Characterization of the effects of the partial dopamine agonist terguride on cocaine self-administration in the rat

One notable functional abnormality in murine and human systemic lupus erythematosus (SLE) is the defect in the production of IL-2 in association with the deficit in naive CD4+ T cells. The mechanism is unknown, but one idea is that naturally occurring autoantibodies with specificities to the naive CD4+ T cell subpopulation are related to this event. We selected hybridoma monoclonal autoantibodies from SLE-prone (New Zealand Black (NZB) x New Zealand White (NZW))F1 mice that reacted with restricted populations of CD4+ T cells. One of these, H32, was specific for L-selectin, as determined by 1) distribution of Ag H32 on lymphoid cells similar to Mel-14, an epitope of L-selectin; 2) shedding of 80-kDa molecules with epitope H32 from the surface of lymph node cells coincidentally with Mel-14, when stimulated with phorbol ester; 3) cross-inhibitory activities on Ag binding between H32 and Mel-14; and 4) reactivity of H32 with recombinant mouse L-selectin. Pretreatment of 51Cr-labeled lymphocytes from BALB/c mice with H32 significantly inhibited their homing to lymph nodes in vivo. The BALB/c splenic H32+ CD4+ T cell subset produced few cytokines except IL-2, thus corresponding to naive ThP-type cells. This subset was markedly selectively depleted in aged (NZB x NZW)F1 mice. There was an age-associated increase in frequencies and titers of anti-L-selectin autoantibodies in sera from (NZB x NZW)F1 mice. Thus, abnormalities of naive CD4+ T cell subset, including IL-2 production in subjects with SLE, are at least partly attributed to the generation of autoantibodies to L-selectin.     

Functional CD4+ T cell subsets defined by expression of CD45RC and NTA260 antigens and age-associated polarization in murine lupus

Using two mAb, one specific to the alternative exon 6-dependent epitope of CD45 molecules (JH6.2) and one a natural thymocytotoxic autoantibody (NTA) with an unknown reactive epitope (NTA260), we subdivided splenic CD4+ T cells from 2-month-old BALB/c mice into five phenotypically distinct subsets. CD45RC+NTA260- (S I) cells were phenotypically analogous to CD4+ T cells predominating in newborn mice and produced a significant amount of IL-2, but not so IL-4, IL-10 or IFN-gamma when stimulated with immobilized anti-CD3 mAb in vitro. They appeared to consist mainly of naive ThP cells. The CD45RC+NTA260+ (S II) subset also produced IL-2, but not other cytokines; however, the IL-2 levels produced were much higher than seen with the S I subset, thereby suggesting the predominance of further maturated ThP cells. The CD45RC-NTA260+ (S III) subset mainly produced IL-4, IL-10, IFN-gamma and less IL-2, and contained memory cells that helped the secondary antibody response to a recall antigen, and hence contained Th2 and probably a mixture of Th0 and Th1 cells. The CD45RC-NTA260- (S IV) subset was a poor responder to the immobilized anti-CD3 mAb. The CD45RCbrightNTA260dull (S V) subset consisted of a small number of cells that were phenotypically analogous to activated CD4+ T cells. While an age-associated decrease in the proportion of S I and less markedly in S II and in turn increase in S III subsets of CD4+ T cells occurred in normal BALB/c mice, autoimmune disease-prone (NZB x NZW)F1 mice showed a marked age-associated decrease in the proportion of not only S I, II but also III subsets. As aged (NZB x NZW)F1 mice carry CD4+ T helper cells for IgG anti-DNA antibody production, such age-associated polarization to the S IV subset appears to be critical in the pathogenesis of autoimmune disease in these mice.     

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.     

