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Immunopathogenesis of dengue virus (DEN) infection remains poorly studied. Identification and characterization of human CD8(+) T-cell epitopes on DEN are necessary for a better understanding of the immunopathogenesis of dengue infection and would facilitate the development of immunotherapy and vaccines to protect from dengue infection. Here, we identified two new HLA-A*0201-restricted CD8(+) T-cell epitopes, DEN-4 NS1(990)(-998) and DEN-4 NS1(997)(-1005) that are conserved in three or four major DEN serotypes, respectively. Unexpectedly, we found that immunization of HLA-A*0201 transgenic mice with DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005) epitope peptide induced de novo synthesis of tumor necrosis factor (TNF)-α and IFN-γ, two important pro-inflammatory molecules that are hard to be detected directly without in vitro antigenic re-stimulation. Importantly, we demonstrated that CD8(+) T cells specifically activated by DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005) epitope peptide induced de novo synthesis of perforin. Furthermore, we observed that DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005)-specific CD8(+) T cells capable of producing large amounts of perforin, TNF-α and IFN-γ preferentially displayed CD27(+)CD45RA(-), but not CD27(-)CD45RA(+), phenotypes. This study, therefore, suggested the importance of synergistic effects of pro-inflammatory cytokines and cytotoxic molecules which were produced by dengue-specific CD8(+) T cells in immunopathogenesis or anti-dengue immunity during dengue infection.

Intraepithelial lymphocytes (IELs) represent the first line of lymphocyte defense against the intestinal bacteria. Although previous studies have demonstrated a protective role of IELs in the development of colitis, the data supporting a regulatory role for IELs are limited. The objective of this study was to examine the suppressive activity of IELs in vitro and in vivo using a mouse model of chronic small and large bowel inflammation. Adoptive transfer of CD8α(+) IELs isolated from small intestines of wild-type (WT) mice into TCR βxδ-deficient (TCR βxδ(-/-)) recipients did not prevent or delay the onset of the disease induced by WT CD4(+)CD45RB(high) T cells. On the contrary, we observed a more rapid onset of wasting and clinical signs of intestinal inflammation when compared with animals injected with CD4(+)CD45RB(high) T cells alone. Histopathological scores of small and large bowel did not differ significantly between the two groups. Transfer of IELs alone did not produce any pathological changes. Real-time PCR analysis of intestinal tissues showed up-regulation of message for T(h)1- and macrophage-derived cytokines in colon and small bowel. Using Foxp3-GFP reporter mice, we were unable to detect any Foxp3(+) cells within the CD8α(+) IELs but did find a small population of Foxp3(+)CD4(+) IELs in the small and large bowel. Using in vitro suppression assay, we found that neither TCRαβ(+)CD8αα(+), TCRαβ(+)CD8αβ(+) nor TCRγδ(+)CD8αα(+) IELs were capable of suppressing CD4(+) T-cell proliferation. Taken together, our data do not support an immunoregulatory role for CD8α(+) IELs in a mouse model of small and large bowel inflammation.

Collaborative interactions between T(h) cells and B cells are necessary for the production of antibody responses to most protein antigens and for the generation of memory B cells in germinal centers (GCs). Although it is well established that T(h) cells are pivotal for the GC reaction, the mechanisms that control the homeostasis of T(h) cells during the GC response remain largely unknown. Here we show that, unlike other effector T cells, a significant number of CD4(+)CD45RO(+)CD57(+) T cells, which are the major T(h) cells residing in the GCs, are undergoing apoptosis in vivo. CD4(+)CD45RO(+)CD57(+) GC T cells exhibit similar sensitivities to apoptotic signals and to caspase inhibitors as immature thymocytes. Moreover, CD4(+)CD45RO(+)CD57(+) GC T cells express a unique profile of genes that control apoptosis and cell cycle, providing possible molecular mechanisms for the high rates of apoptotic death of these T(h) cells in the GCs.

Naive and primed/memory CD4 T cells are distinguished by changes in the expression of activation/adhesion molecules that correspond with an altered function. Adoptively transferred TCR transgenic (tg) CD4 T cells specific for ovalbumin peptide (OVA-pep) were analysed for changing phenotype and the speed of change in vivo following antigen challenge with alum-precipitated (ap) OVA-pep, a conjugate that stimulated a Th2-type cytokine response. The change of CD45RB in relation to number of divisions showed that the transition from CD45RB(hi) (naive) to CD45RB(low) (primed/memory) was incremental; with each cell cycle the number of CD45RB(hi) molecules on the cell surface was diluted by approximately half and replaced by the low-weight isoform. Similarly, the change to CD44(hi) expression increased gradually during four rounds of proliferation. The loss of CD62L expression occurred early and was independent of cell division. CD69 was up-regulated quickly within 1-2 cycles, but down-regulated after about seven divisions. The expression of CD49d was not altered during the early rounds of division, although it was up-regulated on 30-60% of tg T cells dividing repeatedly (or=8 cycles). When analysed on day 3 following stimulation, CD25 was no longer up-regulated. The intra-peritoneal injection of ap-OVA-pep stimulated tg T cells in the spleen and mesenteric lymph node one day in advance of those in more distant peripheral lymph nodes. Evidence indicated that residual antigen persisted for at least 4 weeks and was able to stimulate naive tg T cells. However, residual antigen had no net effect on extending or reducing survival of the transferred population.

CD45 is a receptor tyrosine phosphatase essential for TCR signaling. One isoform, CD45RB, is down-regulated in memory cells and targeting CD45RB with a specific antibody has been shown to inhibit graft rejection. Its role in immunity to infection, however, has not been tested. Here, we report the effect of anti-CD45RB antibody treatment on the induction of anti-influenza CD8+ T cells and viral clearance. Anti-CD45RB-treated mice had delayed pulmonary viral clearance compared with untreated mice whose infection was completely cleared by day 8 post-infection. In anti-CD45RB-treated mice, the total CD4+ and CD8+ T cell numbers in both the lungs and mediastinal nodes were substantially reduced at days 5 and 8; this effect was less marked for the spleen. CD8+ T cells specific for influenza virus were also reduced compared with the control group in all three organs at day 8. By day 11, when both treated and control groups showed no virus remaining in the lungs, specific CD8+ T cell numbers were at similar low levels. Homing to lymph nodes and lung of dye-labeled T cells was greatly inhibited (by80%) by anti-CD45RB treatment. This reduced homing corresponded with reduced CD62L and beta1-integrin expression in both uninfected and infected mice. Since CD62L plays a critical role in homing lymphocytes to lymph nodes, and high levels of CD62L and alpha4beta1-integrin are expressed by lymphocytes that home to bronchus-associated lymphoid tissue, we suggest that reduced expression of these molecules is a key explanation for the delay in immune responses.

The microphthalmic (mi) mouse exhibits deficiencies in the development of osteoclasts, melanocytes, mast cells and marrow B cells. Previously, we demonstrated that the marrow of such mice over-express receptor activator of nuclear factor kappaB (RANK) ligand (RANKL). RANKL has been shown to induce the production of IFN-beta, a type I IFN. Additionally, maturing B cells have been shown to undergo apoptosis in response to type I IFNs including IFN-beta during differentiation. We hypothesized that the loss of B cells in the marrow of mi mice was due to the over-expression of IFN-beta as a result of heightened RANK-RANKL signaling. Creating a mouse with the mi genotype that was non-responsive to IFN-beta (lacking the type I IFNR) allowed us to test this hypothesis. These mice demonstrated an elevated number of marrow B cells and marrow precursor cells compared with mi animals possessing the type I IFNR. Intriguingly, type I IFNR-deficient wild-type animals also demonstrated an increased number of precursor cells in the marrow, but not an expansion of B220-positive pre-B cells, compared with wild type, suggesting that modulation of type I IFN responses directly controls the development of marrow constituents.

