U5A2-13, an antigen originally found on mouse NK-like T cells,is an early inducible cell surface antigen during lymphoid activation

We have previously reported a monoclonal antibody (mAb), U5A2-13 mAb, which originally recognizes a phenotypically and functionally similar population of natural killer (NK)-like T cells. In this study, we found that U5A2-13 antigen (U5A2-13) was expressed not only on NK-like T cells but also on T and B cells during activation. In contrast to the low levels of U5A2-13 on freshly harvested T and B cells, the activation of these cells by various stimuli resulted in high levels of expression of U5A2-13 in vitro and in vivo. Similar to CD69, U5A2-13 is also expressed in most mouse lymphoid cell lines but not in nonhematopoietic cells. U5A2-13 on T cells reached maximal expression by 24h after stimulation and returned to baseline levels after 3 days. However, U5A2-13 differed from CD69 since its expression profile was different on CD4(+)- and CD8(+)-activated T cells, phorbol ester-activated EL-4 cells, and activated splenocytes in CD69-deficient mice. In addition, immunoprecipitation study indicated that U5A2-13 is not identical to CD69. Importantly, the U5A2-13-positive population of CD4(+) T cells exhibited significant levels of cytokine producing activity upon stimulation. Overall, U5A2-13 is an early inducible cell surface antigen that could be involved in lymphocyte activation.     

Monoclonal antibodies to CD2 and lymphocyte function-associated antigen 3 inhibit human thymic epithelial cell-dependent mature thymocyte activation

Recent study of human thymocyte-thymic epithelial (TE) cell interactions has demonstrated that thymocytes bind to TE cells, and a consequence of this binding is the provision of accessory cell signals by TE cells for phytohemagglutinin (PHA)-induced mature thymocyte activation. In this paper we report on studies of the molecules involved in TE cell-dependent mature thymocyte activation. TE-thymocyte interactions necessary for PHA-induced thymocyte activation were inhibited by monoclonal antibodies against the cluster of differentiation (CD)2 antigen on thymocytes and lymphocyte function-associated (LFA)-3 antigen on TE cells. Inhibition of TE accessory cell signals by antibodies against CD2 (alpha CD2) and LFA-3 (alpha LFA-3) antigens occurred early on during thymocyte activation and prevented thymocyte interleukin 2 receptor expression. Further, alpha CD2 and alpha LFA-3 inhibited PHA-induced thymocyte activation in whole thymic explant cultures suggesting a significant role of the CD2 and LFA-3 antigens in thymocyte activation when accessory cell signals for PHA-induced thymocyte triggering were delivered by cells within an intact thymic microenvironment.     

Induction of CD69 activation molecule on human neutrophils by GM-CSF,IFN-gamma,and IFN-alpha

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes but it expression is induced in vitro on cells of most hematopoietic lineages, including neutrophils after stimulation with PMA or fMLP. In this study, we investigated whether CD69 expression on human neutrophils could be modulated by inflammatory or anti-inflammatory cytokines (IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, G-CSF, GM-CSF, TNF-alpha, TGF-beta, IFN-alpha, IFN-gamma). Resting neutrophils from healthy subjects did not express CD69 on the cell surface; moreover, a preformed intracellular pool of CD69 was not evident in these cells. CD69 was barely detectable on these cells after overnight incubation in medium while overnight incubation with GM-CSF, IFN-gamma or IFN-alpha significantly induced CD69 expression on neutrophils with GM-CSF appearing to be the most potent inducer. This induction was dependent on a new protein synthesis as it was significantly inhibited by cycloheximide (about 50% inhibition). CD69 cross-linking on GM-CSF-primed neutrophils sinergized with LPS and increased TNF-alpha production and secretion suggesting a role for CD69-positive neutrophils in the pathogenesis and maintenance of different inflammatory diseases.     

