Regulation of murine B cell Thy-1 expression by IL-4, IFN-gamma, and CD4+ T cell subsets

Interleukin 4 (IL-4) induces the expression of membrane Thy-1 on the vast majority of lipopolysaccharide (LPS)-stimulated normal murine B cells in vitro. This induction is inhibited by interferon-gamma (IFN-gamma). IL-4 and IFN-gamma are required late in culture to effect maximal induction and inhibition of Thy-1 expression by LPS- or LPS + IL-4-stimulated B cells, respectively. IFN-gamma suppresses IL-4-induced Thy-1 expression by inhibiting the induction of steady-state levels of Thy-1-specific mRNA. Three distinct CD4+ Th2 clones, through their release of IL-4, induce B cells to express high levels of Thy-1, by 24 hr, in striking contrast to the 3 days required to induce Thy-1 expression after stimulation with LPS and IL-4. This induction is abrogated by the addition of IFN-gamma. B cells stimulated with three distinct Th1 clones (IFN-gamma- and IL-2-producing) exhibit a modest, non-IL-4-dependent, expression of Thy-1. In contrast to intrinsic expression of Thy-1 by Th2-stimulated B cells. Thy-1 expressed by Th1-stimulated B cells is acquired, having the allotype specificity of the stimulating T cell.     

Characterization of the proliferative response of a CD4-8- thymic T lymphoma cell line to stimulation by thymic cellular elements

E710.2 is a cloned T cell line that was isolated from an AKR/J thymic tumor. This clone expresses Thy-1, heat-stable Ag, and the CD3/TCR complex but does not express CD4 or CD8. When the E710.2 cell line is injected into syngeneic mice, it grows as a malignant tumor in lymphoid organs and the thymus. In contrast, this cell line does not grow in vitro under standard culture conditions. This latter property allowed us to analyze the in vitro responsiveness of this CD4-CD8- cell line to stimulation by pharmacologic agents and cellular elements from the spleen and thymus. E710.2 cells proliferate when stimulated with phorbol esters or when cocultured with thymocytes or splenocytes. We could not detect soluble stimulatory factors in cultures of E710.2 and/or lymphoid cells, suggesting that cell contact might be required for this response. The stimulatory activity in thymus and spleen appears to be broadly expressed, because all cell subsets that were examined from these tissues stimulate this cell line. The stimulation of E710.2 cells is not MHC-restricted and is not inhibited by anti-MHC mAb. Furthermore, the responsiveness of these cells is not decreased when the TCR/CD3 complex is modulated from the cell surface. Similarly, TCR/CD3-deficient E710.2 variant clones retain their responsiveness to thymic and splenic cell stimulation. These findings suggest that there is a TCR-independent pathway of activation in E710.2 that is stimulated by a broadly expressed, non-MHC-encoded molecules(s).     

A murine thymic stromal cell line which may support the differentiation of CD4-8- thymocytes into CD4+8- alpha beta T cell receptor positive T cells.

A fibroblastoid cell line TSt-4 was established from fetal thymus tissue of C57BL/6 mice. When fetal thymus (FT) cells or CD4-8- (DN) cells of adult thymuses were cultured on the monolayer of TSt-4, a considerable proportion of lymphocytes expressed CD4 or both CD4 and CD8 within 1 day, and the CD4+CD8- cells were maintained further while the CD4+8+ cells disappeared by Day 5. A large proportion of cells generated from DN cells but not FT cells was shown to express CD3 and T cell receptor alpha beta. Addition of recombinant interleukin (IL)-7 into the cultures resulted in a marked increase of cell recovery without virtual change in differentiation process of alpha beta lineage. The present work strongly suggests that thymic fibroblasts play an important role in T cell differentiation and IL-7 contributes to supporting proliferation of differentiated cells.     

Peripheral CD4+ T cell maturation recognized by increased expression of Thy-1/CD90 bearing the 6C10 carbohydrate epitope.

The SM6C10 IgM autoantibody recognizes a surface determinant, 6C10, that is highly expressed on all immature thymocytes. In contrast, its expression on peripheral T cells appears developmentally regulated, i.e., absent from most naive T cells in spleen of neonatal mice, but expressed on 40-80% of naive CD4+ T cells in adult. In this paper, we demonstrate that SM6C10 recognizes a carbohydrate epitope on the Thy-1 glycoprotein using immunoprecipitation analysis, by binding to affinity-purified Thy-1 in an ELISA, and by sensitivity to N-glycosidase-F treatment. Retroviral Thy-1 gene transduction experiments into Thy-1- variant T cell lines and a pro-B cell line provide evidence that 6C10 glycosylated Thy-1 expression is not restricted to T cells but depends on the recipient cell. Therefore, differences in 6C10 levels among Thy-1+ T cells in mice likely reflect developmental regulation of posttranslational modification of the Thy-1 glycoprotein. The ability of naive CD4+ T cells to respond to anti-Thy-1 stimulation increases from neonate to adult, and 6C10- naive cells from adult mice respond poorly compared with 6C10+ cells, similar to the cells in neonatal mice. These results suggest that there is functional maturation by peripheral CD4+ T cells that coincides with 6C10 glycosylated Thy-1 up-regulation, and natural autoantibody recognizes this 6C10 carbohydrate epitope.     