Effects of bacterial DNA on cytokine production by (NZB/NZW)F1 mice

Microbial DNA has multiple immune effects including the capacity to induce polyclonal B cell activation and cytokine production in normal mice. We recently described the accelerated induction of anti-DNA Abs in NZB/NZW mice immunized with Escherichia coli (EC) dsDNA; paradoxically these mice developed less renal disease than unimmunized mice or mice immunized with calf thymus DNA. We postulated that alterations in cytokine production induced by bacterial DNA may play a key role in renal protection. To determine the effect of bacterial DNA on cytokine production in NZB/NZW mice, we measured the serum cytokine levels, cell culture supernatant cytokine levels, and number of cytokine-producing splenocytes in NZB/NZW mice injected with EC DNA, calf thymus DNA, or an immune active oligonucleotide. There was a 10- to 25-fold increase in the number of cells secreting IFN-gamma compared with IL-4 in mice immunized with EC DNA. IL-12-secreting cells were also increased by bacterial DNA immunization. In parallel with the increase in IFN-gamma secreting cells, there was a significant rise in serum IFN-gamma levels in mice receiving EC DNA. These results indicate that EC DNA modulates systemic cytokine levels in NZB/NZW mice, selectively increasing IL-12 and IFN-gamma while decreasing IL-4 production. The cytokine response of NZB/NZW mice to bacterial DNA may be of significance in disease pathogenesis and relevant to the treatment of lupus-like disease.     

Impaired Th2 subset development in the absence of CD4

Prior studies in CD4-deficient mice established the capacity of T helper (Th) lineage cells to mature into Th1 cells. Unexpectedly, challenge of these mice with Nippostrongylus brasiliensis, a Th2-inducing stimulus, failed to result in the development of Th2 cells. Additional studies were performed using CD4+ or CD4-CD8- (double-negative) T cell receptor (TCR) transgenic T cells reactive to LACK antigen of Leishmania major. Double-negative T cells were unable to develop into Th2 cells in vivo, and, unlike CD4+ T cells, could not be primed for interleukin-4 production in vitro. Similarly, CD4+ TCR transgenic T cells primed on antigen-presenting cells expressing mutant MHC class II molecules unable to bind CD4 did not differentiate into Th2 cells. These data suggest that interactions between the TCR, MHC II-peptide complex and CD4 may be involved in Th2 development.     

CD8 inhibits signal transduction through the T cell receptor in CD4-CD8- thymocytes from T cell receptor transgenic mice reconstituted with a transgenic CD8 alpha molecule

T cell repertoire selection processes involve intracellular signaling events generated through the TCR. The CD4 and CD8 coreceptor molecules can act as positive regulators of TCR signal transduction during these developmental processes. In this report, we have used TCR transgenic mice to determine whether TCR signaling can be modulated by the CD8 coreceptor molecule. These mice express on the majority of their T cells a TCR specific for the male (H-Y) Ag presented by the H-2Db MHC class I molecule. We show that CD4-CD8-, but not CD4-CD8+, thymocytes expressing the H-Y TCR responded with high intracellular calcium fluxes to TCR/CD3 stimulation without extensive receptor cross-linking. To examine the effects of CD8 expression on intracellular signaling responses in the CD4-CD8- cells, the H-Y TCR transgenic mice were mated with transgenic mice that constitutively expressed the CD8 alpha molecule on all T cells. The expression of the CD8 alpha alpha homodimer in the CD4-CD8-thymocytes led to impaired intracellular calcium responses and less efficient protein tyrosine phosphorylation of substrates after TCR engagement. In male H-2b H-Y transgenic mice, the majority of thymocytes have been deleted with the surviving cells expressing a high density of the transgenic TCR and exhibiting either a CD4-CD8- or CD4-CD8lo phenotype. It has been postulated that these cells escaped deletion by down-regulating the CD8 molecule. In the H-Y TCR/CD8 alpha double transgenic male mice, the CD4-CD8lo cells were completely eliminated as a result of CD8 alpha expression. However, the CD4-CD8- T cells were not deleted despite normal levels of the CD8 alpha transgene expression. These results suggest that the CD4-CD8- thymocytes may not be susceptible to the same deletional mechanisms as other thymocytes expressing TCR-alpha beta.     

Studies using antigen-presenting cells lacking expression of both B7-1 (CD80) and B7-2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production