Synovial tissues are frequent sites of inflammatory disorders in which dendritic cells (DCs) may play an important role. This study examines potential antigen-presenting cells obtained from synovium-rich tissues (SRTs) by vascular perfusion of rat hind limbs with collagenase and further enzymatic digestion of the disarticulated hind paws in vitro. The three sub-populations of interest were: CD45+MHC IIhi, mainly CD11c+ and CD163-; CD45+MHC IIlo, mainly CD11c- and CD163+ and CD45+MHC II-, mainly CD11c- and CD163+. Expression of CD11c and CD163 correlated with ruffled cell-surface (CD11c+CD163-) and highly vacuolated cytoplasm (CD11c-CD163+), respectively. Culture of the CD45+CD163- sub-population in granulocyte macrophage colony-stimulating factor (GM-CSF) yielded CD45+MHC IIhi CD11c+CD163- cells with veiled morphology, while the large vacuolated cells that expressed CD163 resembled type A synoviocytes in both surface antigen phenotype and morphology. These results demonstrate that SRTs contain indeterminate cells that can differentiate into mature DCs in vitro in response to GM-CSF, plus mature synovial lining macrophages.

The protein tyrosine phosphatase CD45 has been shown to be an important regulator of antigen-receptor signaling in both T and B lymphocytes. Lymphocyte populations within the gut mucosa are phenotypically and ontogenetically different from those generally found in secondary lymphoid tissue. In T lymphopenic mice, extrathymic T cell development takes place within the gut. Here we report the characterization of T and B cell populations in the distinct compartments of the gut mucosa and gut-associated lymphoid tissue of CD45-null mice. These data suggest that CD45 is required for the development of the specialized T cell populations within the gut environment as has been previously shown for thymic development. We demonstrate that within the large intestine intraepithelial compartment alphabeta-TCR+ CD4+ T cells are selectively retained by CD45KO mice. T cells and NK cells within the intraepithelium and the gut mucosa associated lymphoid tissue of CD45KO mice frequently possess an activated phenotype, differentiated to produce typical TH1 and TH2 cytokines. These data demonstrate that local environmental differences within the gut can, at least in part, overcome the requirement for CD45 during activation of T cells.

Histamine plays an important role in the regulation of various immunological functions. To evaluate the role of histamine in contact hypersensitivity, contact dermatitis was induced with dinitrofluorobenzene (DNFB) in histidine decarboxylase knockout (HDC-/-) histamine-deficient and wild-type mice. The DNFB-induced increase of the ear thickness was significantly higher in HDC-/- mice than in wild-type mice. Using flow cytometry, significantly lower percentages of CD4+ Th and CD8+ Tc cells, and significantly higher percentages of CD45R+ B cells were observed in the regional lymph nodes in HDC-/- mice than in wild-type mice. In the ear specimens of both groups, the majority of the infiltrating cells were neutrophils and macrophages at 24 and 48 h after challenge. Using immunohistochemistry, we observed significantly more CD45+ leukocytes in HDC-/- mice than in wild-type mice. The expression of Th1 (IL-2, IFN-gamma, TNF-alpha) and Th2 (IL-4) mRNAs was examined by quantitative real time RT-PCR in the ear samples. The levels of Th1 cytokine mRNAs both at 24 and 48 h after challenge and IL-4 mRNA at 48 h showed a significantly higher increase in HDC-/- mice than in wild-type mice. These results suggest that histamine plays a negative immunoregulatory role in DNFB-induced contact hypersensitivity.

Transgenic mice have been constructed expressing high (CD45RABC) and low (CD45R0) molecular weight CD45 isoforms on a CD45 � /� background. Phenotypic analysis and in vivo challenge of these mice with influenza and lymphocytic choriomeningitis viruses shows that T cell differentiation and peripheral T cell function are related to the level of CD45 expression but not to which CD45 isoform is expressed. In contrast, B cell differentiation is not restored, irrespective of the level of expression of a single isoform. All CD45 trangenic mice have T cells with an activated phenotype and increased T cell turnover. These effects are more prominent in CD8 than CD4 cells. The transgenic mice share several properties with humans expressing variant CD45 alleles and provide a model to understand immune function in variant individuals.

The ability of CTLA-4 to inhibit T cell activation may be either negatively or positively regulated by a critical tyrosine at position 201 (Y201) within the CTLA-4 cytoplasmic domain. By binding to the clathrin-associated adaptor complex AP-2 and inducing endocytosis, Y201 reduces the amount of CTLA-4 on the cell surface, thereby down-regulating CTLA-4 inhibitory function. Alternatively, Y201 may function to transmit CTLA-4 inhibitory signals, perhaps through binding to intracellular proteins that oppose TCR- and/or CD28-induced signal transduction. Results from studies performed in vitro have cast doubt on whether this second mechanism contributes significantly to CTLA-4 function. In order to determine if a role existed for Y201 in mediating CTLA-4 inhibitory signaling in vivo, we studied lymphocyte activation and homeostasis in CTLA-4(-/-) mice that were reconstituted with a transgenic CTLA-4 receptor in which Y201 was mutated to valine (Y201V/CTLA-4(-/-)). We found that despite augmented levels of CTLA-4 on the cell surface of T cells, Y201V/CTLA-4(-/-) mice developed a lymphoproliferative syndrome characterized by lymphadenopathy and the accumulation of T cells that secreted IL-4. Mutant T cells exhibited increased cell division when treated with suboptimal doses of mitogenic stimuli in vitro. These results demonstrate that in addition to down-modulating CTLA-4 expression on the cell surface of T cells, the Y201 residue also functions to transmit CTLA-4 inhibitory signals in vivo. Elucidating the biochemical pathways downstream of Y201 will be important for a full understanding of the molecular basis for CTLA-4 function.

Several autoimmune disease models depend on an imbalance in the activation of aggressor T(h)1 and CD4(+)CD25(+) regulatory T (T(reg)) cells. Here we compare the requirement for signals through the co-stimulatory molecules CD28 and inducible co-stimulator (ICOS) in chronic murine colitis, a model for inflammatory bowel disease. We used a colitis model in which disease-causing CD45RB(hi) T cells alone or in combination with CD4(+)CD25(+) T cells from either CD28-deficient or wild-type donors were transferred into T cell-deficient animals, half of which were treated with ICOS-blocking reagents. Blocking ICOS on the surface of CD28-deficient T(h)1 cells abrogated development of colitis, whereas blocking CD28 or ICOS alone had little to no effect on disease induction. In contrast to T(h)1 cells, regulatory T cell functioning depended mostly on CD28 signaling with only a minor contribution for ICOS. We conclude that CD28 and ICOS collaborate to development of murine colitis by aggressor T(h)1 cells, and that CD28 is required for T(reg) cells, which should caution against the use of CD28-blocking reagents in inflammatory bowel disease.