The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner

Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^ 8^ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.     

Thy-1: a lymphoid cell subset marker capable of delivering an activation signal to mouse T lymphocytes

Monoclonal anti-Thy-1 antibodies are capable of activating mouse T cells in the absence of an antigen-specific signal. Therefore, Thy-1 appears to be connected to an alternative signal transduction pathway, operative in thymocytes as well as in neuronal cells, since this molecule is also present on brain. Biochemical data have shown that this molecule is differentially glycosylated with respect to its cellular distribution. Structure and sequence comparisons revealed a strong homology with the immunoglobulin primordial domain. In addition, the Thy-1 glycoprotein has the particularity of being anchored to the membrane via a glycophospholipid tail. Gene transfer experiments in different cell types have been performed to analyze the mechanism of the Thy-1 pathway of activation.     

A hypotetraploid human T lymphoid cell line established by cell fusion.

A human T lymphoid cell line was established by cell hybridization technique from peripheral blood leucocytes of a patient with Sezary syndrome. The cells beared the surface antigens of human T lymphocyte specificity as demonstrated by immune cytolysis tests, but did not form E rosettes with sheep red blood cells. Isozyme patterns of enzymes in this line such as lactate dehydrogenase, glucose 6-phosphate dehydrogenase and esterase were of human type. The line had 79 chromosomes in modal number. This case supports the proposal that the production of tetraploids is favourable for establishment of cell lines.     

Induction of CD4(+)CD25(+)Foxp3(-) regulatory T cells by Thy-1 stimulation of CD4(+) T cells

Thy-1 (CD90) on mouse T cells has been reported to have both T-cell activating and regulatory roles. In this study, we show that monoclonal antibody (mAb)-mediated crosslinking of Thy-1 on CD4(+) mouse T-cells-induced regulatory T (T(reg)) cells that expressed CD25, CD39 and glucocorticoid-induced tumor necrosis factor receptor family-related gene, but not CD73, CD122 or Foxp3. The proliferation of CD4(+) T(responder) cells in response to anti-CD3/anti-CD28mAb-coated T-cell expander beads or syngeneic dendritic cells and soluble anti-CD3mAb was inhibited by Thy-1-induced T(reg) cells, in spite of elevated IL-2 levels in the co-cultures. Interestingly, stimulation with T-cell expander beads caused Thy-1-induced T(reg) cells to synthesize large amounts of interleukin-2 (IL-2). IL-10 was also elevated in co-cultures of activated T(responder) cells and Thy-1-induced T(reg) cells. However, mAb-mediated neutralization of IL-10 did not restore T(responder)-cell proliferation to control levels, which excluded IL-10 as a potential mediator of Thy-1-induced T(reg)-cell suppressor function. In addition, Thy-1-induced T(reg) cells did not inhibit IL-2-dependent proliferation of CTLL-2 cells, suggesting that IL-2 receptor signaling remained intact in the presence of Thy-1-induced T(reg) cells. We suggest that T(reg) cells induced by Thy-1 ligation in vivo may contribute to the maintenance of T-cell homeostasis.     

