Expression Of Heat-stable Antigen Research Articles

LKB1 regulates TCR-mediated PLCγ1 activation and thymocyte positive selection

The serine/threonine kinase LKB1 is a tumour suppressor that regulates cell growth, polarity, and proliferation in many different cell types. We previously demonstrated that LKB1 controls thymocyte survival via regulation of AMPK activation. In this study, we show that LKB1 was also involved in thymocyte positive selection through regulation of T cell receptor (TCR) signalling. Both Lck-Cre- and CD4-Cre-mediated deletion of LKB1 impaired the generation of mature CD4 and CD8 single positive (SP) thymocytes that might have resulted from the attenuated tyrosine phosphorylation of phospholipase C-γ 1 (PLCγ1) in the absence of LKB1. We found that LKB1 was directly phosphorylated by Lck at tyrosine residues 36, 261, and 365 and predominately interacted with LAT and PLCγ1 following TCR stimulation. Loss of LKB1 led to impaired recruitment of PLCγ1 to the LAT signalosome. Correlatively, LKB1-deficient thymocytes failed to upregulate lineage-specifying factors, and to differentiate into SP thymocytes even if their impaired survival was rescued. These observations indicated that LKB1 is a critical component involved in TCR signalling, and our studies provide novel insights into the mechanisms of LKB1-mediated thymocyte development.

The heat-stable antigen determines pathogenicity of self-reactive T cells in experimental autoimmune encephalomyelitis.

Induction of myelin-specific CD4 T cells is a pivotal event in the development of experimental autoimmune encephalomyelitis (EAE). Other checkpoints in EAE pathogenesis have not been clearly defined, although multiple genetic loci are known to influence EAE development. We report here that targeted mutation of the heat-stable antigen (HSA) abrogates development of EAE despite a complete lack of effect on induction of autoimmune T cells. To test whether T-cell expression of HSA is sufficient, we created transgenic mice in which HSA is expressed exclusively in the T-cell lineage. We found that these mice remain resistant to EAE induction. Adoptive transfer studies demonstrate that both T cells and non-T cells must express HSA in order for the pathogenic T cells to execute their effector function. Moreover, HSAIg, a fusion protein consisting of the extracellular domain of the HSA and the Fc portion of immunoglobulin, drastically ameliorates the clinical sign of EAE even when administrated after self-reactive T cells had been expanded. Thus, identification of HSA as a novel checkpoint, even after activation and expansion of self-reactive T cells, provides a novel approach for immunotherapy of autoimmune neurologic diseases, such as multiple sclerosis.

Kinetics and differential expression of heat-stable antigen and GL7 in the normal and Toxoplasma gondii-infected murine brain.

The co-expression of various cell surface molecules by cells of the nervous system and the immune system is a remarkable feature. To identify novel molecules that are shared between cells of the neural and hematopoietic lineage, the expression and regulation of heat-stable antigen (HSA, CD24, nectadrin) and GL7, two hematolymphoid differentiation antigens that are involved in antigen presentation, cell adhesion, signal transduction and activation, was studied in the adult normal and Toxoplasma gondii-infected murine brain by immunohistochemistry and flow cytometry of isolated cerebral leukocytes. In the normal brain ependymal cells, plexus macrophages and a fraction of blood vessel endothelial cells were HSA positive (+), whereas the choroid plexus epithelium was GL7+. This basal expression of HSA and GL7 was not further modified on these cell populations in Toxoplasma encephalitis (TE). In acute and chronic TE, HSA and GL7 were strongly induced on resident brain cells, and activated astrocytes were the predominant HSA+ and GL7+ cell type. FACS analysis additionally identified a minor fraction of HSA+ microglia in the normal brain with a small, but significant increase in TE. The differential expression pattern of HSA and GL7 on distinct resident cell populations in various anatomic compartments of the normal adult brain and their up-regulation in TE may indicate that their intracerebral role is diverse and may include both immunological as well as non-immunological functions.