Neonatal CD4(+) T cells express less CD154 protein and mRNA than adult CD4(+) T cells after activation by calcium ionophore and phorbol ester, but the mechanism for this reduced expression and its relevance to the primary immune response remain unclear. We compared expression of CD154 protein and mRNA and CD154 gene promoter activity by purified naive (CD45RA(high)CD45RO(low)) neonatal and adult CD4(+) T cells after activation by calcium ionophore (ionomycin) and phorbol myristate acetate (PMA) treatment or by engagement of alphabeta TCR-CD3 complex. Substantial and consistent reductions in expression by neonatal cells were found in all cases and were paralleled by decreased CD154-dependent activation of a B cell line. CD69 expression by neonatal CD4(+) T cells after alphabeta TCR-CD3 engagement was also reduced compared to adult cells, which suggested that limitations in activation-induced signaling by neonatal CD4(+) T cells occurred at a point upstream of where the signaling pathways leading to CD154 and CD69 expression diverge. Decreased CD154 expression by neonatal cells after alphabeta TCR-CD3 engagement was paralleled by a lower free intracellular calcium concentration, a key event for CD154 gene transcription. Reduced CD154 promoter activity by neonatal cells persisted when proximal signaling events were bypassed using ionomycin and PMA, suggesting an additional and more distal mechanism for decreased transcription. In contrast, CD154 mRNA stability was similar in neonatal and adult cells after either ionomycin and PMA stimulation or engagement of the alphabeta TCR-CD3 complex. We conclude that decreased CD154 production by neonatal CD4(+) T cells is due to limitations in both proximal and distal activation events, which together ultimately limit CD154 gene transcription.

Radiation-attenuated (RA) schistosomes penetrate the host via the skin where they stimulate intense inflammatory reactions and the release of pro-inflammatory IL-12, important for T(h)1-type immune responses which are partially host protective. However, RA larvae also induce the secretion of regulatory IL-10. We now show that following vaccination of IL-12p40(-/-) mice, abundant IL-10 was produced by in vitro cultured skin biopsies between days 4 and 14, corresponding to the down-regulation of MHC II expression by cells in the dermis and cells that emigrate from the skin. In IL-10(-/-) mice, inflammation of the vaccination site was increased with larger numbers of IL-12p40(+), MHC II(+) and CD86(+) cells in the dermal exudate, and was associated with elevated levels of skin-derived IL-12p40 and IL-1beta. These changes in IL-10(-/-) mice were also reflected by an increased number of cells in the skin-draining lymph nodes (sdLN) and greater levels of lymphocyte proliferation. Moreover, such mice had increased numbers of CD4(+) sdLN cells that were CD25(+), CD28(+) or CD152(+) and accessory cells that were CD40(+) or MHC II(+). Finally, the secretion of IFN-gamma (and IL-12p40) by in vitro cultured sdLN cells was substantially raised in IL-10(-/-) mice, but much reduced in IL-12p40(-/-) mice, resulting in the development of highly polarized T(h)1 and T(h)2 cytokine profiles in the two groups of mice respectively. We conclude that IL-10 has an important role early in the regulation of IL-12-mediated priming of acquired immune responses, and effectively contains excessive dermal inflammation and prevents the development of highly polarized T(h)1-type responses.

B cell autoreactivity is a component of chronic graft versus host (GVH) disease in humans and mice. Chronic GVH driven by I-A disparity results in loss of B cell tolerance in Ig/sHEL tolerant mice. In these mice, B cell anergy is characterized by down-modulation of sIgM mediated by intracellular retention in the endoplasmic reticulum (ER) and/or a block in post-ER processing of IgM receptors. Here, we report that GVH induces a selective increase in post-ER processing of the micro chain and trafficking to the cell surface of IgM receptors in B cells that bind HEL self-antigen. The increase in sIgM was detectable as early as 6 days post-GVH, before the appearance of circulating autoantibodies, and was particularly prominent in B cells that up-regulated surface I-A. A further increase in sIgM was found at later time points, along with circulating anti-HEL autoantibodies and a marked decrease in serum-free HEL, but no significant change in the amounts of HEL bound to B cells in vivo. These findings suggest that (i) abrogation of ER retention of IgM receptors in self-reactive B cells is an early event triggered by allogeneic T cells and (ii) at later stages of GVH disease the appearance of autoantibodies reduces the availability of free autoantigen, which may further escalate anergy escape of self-reactive B cells, and lead to exacerbation and perpetuation of autoimmunity.

Platelet factor 4 (PF4) is a CXC chemokine secreted by activated platelets. PF4 has been shown to promote monocyte survival and induce the differentiation of monocytes into macrophages. However, the effect of PF4 on differentiation of monocytes into dendritic cells (DC) has yet to be determined. As reported previously, monocytes cultured in RPMI medium containing FCS, granulocyte macrophage colony stimulating factor and IL-4 differentiated into CD1a+ DC. When PF4 was added, the expression of CD1a on DC was inhibited. This inhibitory effect was not observed with the other platelet-derived CXC chemokine, beta-thromboglobulin. The relative number of CD1a- DC increased from 17 to 92% when the PF4 concentration was increased from 0 to 10 micro g/ml. The inhibitory effect of PF4 on CD1a expression was reversed by 50 U/ml heparin. DC developed in the PF4-containing media appeared more adhesive to plastic culture wells and had higher light side scatter by flow cytometry. Immunophenotypically, monocyte-derived DC in the presence of increasing concentrations of PF4 proportionally expressed higher CD86 and lower HLA-DR. The levels of CD11c, CD40 and CD80 remained unchanged with or without PF4. Both CD1a+ DC and CD1a- DC were negative for CD14, CD68 and CD83. Functionally, DC developed in the presence of PF4 had their secretion of tumor necrosis factor-alpha and IL-12 reduced by 75 +/- 10 and 79 +/- 13% respectively when they were stimulated by 100 ng/ml lipopolysaccharide and 50 ng/ml IFN-gamma. CD1a- DC developed in the presence of PF4 were not as active as the control CD1a+ DC in stimulating allogeneic T cells to proliferate. In addition, CD1a- DC were less potent in priming naive CD4+ T cells to secrete both type 1 and 2 cytokines. These results indicate that PF4 can influence differentiation and function of monocyte-derived DC.

Pancreatic islet xenotransplantation has been advocated as a way of overcoming the shortage of human donor tissue for the treatment of type 1 diabetes. However, the potent immune response against xenografts is a major barrier to their use. We show that a short course of the anti-CD45RB antibody, MB23G2, prolongs survival of fetal pig pancreas grafts in mice. To investigate this effect further we used an i.p. xenograft model in which both donor pig cells and host inflammatory cells can be expediently recovered and analyzed. Graft prolongation was associated with reduced T cell and macrophage infiltration, and reduced production of both T(h)1 and T(h)2 cytokines at the graft site. Graft survival was further increased and T cell infiltration further reduced by combining anti-CD45RB antibody with co-stimulation blockade. The primary effect of anti-CD45RB antibody may be on CD4 T cells, in keeping with the marked reduction in T cell cytokine production in both spleen and graft sites. This concurs with previous studies in allogeneic models that indicate that this antibody perturbs T cell responses by modifying signaling via the TCR. In addition, anti-CD45RB treatment led to reduced expression of LFA-1 and CD62 ligand (CD62L) on CD4 T cells, independent of antigenic challenge. LFA-1 may enhance co-stimulation, and both LFA-1 and CD62L are involved in T cell trafficking. Their reduced expression provides an explanation why the T cell pool is reduced in lymph nodes. We conclude that modulation of inflammation against xenografts by anti-CD45RB antibody is due to effects on both T cell priming and trafficking.

The ontogeny of thymic B cells was determined by three-color flow cytometry and the presence or absence of B cell progenitors confirmed by cell culture experiments. In the thymus of young normal mice, CD117(+), B220(low) pro- and pre-B cells are present but disappear with age. B220(low), CD5(+), B-1 B cells are present in the thymus of older animals following the appearance of similar cells in the peritoneal cavity and blood. In CD3 epsilon gene-deleted mice, the phenotypic progression and number of thymic B cells remains unaltered, showing that blocking T cell development does not automatically result in an increase of thymic B lymphopoiesis. Pro-B cells in RAG-2 knockout mice are found in the fetal and neonatal blood, spleen and thymus, but with increasing age are only found in the bone marrow. B lymphopoiesis in adult IL-7 transgenic mice is dramatically altered with CD117(+) pro- and pre-B cells present in spleen, lymph node and blood. In the thymus of adult IL-7 transgenic mice, the fraction of CD117(+) thymic B cells is significantly increased. These results show that in the steady state, the phenotype of thymic B cells is critically dependent on both mouse age and the phenotype of circulating B cells.

