Increased in vitro induced CD4+ and CD8+ T cell IFN-gamma and CD4+ T cell IL-10 production in stable relapsing multiple sclerosis

Multiple sclerosis (MS) is presumed to be a T-cell mediated chronic inflammatory disease of the central nervous system. Investigators previously demonstrated increased IFN-gamma (pro-inflammatory) and IL-10 (counterregulatory anti-inflammatory) in MS. The balance of pro-inflammatory and counterregulatory anti-inflammatory cytokines may be important in the stabilization of disease activity. Purified CD4+ and CD8+ T cells from patients with clinically definite, stable relapsing MS (RRMS) were stimulated by anti-CD3 mAb or Con A for 48 hours and cytokine supernatants analysed for production of IL-2, IL-6, IFN-gamma, TNF-alpha (potential pro-inflammatory) and IL-4, IL-10, and TGF-beta (potential counterregulatory anti-inflammatory). Con A activated CD4+ and CD8+ T cell proinflammatory cytokine IL-2 secretion, CD4+ T cell IL-6 secretion, CD4+ and CD8+ T cell TNF-alpha secretion and CD8+ T cell IFN-gamma secretion was decreased significantly in RRMS subjects compared to controls. CD3 activated CD4+ and CD8+ T cell IL-6 secretion and CD4+ T cell TNF-alpha secretion was significantly decreased in MS subjects compared to controls. In contrast, there was increased CD3-induced IFN-gamma in both CD4+ and CD8+ T cells and counterregulatory anti-inflammatory CD3-induced IL-10 secretion in CD4+ T cells in RRMS compared to controls. These data suggest that an equilibrium of a pro-inflammatory (IFN-gamma) and a counterregulatory anti-inflammatory (IL-10) cytokine may define stable clinically definite early RRMS.     

Urocanic acid enhances IL-10 production in activated CD4+ T cells

The immunosuppressive effects of UV radiation have been well documented. This suppression has been attributed to the action of the cis form of urocanic acid (UCA), a photoproduct of trans-UCA, a natural constituent of the skin. Here, we show that mouse spleen cells preincubated with cis-UCA have a diminished proliferative response to allogeneic cells in MLC and to stimulation with anti-CD3 mAb. Cells preincubated with cis-UCA also had a decreased ability to serve as APC and to stimulate the proliferation of allogeneic lymphocytes in MLC. Simultaneously, the production of IL-2 and IFN-gamma by cells preincubated with cis-UCA was decreased. However, IL-10 gene expression and IL-10 protein secretion by spleen cells stimulated in the presence of cis-UCA were significantly enhanced. The principal cell population displaying the cis-UCA-induced elevated production of IL-10 was CD4+ T cells, which were shown to be a direct target of cis-UCA action. This was also supported by the observation that production of IL-10 by stimulated splenic non-T cells or by macrophages was not altered by cis-UCA. The enhanced production of IL-10 by activated CD4+ T cells may represent a novel pathway of UVB radiation-induced, cis-UCA-mediated immunosuppression. We suggest that the elevated production of IL-10 by activated CD4+ T cells may account for the suppressor T cell phenomena described in UV-irradiated recipients.     

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.     

Enrichment for Th1 cells in the Mel-14+ CD4+ T cell fraction in aged mice

CD4+ T cells from young and aged mice were sorted into Mel-14+ cells which are regarded as naive cells and Mel-14- cells which are regarded as memory cells. These subsets were stimulated in short-time cultures with anti-CD3 or anti-CD3/anti-CD28 in order to determine the presence of Th1 and/or Th2 cytokines. Based on the simultaneous production of IL-2, IL-4, IL-10, and IFN-gamma upon anti-CD3 stimulation by Mel-14- cells from young and aged mice, it is concluded that this cell population comprises Th1, Th2, and/or Th0 cells. Mel-14+ cells from young mice only secrete substantial amounts of IL-2 in the presence of anti-CD28 as a costimulatory signal and can therefore be regarded as Th precursor cells. By contrast, Mel-14+ cells from aged mice responded to anti-CD3 alone, not only by the production of IL-2 but also by the production of high amounts of IFN-gamma and minute amounts of IL-4 and IL-10, suggesting that these "naive" cells in aged mice are enriched for Th1 cells. This was not due to lack of CD28 triggering since anti-CD28 enhanced IFN-gamma as well as IL-4 and IL-10 to a similar extent. Our data therefore indicate that Mel-14 is not exclusively expressed on naive CD4+ T cells.     

