-12-induced priming for IFN-gamma, but almost completely suppressed the priming for IL-10 production. A proportion of the clones generated from memory CD45RO+ cells, but not those generated from naive CD45RO- CD4+ T cells, produced some combinations of IFN-gamma, IL-10, and IL-4 even in the absence of IL-12 and IL-4, suggesting in vivo cytokine priming; virtually all CD4+ clones generated from either CD45RO(-) or (+) cells, however, produced high levels of both IFN-gamma and IL-10 when IL-12 was present during expansion. These results indicate that each Th1-type (IFN-gamma) and Th2-type (IL-4 and IL-10) cytokine gene is independently regulated in human T cells and that the dichotomy between T cells with the cytokine production pattern of Th1 and Th2 cells is not due to a direct differentiation-inducing effect of immunoregulatory cytokines, but rather to secondary selective mechanisms. Particular combinations of cytokines induce a predominant generation of T cell clones with anomalous patterns of cytokine production (e.g., IFN-gamma and IL-4 or IFN-gamma and IL-10) that can also be found in a proportion of fresh peripheral blood T cells with "memory" phenotype or clones generated from them and that may identify novel Th subsets with immunoregulatory functions.     

Preferential replication of HIV-1 in the CD45RO memory cell subset of primary CD4 lymphocytes in vitro.

The ability of HIV-1 to establish an infection and replicate to high copy number in CD4 lymphocytes is dependent on both the activation state of the cell and virus-encoded regulatory proteins that modulate viral gene expression. To study these required virus-cell interactions, we have used an in vitro cell model of acute HIV infection of quiescent, primary CD4 lymphocytes and subsequent induction of T cell activation and virus replication by lectin or CD3 receptor cross-linking. Experiments were done to determine if the capacity of HIV to establish infection and complete replication was impacted by the maturational state of the CD4 cell target or the specific signal induction pathway engaged during activation. Primary CD4 cells were FACS-sorted into the major phenotypic subsets representative of memory (CD45RO) and naive (CD45RA) cells. Levels of virus replication were compared between infection with wild-type NL4-3 virus and an isogenic mutant containing a deletion in nef regulatory gene. PHA mitogen stimulation was compared with anti-CD3, with and without anti-CD28 costimulation, for induction of cell proliferation and virus replication. In both infected and uninfected cells, the RA cell subset exhibited significantly greater response to CD3/CD28 stimulation than did the RO cell subset. In contrast, the majority of virus replication occurred consistently in the RO cell subset. Deletion of HIV nef function caused a severe reduction in viral replication, especially in the RA naive cell subset after CD3 induction. PCR analysis of viral DNA formation, during infection of quiescent cells, demonstrated that the observed differences in HIV replication capacity between RO and RA cell subsets were not due to inherent differences in cell susceptibility to infection. Our results indicate that HIV replication is enhanced selectively in CD45RO memory phenotype cells through the probable contribution of specialized cellular factors which are produced during CD3-initiated signal transduction.     

PNH患者淋巴细胞CD59和CD45的表达

目的分析阵发性睡眠性血红蛋白尿症(PNH)患者淋巴细胞及其亚群上糖化肌醇磷脂结合蛋白(CD59)的表达和功能转化.方法采用流式细胞术多色分析法,配合多种细胞特异性抗体,对PNH患者不同类型的淋巴细胞及其亚群上CD59的表达进行分析,并通过细胞刺激实验对CD59表达阴性和部分阳性的异常淋巴细胞(即病态细胞)表面分化抗原CD45异构表型的功能转化进行研究.结果14例PNH患者病态淋巴细胞的百分率较病态的中性粒细胞和红细胞低;所有PNH患者均可检测到病态B淋巴细胞的存在,B淋巴细胞病态比例高于NK细胞和T细胞;在T细胞亚群中,抑制性T细胞病态比例高于辅助性T细胞(P＜0.001);病态T细胞上HLA-DR和CD45RO的表达率明显低于正常T细胞(CD59表达完全阳性的细胞)(P＜0.01),而其CD45RA的表达率明显高于正常T细胞(P＜0.01);病态T细胞经植物血凝素刺激后培养7 d,其CD45RA的表达率明显下降(中位数从82.7%降至26.6%),而CD45RO表达率明显升高(中位数由19.6%升至64.3%).结论PNH患者各种淋巴细胞及其亚群均存在病态细胞,B淋巴细胞上CD59的表达情况可作为诊断PNH的指标,病态的T淋巴细胞以无活性的初始细胞(CD45RA+HLA-DR-)为主,受到抗原刺激后,可获得有活性的记忆细胞表型(CD45RA+HLA-DR+).     

