Differential expression of CD22 (Lyb8) on murine B cells

Previous studies have established the distribution, biochemistry and functional attributes of human CD22, a B cell-restricted glycoprotein. Recently, molecular cloning of the murine CD22 equivalent revealed this molecule to be the same as the previously described Lyb8 alloantigen. Using the anti-Lyb8 mAb Cy34.1.2, the present report documents the expression patterns of CD22 within the murine B cell compartment. The results demonstrate that in the bone marrow, murine CD22 is absent on the surface of pro-B cells, pre-B cells and newly emerging IgM+ B cells. CD22 is present at a low density on immature IgMhi B cells and fully expressed on mature recirculating B cells. In the periphery, murine CD22 is expressed at mature levels on all B cell subsets including follicular, marginal zone, B1 and switched B cells. Further studies showed CD22 to be retained on activated murine B cells for extended periods. Finally, in combination with CD23 and heat stable antigen, CD22 can be used to delineate the immature splenic B cells, and distinguish them from follicular and marginal zone cells. Together, the results demonstrate murine CD22 to be a useful pan marker for all mature B cell subsets.     

Identification of functional human splenic memory B cells by expression of CD148 and CD27

Memory B cells isolated from human tonsils are characterized by an activated cell surface phenotype, localization to mucosal epithelium, expression of somatically mutated immunoglobulin (Ig) variable (V) region genes, and a preferential differentiation into plasma cells in vitro. In spleens of both humans and rodents, a subset of memory B cells is believed to reside in the marginal zone of the white pulp. Similar to tonsil-derived memory B cells, splenic marginal zone B cells can be distinguished from naive follicular B cells by a distinct cell surface phenotype and by the presence of somatic mutations in their Ig V region genes. Although differences exist between human naive and memory B cells, no cell surface molecules have been identified that positively identify all memory B cells. In this study, we have examined the expression of the receptor-type protein tyrosine phosphatase CD148 on human B cells. CD148(+) B cells present in human spleen exhibited characteristics typical of memory B cells. These included an activated phenotype, localization to the marginal zone, the expression of somatically mutated Ig V region genes, and the preferential differentiation into plasma cells. In contrast, CD148(-) B cells appeared to be naive B cells due to localization to the mantle zone, the expression of surface antigens typical of unstimulated B cells, and the expression of unmutated Ig V region genes. Interestingly, CD148(+) B cells also coexpressed CD27, whereas CD148(-) B cells were CD27(-). These results identify CD148 and CD27 as markers which positively identify memory B cells present in human spleen. Thus, assessing expression of these molecules may be a convenient way to monitor the development of memory B cell responses in immunocompromised individuals or in vaccine trials.     

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.     

Association of CD4+ T cell-dependent keratitis with genetic susceptibility to Pseudomonas aeruginosa ocular infection

The role of T lymphocytes in susceptibility to Pseudomonas aeruginosa corneal infection was studied in inbred C57Bl/6 (B6) beta2-microglobulin+/+ (beta2m+/+) and beta2m-/- knockout (KO) mice on a B6 genetic background. The corneas of both B6 and KO mice perforated by 7 days postinfection (p.i.). Histopathology revealed a similar inflammatory response characterized by an infiltration of polymorphonuclear neutrophilic leukocytes by 24 h p.i. in both groups of mice. CD4+ and CD8+ (latter absent in KO) T cells were present in cornea by 3 days p.i., and by 5 days, IL-2R-positive cells were positively immunostained. Corneas of B6 beta2m+/+ mice depleted of CD4+ T cells and infected with P. aeruginosa did not perforate at 7 days p.i. vs mice depleted of CD8+ T cells or treated with an irrelevant mAb. Neutralization of IFN-gamma before infecting B6 mice prevented corneal perforation and was associated with a lower delayed-type hypersensitivity than in B6 mice similarly treated with an irrelevant mAb. These data provide evidence that a CD4+ T cell (Th1)-dominated response following P. aeruginosa corneal infection is associated with genetic susceptibility and corneal perforation in inbred B6 mice.     