IL-7 promotes the survival and maturation but not differentiation of human post-thymic CD4+ T cells

This study examines the influence of IL-7 on post-thymic CD4+ T cells using cord blood as a model system. Survival of naive cord blood T cells in the presence of IL-7 alone was significantly prolonged by up-regulating bcl-2, thereby preventing apoptosis while maintaining maximal cell viability. Cultures without IL-7 showed high rates of apoptosis resulting in 50% cell death by day 5 of culture. Upon phorbol 12-myristate 13-acetate + ionomycin stimulation, accumulation of cytoplasmic IL-2 was similar to that observed in freshly isolated cells, but no IL-4- or IFN-gamma-positive cells were detected. IL-7 maintained the naive T cells in a quiescent state expressing the CD45RA antigen. A significant finding was the loss of CD38 antigen expression on the naive cord blood T cells to levels similar to that observed on adult naive T cells. In contrast to the reduced proliferative response of fresh cord blood T cells to anti-CD2 + CD28 stimulation, the proliferative response of IL-7-treated cells was similar to that of adult naive T cells. This study shows that as well as maintaining the naive T cell pool by enhancing cell survival and up-regulating bcl-2 expression, IL-7 also functions as a maturation factor for post-thymic naive T cells.     

The CD7- T cell subset represents the majority of IL-5-secreting cells within CD4+CD45RA- T cells

Absence of CD7 is a stable phenotype in a subset of normal human T cells. Most circulating CD7- T cells express the CD4CD45RO+CD45RA- memory phenotype. We analysed CD4+CD45RA- peripheral blood lymphocytes that were separated into CD7+ and CD7- for their in vitro cytokine secretion in response to different stimuli. The CD4+CD7- subpopulation was found to secrete significantly higher levels of IL-5 compared with the CD4+CD7- subset upon stimulation with ionomycin/phorbol myristate acetate (PMA) plus anti-CD28 MoAbs. In contrast to IL-5 secretion, IL-4 and interferon-gamma (IFN-gamma) secretion was not significantly different in CD7+ and CD7- T cells upon stimulation in vitro. The data indicate that the CD4+CD7- T cell represents the majority of IL-5-secreting cells within the population of CD4+CD45RA- memory T cells. Since CD4+CD7- T cells were found to be enriched in various skin lesions associated with eosinophilic infiltration, the results of our study support the hypothesis that skin-infiltrating CD7- T cells are one of the major sources of IL-5 responsible for the development of eosinophilic inflammation in certain skin diseases.     

CD4-mediated inhibiton of IL-2 production in activated T cells

The role of CD4 in T cell activation has been attributed to its capacity to increase the avidity of interaction with APC and to shuttle associated Lck to the TCR/CD3 activation complex. The results presented in this study demonstrate that ligation of CD4 inhibits ongoing responses of preactivated T cells. Specifically, delayed addition of CD4-specific mAb is shown to inhibit Ag- or mAb-induced responses of both primary T cells and T cell clonal variants. The Ag responses of the latter are independent of the adhesion provided by CD4; thus the observed inhibition is not due to blocking CD4-MHC interactions. Further, analysis of the clonal variants demonstrates that CD4-associated Lck is not essential for the inhibition observed, as anti-CD4 inhibits responses of clonal variants, expressing a form of CD4 unable to associate with Lck (double cysteine-mutated CD4). The inhibition is counteracted by the addition of exogenous IL-2, demonstrating that the block is not due to a lesion in IL-2 utilization, rather its production. It is demonstrated that the delayed addition of anti-CD4 results in a rapid reduction in steady-state levels of IL-2 mRNA in both primary T cells and clonal variants.     