In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.

Defensins released by neutrophils are able to kill a broad spectrum of microbes. They also induce leukocyte migration in vitro and elicit inflammatory leukocyte responses at s.c. injection sites in mice. In vitro experiments showed that human defensins enhanced concanavalin A-stimulated murine spleen cell proliferation and IFN-gamma production. This led us to examine the effects of human defensins on specific immune responses in vivo. BALB/c mice were immunized with 50 microg of keyhole limpet hemocyanin (KLH) adsorbed to aluminum hydroxide and administered with defensins in aqueous solution. Intraperitoneal administration of defensins significantly increased the production of KLH-specific IgG1, IgG2a and IgG2b antibodies 14 days after immunization. In vitro splenic KLH-specific proliferative responses were higher in mice treated with KLH and defensins than in those treated with KLH alone. Increased IFN-gamma and, to a lesser extent, IL-4 production were also detected in the supernatants of ex vivoKLH-activated spleen cells from mice treated with defensins. Finally, defensins significantly enhanced the antibody response to a syngeneic tumor antigen, lymphoma Ig idiotype and also augmented resistance to tumor challenge. These results indicate that defensins act as potent immune adjuvants by inducing the production of lymphokines, which promote T cell-dependent cellular immunity and antigen-specific Ig production. Thus, defensins appear to function as neutrophil-derived signals that promote adaptive immune responses.

Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo. The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52-TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases.

The transmembrane tyrosine phosphatase CD45 regulates the activity of src family protein tyrosine kinases (PTK) and thereby influences the signaling via such receptors as T and B cell antigen receptors associated with these PTK. However, its implication in signaling through the mast cell receptor with high affinity for IgE (FcepsilonRI) is less clear, although Lyn, a member of the src family, plays an important role in FcepsilonRI-mediated signaling. To define a role for CD45 in FcepsilonRI signal transduction, we established CD45 high expressing rat basophilic leukemia cell lines (RBL-CD45H) and cell lines expressing trace amounts of CD45 (RBL-CD45L). We demonstrate that although all RBL-CD45L cell lines degranulate following IgE- and antigen-induced FcepsilonRI aggregation, the response is significantly reduced at a low dose of antigen. The cells show a delayed and slowed Ca(2+) mobilization even though at a higher dose where the cells degranulate to a similar extent as RBL-CD45H. This diminished Ca(2+) response is restored by reconstitution of RBL-CD45L with a chimeric molecule containing the cytoplasmic phosphatase domains of rat CD45. Furthermore, tyrosine phosphorylation of FcepsilonRI, association of FcepsilonRI with Lyn and PTK activity associated with FcepsilonRI, all of which are enhanced upon FcepsilonRI aggregation in RBL-CD45H, are impaired in RBL-CD45L. Finally, we show that FcepsilonRI is physically associated with CD45 in RBL-CD45H prior to receptor aggregation. Thus, we propose that, although not indispensable in mast cell degranulation, CD45 positively regulates the signaling through FcepsilonRI by promoting the activation of FcepsilonRI-associated Lyn.

The aim of the present study was to better characterize the expansion of double-negative (DN) T cells in vivo in AIDS patients and to ascertain the discrepant response of an immunodepressed immune system towards two distinct mycobacterial infections. In a large cohort of HIV-1 seropositive patients with low CD4(+) T cell counts (

The selective migration of functional T(h) lymphocyte subsets with different cytokine production profiles into inflamed tissue is likely to depend on the state of activation of the cells, as well as on the differential expression of various adhesion molecules and chemokine receptors. In this study, we have analyzed the effect of allergen-specific activation on the expression of the chemokine receptor CXCR4 on T lymphocytes. We show that stimulation of peripheral blood mononuclear cells from atopic patients with the allergen Der p results in down-regulation of CXCR4 surface expression on Der p-activated CD25(+)CD45RO(+) antigen-specific memory cells which was caused by a decrease in CXCR4 gene transcription and did not seem to be mediated by endogenous cytokines, such as IFN-gamma. In contrast, however, CXCR4 surface expression was enhanced on naive CD25(-)CD45RO(-) and resting CD25(-)CD45RO(+) memory T cells, as a result of the presence of endogenous IL-4, most likely produced by Der p-activated memory T cells. Antigen-specific CD25(+)CD45RO(+) T lymphocytes, purified 7 days after stimulation with Der p, had a strongly reduced capacity to migrate in response to stimulation with stromal cell-derived factor (SDF)-1, the ligand for CXCR4. Together, these results suggest that differential expression of CXCR4 on activated and resting T cells is due to the counteracting effects of TCR-mediated down-regulation and IL-4-mediated up-regulation of this chemokine receptor respectively, and furthermore indicate that antigen-activated memory T cells are unlikely to migrate into inflamed tissue in response to SDF-1.

A pre-TCR-CD3 signal is required for the efficient maturation of CD4- CD8- thymocytes to the CD4+ CD8+ stage. This study addressed whether a similar signal is required for maturation of intestinal intraepithelial lymphocytes (IEL) that may develop extrathymically. We have shown previously that IEL from mice deficient for CD3- associated zeta chains include an immature population of CD3- CD8alphaalpha+ cells expressing cytoplasmic TCR beta chains but lacking detectable surface TCRalphabeta, CD16 and B220. Here we stimulated the appearance of such IEL in epsilon+/- zeta-/- mice by expression of an activated Lck transgene or in vivo treatment with anti-CD3epsilon. Anti-CD3epsilon treatment of RAG-deficient animals also yielded CD16- B220- IEL. In contrast, expression of a TCRbeta transgene in rag-1(-/-) mice did not stimulate the appearance of CD3- CD8alphaalpha+ CD16- B220- cells. Taken together these data indicate that although anti-CD3epsilon treatment and LckF505 assist in catalyzing a CD16+ B220+ --CD16- B220- transition, these manipulations are not equivalent to a pre-TCR signal in IEL lymphocytes.

Helper T cells are classified into Th1 and Th2 subsets based on their profiles of cytokine production. Th1 cells are involved in cell-mediated immunity, whereas Th2 cells induce humoral responses. Selective recruitment of these two subsets depends on specific adhesion molecules and specific chemoattractants. Here, we demonstrate that the T cell-directed CC chemokine thymus and activation-regulated chemokine (TARC) was abundantly produced by monocytes treated with granulocyte macrophage colony stimulating factor (GM-CSF) or IL-3, especially in the presence of IL-4 and by dendritic cells derived from monocytes cultured with GM-CSF + IL-4. The receptor for TARC and another macrophage/dendritic cell-derived CC chemokine macrophage-derived chemokine (MDC) is CCR4, a G protein-coupled receptor. CCR4 was found to be expressed on approximately 20% of adult peripheral blood effector/memory CD4+ T cells. T cells attracted by TARC and MDC generated cell lines predominantly producing Th2-type cytokines, IL-4 and IL-5. Fractionated CCR4+ cells but not CCR4- cells also selectively gave rise to Th2-type cell lines. When naive CD4+ T cells from adult peripheral blood were polarized in vitro, Th2-type cells selectively expressed CCR4 and vigorously migrated toward TARC and MDC. Taken together, CCR4 is selectively expressed on Th2-type T cells and antigen-presenting cells may recruit Th2 cells expressing CCR4 by producing TARC and MDC in Th2-dominant conditions.

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.