Delta -tetrahydrocannabinol suppresses macrophage costimulation by decreasing heat-stable antigen expression

Delta 9-tetrahydrocannabinol (THC) suppresses several immunologic functions of macrophages. The costimulatory activity of a THC-exposed macrophage hybridoma was investigated by its ability to elicit interleukin-2 secretion by a helper T cell hybridoma activated with immobilized monoclonal anti-CD3 antibody. THC added at culture initiation inhibited the T cell response in a dose-dependent manner. When the macrophages were fixed with paraformaldehyde before culture, THC had no effect on T cell stimulation. However, macrophages, which were preincubated with THC and then fixed, were impaired in delivering costimulatory signals to T cells cultured without THC. The drug’s inhibitory effect on macrophage costimulatory activity was reversible. THC exposure also decreased macrophage expression of heat-stable antigen (HSA). Antibody blocking experiments showed that HSA expressed on the macrophages provided an important costimulatory signal, whereas B7-1 and B7-2 molecules had a minor role. Treatment of the macrophages with phosphatidylinositol-specific phospholipase C cleaved HSA, but not the transmembrane B7 molecules, from the cell surface. Similar to THC, enzyme treatment significantly diminished macrophage costimulatory activity, which was also reversible. After drug or enzyme removal, HSA expression returned to the control level by 4 h. Therefore, THC suppresses macrophage costimulatory activity by diminishing cell surface expression of HSA.

Regulation of the stability of heat-stable antigen mRNA by interplay between two novel cis elements in the 3' untranslated region.

The heat-stable antigen (HSA) is a costimulatory molecule for T-cell activation. Its expression is strictly regulated during lymphocyte development and differentiation. Recent studies using HSA-transgenic mice have demonstrated that this regulated expression is critical for normal development of T and B lymphocytes. However, the mechanisms that control the expression of HSA are largely unknown. HSA mRNA is comprised of a 0.23-kb open reading frame and a 1.5-kb 3' untranslated region (3'UTR). The function of the long 3'UTR has not been addressed. Here we investigate the role of the 3'UTR of HSA mRNA. We show that a 160-bp element, located in the region of nucleotides 1465 to 1625 in the 3'UTR of HSA mRNA, promotes RNA degradation and that this effect is neutralized by a 43-bp fragment approximately 1 kb upstream of the negative cis element. Both positive and negative cis elements in the HSA mRNA are distinct from other sequences that are known to modulate mRNA stability. These results provide direct evidence that the interplay between two novel cis elements in the 3'UTR of HSA mRNA determines cell surface HSA expression by modulating its RNA stability.

The Porphyrin Photosensitizer Photofrin® Elevates Murine Splenic Erythropoiesis

Changes occurring within the spleens of the genetically distinct DBA/1 and DBA/2 mouse strains produced by the photosensitizer Photofrin® in the absence of direct light exposure were analyzed. Photofrin® significantly increased spleen weight, cellularity and erythroid progenitor levels in both mouse strains tested. The expression of heat stable antigen (HSA), a marker present upon different immature leukocytes as well as certain fully-differentiated cell types including erythrocytes, was increased in the spleens of mice given Photofrin®. It was shown for Photofrin®-injected DBA/1 mice that a spleen cell population which expressed high levels of HSA also bound the iron transport protein transferrin. Photofrin® increases the splenic demand for iron by promoting erythropoietic activity within the tissue.

Homotypic interaction of the heat-stable antigen is not responsible for its co-stimulatory activity for T cell clonal expansion.

The heat-stable antigen (HSA) is an important co-stimulatory molecule on antigen-presenting cells (APC). However, the receptor on T cells that receives the co-stimulatory signal from HSA has not been identified. Because the HSA is transiently expressed on T cells after the T cell receptor/CD3 complex is engaged, and because it can bind to itself in a homotypic fashion, it has been proposed that homotypic interaction of HSA is responsible for its co-stimulatory activity. Here we test this hypothesis using mice that have a targeted mutation of the HSA gene, as well as novel transgenic mice that constitutively express HSA on T cells. We show that HSA-deficient T cells remain responsive to co-stimulation by HSA. Furthermore, constitutive expression of HSA does not enhance T cell response to co-stimulatory by HSA. Taken together, our results demonstrate that homotypic interaction of HSA is not responsible for co-stimulation mediated by HSA expressed on APC.