Antigen-specific B cells preferentially induce CD4+ T cells to produce IL-4

The bone marrow microenvironment influences whether a given B cell proliferates, differentiates, or undergoes apoptosis. In this report, we demonstrate that apoptosis of primary murine B lymphocyte precursors can be regulated either positively or negatively by stroma. Several stromal lines that support lymphocyte outgrowth suppressed the spontaneous apoptosis of pre-B cells by as much as 90%. Direct contact with stromal cells more effectively protected lymphocytes than did stromal cell-CM or a collection of recombinant cytokines. In contrast, one unique stromal cell clone actually induced lymphocyte apoptosis, and a second line appeared inert. A survey of adherent cell lines suggested that expression of life-sparing molecules is widespread but not ubiquitous. Experiments with neutralizing Abs to CD44, vascular cell adhesion molecule-1 (VCAM-1), CD9, intercellular adhesion molecule-1 (ICAM-1), or ICAM-2 suggested that these interaction molecules do not deliver short-term survival signals to B cell precursors. Of particular interest, direct interaction with lymphocyte-supportive stromal cells minimized the negative regulatory effects of IL-1alpha, and a glucocorticoid, but not IFN-beta or PGE2. These results demonstrate that the effect of negative regulators depends upon the context in which these signals are presented. As molecules that influence B lymphopoiesis are better defined, it will be important to consider the role of each in combination with other stimuli.     

CD4-CD8-C.B-17 SCID thymocytes enter the CD4+CD8+ stage in the presence of neonatally grafted T cells

Our previous studies in iron-loaded rat heart cells showed that in vitro iron loading results in peroxidative injury, manifested in a marked decrease in rate and amplitude of heart cell contractility and rhythmicity, which is correctable by treatment with deferoxamine (DF). In the present studies we explored the role of mitochondrial damage in myocardial iron toxicity. Iron loading by 24-hour incubation with 0.36 mmol/L ferric ammonium citrate resulted in a decrease in the activity of nicotinamide adenine dinucleotide (NADH)-cytochrome c oxidoreductase (complex I+III) to 35.3%+/-11.2% of the value in untreated controls; of succinate-cytochrome c oxidoreductase (complex II+III) to 57.4%+/-3.1%; and of succinate dehydrogenase to 63.5%+/-12.6% (p < 0.001 in all cases). The decrease in activity of other mitochondrial enzymes, including NADH-ferricyanide reductase, succinate ubiquinone oxidoreductase (complex II), cytochrome c oxidase (complex IV), and ubiquinol cytochrome c oxidoreductase (complex III), was less impressive and ranged from 71.5%+/-15.8% to 91.5%+/-14.6% of controls. That the observed loss of respiratory enzyme activity was a specific effect of iron toxicity was clearly demonstrated by the complete restoration of enzyme activities by in vitro iron chelation therapy. Sequential treatment with iron and doxorubicin caused a loss of complex I+III and complex II+III activity that was greater than that seen with either agent alone but was only partially correctable by DF treatment. Alterations in cellular adenosine triphosphate measurements paralleled very closely the changes observed in respiratory complex activity. These findings demonstrate for the first time the impairment of cardiac mitochondrial respiratory enzyme activity caused by iron loading at conditions formerly shown to produce severe abnormalities in contractility and rhythmicity.     