CD83 expression is a sensitive marker of activation required for B cell and CD4+ T cell longevity in vivo

CD83 is a surface marker that differentiates immature and mature human dendritic cell populations. Thymic epithelial cell expression of CD83 is also necessary for efficient CD4+ T cell development in mice. The altered phenotypes of peripheral B and CD4+ T cells, and the reduction of peripheral CD4+ T cells in CD83-/- mice, suggest additional functions for CD83. To assess this, a panel of mAbs was generated to characterize mouse CD83 expression by peripheral leukocytes. As in humans, activation of conventional and plasmacytoid murine dendritic cell subsets led to rapid up-regulation of CD83 surface expression in mice. In primary and secondary lymphoid compartments, a subset of B cells expressed low-level CD83, while CD83 was not detected on resting T cells. However, CD83 was prominently up-regulated on the majority of spleen B and T cells within hours of activation in vitro. In vivo, a low dose of hen egg lysozyme (1 microg) induced significant CD83 but not CD69 expression by Ag-specific B cells within 4 h of Ag challenge. Although B cell development appeared normal in CD83-/- mice, B and CD4+ T cell expression of CD83 was required for lymphocyte longevity in adoptive transfer experiments. Thus, the restricted expression pattern of CD83, its rapid induction following B cell and T cell activation, and its requirement for B cell and CD4+ T cell longevity demonstrate that CD83 is a functionally significant and sensitive marker of early lymphocyte activation in vivo.     

CD69 molecule in human neutrophils: its expression and role in signal-transducing mechanisms.

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes and it is constitutively expressed on thymocytes and platelets. Here we report its presence on neutrophils and on bone marrow-derived myeloid precursors. Indeed, promyelocytic cells are CD69+ on the cell membrane, while in resting neutrophils this molecule is located inside the cell. However, intracellular CD69 molecules are rapidly mobilized to the cell surface upon activation by PMA or fMLP. This translocation is independent on a new protein synthesis, as it is not inhibited by cycloheximide; furthermore, CD69 molecules are likely stored in a trans-Golgi structure since their expression is not affected by brefeldin A, a drug that blocks molecular trafficking from ER to Golgi vesicles. Immunoprecipitation of CD69 molecules either from activated neutrophils or from bone marrow cells showed that this protein has the same molecular size (28-34 kDa) as observed in platelets, T and B lymphocytes, and thymocytes. This similarity is reflected also in the functional role played by this molecule: in neutrophils as well as in lymphocytes and platelets, CD69 stimulation induced Ca2+ influx through cellular membrane; furthermore, the perturbation of the CD69 antigen on PMA-activated neutrophils enhances the lysozyme release, suggesting a role of this molecule in the regulation of granule exocytosis, probably through a Ca(2+)-dependent mechanism.     

Tyrosine phosphorylation of CD19 in pre-B and mature B cells.

Cross-linking of B cell surface immunoglobulins (sIg) results in activation of mature B cells and stimulates a molecular signaling mechanism for antigen-specific B cell expansion and differentiation. This signaling pathway is dependent on tyrosine (Tyr) phosphorylation and results in the activation of sIg-associated src family kinases and p72SYK. Rapid Tyr phosphorylation occurs on multiple protein substrates. Here we show that activation of B cells by cross-linking sIg results in an increase in Tyr phosphorylation of the lineage-restricted B cell surface antigen CD19, and show that it is a major substrate of activated Tyr kinase following sIg stimulation. Lower levels of constitutive CD19 Tyr phosphorylation occurred in most sIg+ mature B cell lines examined and in normal dense tonsillar B cells. We also find that when CD19 is Tyr-phosphorylated it becomes competent to interact with SH2 domains suggesting a mechanism whereby, following B cell activation, CD19 could be linked to intracellular signaling pathways. In sIg- pre-B cell lines, CD19 was expressed but was not constitutively phosphorylated on tyrosine. Upon CD19 cross-linking, Tyr phosphorylation of CD19 was induced in sIg- pre-B cell lines. CD19 cross-linking also directly induced Tyr phosphorylation of CD19 and other substrates in mature B cells. The ability of CD19 to signal in the absence of sIg expression may provide important stimulation in pre-B cell development.     