Interaction of murine dendritic cells with collagen up-regulates allostimulatory capacity, surface expression of heat stable antigen, and release of cytokines.

In vivo, dendritic cells (DC) reside in direct proximity to extracellular matrix (ECM) proteins. Because ECM proteins affect morphology and function of a number of cell types, this study investigated potential effects of ECM proteins on functional properties of DC. DC were generated from murine bone marrow cultures, supplemented with granulocyte-macrophage colony-stimulating factor, and subsequently cultured on tissue culture plates coated with various ECM proteins. Among the ECM proteins tested, collagen (COL) up-regulated the T cell stimulatory capacity of DC. This effect was accompanied by sustained surface expression of the co-stimulatory molecule heat stable antigen on DC and by enhanced release of interleukin-1 and interleukin-6, respectively. Because fibronectin or solubilized COL were unable to cause similar changes in DC phenotype or function, we conclude that adherence to COL interferes specifically with DC function. These data suggest that ECM proteins may be involved in regulation of DC phenotype as well as in their functional activation.

Hematopoietic Supportive Functions of Mouse Bone Marrow and Fetal Liver Microenvironment: Dissection of Granulocyte, B-Lymphocyte, and Hematopoietic Progenitor Support at the Stroma Cell Clone Level

We dissected the functions of the microenvironment of bone marrow (BM) and fetal liver (FL) at the cellular level by cloning individual stromal calls and characterizing their phenotypical and functional features. Stromal cell clones derived from FL are large in size (mean forward light scatter intensity [mFSC] of 450), express the surface antigen Thy-1 but not Sca-1 and 6 out of 6 are able to differentiate into fat accumulating adipocytes. BM derived stromal cell clones are either small (mFSC of 250) or large (mFSC of 450), express Sca-1 but not Thy-1 and only 2 out of 7 differentiate towards adipocytes. Heterogeneity in terms of vascular adhesion molecule-1, intracellular adhesion molecule-1 and heat stable antigen expression was found among the different cell clones. Functional assays using long- and short-term cocultures of stromal and hematopoietic calls revealed: (1) the capacity of 8 out of 12 stromal cell clones to support the expansion of primitive hematopoietic progenitors (colony forming unit spleen day 12) more than 10 weeks. Fat accumulation but not expression of stem cell factor by stromal cells did correlate with this supportive function. (2) Better support of granulocyte maturation and proliferation by BM- compared to FL-derived stromal cell clones. However, stromal cell clones from both organs expressed macrophage-colony stimulating factor. (3) The ability of 4 out of 12 stromal cell clones (derived from both, FL and BM) to support the expansion of Interleukin-7 dependent pre-B cells from the BM. Pre-B cell growth stimulating factor was not restricted to supporters. (4) Mutual exclusiveness of myeloid and lymphoid support in that a given stromal cell clone supported either pre B-cell or granulocyte expansion. Experiments comparing the support of BM- and FL-derived hematopoietic progenitors showed identical responses of late (B220+/c-kit-) but strikingly different responses of early (B220+/c-kit+) pre-B cells, revealing different proliferation requirements for FL- versus BM- derived early pre-B cells in vitro.

Heat-stable antigen is expressed by murine keratinocytes and delivers costimulatory signals in T-cell activation.

Heat-stable antigen (HSA), expressed by various antigen-presenting cells (APC), has been described as a costimulatory molecule for CD4+ T cells. Recently, we observed that HSA also serves as an important costimulatory molecule on epidermal Langerhans cells (LC). During these studies, low levels of HSA staining were also detected on normal murine keratinocytes (KC). To investigate whether HSA also is involved in T-cell activation by KC, normal murine KC or the spontaneously transformed KC cell-line PAM 212 were treated with PDB or PMA to induce HSA-expression. FACS analyses showed induction of HSA expression on normal murine KC, as well as PAM 212 cells. In functional assays PDB or PMA-treated normal or transformed KC were far more potent inducers of primary allogeneic T-cell responses than untreated KC. Addition of anti-HSA-specific mAb 20C9 specifically inhibited the costimulatory activity of KC, an effect that was even more pronounced when CTLA-4Ig was added to the cultures. Cleavage of HSA on KC surfaces by a phosphoinositol-specific phospholipase C (PI-PLC) also significantly inhibited the costimulatory capacity of KC for naive CD4+ T cells. In aggregate, our data indicate that expression of HSA on activated KC contributes to the capacity of these cells to induce proliferation of allogeneic T cells.