IL-1 and IL-4 modulate IL-1 receptor expression in a murine T cell line

The combination of IL-1 and IL-4 stimulates the proliferation of certain murine T cell populations. Although this effect has been best characterized for a number of murine type 2 Th cell (Th2) clones, the mechanism(s) by which these cytokines effect this response is unclear. We have examined the effects of IL-1 and IL-4 on IL-1R expression by MD10 cells, and IL-1-responsive murine T cell line. These cells bear specific IL-1R, which bind human and murine IL-1 alpha and -beta. The measured apparent IL-1R dissociation constant ranged from 41 to 255 pM using 125I-HrIL-1 alpha. Cross-linking studies demonstrated two different 125I-HrIL-1 alpha binding complexes having Mr of 70,000 and 130,000 to 156,000. When removed from passage conditions and placed in non-growth factor-supplemented media, MD10 IL-1R expression spontaneously increased two- to fourfold over the first 11 to 12 h of culture followed by a decline. This phenomenon is partially inhibitable by cycloheximide suggesting that protein synthesis is involved. In agreement with other reports, HrIL-1 alpha down-regulated the expression of its own receptor with an ED50 of between 1 and 10 pM HrIL-1 alpha for this effect. In most experiments, low amounts of HrIL-1 alpha (1.0, 0.1 pM) significantly augmented IL-1R expression. Scatchard analysis of data obtained with all HrIL-1 alpha treatment conditions showed that the effects were due to a change in receptor number, not affinity. Significantly, purified murine IL-4 (MpIL-4) augmented MD10 IL-1R expression in both a time- and dose-dependent fashion. In the presence of 50 U/ml MpIL-4, MD10 IL-1R expression increased two- to threefold after 24 h without a change in receptor affinity. When MpIL-4 (50 U/ml) and various amounts of HrIL-1 alpha (.01-1000 pM) were co-added, the down-regulatory effect of high levels of HrIL-1 alpha was significantly antagonized. When added to cultures after 24 h of HrIL-1 alpha (100 pM) treatment, MpIL-4 reversed the IL-1R down-regulatory effect induced by high levels of HrIL-1 alpha. Finally, when combined in MD10 proliferation assays, MpIL-4 synergistically enhanced the proliferation of MD10 cells treated with suboptimal levels of HrIL-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ageing combines CD4 T cell lymphopenia in secondary lymphoid organs and T cell accumulation in gut associated lymphoid tissue

Background

CD4 T cell lymphopenia is an important T cell defect associated to ageing. Higher susceptibility to infections, cancer, or autoimmune pathologies described in aged individuals is thought to partly rely on T cell lymphopenia. We hypothesize that such diverse effects may reflect anatomical heterogeneity of age related T cell lymphopenia. Indeed, no data are currently available on the impact of ageing on T cell pool recovered from gut associated lymphoid tissue (GALT), a crucial site of CD4 T cell accumulation.

Results

Primary, secondary and tertiary lymphoid organs of C57BL/6 animals were analysed at three intervals of ages: 2 to 6 months (young), 10 to 14 months (middle-aged) and 22 to 26 months (old). We confirmed that ageing preferentially impacted CD4 T cell compartment in secondary lymphoid organs. Importantly, a different picture emerged from gut associated mucosal sites: during ageing, CD4 T cell accumulation was progressively developing in colon and small intestine lamina propria and Peyer’s patches. Similar trend was also observed in middle-aged SJL/B6 F1 mice. Interestingly, an inverse correlation was detected between CD4 T cell numbers in secondary lymphoid organs and colonic lamina propria of C57BL/6 mice whereas no increase in proliferation rate of GALT CD4 T cells was detected. In contrast to GALT, no CD4 T cell accumulation was detected in lungs and liver in middle-aged animals. Finally, the concomitant accumulation of CD4 T cell in GALT and depletion in secondary lymphoid organs during ageing was detected both in male and female animals.

Conclusions

Our data thus demonstrate that T cell lymphopenia in secondary lymphoid organs currently associated to ageing is not sustained in gut or lung mucosa associated lymphoid tissues or non-lymphoid sites such as the liver. The inverse correlation between CD4 T cell numbers in secondary lymphoid organs and colonic lamina propria and the absence of overt proliferation in GALT suggest that marked CD4 T cell decay in secondary lymphoid organs during ageing reflect redistribution of CD4 T cells rather than generalized CD4 T cell decay. Such anatomical heterogeneity may provide an important rationale for the diversity of immune defects observed during ageing.

     

MS4A1 dysregulation in asbestos-related lung squamous cell carcinoma is due to CD20 stromal lymphocyte expression

Asbestos-related lung cancer accounts for 4-12% of lung cancers worldwide. We have previously identified ADAM28 as a putative oncogene involved in asbestos-related lung adenocarcinoma (ARLC-AC). We hypothesised that similarly gene expression profiling of asbestos-related lung squamous cell carcinomas (ARLC-SCC) may identify candidate oncogenes for ARLC-SCC. We undertook a microarray gene expression study in 56 subjects; 26 ARLC-SCC (defined as lung asbestos body (AB) counts >20AB/gram wet weight (gww) and 30 non-asbestos related lung squamous cell carcinoma (NARLC-SCC; no detectable lung asbestos bodies; 0AB/gww). Microarray and bioinformatics analysis identified six candidate genes differentially expressed between ARLC-SCC and NARLC-SCC based on statistical significance (p<0.001) and fold change (FC) of >2-fold. Two genes MS4A1 and CARD18, were technically replicated by qRT-PCR and showed consistent directional changes. As we also found MS4A1 to be overexpressed in ARLC-ACs, we selected this gene for biological validation in independent test sets (one internal, and one external dataset (2 primary tumor sets)). MS4A1 RNA expression dysregulation was validated in the external dataset but not in our internal dataset, likely due to the small sample size in the test set as immunohistochemical (IHC) staining for MS4A1 (CD20) showed that protein expression localized predominantly to stromal lymphocytes rather than tumor cells in ARLC-SCC. We conclude that differential expression of MS4A1 in this comparative gene expression study of ARLC-SCC versus NARLC-SCC is a stromal signal of uncertain significance, and an example of the rationale for tumor cell enrichment in preparation for gene expression studies where the aim is to identify markers of particular tumor phenotypes. Finally, our study failed to identify any strong gene candidates whose expression serves as a marker of asbestos etiology. Future research is required to determine the role of stromal lymphocyte MS4A1 dysregulation in pulmonary SCCs caused by asbestos.     