CD69 in resting and activated T lymphocytes. Its association with a GTP binding protein and biochemical requirements for its expression.

CD69 is a phosphorylated disulfide-linked homodimer that appears on the surface of human T, B cells and thymocytes in the early steps of activation; its molecular mass is 28 to 34 kDa under reducing conditions. This molecule is able to mediate positive signals to the lymphocytes as the anti-CD69 mAb (MLR3, AIM, Leu 23) in synergism with phorbol esters induce IL-2 production and proliferation of lymphocytes. Here we show that this molecule is associated to a GTP binding protein that is a substrate for Bordetella pertussis toxin. The relevance of CD69 in the activation process is also suggested by the broad range of signals able to modulate CD69 on T cells. In fact, not only the mitogens or the CD3-promoted activation, but also the alternative pathways mediated by CD2 or CD28 are accompanied by CD69 expression; moreover a very rapid and transient appearance of CD69 on the cell surface is observed also in response to a stimulus not specifically involved in T cell activation such as heat shock. Finally we demonstrate that CD69 is present in the cytoplasm of nonactivated T cells; accordingly its surface expression at the onset of activation is independent on a new RNA or protein synthesis.     

The radiation-sensitive costimulatory factors involved in B-cell-dependent T-cell activation by minor lymphocyte stimulating antigen

The regulation of CD28/B7 is important in T-cell activation. It has been argued that its aberrant expression is involved in the radiosensitivity of B-cell-stimulated T-cell response. Here, this possibility is studied in the mixed lymphocyte reaction (MLR) induced by minor lymphocyte-stimulating (Mls) antigen-presenting irradiated B cells. By using anti-CD28 antibody, the CD28/B7-2-, LFA-1/ICAM-1-dependent Mls-MLR was found to be restored. By flow cytometry, approximately 70% B cells were lost but with unaffected B7-2 expression, indicating that the moderate CD28 costimulation was caused by mortality of antigen presenting cells. Despite of costimulatory deficiency, T cells were shown primed. However, the expression of early activation markers CD25 and CD69, which was shown unaffected by B7/CD28 blocking, was found partially inhibited. To further understand the regulation, we examined the ICAM-1 expression, and found that it was again not altered on irradiated B cells. Thus, the radiation-induced rapid loss of resting B cells may be the basic mechanism causing insufficient costimulatory activity in radiosensitive B-T interaction. Furthermore, the presence of an element, other than B7-2, involving in controlling early T-cell response is suggested.     

Post-Transplant Immunosuppression: Regulation of the Efflux of Allospecific Effector T Cells from Lymphoid Tissues

A functional sphingosine-1-phosphate (S1P) receptor antagonist specifically inhibited the egress of activated allospecific T cells from draining popliteal lymph nodes in alloantigen-sensitised mice. The level of S1P receptor 1 (S1PR1) mRNA was similarly reduced 1 and 3 days after mitogenic activation of T cells. However, the response of these cells to the S1PR1-specific agonist SEW2871 was only reduced on the first day after T cell activation with normal receptor-mediated Akt-phosphorylation restored by day 3. Longitudinal analysis of CD69 expression showed that almost all T cells expressed this antigen on days 1 and 3 after activation. However, the absolute level of cell-surface expression of CD69 peaked on undivided T cells and was then halved by each of the first 3 cycles of mitosis. CD69-specific small interfering RNA (siRNA) reduced the maximal level of CD69 expression by undivided, mitogen-stimulated T cells. These cells retained their capacity to phosphorylate Akt in response to stimulation with SEW2871. These data show that S1P receptors are involved in controlling the egress of activated T cells from lymph nodes, and that S1PR1 function is regulated by the level of T cell surface CD69. They suggest a potential for augmentation of this process to deplete alloreactive effector cells after organ transplantation.     