Mechanism of enhanced antigen presentation by B cells activated with anti-mu plus interferon-gamma: role of B7-2 in the activation of naive and memory CD4+ T cells.

B cells activated with anti-mu antibody plus interferon (IFN)-gamma exerted strong antigen presentation activity for T cell proliferation. The enhanced antigen presentation function was shown to be due to the increase in B7-2 expression. When B cells were stimulated with anti-mu, expression of MHC major histocompatibility complex class II, heat-stable antigen (HSA), ICAM-1 and B7-2 was increased. The presence of IFN-gamma further augmented the expression of B7-2 on anti-mu-stimulated B cells. B7-1 was not expressed on B cells under these conditions. The participation of B7-2 in the elicitation of the proliferative response of T cells was confirmed by the inclusion of anti-B7-2 antibody in cultures. The enhanced expression of either HSA or ICAM-1 was shown not to play a major role in the increased B cell antigen presentation capacity. The major T cell population responding to this activated B cell antigen presentation was shown to be CD44low naive CD4+ T cells, whereas CD45RBlow memory CD4+ T cells responded only weakly. The difference in proliferative responses between naive and memory CD4+ T cells was explained by the different efficiency in IL-2 production of these cell populations in response to antigen presentation by B cells activated by anti-mu plus IFN-gamma. These results suggest that IFN-gamma plays an important role in recruitment of naive T cells for an immune response.

B-cell maturation in chimaeric mice deficient for the heat stable antigen (HSA/mouse CD24)

The murine differentiation marker heat stable antigen (HSA) is a GPI-anchored surface glycoprotein showing strong expression on immature B- and T-lymphocytes and gradually reduced expression during maturation. Although HSA has been suggested to be involved in adhesion and/or signalling, its function has not been clearly demonstrated so far. In order to elucidate the function of HSA, we analysed chimaeric mice that were generated by targeted disruption of both HSA alleles in ES cells. These mice contain normal numbers of peripheral B-cells and normal serum IgM and IgG titres of ES cell-derived allotype, demonstrating that HSA expression on B-cells is not an absolute requirement for their maturation. However, a reduction in immature B-cells in the bone marrow and an altered degree of bone marrow and blood chimaerism suggest that HSA expression influences the maturation of B-cells.

The heat-stable antigen can alter very late antigen 4-mediated adhesion.

The integrin very late antigen, (VLA-4) alpha 4 beta 1 and its counter receptor vascular cell adhesion molecule 1 (VCAM-1) are involved in B cell maturation and pre-B cell attachment to bone marrow stroma cells. We have analyzed whether heat-stable antigen (HSA), a marker for immature leukocytes, is involved in such cell adhesion phenomena. HSA is a glycolipid-anchored, highly glycosylated surface protein differentially expressed on cells during the maturation of both the hematopoietic and nervous systems. We found that pre-B cells lacking HSA (due to targeted disruption of both alleles) can still bind via VLA-4 to tumor necrosis factor alpha-stimulated endothelioma cells. This binding, however, cannot be blocked by an anti-VCAM-1 antibody. Restoration of HSA expression restores the inhibitable VCAM-1 binding. We also found that pre-B cells lacking HSA did not bind to the FN40 fragment of fibronectin but reexpression of HSA restored VLA-4-mediated binding to fibronectin. Thus, expression of HSA on pre-B cells modifies the binding specificity of VLA-4 for two known ligands.

Induction of B cell costimulatory function by recombinant murine CD40 ligand.