Human CD4+ CD25hi Foxp3+ regulatory T cells are derived by rapid turnover of memory populations in vivo

While memory T cells are maintained by continuous turnover, it is not clear how human regulatory CD4+ CD45RO+ CD25hi Foxp3+ T lymphocyte populations persist throughout life. We therefore used deuterium labeling of cycling cells in vivo to determine whether these cells could be replenished by proliferation. We found that CD4+ CD45RO+ Foxp3+ CD25hi T lymphocytes were highly proliferative, with a doubling time of 8 days, compared with memory CD4+ CD45RO+ Foxp3- CD25- (24 days) or naive CD4+ CD45RA+ Foxp3- CD25- populations (199 days). However, the regulatory population was susceptible to apoptosis and had critically short telomeres and low telomerase activity. It was therefore unlikely to be self regenerating. These data are consistent with continuous production from another population source. We found extremely close TCR clonal homology between regulatory and memory CD4+ T cells. Furthermore, antigen-related expansions within certain TCR Vbeta families were associated with parallel numerical increases of CD4+ CD45RO+ CD25hi Foxp3+ Tregs with the same Vbeta usage. It is therefore unlikely that all human CD4+ CD25+ Foxp3+ Tregs are generated as a separate functional lineage in the thymus. Instead, our data suggest that a proportion of this regulatory population is generated from rapidly dividing, highly differentiated memory CD4+ T cells; this has considerable implications for the therapeutic manipulation of these cells in vivo.     

Induction of a virus-specific effector-memory CD4+ T cell response by attenuated SIV infection

We investigated simian immunodeficiency virus (SIV)-specific CD4+ T cell responses in rhesus macaques chronically infected with attenuated or pathogenic SIV strains. Analysis of SIVDeltanef-infected animals revealed a relatively high frequency of SIV-specific CD4+ T cells representing 4-10% of all CD4+ T lymphocytes directed against multiple SIV proteins. Gag-specific CD4+ T cells in wild-type SIV-infected animals were 5-10-fold lower in frequency and inversely correlated with the level of plasma viremia. SIV-specific CD4+ cells from SIVDeltanef animals were predominantly CD27-CD28-CD45RAlowCCR7-CCR5-, consistent with an effector-memory subset, and included a fully differentiated CD45RA+CCR7- subpopulation. In contrast, SIV-specific CD4+ T cells from SIV-infected animals were mostly CD27+CD28+CD45RA-CCR7+CCR5+, consistent with an early central memory phenotype. The CD45RA+CCR7-CD4+ subset from SIVDeltanef animals was highly enriched for effector CD4+ T cells, as indicated by the perforin expression and up-regulation of the lysosomal membrane protein CD107a after SIV Gag stimulation. SIV-specific CD4+ T cells in attenuated SIV-infected animals were increased in frequency in bronchioalveolar lavage and decreased in lymph nodes, consistent with an effector-memory T cell population. The ability of SIVDeltanef to induce a high frequency virus-specific CD4+ T cell response with direct effector function may play a key role in protective immunity produced by vaccination with attenuated SIV strains.     

Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells

Abnormalities in CD4(+)CD25(+)Foxp3(+) regulatory T (T reg) cells have been implicated in susceptibility to allergic, autoimmune, and immunoinflammatory conditions. However, phenotypic and functional assessment of human T reg cells has been hampered by difficulty in distinguishing between CD25-expressing activated and regulatory T cells. Here, we show that expression of CD127, the alpha chain of the interleukin-7 receptor, allows an unambiguous flow cytometry-based distinction to be made between CD127(lo) T reg cells and CD127(hi) conventional T cells within the CD25(+)CD45RO(+)RA(-) effector/memory and CD45RA(+)RO(-) naive compartments in peripheral blood and lymph node. In healthy volunteers, peripheral blood CD25(+)CD127(lo) cells comprised 6.35 +/- 0.26% of CD4(+) T cells, of which 2.05 +/- 0.14% expressed the naive subset marker CD45RA. Expression of FoxP3 protein and the CD127(lo) phenotype were highly correlated within the CD4(+)CD25(+) population. Moreover, both effector/memory and naive CD25(+)CD127(lo) cells manifested suppressive activity in vitro, whereas CD25(+)CD127(hi) cells did not. Cell surface expression of CD127 therefore allows accurate estimation of T reg cell numbers and isolation of pure populations for in vitro studies and should contribute to our understanding of regulatory abnormalities in immunopathic diseases.     

Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production

Chemokines and their receptors have been identified as major regulators controlling the functional organization of secondary lymphoid organs. Here we show that expression of CXC chemokine receptor 5 (CXCR5), a chemokine receptor required for B cell homing to B cell follicles, defines a novel subpopulation of B helper T cells localizing to follicles. In peripheral blood these cells coexpress CD45RO and the T cell homing CC chemokine receptor 7 (CCR7). In secondary lymphoid organs, CD4(+)CXCR5(+) cells lose expression of CCR7, which allows them to localize to B cell follicles and germinal centers where they express high levels of CD40 ligand (CD40L), a costimulatory molecule required for B cell activation and inducible costimulator (ICOS), a recently identified costimulatory molecule of the CD28 family. Thus, when compared with CD4(+)CD45RO(+)CXCR5(-) cells, CD4(+)CD45RO(+)CXCR5(+) tonsillar T cells efficiently support the production of immunoglobulin (Ig)A and IgG. In contrast, analysis of the memory response revealed that long-lasting memory cells are found within the CD4(+)CD45RO(+)CXCR5(-) population, suggesting that CXCR5(+)CD4 cells represent recently activated effector cells. Based on the characteristic localization within secondary lymphoid organs, we suggest to term these cells "follicular B helper T cells" (T(FH)).     

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.     

Chemokine receptor expression identifies Pre-T helper (Th)1, Pre-Th2, and nonpolarized cells among human CD4+ central memory T cells

We previously reported that central-memory T cells (T(CM) cells), which express lymph node homing receptors CCR7 and CD62L, are largely devoid of effector functions but acquire characteristics of effector-memory T cells (T(EM) cells) (i.e., CCR7(-) T helper [Th]1 or Th2 cells) after stimulation with T cell receptor agonists or homeostatic cytokines. Here we show that three chemokine receptors identify functional subsets within the human CD4(+) T(CM) cell pool. T(CM) cells expressing CXCR3 secreted low amounts of interferon gamma, whereas CCR4(+) T(CM) cells produced some interleukin (IL)-4, but not IL-5. In response to IL-7 and IL-15, CXCR3(+) T(CM) and CCR4(+) T(CM) cells invariably generated fully differentiated CCR7(-) Th1 and Th2 cells, respectively, suggesting that they represent pre-Th1 and pre-Th2 cells. Conversely, CXCR5(+) T(CM) cells lacking CXCR3 and CCR4 remained nonpolarized and retained CCR7 and CD62L expression upon cytokine-driven expansion. Unlike naive cells, all memory subsets had a low T cell receptor rearrangement excision circle content, spontaneously incorporated bromodeoxyuridine ex vivo, and contained cells specific for tetanus toxoid. Conversely, recall responses to cytomegalovirus and vaccinia virus were largely restricted to CXCR3(+) T(CM) and T(EM) cells. We conclude that antigen-specific memory T cells are distributed between T(EM) cells and different subsets of T(CM) cells. Our results also explain how the quality of primary T cell responses could be maintained by T(CM) cells in the absence of antigen.     

A subset of memory CD4+ helper T lymphocytes identified by expression of Pgp-1

The Pgp-1 glycoprotein (Ly-24 antigen) is acquired by mature murine T lymphocytes at the time of primary antigen stimulation Pgp-1 was previously shown to be a useful cell surface marker for distinguishing antigen-specific memory CD8+ T lymphocytes after immunization. Here we demonstrate that this observation extends to CD4+ T lymphocytes. Antigen-specific CD4+ T cells in mice immunized with sperm whale myoglobin or keyhole limpet hemocyanin were contained nearly exclusively in the minor Pgp-1+ subset.     