NK cells not only function as cytotoxic effector cells, but also have immunoregulatory roles including the enhancement of Ig secretion. To have a stable and uniform population of NK cells to study their role in Ig secretion, we generated murine NK clones. Thus, culture of splenocytes from mice that were homozygous for a mutation in the p53 tumor suppressor gene (p53-KO) with IL-2 and poly(IC) resulted in a long-term NK line, from which four stable clones were derived. This approach also yielded a long-term NK line from splenocytes of normal C57BL/6 mice. Identification of the clones as members of the NK lineage was based on large granular morphology, expression of NK-TR and absence of TCR gene rearrangement. Flow cytometry revealed that all clones expressed IL-2R alpha and beta, chains and B220, but no CD3, NK1.1, DX5 or Ly-49. RT-PCR analysis showed heterogeneity in NK1.1 gene expression, and demonstrated expression of perforin and several granzymes in all clones. Three out of four clones lysed YAC-1, but not P815 target cells, corresponding to a pattern of NK specificity. All NK clones enhanced Ig secretion in an in vitro model for T cell-independent type 2 antigens, albeit to varying degrees. We found no correlation between the degree of helper activity of the NK clones and the level of their cytotoxic activity on YAC-1 targets. Thus, we established murine NK clones, and show that they mediate both cytotoxicity and enhancement of Ig secretion.

In human and mouse lymphocytes the protein tyrosine phosphatase CD45, a key molecule involved in T cell activation, non-covalently associates with the tyrosine kinase p56(lck) and lymphocyte phosphatase-associated phosphoprotein (LPAP), a 32 kDa phosphoprotein of unknown function. In order to gain insight into the function of LPAP we have generated an LPAP-deficient Jurkat variant by means of antisense strategies. Analysis of the CD45-p56(lck) molecular complex in this cell line revealed that loss of LPAP does not alter the expression or the enzymatic activity of CD45 or p56(lck). In addition, the association between CD45 and p56(lck) is not affected in LPAP-deficient T cells. These data suggest that LPAP does not regulate the enzymatic activity of CD45 or p56(lck) and is not required for the association between these two proteins. In order to identify polypeptides that preferentially associate with LPAP we established a Jurkat variant expressing a chimeric receptor which was composed of the extracellular portion of the human HLA-A2.1 molecule and the full-length LPAP protein. Comparative two-dimensional analysis of CD45 and HLA-A2 immunoprecipitates obtained from these cells following metabolic labeling resulted in the identification of a 43 kDa protein that preferentially associates with LPAP under mild detergent conditions.

HLA molecules influence the selection of naive CD4+ T cells as demonstrated by HLA-DR-dependent differences in BV8-BJ frequencies. The repertoire of mature peripheral T cells utilized in antigen responses is shaped by additional factors such as antigens encountered in the environment. To identify mechanisms underlying the formation of the memory repertoire, differences in the BV8-BJ repertoire of CD45RO- and CD45RO+ CD4+ T cells were examined in 21 normal donors. The naive and memory CD4+ compartments displayed unique BV8-BJ repertoires in all individuals, demonstrating that the recruitment of CD4+ T cells into the memory population is a non-random process. The frequencies of selected BV8-BJ combinations were increased among CD45RO+ T cells. Size fractionation of such expanded BV8-BJ populations demonstrated that most of them were polyclonal in nature. Twenty-five percent of the expanded BV-BJ combinations included a dominant TCR sequence, indicating monoclonal proliferation. Selection of BV8-BJ combinations for preferential use among memory T cells was HLA dependent. HLA-DR1/4+ individuals were characterized by an increased usage of BV8-BJ2S7+ TCR, and decreased usage of BV8-BJ2S1 + and BV8-BJ2S2+ TCR, whereas HLA-DR3/7+ individuals preferentially recruited BV8-BJ2S5+ T cells, and disfavored BV8-BJ2S3+ and BV8-BJ2S7+ T cells. HLA-imposed effects on the naive and memory repertoire were distinct. The BV-BJ frequencies of CD45RO+ T cells could not be predicted from the pattern of TCR found in naive CD4+ T cells, suggesting that the HLA-DR polymorphisms influence thymic selection processes differently than peripheral selection forces.

CD4+ CD8+ double-positive thymocytes differentiate into CD4+ and CD8+ single-positive T cells during thymic positive selection. This process requires the interaction between the TCR and self MHC molecules. In this context we have analyzed the expression of CD45, an abundant transmembrane protein tyrosine phosphatase, and describe here its differential surface expression during T cell maturation. Using four-color FACS analysis of thymocytes from normal as well as TCR-transgenic mice we demonstrate that CD45 is up-regulated only during positive selection concomitantly with the TCR-CD3 complex and the transient early activation marker CD69, but that this up-regulation precedes heat stable antigen down-regulation. The tight linkage of the up-regulation of the TCR-CD3 complex and CD45 may be required because the CD45 tyrosine phosphatase plays a role in modulating signal transduction by the TCR-CD3 complex during positive selection. In addition, our findings argue for a regulation mechanism that adapts the CD45 levels to increasing antigen receptor levels on mature T cells and B cells.

The leukocyte common antigen isoforms CD45RA and CD45RO have long been used to discriminate human naive and memory T cells respectively. This model was largely based on the observation that CD45RO+ T cells respond preferentially to and show a higher frequency of precursors specific for recall antigens. However, CD45RA+ T cells have more stringent requirements for stimulation and standard in vitro assays may favour CD45RO+ cells in this respect. We tested the hypothesis that CD45RA+ T cells respond poorly to in vitro stimulation with recall antigens because of inadequate stimulation rather than a lack of precursors. Limiting dilution analyses (LDA) for tetanus toxoid (TT)-specific T cells were performed in the presence or absence of exogenous anti-CD28 antibody. Addition of anti-CD28 yielded no proliferation in the absence of specific antigen. The precursor frequency for TT in the CD4+ CD45RO+ population was approximately 1:4000, while the frequency of CD4+ CD45RA+ T cells specific for TT was 4- to20-fold lower. Addition of anti-CD28 antibody did not significantly alter the apparent precursor frequency for CD45RO+ cells but yielded an enhancement of the value for CD45RA+ cells by 3- to5-fold. No enhancement of antigen-specific proliferation by anti-CD28 was observed with CD45RA+ T cells derived from cord blood, although phytohemagglutinin responses of these cells were amplified by CD28 antibody. These results indicate that conventional LDA underestimate the true precursor frequency of antigen-specific cells within the adult CD45RA+ population and support the possibility that a small number of cells revert from a primed (CD45RO+) to an unprimed (CD45RA+) state. The majority of memory T cells, however, appear to reside in the CD45RO+ population.

CD45 is a transmembrane protein tyrosine phosphatase expressed by all lymphoid cells including T cells. Substantial experimental data has shown that CD45 maintains a permissive state for TCR signaling. The highly glycosylated extracellular domain of CD45 may be the site of interaction with regulatory lectin-like counter-receptors on antigen-presenting cells. The mAb NDA5, recognizing a unique but broadly distributed epitope of CD45, was used to study the possible immunoregulatory role of CD45 during anti-CD3 and antigen-specific CD4+ T cell activation. In vitro priming of peripheral blood mononuclear cells with peptide antigens in the presence of mAb NDA5 results in a higher frequency of antigen-specific T cells. The responses of both naive and memory T cells to peptide antigens were sensitive to mAb NDA5-enhanced priming. Anti-CD3 activation of normal resting T cells, in the presence of mAb NDA5, resulted in enhancement of tyrosine phosphorylation of specific intracellular proteins associated with TCR signal transduction. In cultures without antigen, mAb NDA5 down-regulated the cell surface expression of both CD3 and CD4, yet did not stimulate proliferation of resting T cells. Together these results suggest that engagement of CD45 during in vitro priming has a significant effect on the development of antigen-specific T cell populations.