CD11b+CD28-CD4+ human T cells: activation requirements and association with HLA-DR alleles

Engagement of CD28 induces a major costimulatory pathway required by many CD4+ T cells in addition to activation via the TCR. In the absence of signals provided by CD28, ligation of the TCR alone can induce anergy or apoptosis in CD28+ cells. However, we report here characterization of a distinct subset of CD4+ T cells that are CD28-. Three autoreactive CD4+ human T cell clones that could be activated to produce IL-2 and proliferate by anti-CD3 alone were found to lack expression of CD28. CD28- clones that were activated with anti-CD3 alone were not anergic to restimulation via CD3. The presence of CD28-CD4+ T cells was verified in peripheral blood, and their frequency ranged from 0% to >22% of CD4+ T cells in different individuals. The percentage of CD28-CD4+ T cells in the peripheral blood of 57 individuals was significantly correlated with specific class II MHC alleles. Persons with HLA-DRB1*0401 and DR1 alleles had significantly higher numbers of CD28- T cells, while individuals with HLA-DR2(15) had significantly fewer CD28-CD4+ T cells than the mean. Like the CD28- clones, CD28-CD4+ T cells isolated from peripheral blood proliferated upon CD3 cross-linking in the absence of costimulation. The finding that CD28-CD4+ T cells resist induction of anergy following engagement of the TCR in the absence of conventional costimulation demonstrates one mechanism by which autoreactive T cells can escape processes that censor self-reactivity. The MHC associations observed suggest a relationship with autoimmunity and loss of self-tolerance.     

CD4+ T cells inhibit growth of Epstein-Barr virus-transformed B cells through CD95-CD95 ligand-mediated apoptosis

Greater than 90% of the human population acquire Epstein-Barr virus (EBV) in infancy and retain a lifelong latent infection without any clinical consequences. Nevertheless EBV has been identified as the causal agent of infectious mononucleosis, and is associated with several tumours including endemic Burkitt's lymphoma and B cell lymphomas in immunosupressed patients. B cells infected with EBV are transformed in vitro and grow continuously as lymphoblastoid cell lines. The growth of EBV-transformed B cells in vivo is controlled by the immune system. Studies on immunity to EBV have mainly focused on MHC class I-restricted CD8+ cytotoxic T cells specific for viral latent antigens. Here it is reported that in vitro stimulation of peripheral blood lymphocytes by autologous EBV-infected B cells, which have been induced to express lytic cycle antigens, gives rise to a predominantly CD4+ T cell response. Furthermore, the growth of EBV-infected B cells can also be regulated by these activated CD4+ T cells through apoptosis mediated by CD95-CD95 ligand (CD95L). CD95-CD95L-mediated apoptosis is an important mechanism of normal B cell growth regulation. As EBV-transformed B cells remain susceptible to this mechanism, the control of EBV in vivo may be not only by virus-specific CD8+ cytotoxic T cell immunity but also by normal mechanisms of immune regulation of B cell growth.     

Increase of circulating CD3+CD4-CD8-CD19+ cells in the latent phase of HIV-1 infection

Having reported that HIV-1-infected T cell lines are rescued as CD4- from cytolysis by human complement factor B, we now show the presence of an in vivo counterpart of such CD4- T cells by demonstrating the circulating CD3+ CD4- CD8- CD29+ cells in the blood of seropositive subjects (n = 91, classified by the immunologic scale scores 0, 1, 2 and 3). The cell population was found to be significantly increased in the early phase of infection in score 0: 195/mm3 (p < 0.005) and in score 1:376/mm3 (p = 0.001). With the infection progressing to score 2, the cells decreased to 220/mm3 (p < 0.001) and finally to the same range: 101/mm3, as that of uninfected subjects. Further elucidation of the mechanism of the appearance and disappearance of that population in vivo could help to elucidate protective immunologic processes.     

Induction of Th2 cell differentiation in the primary immune response: dendritic cells isolated from adherent cell culture treated with IL-10 prime naive CD4+ T cells to secrete IL-4