CD40 activation boosts T cell immunity in vivo by enhancing T cell clonal expansion and delaying peripheral T cell deletion

In this report we show that activation of APC with an agonist anti-CD40 mAb profoundly alters the behavior of CD4 T cells in vivo. Stimulation of mice with anti-CD40 2 days before, but not 1 day after, administration of superantigen (SAg) enhanced CD4 and CD8 T cell clonal expansion by approximately threefold. Further, CD40 activation also delayed peripheral T cell deletion after activation. Dying, activated T cells were quantitated by detecting extracellular phosphatidylserine with concomitant staining for SAg-reactive T cells using a TCR Vbeta-specific mAb. Upon close examination, it was shown that CD40 activation delayed the death of the activated T cells. Additionally, it was found that enhanced survival of CD4 T cells was equally dependent on APC expression of B7-1 and B7-2. This is in contrast to CD8 T cells, which did not depend as much on B7-1 as B7-2. Thus, CD40 activation indirectly promotes T cell growth and delays the death of SAg-stimulated CD4 T cells in vivo. These data suggest that one way CD40 activation promotes a more robust immune response is by indirectly increasing the production of effector T cells and by keeping them alive for longer periods of time.     

Lymphotoxin alpha/beta and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen

Mice deficient in the cytokines tumor necrosis factor (TNF) or lymphotoxin (LT) alpha/beta lack polarized B cell follicles in the spleen. Deficiency in CXC chemokine receptor 5 (CXCR5), a receptor for B lymphocyte chemoattractant (BLC), also causes loss of splenic follicles. Here we report that BLC expression by follicular stromal cells is defective in TNF-, TNF receptor 1 (TNFR1)-, LTalpha- and LTbeta-deficient mice. Treatment of adult mice with antagonists of LTalpha1beta2 also leads to decreased BLC expression. These findings indicate that LTalpha1beta2 and TNF have a role upstream of BLC/CXCR5 in the process of follicle formation. In addition to disrupted follicles, LT-deficient animals have disorganized T zones. Expression of the T cell attractant, secondary lymphoid tissue chemokine (SLC), by T zone stromal cells is found to be markedly depressed in LTalpha-, and LTbeta-deficient mice. Expression of the SLC-related chemokine, Epstein Barr virus-induced molecule 1 ligand chemokine (ELC), is also reduced. Exploring the basis for the reduced SLC expression led to identification of further disruptions in T zone stromal cells. Together these findings indicate that LTalpha1beta2 and TNF are required for the development and function of B and T zone stromal cells that make chemokines necessary for lymphocyte compartmentalization in the spleen.     

CD40 ligand expressed on B cells in the BXSB mouse model of systemic lupus erythematosus

Male BXSB mice, unlike female BXSB, develop a severe early onset lupus-like disease that has been linked to an intrinsic B cell defect. In investigating this B cell defect the present study showed that male, but not female, BXSB contained a higher percentage of large, activated splenic B cells that were more responsive to anti-CD40 mAb-induced proliferation. The hyperactivity of the large B cells from the male mice was also observed in the absence of anti-CD40 mAb or any other stimuli. In examining the mechanism of the B cell hyperactivity, it was found that 20% of unstimulated large B cells from male mice, unlike large B cells from female mice, expressed CD40 ligand (CD40L), a molecule normally expressed on activated CD4+ cells. The percentage of large B cells from the male BXSB that expressed CD40L was increased to 43% by stimulation with LPS. A functional role for CD40L expression on B cells was confirmed by showing that CD40-Ig blocked the spontaneous proliferation of the large B cells from male mice. In addition, the stimulatory capacity of the large B cells from the male mice was demonstrated by their ability to induce DNA synthesis in small B cells in a CD40L-dependent manner. These results demonstrated that large B cells from male BXSB expressed functionally active CD40L. It is likely that the B cell CD40L expression and increased susceptibility to CD40 signaling due to an intrinsic B cell hyperactivity promotes autoimmune disease in BXSB mice.     