alpha-galactosylceramide induces early B-cell activation through IL-4 production by NKT cells

alpha-Galactosylceramide (alpha-GalCer), a glycolipid antigen, specifically activates natural killer T (NKT) cells by a CD1d-restricted mechanism. In this work, we found that in vivo administration of alpha-GalCer resulted in the activation of B cells in addition to NKT cells, namely, alpha-GalCer administration caused upregulation of the early activation marker, CD69, on both NKT and B cells. In addition, expression of B7.2 and I-A(b) on B cells was greatly upregulated by alpha-GalCer. However, serum levels of IgE, IgG1, and IgG2a were not significantly changed within 48 h. In the present experiments, it was also demonstrated that the upregulation of CD69 expression by alpha-GalCer was strongly blocked by anti-IL-4 monoclonal antibody. Moreover, B-cell activation by alpha-GalCer was not observed in NKT-deficient mice. These results suggested that antigen-stimulated NKT cells might play a critical role not only in early defense mechanisms but also in early B-cell activation through IL-4 production.     

Expression of mouse and human fibronectin in mouse-human somatic cell hybrids

Using antisera to the human and mouse fibronectin (Large External Transformation Sensitive (LETS) protein), we studied the expression of mouse and human fibronectin in mouse-human somatic cell hybrids segregating either human or mouse chromosomes. The results of this study show that, in such somatic cell hybrids, mouse and human fibronectin are coexpressed. In addition, activation of the synthesis of mouse fibronectin was observed in somatic cell hybrids between mouse peritoneal macrophages, which do not produce detectable amounts of fibronectin, and either SV40-transformed human cells or cells derived from a human fibrosarcoma.     

Higher Sensitivity of Foxp3+ Treg Compared to Foxp3- Conventional T Cells to TCR-Independent Signals for CD69 Induction

T lymphocytes elicit specific responses after recognizing cognate antigen. However, antigen-experienced T cells can also respond to non-cognate stimuli, such as cytokines. CD4⁺ Foxp3⁺ regulatory T cells (Treg) exhibit an antigen-experienced-like phenotype. Treg can regulate T cell responses in an antigen-specific or bystander way, and it is still unclear as to which extent they rely on T cell receptor (TCR) signals. The study of the antigen response of Treg has been hampered by the lack of downstream readouts for TCR stimuli. Here we assess the effects of TCR signals on the expression of a classical marker of early T cell activation, CD69. Although it can be induced following cytokine exposure, CD69 is commonly used as a readout for antigen response on T cells. We established that upon in vitro TCR stimulation CD69 induction on Foxp3⁺ Treg cells was more dependent on signaling via soluble factors than on TCR activation. By contrast, expression of the activation marker Nur77 was only induced after TCR stimulation. Our data suggest that Treg are more sensitive to TCR-independent signals than Foxp3^- cells, which could contribute to their bystander activity.     

Assignment of gene(s) coding for antigen defined by monoclonal antibody 2B2

A mouse monoclonal antibody (2B2) recognizes an antigen which is present on most human peripheral blood leukocytes but is absent from most proliferating cells. The antibody precipitated two surface-labeled membrane glycopolypeptides with molecular weights of 86,000 and 145,000, and it was strongly mitogenic to normal human lymphocytes. Somatic cell hybrids have been used for assigning the genes coding for these membrane glycoproteins to human chromosome 21. The assignment was based on correlation of antigen expression on mouse-human T-lymphocyte hybrids with the presence of human chromosomes in the same hybrid clones.     

Different sensitivity to interleukin 4 of interleukin 2- and interferon alpha-induced CD69 antigen expression in human resting NK cells and CD3+, CD4-, CD8- lymphocytes.