Rates of hormone replacement therapy (HRT) in women have varied substantially over the last 25 years. Data on the impact of recent recommendations for widespread use to prevent cardiovascular disease and osteoporosis and factors that influence use are needed. We attempted to (1) describe recent trends in HRT use, (2) investigate the relationship between HRT use and prepaid drug benefit, and (3) detail prescribing frequencies by provider specialty. We conducted a cross-sectional analysis of annual HRT pharmacy dispensings from 1986 to 1995 in a large HMO to all female HMO members aged 45 years and older. HRT rates increased among all age categories, although the magnitude of change varied by age. Highest rates of use were found in those 50-59 years old. Although combined estrogen-progestin use increased, 57% of all estrogen users did not receive progestin in 1995. Unopposed estrogen use was largely limited to hysterectomized women. Women of all ages with no prepaid drug benefit as part of their HMO coverage had the lowest HRT rates. Internal medicine, obstetrics/gynecology, and family practice providers prescribed over 90% of HRT, and prescriber specialty varied with user age. HRT use increased in the HMO from 1986 to 1995, especially among younger women. In 1995, about half of women aged 50-64 years received one or more HRT dispensings. As the benefits, risks, and cost effectiveness of HRT depend on the duration of use, additional information on current use duration is needed. Combined estrogen-progestin use increased and appeared appropriate to hysterectomy status. Research is needed to determine if lower HRT use rates among women without a prepaid drug benefit indicate less prophylactic HRT use, particularly among younger women, for whom this lack of coverage was relatively common.     

Reciprocal regulation of CD30 expression on CD4+ T cells by IL-4 and IFN-gamma

Prior studies have implicated CD30 as a marker for Th2 cells, but the mechanism that underlies this correlation was unknown. We show here that CD30 was expressed on activated CD4+ T cells in the presence of IL-4. In the absence of endogenously produced IL-4, however, even Th2 lineage cells lost CD30 expression. Thus, CD30 is not an intrinsic marker of Th2 cells, but is inducible by IL-4. CD30 was also found to be down-regulated by IFN-gamma. Committed Th1 effector cells do not express CD30, although differentiating Th1 lineage cells temporarily express CD30. The transient expression of CD30 on differentiating Th1 lineage cells was mainly the result of endogenously produced IL-4 induced by IL-12. Culture of IL-12-primed cells under conditions that reverse the phenotype (Ag plus IL-4) resulted in two cell populations based upon their ability to express CD30. One population responded to IL-4 upon restimulation and became a CD30-positive, Th0-like cell population, while the other remained CD30 negative and synthesized only IFN-gamma. Thus, CD30 expressed on CD4+ T cells reflected the ability of CD4+ T cells to respond to IL-4.     

IL-10 impacts autoimmune diabetes via a CD8+ T cell pathway circumventing the requirement for CD4+ T and B lymphocytes

IL-10 is essential for an early phase of diabetes in nonobese diabetic (NOD) mice, but later becomes protective against its development. The mechanism by which IL-10 mediates the pathway to diabetes in these mice is unknown. Herein, we dissected the cellular and costimulation requirements for diabetes in transgenic (tg) NOD mice that expressed IL-10 in their pancreatic islets (IL-10-NOD mice). We found that IL-10 alone did not cause diabetes because the offspring (IL-10-NOD-scid mice) from back-crosses of IL-10-NOD mice with NOD-scid mice had no diabetes. Moreover, these IL-10-NOD-scid mice were free of lymphocytic infiltration. Treatment of IL-10-NOD mice with depleting anti-CD4 mAb or control mAb had no effect on diabetes. Surprisingly, depletion of CD8+ T cells by treatment with the corresponding mAb inhibited diabetes without attenuating insulitis, demonstrating a critical role for CD8+ T cells in the disease process. Interestingly, B cell-deficient IL-10-NOD mice readily developed diabetes with kinetics and incidence similar to those observed in wild-type mice, demonstrating that B lymphocytes as APCs were not required in the disease process. Administration of anti-CD40 ligand (CD40L) mAb did not prevent disease, indicating that CD40/CD40L costimulation is not required for diabetes in IL-10-NOD mice. Immunization of IL-10-NOD mice with CFA or heat-shock protein 65, known to block diabetes in NOD mice, had no effect on their diabetes. We demonstrate that IL-10 contributes early to the pathology of diabetes via a CD8+ T cell pathway, eliminating the requirement for B lymphocytes and CD40-CD40L costimulation. Our findings provide a mechanism for the participation of IL-10 in the early development of diabetes.     