CD73 expression and fyn-dependent signaling on murine lymphocytes

CD73 is a glycosyl phosphatidylinositol-anchored protein with both ecto-enzyme activity (ecto-5'-nucleotidase) and signal transducing capabilities for human T lymphocytes. We now report an analysis of the distribution and function of CD73 in murine lymphoid tissues made possible by the development of the first monoclonal antibodies (mAb) specific for murine CD73. Subsets of T and B lymphocytes are CD73+ and the level of expression increases with lymphocyte maturation in both species. Among B cells, CD73 is largely restricted to cells which have undergone isotype switching. The signal transmitting function of CD73 is also conserved, as splenic T cells treated with anti-CD73 mAb plus phorbol 12-myristate 13-acetate proliferate and secrete IL-2. Fyn-/- mice are unresponsive to CD73 ligation, however, demonstrating the requirement for this tyrosine kinase in CD73-mediated signal transduction. CD73 is down-regulated after mAb plus cross-linking, suggesting that expression may be controlled by interaction with a ligand. Only small numbers of thymocytes are CD73+, so CD73 receptor functions are unlikely to be important for developing T cells. However, immunohistochemical analysis reveals that reticular and vascular cells throughout the thymus and other lymphoid tissues are markedly CD73+. Therefore, CD73 might mediate lymphocyte-stromal cell interactions or condition the local microenvironment to facilitate lymphocyte development and/or function.     

Mouse germinal center B cells with the xid mutation retain responsiveness to antimouse CD40 antibodies but diminish IL-5 responsiveness

The germinal center (GC) develops in secondary lymphoid tissues in response to thymus-dependent (TD) antigens. To investigate the molecular mechanism of B cell differentiation in GC, we enriched GC B cells from spleen of TD antigen-immunized wild-type and X-linked immunodeficient (XID) mice, and examined the differentiation of GC B cells into antigen-specific IgG1 antibody-forming cells (AFC) in response to anti-CD40 mAb and cytokines. A significant proportion of freshly purified GC B cells expressed receptors for IL-4 and IL-5. Anti-CD40 mAb sustained the viability of GC B cells and IL-4 co-operated with anti-CD40 mAb for further enhancement of the cell viability. Anti-CD40 mAb and IL-4 were essential for inducing differentiation of GC B cells into antigen-specific IgG1-AFC and IL-5 efficiently enhanced their differentiation. GC B cells with the xid mutation responded for proliferation to CD40 ligation to a lesser extent and for the IgG1-AFC response to anti-CD40 mAb together with IL-4, but they showed impaired responsiveness to IL-5, regardless of enhanced expression of IL-5R in response to anti-CD40 mAb and IL-4. These results suggest that anti-CD40 mAb, IL-4 and IL-5 play a critical role in the differentiation of mouse GC B cells. The GC B cells from XID mice show a functional defect with respect to IL-5-mediated differentiation.     

Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses

PD-1, an Ig superfamily member, contains an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic tail. It is expressed in a minor fraction of CD4-CD8- normal thymocytes and induced in peripheral lymphocytes following activation. To assess the possible roles of PD-1 in the immune responses, PD-1-deficient (PD-1-/-) mice were generated by a gene-targeting strategy. PD-1-4- mice developed and grew normally. Although the thymus was apparently normal, PD-1-/- mice showed moderate but consistent splenomegaly, which reflected the increased cellularity of both lymphoid and myeloid cells. The proliferative response of B cells by anti-IgM antibodies, but not of T cells by an anti-CD3 (145-2C11) mAb in vitro, was augmented in PD-1-/- mice as compared with control littermates. PD-1-/- mice showed increased serum levels of IgG2b, IgA and most strikingly IgG3, while those of IgM and IgG1 were comparable with control mice. Furthermore, PD-1-/- mice exhibited significantly augmented IgG3 anti-DNP antibody response to a type 2 T-independent antigen, DNP-Ficoll, with comparable IgM and IgG1 antibody responses with littermate controls. In the peritoneal cavity, the B-1 cell population in PD-1-/- mice exhibited significantly reduced expression of CD5, a negative regulator of B-1 cell activation, despite a marginal increase in the number of B-1 cells. Thus, PD-1 was suggested to be involved in the negative regulation for particular aspects of B cell proliferation and differentiation including class switching.     