T cell-dependent regulation of B cell growth and differentiation involves an interaction between CD40, a B cell surface molecule, and the CD40 ligand (CD40L) which is expressed on activated CD4+ T cells. In the current study, we show that recombinant membrane-bound murine CD40L induces B cells to express costimulatory function for the proliferation of CD4+ T cells. CD40L- or lipopolysaccharide (LPS)-activated, but not control-cultured B cells were strong costimulators of anti-CD3 or alloantigen-dependent T cell responses. The molecular interactions responsible for the increased costimulatory functions were examined by analyzing the activated B cells for changes in the expression of two costimulatory molecules, B7 and heat-stable antigen (HSA), as well as by the use of antagonists of B7 and HSA (CTLA4.Fc and 20C9, respectively). The expression of both B7 and HSA was enhanced on B cells activated with LPS. As observed in previous studies, the costimulatory activity of the LPS-activated B cells was dependent on both B7 and HSA and was completely inhibited in the presence of a combination of CTLA4.Fc and 20C9. In contrast, activation of B cells with CD40L induced the expression of B7 but did not enhance the expression of HSA. In addition the costimulatory activity of the CD40L-activated B cells was partially, but not completely, inhibited by the combination of CTLA4.Fc and 20C9. These results demonstrate that CD40L regulates costimulatory function of B cells in part by inducing the expression of B7 and suggest that CD40L-activated B cells express an additional costimulatory activity that is not associated with LPS-activated B cells.

Defective development of thymocytes overexpressing the costimulatory molecule, heat-stable antigen.

Heat-stable antigen (HSA) is a small, glycosyl phosphatidylinositol-anchored protein that can act as a costimulatory molecule for antigen-dependent activation of helper T cells. In addition to being expressed on antigen-presenting B cells, HSA is also expressed during the initial stages of T cell development in the thymus. HSA levels are very high on immature CD4-, CD8- double negative thymocytes, but are reduced on CD4+, CD8+ double positive cells undergoing selection in the thymus, and are entirely eliminated when these cells differentiate into immunologically competent CD4+ or CD8+ single positive T cells. To examine the potential roles of this molecule in T cell development and selection, we generated transgenic mice in which HSA was highly expressed on all classes of thymocytes. The consequence of deregulated HSA expression was a pronounced reduction in the numbers of double positive and single positive thymocytes, whereas the numbers of their double negative precursors were largely unaffected. These results demonstrate that downregulation of HSA expression at the double positive stage is a critical event in thymocyte development. The depletion of thymocytes resulting from HSA overexpression begins at the same time as the onset of negative selection, suggesting that HSA may provide signals that contribute to determining the efficiency of this process.

Development of gamma delta T cells in the adult murine thymus.

The development of T cells belonging to the gamma delta lineage is not well understood. We have analyzed the cells in the adult murine thymus which express the gamma delta TcR on the surface in order to learn more about this process. Our data demonstrate a number of clear subpopulations of gamma delta expressing cells in the thymus based on the expression of Thy-1 and HSA (heat-stable antigen). Only one of these subpopulations, the one expressing both Thy-1 and HSA, contains dividing cells or has a significant rate of turnover. Together with the fact that emigrant gamma delta cells are HSA+Thy-1+, this suggests that this thymic subpopulation is the sole, or major, source of exported cells. However, the turnover of cells from this population is 5 x 10(4) - 10 x 10(4) cells per day, while previous estimates of the rate of export of gamma delta cells are in the order of 10(4) cells per day. Furthermore the V gamma profile of recent gamma delta+ emigrants differs from that of the thymic HSA+Thy-1+ cells. This raises the possibility that only a selected subpopulation of the thymic gamma delta+HSA+Thy-1+ population is exported, and that some gamma delta cells may die in situ in the thymus. The function of the other gamma delta thymic subpopulations, which are turning over very slowly or not at all, (i.e. the HSA-Thy-1- and HSA-Thy-1+ subpopulations) remains unclear.

Direct demonstration of MHC class II surface expression on murine pre-B cells.