Differential coreceptor expression allows for independent evolution of non-syncytium-inducing and syncytium-inducing HIV-1

We demonstrated previously that CD45RA(+) CD4(+) T cells are infected primarily by syncytium-inducing (SI) HIV-1 variants, whereas CD45RO(+) CD4(+) T cells harbor both non-SI (NSI) and SI HIV-1 variants. Here, we studied evolution of tropism for CD45RA(+) and CD45RO(+) CD4(+) cells, coreceptor usage, and molecular phylogeny of coexisting NSI and SI HIV-1 clones that were isolated from four patients in the period spanning SI conversion. NSI variants were CCR5-restricted and could be isolated throughout infection from CD45RO(+) CD4(+) cells. SI variants seemed to evolve in CD45RO(+) CD4(+) cells, but, in time, SI HIV-1 infection of CD45RA(+) CD4(+) cells equaled infection of CD45RO(+) CD4(+) cells. In parallel with this shift, SI HIV-1 variants first used both coreceptors CCR5 and CXCR4, but eventually lost the ability to use CCR5. Phylogenetically, NSI and SI HIV-1 populations diverged over time. We observed a differential expression of HIV-1 coreceptors within CD45RA(+) and CD45RO(+) cells, which allowed us to isolate virus from purified CCR5(+) CXCR4(-) and CCR5(-) CXCR4(+) CD4(+) cells. The CCR5(+) subset was exclusively infected by CCR5-dependent HIV-1 clones, whereas SI clones were preferentially isolated from the CXCR4(+) subset. The differential expression of HIV-1 coreceptors provides distinct cellular niches for NSI and SI HIV-1, contributing to their coexistence and independent evolutionary pathways.     

Bonzo/CXCR6 expression defines type 1-polarized T-cell subsets with extralymphoid tissue homing potential

Chemokine receptor expression is finely controlled during T-cell development. We show that newly identified chemokine receptor Bonzo/CXCR6 is expressed by subsets of Th1 or T-cytotoxic 1 (Tc1) cells, but not by Th2 or Tc2 cells, establishing Bonzo as a differential marker of polarized type 1 T cells in vitro and in vivo. Priming of naive T cells by dendritic cells induces expression of Bonzo on T cells. IL-12 enhances this dendritic cell-dependent upregulation, while IL-4 inhibits it. In blood, 35-56% of Bonzo+ CD4 T cells are Th1 cells, and 60-65% of Bonzo+ CD8 T cells are Tc1 cells, while few Bonzo+ cells are type 2 T cells. Almost all Bonzo+ Tc1 cells contain preformed granzyme A and display cytotoxic effector phenotype. Most Bonzo+ T cells lack L-selectin and/or CCR7, homing receptors for lymphoid tissues. Instead, Bonzo+ T cells are dramatically enriched among T cells in tissue sites of inflammation, such as rheumatoid joints and inflamed livers. Bonzo may be important in trafficking of effector T cells that mediate type 1 inflammation, making it a potential target for therapeutic modulation of inflammatory diseases.     

Role of interleukin 6 for differential responsiveness of naive and memory CD4+ T cells in CD2-mediated activation

The present study was undertaken to elucidate different requirements for CD2-mediated activation of naive (CD45RO-) and memory (CD45RO+) CD4+ T cells. A mitogenic combination of anti-CD2 (anti-T11(2) and anti-T11(3] mAbs could effectively induce the proliferation of memory CD4+ T cells even in the absence of monocytes. In marked contrast, naive CD4+ T cells did not disclose any proliferative responses to anti-CD2 mAbs, when monocytes were absent in culture. This differential responsiveness of naive and memory CD4+ T cells appeared to be related largely to a difference in IL-6-producing ability between both populations. IL-6 among monocyte-derived cytokines could correct unresponsiveness of naive CD4+ T cells to anti-CD2 stimulation. Unlike naive CD4+ T cells, memory CD4+ T cells produced IL-6 by themselves, with its mRNA being expressed on anti-CD2 stimulation. Anti-IL-6R mAb significantly inhibited proliferation of memory CD4+ T cells seen in the anti-CD2-stimulated cultures without monocytes, indicating the involvement of their own production of IL-6 in CD2-mediated activation. The results suggest an essential role of IL-6 for triggering of CD4+ T cells via the CD2 molecule.     