The effect of rIL-4 on CD69 antigen expression induced by rIL-2 or by rINF-alpha on human resting NK cells and CD3+, CD4-, CD8- T lymphocytes has been investigated. rIL-4 drastically inhibited CD69 antigen expression induced by rIL-2 in both cell types. In contrast, rIL-4 did not alter rINF-alpha-induced CD69 antigen expression. Consistent results were obtained evaluating the cytolytic activity of NK cells against the Raji target cell line: rINF-alpha-induced lytic activity was not inhibited by rIL-4, while rIL-2-induced lytic activity was drastically inhibited. Proliferative activity of NK cells induced by rIL-2, in contrast, was only slightly reduced by rIL-4. rIL-4 did not alter the expression of the beta chain of IL-2 receptor, evaluated in NK cells by indirect immunofluorescence. Expression of the alpha chain of IL-2 receptor could not be detected in NK cells by indirect immunofluorescence. It can therefore be suggested that the selective inhibitory effect of rIL-4 on rIL-2-induced activation of NK cells is not mediated by downregulation of alpha and beta chains of IL-2 receptor.     

Mesenchymal stem cells promote the sustained expression of CD69 on activated T lymphocytes: roles of canonical and non-canonical NF-κB signalling

Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3(+) T cells were activated and cultured in the presence or absence of MSCs. CD4(+) cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69(+) cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69(+) cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling.     

CD1d-independent activation of mouse and human iNKT cells by bacterial superantigens

Invariant NKT (iNKT) cells are infrequent but important immunomodulatory lymphocytes that exhibit CD1d-restricted reactivity with glycolipid Ags. iNKT cells express a unique T-cell receptor (TCR) composed of an invariant α-chain, paired with a limited range of β-chains. Superantigens (SAgs) are microbial toxins defined by their ability to activate conventional T cells in a TCR β-chain variable domain (Vβ)-specific manner. However, whether iNKT cells are directly activated by bacterial SAgs remains an open question. Herein, we explored the responsiveness of mouse and human iNKT cells to a panel of staphylococcal and streptococcal SAgs and examined the contribution of major histocompatibility complex (MHC) class II and CD1d to these responses. Bacterial SAgs that target mouse Vβ8, such as staphylococcal enterotoxin B (SEB), were able to activate mouse hybridoma and primary hepatic iNKT cells in the presence of mouse APCs expressing human leukocyte antigen (HLA)-DR4. iNKT cell-mediated cytokine secretion in SEB-challenged HLA-DR4-transgenic mice was CD1d-independent and accompanied by a high interferon-γ:interleukin-4 ratio consistent with an in vivo Th1 bias. Furthermore, iNKT cells from SEB-injected HLA-DR4-transgenic mice, and iNKT cells from SEB-treated human PBMCs, showed early activation by intracellular cytokine staining and CD69 expression. Unlike iNKT cell stimulation by α-galactosylceramide, stimulation by SEB did not induce TCR downregulation of either mouse or human iNKT cells. We conclude that Vβ8-targeting bacterial SAgs can activate iNKT cells by utilizing a novel pathway that requires MHC class II interactions, but not CD1d. Therefore, iNKT cells fulfill important effector functions in response to bacterial SAgs and may provide attractive targets in the management of SAg-induced illnesses.     

Costimulatory Effect of Fas in Mouse T Lymphocytes

To induce proper immune responses, T lymphocytes require two types of stimuli, antigen-specific and costimulatory signals. Among costimulatory molecules, CD28-engagement promotes the survival and proliferation of both naive and memory T cells. In addition, it is now believed that Fas may play a role in T cell activation in the human system. It is, however, controversial whether Fas can act as a costimulatory signal in the murine system. Thus, we investigated fundamental differences in the capacity to induce proliferation of T cells between Fas and CD28 in mice. Fas-mediated T cell proliferation was observed only with a full mitogenic dose of anti-CD3 antibodies, whereas CD28 engagement was able to enhance T cell proliferation in the presence of a suboptimal level of anti-CD3 antibody. Furthermore, Fas-engaged T cells showed faster response in the upregulation of CD25 and CD69 expression than CD28-engaged ones. Here, we report that Fas might play a role in mature T cell activation in the mouse system through a different mechanism from that in CD28 costimulation.