Infection of susceptible strains of mice with the Duplan strain of murine leukemia viruses induces a syndrome called MAIDS (murine acquired immunodeficiency syndrome) characterized by immunodeficiency and lymphoproliferation. In addition to a complete refractoriness of most subsets of lymphocytes to mitogen stimulation, the development of phenotypic abnormalities occurs such as the appearance of an abnormal CD4 + T cell subset lacking membrane Thy-1. This study was performed to compare the calcium responses during the early stages of MAIDS (week 9 or earlier) between T cells and B cells and between CD4 + Thy-1 − and CD4 + Thy-1 + T cells. B cells were strikingly less affected than T cells: their baseline [Ca2 + ] i did not significantly increase, and their calcium response to anti-lgM antibody and concanavalin A (Con A) was partially maintained. In contrast, the response to Con A was completely abolished in T cells. Interestingly, calcium mobilization in response to membrane receptor-independent stimuli such as ionophores and thapsigargin was strongly inhibited in T cells, while no such inhibition was found in B cells. In comparison with their CD4 + Thy-l + counterparts, CD4 + Thy-1 − T cells had blunted calcium responses in controls, as well as in infected mice. However, CD4 + Thy-1 + T cells were also strikingly altered, suggesting that the loss of membrane Thy-1 could be associated with, but not directly responsible for abnormalities of calcium responses in CD4 + T cells from RadLV-Rs-infected mice.

In this study the effects of CD45 engagement on CD3-TCR-driven stimulation of CD1+ human immature thymocytes have been analyzed. Simultaneous cross-linking of CD45 and CD3 antigens on highly purified CD1+ thymocytes reduced the number of cells undergoing apoptosis after 16 h of in vitro culture. This cell population might represent immature thymocytes committed in vivo to die by programmed cell death (PCD). CD45 engagement could also increase the number of cycling CD1+ thymocytes; of note, the large majority (> 95%) of dividing cells expressed the CD1 molecule at the cell surface, indicating that proliferating cells were actually represented by immature thymocytes. These data suggest that the CD45 molecule might play a role in the rescue of immature thymocytes from PCD during differentiation. Along this line, we found that activation of CD1+ thymocytes via the CD3-TCR complex could be enhanced by CD45, both in terms of transcription and surface expression of IL-2R. These effects might be explained by the finding that the CD45 molecule (but not its isoforms CD45RO and RA) was physically associated with the CD3-TCR complex at the cell surface of CD1+ human thymocytes, as shown by co-precipitation and co-capping experiments. Finally, cross-linking of CD45 and CD3 antigens led to the expansion of CD3+ thymocytes co-expressing CD4 and CD8, indicating that simultaneous engagement of CD45 and CD3 molecules can block CD1+ cells at the double-positive (CD3+CD4+CD8+) differentiation stage. On the other hand, stimulation through CD3 resulted in the expansion of thymocytes showing a mature phenotype (CD3+CD4+ or CD3+CD8+). Altogether, these findings suggest that the CD45 molecule is involved both in early activation and in the regulation of CD1+ thymocyte differentiation.

Apoptosis is a feature of the synovium of rheumatoid arthritis (RA). We have recently shown that RA synoviocytes were susceptible to anti-Fas mAb and undergo apoptosis in vitro. To investigate whether infiltrating mononuclear cells also undergo Fas-dependent apoptosis, double-labeling techniques combined with immunohistochemical examination with anti-CD3 mAb and the TdT-mediated dUTP-blotin nick end labeling (TUNEL) method to detect apoptotic cells, or in situ RT assay to detect Fas mRNA, were performed using frozen tissue sections. We also examined the in vitro induction of Fas-dependent apoptosis in freshly isolated synovium infiltrating mononuclear cells (SIM), synovial stromal cells (SSC) and peripheral blood lymphocytes (PBL) using tissues from nine patients with RA and three with osteoarthritis (OA). The results showed expression of Fas antigen and apoptotic cells in a number of CD3-bearing cells in RA synovial tissues. In vitro treatment with anti-Fas mAb produced a significant apoptosis of RA SIM and SSC, while none of PBL, and neither SIM nor SSC from OA exhibited apoptosis. Moreover, approximately 50% of CD4+, CD3+ and CD45RO+ cells, and > 90% of Fas-expressing cells of RA SIM underwent apoptosis in response to anti-Fas mAb, as detected by flow cytometry. Our results suggest that RA synovial infiltrating lymphocytes acquire high susceptibility to anti-Fas mAb and undergo apoptosis. Such a phenomenon of infiltrating T cells in RA synovium may play an important pathophysiological role and suggest a possible therapeutic effect for anti-Fas mAb in RA.

We have recently shown that CD45RA+CD4+ naive T cells can be activated to proliferate by a combination of IL-2, TNF-alpha and IL-6, but, at variance with TCR-mediated activation, they do not acquire the CD45RO molecule. This prompted us to investigate the phenotype of these cells and the functional features they display upon TCR stimulation. Naive T cells expanded by cytokines, though remaining CD45RA+, express a variety of activation and adhesion molecules which are peculiar to effector or memory T cells. Naive cells primed by cytokines, when activated with anti-CD3 mAb, produce a broad spectrum of cytokines, express CD40 ligand, but are unable to help B cells for Ig synthesis. A subset of CD4+CD45RA+RO-T cells with a phenotype (HLA-DR-, VLA-2+ or IL-2R+) similar to that of cells activated by cytokines in vitro can be found in vivo. These results demonstrate that activation signals delivered by cytokines, in the absence of TCR stimulation, can activate naive T cells to proliferate and differentiate into a 'split phenotype' with elements common to both naive and memory T cells. This novel antigen-independent activation may help to maintain the naive T cell repertoire and facilitate the antigen-responsiveness of naive T cells.

The nature of inflammatory lymphocytes recruited to the CNS has been studied in a model of chronic inflammation. Injection of killed Corynebacterium parvum into the cortex of the mouse brain produces a circumscribed inflammatory cellular infiltrate around the injection site, and recruited mononuclear inflammatory cells (IC) can be isolated for flow cytometric analysis. The majority of IC were T cells. In comparison with the predominant naive population of mesenteric lymph node T cells, IC T cells express much higher levels of CD44, LFA-1 and ICAM-1, and lower levels of CD45RB, features commonly associated with memory (previously activated) cells. In addition, in contrast to the L-selectin+ alpha 6-integrinlow phenotype of naive lymph node T cells, IC T cells lacked L-selectin and were alpha 6-integrin-. Mac-1, recently proposed as another marker of memory T cell differentiation, was not displayed by IC T cells, suggesting that Mac-1 expression may be heterogeneous among memory T cell subsets. A subset of mesenteric lymph node (MLN) T cells, probably representing activated T cells undergoing the naive to memory transition, but not of IC T cells, expressed high levels of alpha 6-, beta 7- and alpha E-integrin. IC and MLN naive T cells expressed comparable levels of alpha 4-integrin, but IC T cells stain poorly with anti-beta 7 mAbs and with mAb DATK 32, specific for the alpha 4 beta 7 heterodimeric lymphocyte homing receptor for the mucosal addressin MAdCAM-1, suggesting that these inflammatory cells express more alpha 4 beta 1 than alpha 4 beta 7. Consistent with this, in in vitro adhesion assays, brain IC bound better than MLN cells to the alpha 4 beta 1 integrin ligand VCAM-1 and the LFA-1 ligand ICAM-1 but adhered very poorly to the alpha 4 beta 7 ligand MAdCAM-1. These findings are consistent with and extend previous immunohistological studies of T cells in murine experimental autoimmune encephalomyelitis, and demonstrate a distinctive phenotype for lymphocytes being present in the chronically inflamed brain.