The differentiation of CD4+ T cells into a Th1 vs Th2 phenotype profoundly influences the outcome of autoimmune and infectious diseases. B7 costimulation has been shown to affect the production of both Th1 and Th2 cytokines, depending on the system studied. There is, consequently, great interest in manipulating the B7 costimulatory signal for therapeutic purposes. To optimally manipulate this key immunoregulatory pathway, the contribution of B7 costimulation to cytokine production requires further clarification. We have compared the B7 requirement for cytokine production by naive vs previously activated T cells using DO11.10 TCR transgenic CD4+ T cells and splenic APCs from mice lacking B7 expression. Our data indicate that induction of IL-4 production and Th2 differentiation by naive T cells is highly dependent on B7 molecules, whereas IL-4 production by previously activated T cells is B7 independent. The predominant contribution of B7-mediated signals to Th1 cytokine production by both naive and primed T cells is upon IL-2 production (and expansion) rather than IFN-gamma (effector cytokine) production. Thus, our studies demonstrate that the antigenic experience of a T cell at the time of B7 blockade may determine whether blockade predominantly affects T cell expansion, differentiation, or effector cytokine production. These differential effects of B7 costimulation on IL-2 vs IFN-gamma production and on IL-4 production by naive vs primed T cells have important implications for understanding how B7:CD28/CTLA4 blockade can be effectively used to manipulate cytokine production in vivo.     

Cross-linking of the CD40 ligand on human CD4+ T lymphocytes generates a costimulatory signal that up-regulates IL-4 synthesis

Although there is good evidence that the induction of IL-4 synthesis in CD4+ T lymphocytes is favored by Ag presentation by B cells and not macrophages, the precise molecular signals provided by B cells to T cells that enhance IL-4 synthesis are not clear. To examine this issue, we established an APC-independent system to activate highly purified T cells and induce cytokine synthesis, using immobilized mAbs against several T cell surface molecules, including CD3, CD28, and the CD40 ligand (CD40L). The counter-receptors for all three of these molecules are expressed on B cells, and include CD40, which is expressed primarily on B cells, but also on dendritic cells and thymic epithelium. We found that IL-4 synthesis was greatly enhanced by triggering of CD40L on the T cell surface in conjunction with ligation of CD3/TCR and CD28, whereas ligation of CD3/TCR and CD28 in the absence of CD40L triggering resulted in little or no IL-4 synthesis. CD40L costimulation greatly enhanced IL-4 synthesis both in T cells from normal nonallergic adult subjects as well as in naive T cells from cord blood. Furthermore, we demonstrated that IL-4 synthesis was optimally enhanced when the strength of the CD3/TCR signal was limiting, while IL-4 synthesis was inhibited when CD3/TCR stimulation was maximal. These studies confirm that IL-4 synthesis can be induced in normal T lymphocytes in the absence of exogenous IL-4, and demonstrate that CD40L costimulation is of fundamental importance in regulation of IL-4 production. In addition, these findings provide a mechanism by which B cells preferentially enhance IL-4 synthesis in T cells at low Ag concentrations.     

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.     

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.     

IL-2 and IL-7 differentially induce CD4-CD8- alpha beta TCR+NK1.1+ large granular lymphocytes and IL-4-producing cells from CD4-CD8- alpha beta TCR+NK1.1- cells: implications for the regulation of Th1- and Th2-type responses

Effector functions of CD4-CD8- double negative (DN) alpha beta TCR+ cells were examined. Among mouse DN alpha beta TCR+ thymocytes, NK1.1+ cells expressing a canonical V alpha 14/J alpha 281 TCR but not NK1.1- cells produce IL-4 upon TCR cross-linking and IFN-gamma upon cross-linking of NK1.1 as well as TCR. Production of IL-4 but not IFN-gamma from DN alpha beta TCR+NK1.1+ cells was markedly suppressed by IL-2. Whereas V alpha 14/J alpha 281 TCR+ cells express NK1.1+, these cells are not the precursor of DN alpha beta TCR+NK1.1+CD16+B220+ large granular lymphocytes (LGL). IL-2 induces rapid proliferation and generation of NK1.1+ LGL from DN alpha beta TCR+NK1.1- but not from DN alpha beta TCR+NK1.1+ cells. LGL cells exhibit NK activity and produce IFN-gamma but not IL-4 upon cross-linking of surface TCR or NK1.1 molecules. In contrast to IL-2, IL-7 does not induce LGL cells or NK activity from DN alpha beta TCR+NK1.1- cells but induces the ability to produce high levels of IL-4 upon TCR cross-linking. Our results show that DN alpha beta TCR+ T cells have several distinct subpopulations, and that IL-2 and IL-7 differentially regulate the functions of DN alpha beta TCR+ T cells by inducing different types of effector cells.     

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.     