Differential expression of CD45RO (UCHL1) and its functional relevance in two subpopulations of circulating TCR-gamma/delta+ lymphocytes

We examined the developmental profile of TCR-gamma/delta+ cells with respect to CD45RO expression. Although total TCR-gamma/delta+ cells were negligible in the neonatal blood and increased with advancing age, most blood TCR-gamma/delta+ cells markedly expressed CD45RO without a distinction of age, probably reflecting a different CD45RO expression of two subsets defined by BB3 and delta TCS1 mAbs. The vast majority of BB3+ cells expressed CD45RO, whereas expression of CD45RO was virtually absent in the delta TCS1+ population. Functional studies revealed that, while both TCR-gamma/delta+ cell subsets showed CD3-mediated activation, only BB3+ (or Ti gamma A+) cells, but not delta TCS1+ cells, appeared to proliferate in response to PPD in PPD-reactive individuals. The results suggested that the CD45RO+ (BB3+ or Ti gamma A+) subset among blood TCR-gamma/delta+ cells may be mainly involved in the memory or primed component of the immune system responding to some foreign antigens.     

Interleukin (IL) 4, in the absence of antigen stimulation, induces an anergy-like state in differentiated CD8+ TC1 cells: loss of IL-2 synthesis and autonomous proliferation but retention of cytotoxicity and synthesis of other cytokines

Naive T cells in the periphery mainly secrete interleukin (IL) 2 upon activation. After stimulation in the presence of appropriate costimulators, both CD4+ and CD8+ T cells differentiate into effector cells secreting distinct T helper (Th) 1- and Th2-like cytokine patterns. Subsequent to differentiation, both CD4+ (Th1 and Th2) and CD8+ (TC1 and TC2) cells are stable and cannot be induced to differentiate into the opposite pattern or revert to the naive cytokine secretion pattern. We now show that IL-4 caused committed TC1 bulk populations or clones to lose the ability to synthesize IL-2. The cells retained the ability to secrete interferon (IFN) gamma, granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor, did not synthesize any Th2 cytokines, and did not alter cell surface marker expression. IL-4 rapidly inhibited IL-2-synthesizing ability in the absence or presence of antigen-presenting cells, thus demonstrating that IL-4 acted directly on TC1 cells. The defect in IL-2 synthesis could not be reversed by subsequent stimulation with potent antigen-presenting cells in the presence of IL-2 and anti-IL-4, or with anti-CD3 plus anti-CD28 antibodies. Both IL-2+ and IL-2- TC1 cells were strongly cytotoxic toward allogeneic but not syngeneic targets. However, IL-2- TC1 cells were unable to proliferate unless exogenous IL- 2 was provided. TC1 cells that lose IL-2 synthesis but retain IFN-gamma synthesis and cytotoxicity may be similar to the "anergic" cells induced by stimulation of CD4+ or CD8+ cells in the absence of costimulators. These results suggest that during a mixed type 1/type 2 response in vivo, IL-4 may induce the IL-2+ TC1-->IL-2-TC1 conversion, and thus curtail the expansion of the TC1 response without impairing short-term effector function.     

Distinct functional lineages of human V(alpha)24 natural killer T cells

CD1d-restricted autoreactive natural killer (NK)T cells have been reported to regulate a range of disease conditions, including type I diabetes and immune rejection of cancer, through the secretion of either T helper (Th)2 or Th1 cytokines. However, mechanisms underlying Th2 versus Th1 cytokine secretion by these cells are not well understood. Since most healthy subjects express <1 NKT cell per 1,000 peripheral blood lymphocytes (PBLs), we devised a new method based on the combined used of T cell receptor (TCR)-specific reagents alpha-galactosylceramide (alphaGalCer) loaded CD1d-tetramers and anti-V(alpha)24 monoclonal antibody, to specifically identify and characterize these rare cells in fresh PBLs. We report here that CD4(+) and CD4(-)CD8(-) (double negative [DN]) NKT cell subsets represent functionally distinct lineages with marked differences in their profile of cytokine secretion and pattern of expression of chemokine receptors, integrins, and NK receptors. CD4(+) NKT cells were the exclusive producers of interleukin (IL)-4 and IL-13 upon primary stimulation, whereas DN NKT cells had a strict Th1 profile and prominently expressed several NK lineage receptors. These findings may explain how NKT cells could promote Th2 responses in some conditions and Th1 in others, and should be taken into consideration for intervention in relevant diseases.