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.

Human aging is associated with immunosenescence, a process characterized by alterations in numerical and functional features of immune system components. Dendritic cells (DCs) are the main antigen-presenting cells, playing a pivotal role in adaptive and innate immunity. Therefore, we investigated the distribution of human circulating DCs throughout the life, in order to contribute to the knowledge of the physiological background underlying the aging of immune system. Cytofluorimetric analysis of peripheral blood samples by all-aged healthy population showed a significant decrease of circulating DCs and of their two main subsets among age. This reduction was limited to the plasmacytoid cell subtype when young and old subjects were analyzed separately. The analysis of circulating Treg cell number in a cohort of the subjects showed a significant reduction with increasing age and a positive significant correlation to myeloid or plasmacytoid absolute numbers. In conclusion, this work provides a large set of data of normal reference values of peripheral blood dendritic cells in healthy population suitable for comparative clinical studies concerning pathological immune dysfunctions.

Due to alternate mRNA splicing of exons 4, 5 and 6 (or A, B and C respectively), the CD45 cell surface glycoprotein is structurally heterogeneic in lymphoid cells of different lineage or stage of activation. Previous studies show that in vivo induced allo- and superantigen reactive rat cytolytic T lymphocytes (CTL) preferably belong to the CD45RClow subset, whereas tumour selective CTL express high amounts of CD45RC cell surface molecules. In this paper, reverse transcription polymerase chain reaction technique (RT-PCR) was utilized to evaluate CD45 isoform expression of rat lymphoid cells and in vivo activated rat CTL with distinct specificity and CD45RC profile. Cells from lymphoid organs expressed six CD45 mRNA isoforms, exon(45678), exon(5678), exon (578), exon(678), exon(78) and a novel extensively spliced exon(8) variant. In vivo activated TCR alpha beta + CD8+ cells sorted as CD45RClow expressed exon(78), exon(578) and exon(8), whereas TCR alpha beta + CD8+CD45RChigh cells expressed exon(78), exon(578), exon(5678) and full-length exon(45678). Triple-colour staining indicated high expression of LFA-1 in the cytotoxic CD45RCintermediate and CD45RClow cells, and low expression of LFA-1 in CD45RChigh non-cytotoxic cells from allo- and superantigen activated rats. In contrast, tumour activated TCR alpha beta +CD45RChigh cells were divided in LFA-1high and LFA-1low subsets, and sorting of these subsets revealed that tumour-selective cytotoxicity was confined to the LFA-1high effector cell subset. Furthermore, it was evident that the LFA-1high effector cell subset expressed high levels of exon(5678), exon(578) and exon(78) isoforms and, in contrast to the LFA-1low subpopulation, lacked expression of exon 4 containing full-length CD45 mRNA transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies recognising the products of alternatively spliced exons near the N-terminus of the leukocyte common antigen, CD45, have been widely used to distinguish populations of lymphocytes with different functional properties. These alternatively spliced regions contain a high content of serine and threonine residues (average 35%) and are heavily O-glycosylated. Despite evidence that the O-glycosylation contributes significantly to the antigenic character of this region of CD45, work with leukosialin and mucin glycoproteins leads to the prediction that the majority of epitopes in the N-terminal exons should be linear protein determinants. In this study the exons of CD45 were expressed in Escherichia coli as non-glycosylated proteins fused to glutathione S-transferase (GST). Fourteen out of 17 mAbs specific for human CD45R reacted with a fusion protein containing exons 4, 5 and 6 (ABC) of human CD45, and four out of six mAbs specific for rat CD45R reacted with an equivalent rat protein. mAbs recognising the product of rat exon B are reported for the first time. Kinetic analysis of MRC OX22 antibody binding to spleen CD45 and to GST fusion proteins showed that the carbohydrate affected the kinetics of binding of antibodies to the protein backbone. In conclusion, heterogeneity in the glycosylation of heavily O-glycosylated cell surface proteins can affect interactions of these proteins both directly through the carbohydrate and indirectly through effects on the protein backbone.

Transforming growth factor-beta 1 (TGF-beta 1) can have stimulatory or inhibitory effects on cell growth. For several cell types, the effect of TGF-beta 1 was found to correlate with the differentiation stage of the cells and the presence of other cytokines. We have studied here the influence of TGF-beta 1 on CD4+ T cell activation in relation to the differentiation stage of the cells by evaluating the effect of TGF-beta 1 on the proliferative responses of purified CD4+CD45RA+ (unprimed) and CD4+CD45RO+ (primed) lymphocytes. Under certain conditions, TGF-beta 1 exerted a co-stimulatory effect on peripheral blood CD4+CD45RA+ T cells whereas the outgrowth of CD4+CD45RO+ T cells was suppressed in any activation system tested. The enhancement of proliferative responses by TGF-beta 1 in TCR/CD3 or CD2 stimulated cultures of CD45RA+ cells involved up-regulation of CD25 expression and was dependent on the presence of exogenous IL-2 or CD28 mAbs; IL-7 driven proliferative responses were suppressed by TGF-beta 1. These observations were confirmed in experiments with purified cord blood (CB) CD4+ T cells inasmuch as addition of TGF-beta 1 caused a 2- to 7-fold increase in IL-2 driven proliferative responses of these cells. Finally we show that, in contrast to the effect of TGF-beta 1 during primary stimulation of CD CD4+ T cells, TGF-beta 1 suppressed T cell proliferation for approximately 40% in secondary cultures of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-color flow cytometric analysis was used to define surface markers which identify the Th2-type CD4+ cells responsible for the eosinophilia and elevated serum IgE typical of tissue invasive helminth infections. A group of six mAbs to well known cell surface markers were screened for differential expression on CD4+ CD45RO+ lymphocytes from normal individuals (NL; n = 6) and filaria-infected patients (PT; n = 10). The majority of markers were expressed equally by both groups, but the CD4+ CD45RO+ cells in the PTs showed significantly higher levels of expression of HLA-DR than those of NLs (P = 0.014). This CD4+ HLA-DR+ subpopulation was then studied further for its expression of an additional 10 activation and adhesion molecules. CD27 showed a trend for lower intensities of expression on PT CD4+ HLA-DR+ cells than on those of NLs. Analysis of the serum from both NLs and PTs revealed that PTs had significantly higher levels of soluble CD27 and CD25 (IL-2R) in the serum than NLs (P < 0.01 and P = 0.022 respectively) indicating a general state of immune activation and differentiation. Functional analysis of the CD4+ HLA-DR+ and the CD4+ CD27- subpopulations revealed that the CD4+ HLA-DR+ cells produced significantly higher levels of IL-5 than the CD4+ HLA-DR- cells (P = 0.04), and the CD4+ CD27- cells produced significantly higher levels of both IL-4 and IL-5 than the CD4+ CD27+ cells (P < 0.05 and P < 0.001 respectively). Thus, while the CD4+ CD27- and CD27+ subpopulations contain Th1 and Th0 cells, only the CD4+ CD27- population contains the Th2 cells (producing both IL-4 and IL-5).