Factors involved in the differentiation of TGF-beta-producing cells from naive CD4+ T cells: IL-4 and IFN-gamma have opposing effects, while TGF-beta positively regulates its own production

TGF-beta has been shown to play a central role in regulating inflammatory responses; thus, understanding the factors involved in the generation of TGF-beta-producing cells could lead to interventions that are useful in effecting disease progression. In initial studies, the capacity of naive CD4+ T cells from TCR transgenic (Tg) mice to produce TGF-beta following primary and secondary stimulation was assessed. TGF-beta, IL-4, or IFN-gamma production could not be detected from highly purified naive CD4+/lymphocyte endothelial cell adhesion molecule (LECAM)-1high cells following primary stimulation for 36 h with plate-bound anti-CD3, anti-CD28, and IL-2. This population was subsequently used to study the differentiation of TGF-beta-producing CD4+ T cells. In further studies, naive CD4+/LECAM-1high cells from TCR transgenic mice of both the BALB/c and B10.A backgrounds were stimulated with T-depleted spleen cells (TDS) and specific peptide in the presence of various cytokines and/or cytokine antagonists for 5 days, restimulated, and TGF-beta, IL-4, and IFN-gamma production were measured. Priming conditions favoring high IL-4 production and/or low IFN-gamma production greatly enhanced TGF-beta production in secondary cultures. Furthermore, the presence of IL-10 in cultures was associated with an increase in TGF-beta production following restimulation. The importance of IL-4 and IFN-gamma in regulating TGF-beta production was confirmed in studies showing that cells from IFN-gamma(-/-) mice produced more TGF-beta, while cells from IL-4(-/-) mice produced less TGF-beta compared with wild-type controls. Finally, the addition of exogenous TGF-beta to priming cultures significantly enhanced the production of TGF-beta upon restimulation, demonstrating that TGF-beta has a role in self-regulating its own production.     

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.     

Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15

Proliferation of memory-phenotype (CD44hi) CD8+ cells induced by infectious agents can be mimicked by injection of type I interferon (IFN I) and by IFN I-inducing agents such as lipopolysaccharide and Poly I:C; such proliferation does not affect naive T cells and appears to be TCR independent. Since IFN I inhibits proliferation in vitro, IFN I-induced proliferation of CD8+ cells in vivo presumably occurs indirectly through production of secondary cytokines, e.g., interleukin-2 (IL-2) or IL-15. We show here that, unlike IL-2, IL-15 closely mimics the effects of IFN I in causing strong and selective stimulation of memory-phenotype CD44hi CD8+ (but not CD4+) cells in vivo; similar specificity applies to purified T cells in vitro and correlates with much higher expression of IL-2Rbeta on CD8+ cells than on CD4+ cells.     

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.     

Development of CD4 and CD8 single positive T cells in human thymus organ culture: IL-7 promotes human T cell production by supporting immature T cells

This paper describes novel model systems to study the development of human T cells. Fragments of neonatal human thymus (HUNT) can be cultured in vitro; the initial majority population of CD4, CD8 double-positive (DP) thymocytes is not maintained in organ culture. These cells are rapidly replaced by populations of CD4 or CD8 single-positive (SP) T cells. In addition, allogeneic thymic chimeras can be established by the addition of human cord blood (HUCB) mononuclear cells as a source of T progenitor cells to the organ cultures. Culture results in the acquisition of a mature SP T cell phenotype by the donor cells similar to that found when HUCB is allowed to develop in xenogeneic murine scid/scid fetal thymus organ culture. The number of immature and mature T cells produced by organ cultures can be differentially increased by the addition of exogenous IL-7, stem cell growth factor, IL-1, or GM-CSF. Anti-IL-7 antibody inhibits T cell production. Taken together, the results suggest that human T cell development occurs in these in vitro systems in a similar manner, regardless of the species origin of the thymic stromal cells in the culture, and that exogenous cytokines can be used to expand subpopulations of developing T cells.