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

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

Interleukin-17

The particular interest of IL-17, a homodimeric cytokine of about 32 kDa, is the strict requirement for an activation signal to induce its expression from a rather restricted set of cells, human memory T cells or mouse alpha beta TCR+CD4-CD8- thymocytes. In contrast with the tightly controlled expression pattern of this gene, the IL-17 receptor, a novel cytokine receptor, is ubiquitously distributed but apparently more abundant in spleen and kidney. In addition to its capture by the T lymphotropic Herpesvirus Saimiri (HVS), this cytokine is inducing the secretion of IL-6, IL-8, PGE2, MCP-1 and G-CSF by adherent cells like fibroblasts, keratinocytes, epithelial and endothelial cells. IL-17 is also able to induce ICAM-1 surface expression, proliferation of T cells, and growth and differentiation of CD34+ human progenitors into neutrophils when cocultured in presence of irradiated fibroblasts. In vitro, IL-17 synergizes with other proinflammatory signals like TNF alpha for GM-CSF induction, and with CD40-ligand for IL-6, IL-8, RANTES and MCP-1 secretion from kidney epithelial cells. In vivo, injection of IL-17 induces a neutrophilia, except in IL-6-KO mice. The involvement of IL-17 in rejection of kidney graft has also been demonstrated. The role of this T cell secreted factor in various inflammatory processes is presently investigated.     

Cellular mechanisms involved in protection against influenza virus infection in transgenic mice expressing a TCR receptor specific for class II hemagglutinin peptide in CD4+ and CD8+ T cells

Mice transgenic for a TCR that recognizes peptide110-120 of hemagglutinin of PR8 influenza virus in the context of MHC class II I-Ed molecules express the transgenes in both CD4+ and CD8+ T cells. We have found that these TCR-hemagglutinin (TCR-HA) transgenic mice display a significantly increased resistance to the primary infection with PR8 virus compared with the wild-type mice. The TCR-HA transgenic mice mounted significant MHC type II and enhanced MHC type I-restricted cytotoxicity as well as increased cytokine responses in both spleen and lungs after infection with PR8 virus. In contrast, the primary humoral response against PR8 virus was not significantly different from that of the wild-type mice. In vivo depletion and adoptive cell transfer experiments demonstrated that both CD4+ and CD8+ TCR-HA+ T cell subsets were required for the complete clearance of pulmonary virus following infection with a dose that is 100% lethal in wild-type mice. Whereas CD4+ TCR-HA+ T cells were necessary for effective activation and local recruitment of CD8+ T cells, CD8+ TCR-HA+ T cells showed a Th1-biased pattern and MHC type II-restricted cytotoxicity. However, in the absence of in vivo expression of MHC type I molecules on the infected cells, the protection conferred by the TCR-HA+ T cells was impaired, indicating that the enhanced MHC class I-restricted cytotoxicity due to TCR-HA+ CD4+ Th cells was a critical element for clearance of the pulmonary virus by the transgenic mice.     

Signaling through a CD3 gamma-deficient TCR/CD3 complex in immortalized mature CD4+ and CD8+ T lymphocytes

The biologic role of each CD3 chain and their relative contribution to the signals transduced through the TCR/CD3 complex and to downstream activation events are still controversial: they may be specialized or redundant. We have immortalized peripheral blood CD4+ and CD8+ T lymphocytes from a human selective CD3 gamma deficiency using Herpesvirus saimiri. The accessibility of the mutant TCR/CD3 complex to different Abs was consistently lower in immortalized CD8+ cells when compared with CD4+ cells, relative to their corresponding CD3 gamma-sufficient controls. Several TCR/CD3-induced downstream activation events, immediate (calcium flux), early (cytotoxicity and induction of surface CD69 or CD40L activation markers or intracellular TNF-alpha) and late (proliferation and secretion of TNF-alpha), were normal in gamma-deficient cells, despite the fact that their TCR/CD3 complexes were significantly less accessible than those of controls. In contrast, the accumulation of intracellular IL-2 or its secretion after CD3 triggering was severely impaired in gamma-deficient cells. The defect was upstream of protein kinase C activation because addition of transmembrane stimuli (PMA plus calcium ionophore) completely restored IL-2 secretion in gamma-deficient cells. These results suggest that the propagation of signals initiated at the TCR itself can result in a modified downstream signaling cascade with distinct functional consequences when gamma is absent. They also provide evidence for the specific participation of the CD3 gamma chain in the induction of certain cytokine genes in both CD4+ and CD8+ human mature T cells. These immortalized mutant cells may prove to be useful in isolating cytosolic signaling pathways emanating from the TCR/CD3 complex.     