Regulation of surface and intracellular expression of CTLA4 on mouse T cells

CTLA4 is a cell surface molecule that shares 30% homology with CD28 and binds B7 family members with high affinity. Analysis of surface expression on murine T cells revealed up-regulation after stimulation with anti-CD3 mAb in vitro and further augmentation after the addition of exogenous IL-2 or anti-CD28 mAb. The effects of IL-2 and anti-CD28 mAb were additive and in part independent, as anti-CD28 mAb increased anti-CD3 mAb-induced T cell CTLA4 expression in IL-2-deficient mice. In contrast, CTLA4 expression was only minimally augmented by the addition of IL-4, IL-6, IL-7, or IL-12. Expression of CTLA4 induced by anti-CD3 mAb was inhibited by anti-IL-2 plus anti-IL-2R mAbs. Inasmuch as these agents prevented T cell proliferation, the effects of cell cycle inhibitors also were examined. Drugs blocking at G1 (cyclosporin A, mimosine) or S (hydroxyurea) phase inhibited the up-regulation of CTLA4 induced by anti-CD3 mAb, suggesting that entry into the cell cycle was necessary to increase the expression of CTLA4. The kinetics of intracellular expression of CTLA4 after stimulation with anti-CD3 mAb paralleled those of surface expression, but surprisingly, much more CTLA4 was localized in the cytoplasm of T lymphocytes than on the cell surface at each time point. Importantly, surface CTLA4 was rapidly internalized intracellularly, which may explain the low levels of expression generally detected on the cell surface. We conclude that both CD28 and IL-2 play important roles in the up-regulation of CTLA4 expression. In addition, the cell surface accumulation of CTL4 appears to be primarily regulated by its rapid endocytosis.     

Stimulation of basal and L-DOPA-induced motor activity by glutamate antagonists in animal models of Parkinson''s disease

Peripheral T-cell antigen receptor V beta (TCRV beta) repertoire is influenced by clonal deletion both in the thymus and periphery. Developing thymocytes expressing certain TCRV beta are deleted by endogenous superantigens presented on major histocompatibility complex (MHC) molecules in the thymus. Likewise, mature T cells bearing particular TCRV beta chains can be clonally deleted by superantigens in the periphery. The efficiency with which T cells expressing particular V beta subunits are deleted differs depending upon which coreceptor is expressed. Indeed, while deletion of V beta 11+ splenic T cells in CBA/J (Mls-1, a I-E, + MTV 9+) mice is quite efficient for CD4+ spleen T cells, it is much less efficient for CD8+ splenic T cells. If the difference in the efficiency of deletion is due solely to the coreceptor expressed, then a transgene encoding CD4 should increase the efficiency with which CD8+ cells are deleted. To address this question, we have produced CD4 transgenic (TG) mice that express physiologic levels of CD4 on all thymocytes and peripheral CD8 T cells. CD4 molecules expressed on CD8+ splenic T cells were associated with P56lck tyrosine kinase, and were functional as evidenced by their ability to facilitate class II alloreactivity. Furthermore, we found that ectopic expression of TG CD4 molecules on CD8+ cells was able to affect the efficiency of deletion in response to superantigen stimulation. In particular, deletion of TCRV beta 11+ T cells was much less efficient for CD8+ than for CD4+ T-cell subpopulations in (CBA/J x B6) F1 mice. However, expression of the CD4 transgene on CD8+ splenic T cells from these mice increased the efficiency of deletion in the CD8+ V beta 11 T cells. Interestingly, this effect was not observed in a mature CD8+ thymocyte subpopulation. The results in this report demonstrate that CD4 molecules are involved in peripheral deletion of TCRV beta 11+ T cells in (CBA/J x B6) F1 mice, and that the TCRV beta repertoire can be altered by ectopic expression of CD4 on all T-lineage cells.     

Variant effects of non-native kissing-loop hairpin palindromes on HIV replication and HIV RNA dimerization: role of stem-loop B in HIV replication and HIV RNA dimerization

Splenic marginal-zone B cells, marginal-zone B cells of Peyer's patches in the gut, and nodal marginal-zone B cells (also identified as monocytoid B cells) share a similar morphology and immunophenotype. These cells likely represent a distinct subset of B cells in humans and rodents, but their precise ontogenetic relationship as well as their origin from B cells of the germinal center is still debated. To study this, we performed a mutation analysis of the rearranged immunoglobulin variable genes (VH) of microdissected single nodal and splenic marginal-zone cells. In addition, we investigated the presence of proliferating cells and B-cell clones in the human splenic and nodal marginal zone as well as adjacent germinal centers. This was performed by immunohistochemical staining for the Ki-67 antigen and denaturing gradient gel analysis of amplified immunoglobulin heavy chain genes' complementarity determining region 3 of microdissected cell clusters. A variable subset of nodal and splenic marginal-zone B cells showed somatic mutations in their rearranged VH genes, indicating that both virgin and memory B cells are present in the nodal and splenic marginal zone. Nodal and splenic marginal-zone B cells preferentially rearranged VH3 family genes such as DP47, DP49, DP54, and DP58. A preferential rearrangement of the same VH genes has been shown by others in the peripheral CD5(-) IgM+ B cells. These data suggest that the splenic and nodal marginal-zone B cells are closely related B-cell subsets. We also showed that marginal-zone B cells may cycle and that clones of B cells are frequently detected in the nodal as well as the splenic marginal zone. These clones are not related to those present in adjacent germinal centers. These data favor the hypothesis that clonal expansion occurs in the marginal zone. Whether the somatic hypermutation mechanism is activated during the clonal expansion in the marginal zone and which type of immune response triggers the clonal expansion need to be elucidated.     

Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation

We recently reported that previously activated T cells, irrespective of the nature of the first stimulus they encountered, are unable to respond to Staphylococcal enterotoxin B (SEB), nor to soluble anti-CD3 monoclonal antibody (mAb) presented by splenic antigen-presenting cells (APC). Such previously activated T cells are, however, fully capable of responding to plate-bound anti-CD3 plus splenic APC. These data suggest differential integration of the T-cell receptor (TCR) and co-stimulatory signalling pathways in naive versus antigen-experienced T cells. Consistent with this hypothesis, anti-CD28 mAb restores the proliferative capacity of resting ex vivo CD45RBlo CD4+ T cells (representing previously activated T cells) to both soluble anti-CD3 mAb and SEB. Interestingly, mAb-mediated engagement of cytotoxic T-lymphocyte antigen-4 (CTLA-4) completely negates the rescue effects mediated by anti-CD28 mAb in CD45RBlo cells. Nevertheless, the non-responsiveness of CD45RBlo CD4+ T cells cannot be reversed by anti-CTLA-4 Fab fragments, indicating that it is not related to negative regulatory effects of CTLA-4 engagement itself. Interestingly, the addition of interleukin-2 (IL-2) restores the proliferative capacity of CD45RBlo CD4+ T cells to SEB and soluble anti-CD3 mAb. Moreover, when rescued by IL-2, the cells are less susceptible to the negative regulatory effects of CTLA-4 engagement. Together, these findings suggest that the non-responsiveness of CD45RBlo CD4+ T cells to certain stimuli may be related to inadequate TCR signalling, primarily affecting IL-2 production.     

Transfer of lymphocytic choriomeningitis disease in beta 2-microglobulin-deficient mice by CD4+ T cells

In this study we have investigated the role of CD4+, MHC class II-restricted cytotoxic T lymphocytes (CTLs) in the disease caused by lymphocytic choriomeningitis virus (LCMV) in beta 2-microglobulin deficient (beta 2m-) mice. Intracranial (i.c.) infection with LCMV resulted in death of six out of 11 beta 2m- mice. Mice that survived showed a marked loss in body weight. Death and loss of body weight could be prevented by immunosuppressing the mice with irradiation or cyclosporine prior to i.c. injection of LCMV. This treatment also prevented induction of virus-specific, MHC class II-restricted CTL following peripheral inoculation with LCMV. In vivo depletion of CD4+ cells with antibody also prevented death following i.c. injection whereas in vivo depletion of CD8+ cells had no effect. Disease could be transferred to recipient beta 2m- mice by adoptive transfer of beta 2m- derived immune spleen cells. Transfer of non-immune spleen cells did not result in illness. In vitro treatment of immune spleen cells with anti-CD4 antibody and complement eliminated class II-restricted CTL activity and also prevented mortality of recipients after adoptive transfer. Treatment with anti-CD8 antibody had no effect. We were unable to transfer LCM disease to beta 2m- recipients by adoptive transfer of immune spleen cells from C57BL/6 mice. These results suggest that, unlike normal mice, the pathology of LCM disease in beta 2m- mice is dependent upon virus-specific, CD4+CD8-, MHC class II-restricted T cells.     