The immune response of peripheral blood lymphocytes (PBL) of non-exposed human individuals to the Nef protein of HIV-1 was studied. Nef is a regulatory protein of HIV which is immediately expressed after infection and which seems to be important in the pathogenicity of HIV. Nef may therefore serve as a potential target for effective immunity against HIV infection. Epstein-Barr (EBV)-transformed lymphoblastoid B cell lines (LCL) were established from four healthy young seronegative adults and transfected with the Nef gene. These cells served as stimulator cells for autologous PBL in vitro and as target cells for CTL. CTL responses were readily generated against Nef-transfected LCL, consisting of Nef-specific and putative EBV-specific CTL. Nef-specific CTL were generated exclusively from CD8+ cells and were MHC class I restricted. Since a vigorous Nef-specific CTL response in non-infected individuals was unexpected, CTL precursor frequencies were determined by limiting dilution analyses in non-fractionated PBL and in PBL separated into the CD45RO- (naive) and CD45RO+ (memory) T cell populations. As expected, the putative EBV-specific CTL precursors were predominantly found in the CD45RO+ subset at frequencies typical for memory T cells. Nef-specific CTL precursors, in contrast, were found predominantly in the CD45RO- population, at even higher frequencies of approximately 1/1000-1/3000. Nef may thus display either an unusually high number of immunogenic peptides or a limited number of peptides presented in a very efficient way, so that many T cells including low affinity cells, would be triggered.

T cell activation appears to be regulated by an interplay between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). p56lck and p59fyn have been found to associate with CD4 and TCR-CD3 respectively. The CD45 family of transmembrane PTPases has been shown to be able to regulate the activities of these receptor-associated PTKs in vitro. In man, CD45 contains five different isoforms whose distribution defines subsets of T cells having distinct activation requirements and in vitro functions. Several groups have reported a physical interaction between distinct isoforms of CD45 and CD2, CD4, and the TCR-CD3 complex. Given the potential regulatory interaction between CD45 and PTKs in CD4+ subsets expressing different CD45 isoforms, we have examined CD4 associated and TCR-CD3- associated PTK activities, associated phosphatidyl inositol (PI) kinases and substrates of tyrosine phosphorylation in CD45RA+ and CD45RA- CD4+ T cell lines derived from peripheral blood. Both subsets express CD4-associated p56lck and TCR-CD3-associated p59fyn kinases which exhibit identical in vitro phosphorylation at the Y-394 and Y-420 autophosphorylation sites respectively. Further, both subsets exhibited PI kinases activity associated with CD4-p56lck. Consistent with these observations, anti-CD3 crosslinking induced the phosphorylation of a similar spectrum of intracellular substrates in these CD45RA+ and CD45RA- CD4+ T cell lines. These observations indicate that despite the possible interaction between CD45 isoforms and CD4 or TCR-CD3, the mere expression of the CD45RA isoform does not in and of itself alter the presence of receptor-associated kinases or their intracellular targets.

The balance between effector CD4(+) T cells secreting IL-17 (T(h)17) and regulatory T cells (Treg) plays an important role in autoimmune disorders that include rheumatoid arthritis (RA) and Crohn's disease. Tumor necrosis factor (TNF)-alpha is a key pro-inflammatory cytokine that contributes to disease pathogenesis. We investigated the interplay between CD45RA(+) Treg and TNF-alpha in the regulation of human T(h)17 differentiation. We found that CD45RA(+) Treg promoted while TNF-alpha inhibited naive CD4(+) T-cell differentiation into IL-17 and CCL20 co-expressing T(h)17 cells without influencing their IL-22 release. Unexpectedly, CD45RA(+) Treg depletion abrogated TNF-alpha suppressive function. Finally, dendritic cell-derived TNF-alpha suppressed the development of IL-17(+)CCL20(+) expressing T(h)17 cells. In conclusion, CD45RA(+) Treg positively governs human T(h)17 development, which is impaired by TNF-alpha. We propose that TNF-alpha may represent a negative feedback mechanism to control IL-17/CCL20- but not IL-22-associated autoimmune pathologies.

We prepared a novel rat mAb specific for CD45 molecules bearing the epitope coded for by the alternative exon 6 of the murine CD45 gene (CD45RC). Together with available mAbs to alternative exon 4- and 5-dependent epitopes (CD45RA and CD45RB respectively), we found that the three alternative exons show differential expressions on murine lymphocytes. Flow cytometry analysis revealed that although B cells were homogeneously CD45RA+B+C+, the CD4+ T cells clearly included two populations, CD45RA-B+C- and CD45RA-B+C+. The CD8+ T cells were separated into CD45RA-B+C+ and CD45RA+B+C+ populations. Such features of epitope expression on the cell surface correlate well with message levels of corresponding alternative exons. In the CD4+ T cells, messages of alternative exons were associated with either one or two exon forms of the CD45 transcript. In CD8+ T cells, there were transcripts with one, two, or three alternative exons. When stimulated by an immobilized CD3 mAb, the CD45RC+CD4+ T cell subset preferentially secreted IL-2 and CD45RC-CD4+ T cells produced IL-4. Upon stimulation with concanavalin A, CD45RC-CD4+ T cells converted to CD45RC+ cells, and the level of CD45RC expression on the CD45RC+CD4+ T cell subset was up-regulated. These changes were unidirectional and irreversible. Therefore, differential expression of CD45RC probably delineates the functional heterogeneity of murine CD4+ T cells that is associated with the stages of CD4+ T cell maturation or activation.

Tyrosine phosphorylation of several intracellular proteins is observed very early during T cell activation. p56lck, a non-receptor src-like protein tyrosine kinase (PTK) which is associated with the intracellular domains of CD4 and CD8 co-receptors, has been implicated in these early signal transduction events. Furthermore, recent experiments indicate that the receptor phosphotyrosine phosphatase, CD45, might be important in the regulation of p56lck PTK activity and that its expression is required for the generation of second messenger molecules following TCR triggering. Here, using co-capping experiments and double indirect immunofluorescence microscopy in functional human T lymphocytes, a specific co-distribution of a significant fraction of p56lck with CD45, but not with several other cell surface proteins, has been revealed. This is the first demonstration of a physical interaction between a receptor phosphotyrosine phosphatase and a PTK under physiologically relevant conditions. In addition, after antibody-induced capping of CD4, both a co-localization of p56lck and CD4, and concomitantly a significant increase in intracellular phosphotyrosine at the sites of CD4 caps were observed. In strong contrast to these results, co-clustering of CD4 with CD45 did not result in any detectable intracellular phosphotyrosine at the cap sites. These data indicate that CD45 can act on CD4-associated phosphoproteins in viable human T lymphocytes. Further, this provides evidence that p56lck PTK is a substrate of CD45 phosphotyrosine phosphatase in vivo and thereby supports the idea that CD45 is an early regulator of T cell activation involved in the modulation of the coupling of receptor-triggered events to intracellular signalling pathways.

We found that naive (CD45RA+) CD4 T cells have a lower capacity of adhesion to Epstein-Barr virus (EBV) immortalized B cells than memory (CD45RO+) CD4 T cells, as judged by conjugate formation. This would appear to be due to differences in the expression of adhesion molecules [lymphocyte function-associated antigen (LFA)-1, CD2]. However, kinetic studies showed that the degree of adhesion of naive T cells to B cells was stable over 60 min while that of memory T cells, like that of unseparated CD4 T cells, was characterized by a rapid formation and rapid dissociation of conjugates. This could be explained by a difference in the sensitivity of naive and memory CD4 T cells to down-regulation of antigen-independent adhesion by CD4-MHC class II interaction. Indeed, memory T cells also adhered stably to MHC class II(-) B cells. The adhesion of memory T cells, but not naive T cells, to MHC class II(+) B cells was sensitive to inhibition by OKT4a an anti-CD4 antibody, human immunodeficiency (HIV) gp160 (env) protein and a 12-mer peptide encompassing the 35-46 sequence of the HLA, DR beta 1 domain and previously shown to inhibit activation of HLA class II-restricted CD4 T cell responses. Since MHC class II expression did not influence the degree of conjugate formation by naive or memory CD4 T cells with B cells, CD4-MHC class II interaction does not appear to be involved in binding itself, but may down-regulate the adhesion of memory but not naive CD4 T cells.(ABSTRACT TRUNCATED AT 250 WORDS)