Alterations in CD4-binding regions of the MHC class II molecule I-Ek do not impede CD4+ T cell development

The T cell coreceptors CD4 and CD8 enhance T cell responses to TCR signals by participating in complexes containing TCR, coreceptor, and MHC molecules. These ternary complexes are also hypothesized to play a seminal role during T cell development, although the precise timing, frequency, and consequences of TCR-coreceptor-MHC interactions during positive selection and lineage commitment remain unclear. To address these issues, we designed transgenic mice expressing mutant I-Ek molecules with reduced CD4-binding capability. These transgenic lines were crossed to three different lines of I-Ek-specific TCR transgenic mice, and the efficiency of production of CD4+ lineage cells in the doubly transgenic progeny was assessed. Surprisingly, replacing wild-type I-Ek molecules with these mutant molecules did not affect the production of CD4+CD8- thymocytes or CD4+ peripheral T cells expressing any of the three TCRs examined. These data, when considered together with other experiments addressing the role of coreceptor during development, suggest that not all MHC class II-specific thymocytes require optimal and simultaneous TCR-CD4-MHC interactions to mature. Alternatively, it is possible that these particular alterations of I-Ek do not disrupt the CD4-MHC interaction adequately, potentially indicating functional differences between I-A and I-E MHC class II molecules.     

Regulated expression of gp130 and IL-6 receptor alpha chain in T cell maturation and activation

The functional receptor for the inflammatory cytokine IL-6 is composed of the ligand binding IL-6 receptor alpha chain (IL-6R alpha) and the signal transducing chain gp130, which is a shared component of multiple cytokine receptors. We analyzed the surface expression of gp130 and IL-6R alpha in thymocytes and peripheral T cells. While all thymocytes expressed gp130 throughout thymic maturation, they gained expression of IL-6R alpha at the CD4 or CD8 single-positive stage. Approximately 10-30% of the CD4-CD8+ and 40-50% of the CD4+CD8- thymocytes expressed IL-6R alpha. Within the CD4+CD8- population, the IL-6R alpha- subpopulation was cortisone sensitive, appeared immature according to the cell surface markers expressed and failed to proliferate after TCR cross-linking. Peripheral T cells were predominantly gp130+ and IL-6R alpha+, but down-regulated gp130 and IL-6R alpha expression upon TCR engagement in vitro and in vivo. Peripheral gp130low/-IL-6R alphalow/- T cells expressed surface markers characteristic of memory T cells. We show that gp130 and IL-6R alpha are expressed in a regulated manner in T cells, depending on the developmental and functional stage.     

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.     

Maturation of CD4+ lymphocytes in the aged microenvironment results in a memory-enriched population

With advancing age the CD4+ T lymphocyte compartment becomes enriched for memory cells in both humans and experimental animals. Although it has been assumed that the shift from a naive to a memory-dominant population is due to a lifetime of antigenic exposure and selection as well as a loss of naive cell input due to reduced thymopoiesis, the present data suggest that the aged microenvironment influences the maturation of newly produced CD4+ T cells. In two models, aged and young mice were compared for the ability to reconstitute their peripheral CD4+ T cell pools following depletion, and both age groups were found to be competent to renew this population. However, the phenotype and lymphokine profile of populations arising in aged animals were distinctly different from those in the young mice. In contrast to the expectation that depletion and reconstitution might give rise to a naive-dominant T cell pool, aged mice reconstituted a population nearly indistinguishable from that found in control age-matched individuals. The majority of the CD4+ pool were CD44(high) CD45RB(low) Mel-14(low) and upon activation with anti-CD3 these CD4+ T cells produced mRNA for IL-2, IL-4, IL-5, and IFN-gamma. In aged bone marrow-transplanted mice, the same phenotypic profile and cytokine mRNA pattern were found in CD4+ T cells of host and donor origin. In contrast, the majority of CD4+ T cells in young reconstituted mice were CD44(low) CD45RB(high) Mel-14(high). These lymphocytes, when activated, produced high levels of mRNA for IL-2, with little or no IL-4, IL-5, or IFN-gamma mRNA.