Using multi-parameter flow cytometry, MHC class II (Ia) antigens were found to be expressed at readily detectable levels on the majority of surface IgM- B lineage cells in the bone marrow of adult mice. Approximately 90% of the pre-B cells (B220+IgM-) in BALB/c bone marrow (BM) were MCH class II positive, while in C57BL/6 this figure was somewhat less, approximately 60%. Analysis of adult BM for the correlated expression of Ia both with heat stable antigen (HSA) and BP-1 on B lineage cells revealed in both cases a small fraction of cells expressing either the HSA or BP-1 but lacking Ia. This result suggests that the onset of expression of both HSA and BP-1 precede that of Ia in B cell development. When the B220loCD43+ pro-B cell compartment from BALB/c BM was analysed for the expression of Ia very few, if any, cells were found to be positive. Of the remaining B220+ cells, > 90% express detectable Ia. Thus the onset of Ia expression would appear to be soon after the pro-B cell compartment. The results presented here resolve the previous discrepancy between human and murine pre-B cell differentiation of when Ia is first expressed, and additionally, by identifying a new pre-B cell antigen, allow for the refinement of existing schemes of murine B cell development.

Subpopulations of mature murine thymocytes: Properties of CD4 −CD8 + and CD4 +CD8 − thymocytes lacking the heat-stable antigen

The heat-stable antigen (HSA), recognized by the monoclonal antibodies M1/69, B2A2, and J11d, is low or absent on the surface of most murine peripheral T cells but present on all but 3% of thymocytes. The CD4 −CD8 + and CD4 +CD8 − or “single positive” thymic populations may be divided into further subgroups based on surface HSA expression. One group, CD4 −CD8 + and expressing very high levels of HSA (HSA ++), is an immature, T cell antigen receptor (TcR) negative, outer cortical blast cell. However, a further subdivision of CD4 −CD8 + and CD4 +CD8 − single positives may be made, into those negative to low for HSA (HSA −) and those expressing moderate amounts of HSA (HSA +). The proportion of HSA − single positives is low in the thymus of young mice, whereas the proportion of HSA + single positives is similar to that of the adult. Both the HSA − and the HSA + subsets of single positive thymocytes from adult mice are CD3 + and express the normal peripheral T cell incidence of Vβ8 determinants on the TcR. On stimulation with concanavalin A in limit-dilution culture both HSA − and HSA + subsets of single positive thymocytes give a high frequency of proliferating clones, and the clones from both HSA − and HSA + subsets of CD4 −CD8 + thymocytes are cytotoxic. Thus both HSA − and HSA + single positive thymocytes are functionally mature. The HSA − subsets of single positive thymocytes differ from the HSA + subsets in being slightly larger in size, in expressing higher levels of MEL-14, in binding more peanut agglutinin, and in including a proportion of cells expressing high levels of the Pgp-1 glycoprotein. It is suggested that HSA − CD4 −CD8 + and HSA − CD4 +CD8 − thymocytes are more mature than their HSA + counterparts, and might represent a previously activated or “memory” thymic subpopulation.

The surface phenotype of activated T lymphocytes.

The surface phenotype of T cells reflects both their relative maturity and their activation state. To determine the pattern of surface markers characteristic of activated T cells, purified mature T cells were stimulated in vitro for periods of 0.5-5 days with Concanavalin-A (Con-A) or phorbol myristic acetate (PMA) and ionomycin, fluorescein-labelled with monoclonal antibodies, then analysed by flow cytometry. The level of expression of the function-associated antigens CD4 (L3T4) and CD8 (Ly-2) decreased transiently early after activation with PMA/ionomycin, but not after stimulation with Con-A. Both stimuli caused a small drop in the level of CD3 and the T cell antigen receptor (TCR). At no time was CD3, CD4 or CD8 completely lost from the surface. Following activation Pgp-1, the interleukin-2 (IL-2) receptor (as detected by the monoclonal antibody 7D4) and the peanut agglutinin (PNA) receptor were gained by a proportion of cells, MEL-14 was lost by a proportion of cells, and no change was observed in the expression of heat stable antigen. Thy-1 or Ly-1. From the present data no evidence has been found for the generation of the 'immature', CD4- CD8- phenotype found in the thymus by activation of mature T cells. During T cell development, however, changes in expression of Pgp-1, MEL-14, the IL-2 receptor and the PNA receptor may be associated with activation, rather than differentiation per se.