Specific regulation of VLA-4 and alpha 4 beta 7 integrin expression on human activated T lymphocytes

Modulation of VLA integrins was studied in several human T cell clones upon specific and nonspecific cellular activation. Human activated T lymphocytes down-regulated both alpha 4 beta 1 and alpha 4 beta 7 integrins upon specific recognition of alloantigens (cytotoxic T cells) or in the presence of Staphylococcus enterotoxin B (superantigen recognizing noncytotoxic T cells). In contrast, the expression of other membrane integrins, such as VLA-1 and VLA-5 integrins, was not modified. Down-regulation of alpha 4 beta 1 and alpha 4 beta 7 integrins was observed as early as 3 h after stimulation, lasted later than 72 h and was partially inhibited by cytochalasin D. Interestingly, neither target cells nor NK cells modulated CD49d expression after interaction with T cells of K562, respectively, suggesting that CD49d expression was linked to specific T cell activation. The down-regulation of the CD49d chain in T cell clones stimulated with immobilized anti-CD3 mAbs confirmed the role of TCR-mediated activation in CD49d regulation. However, the CD3-independent cellular aggregation induced by soluble anti-CD43 mAb was also able to strongly down-regulate alpha 4 beta 1 and alpha 4 beta 7. The present work shows the first evidence that CD49d subunit-bearing integrin expression is distinctly regulated from other integrins after Ag or superantigen recognition by human activated T cells. CD49d modulation may be relevant for the traffic and tissue localization of locally activated T cells during immune responses.     

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

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

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.     

TCR-gamma delta cells in CD3 zeta-deficient mice contain Fc epsilon RI gamma in the receptor complex but are specifically unresponsive to antigen

Unlike TCR-alpha beta cells, TCR-gamma delta cells express a distinct member of the zeta family, the gamma-chain of Fc epsilon RI (Fc epsilon RI gamma) within the TCR complex. To study the role of the Fc epsilon RI gamma-chain in TCR-gamma delta cells, a TCR-gamma delta transgenic mouse (G8) has been crossed with CD3 zeta-chain-deficient mice (G8.zeta-/-). Thy-1+ spleen and lymph node cells of these animals expressed low levels of CD3/TCR. These results suggested that the zeta-chain is required for effective TCR transport to the cell surface. In contrast, intraepithelial TCR-gamma delta cells of G8.zeta-/- mice expressed high levels of TCR. Immunoprecipitation with anti-CD3 showed that Fc epsilon RI gamma-chains were associated with the TCR complex in T cells isolated from zeta-deficient mice. Although the Fc epsilon RI gamma-expressing T cells proliferated in response to stimulation by TCR-specific Abs including anti-CD3 epsilon, anti-pan gamma delta, and anti-V gamma 2 mAb, the G8.zeta-/- T cells did not respond to the G8-specific Ag (T10b), anti-Thy-1 mAb, or Con A. The unresponsiveness to the Ag was not due to the reduced TCR expression, because intraepithelial TCR-gamma delta cells from the zeta-deficient mice did not respond to Ag. The inability of the G8.zeta-/- T cells to respond to Ag could not be overcome by providing an anti-CD28 costimulatory signal or by adding exogenous rIL-2. Taken together, our data suggest that the Fc epsilon RI gamma-chain associates with the TCR-gamma delta complex in the absence of the zeta-chain, but it is not able to substitute for the zeta-chain for effective transport of TCR to the cell surface or functional responses to Ag.     

The monoclonal antibody CZ-1 identifies a mouse CD45-associated epitope expressed on interleukin-2-responsive cells

We have previously described a monoclonal antibody (mAb), CZ-1, which reacts with an epitope expressed on most peripheral basophils, natural killer cells, B cells, and CD8+ T cells, but not with most thymocytes or peripheral CD4+ T cells. Here we show that mAb CZ-1 defines a sialic acid-dependent epitope associated with a subpopulation of CD45 molecules. This conclusion is based on the ability to block binding of mAb CZ-1 by sialic acid, neuramin-lactose, neuraminidase, and mAb to CD45RB, and by expression of the epitope on transfected psi 2 cells expressing exon B of CD45. The results suggest that the CZ-1 epitope is a post-translational modification expressed on a subpopulation of the CD45 molecules also expressing the B exon. Expression of the CZ-1 epitope was required for freshly isolated lymphocytes to respond to interleukin-2 (IL-2). Depletion of CZ-1+ cells by C' or by cell sorting of thymocytes or splenocytes eliminated the IL-2 responsive cells. The subpopulations of thymocytes and CD4+ splenocytes responding to IL-2 were exclusively within the small CZ-1+ subpopulation. mAb CZ-1 was also used to subdivide CD45+ and CD45RB+ splenocytes into IL-2-responsive and -nonresponsive subpopulations. The CZ-1 epitope was also expressed on virtually all lymphokine-activated killer cell precursors. These data, thus, indicate that cells responsive to IL-2 express this sialated modification of CD45.