Cutting edge: stromal cell-derived factor-1 is a costimulator for CD4+ T cell activation

Stromal cell-derived factor (SDF)-1 is a chemoattractant for T cells, precursor B cells, monocytes, and neutrophils. SDF-1alpha was also found to up-regulate expression of early activation markers (CD69, CD25, and CD154) by anti-CD3-activated CD4+ T cells. In addition, SDF-1alpha costimulated proliferation of CD4+ T cells and production of IL-2, IFN-gamma, IL-4, and IL-10. Stimulation with SDF-1alpha alone did not induce activation marker expression, proliferation, or cytokine production by the CD4+ T cells. SDF-1alpha-mediated costimulation was blocked by anti-CXC chemokine receptor-4 mAb. RANTES also increased activation marker expression by anti-CD3-stimulated peripheral CD4+ T cells, but less effectively than SDF-1alpha did, and did not up-regulate IL-2 production and proliferation. These results indicate that SDF-1 and CXC chemokine receptor-4 interactions not only play a role in T cell migration but also provide potent costimulatory signals to Ag-stimulated T cells.     

Human CD4 expression at the late single-positive stage of thymic development supports T cell maturation and peripheral export in CD4-deficient mice

Positive selection of developing thymocytes is initiated at the double-positive (DP) CD4(+)CD8(+) stage of their maturation. Accordingly, expression of a human CD4 (hCD4) transgene beginning at the DP stage has been shown to restore normal T cell development and function in CD4-deficient mice. However, it is unclear whether later onset CD4 expression would still allow such a restoration. To investigate this issue, we used transgenic mice in which a hCD4 transgene is not expressed on DP, but only on single-positive cells. By crossing these animals with CD4-deficient mice, we show that late hCD4 expression supports the maturation of T cell precursors and the peripheral export of mature TCRalphabeta(+) CD8(-) T cells. These results were confirmed in two different MHC class II-restricted TCR transgenic mice. T cells arising by this process were functional in the periphery because they responded to agonist peptide in vivo. Interestingly, thymocytes of these mice appeared refractory to peptide-induced negative selection. Together, these results indicate that the effect of CD4 on positive selection of class II-restricted T cells extends surprisingly late into the maturation process by a previously unrecognized pathway of differentiation, which might contribute to the generation of autoreactive T cells.     

Transcriptional regulation of CD4 gene expression by T cell factor-1/beta-catenin pathway

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.     

Thymic stromal cells in culture. 2. Binding of normal thymocytes to a cloned thymic stromal cell line.

Thymic stromal cell line TS-9 was found to selectively bind a subpopulation of normal murine thymocytes. Selective binding allowed the isolation and phenotypic characterization of the adherent and nonadherent subpopulations of thymocytes. Flow cytometric analysis of fluorescently labeled thymocytes revealed that the adherent and nonadherent populations differ in maturity, with the adherent population enriched in immature thymocytes of the PNAhi, Thy-1hi, CD3-/lo, and CD4+/CD8+ double positive surface phenotype. A quantitative microwell assay was developed to measure the binding of thymocytes to TS-9. Thymocytes labeled with vital DNA stain Hoechst 33342 were allowed to bind to TS-9 in microwells and the intense fluorescence of this label was readily detected with a scanning fluorometer. The binding was trypsin-sensitive and hyaluronidase and PI-PLC resistant. The binding was also temperature dependent and sensitive to cytochalasin B. A panel of monoclonal antibodies to cell surface antigens including CD2, LFA-I/ICAM-I, and Thy-1 was screened in a quantitative binding assay for their ability to inhibit the binding of thymocytes to TS-9. The binding was partially inhibited by the C3C12 monoclonal antibody which recognizes the recently identified and apparently unique gp23,gp45 complex expressed on murine stromal cells.     

Cutting edge: direct action of thymic stromal lymphopoietin on activated human CD4+ T cells

Thymic stromal lymphopoietin (TSLP) is a cytokine that promotes CD4(+) T cell homeostasis and contributes to allergic and inflammatory responses. TSLP can act directly on mouse CD4(+) T cells, but in humans, the available data have indicated that TSLP receptors are not expressed on CD4(+) T cells and that TSLP instead activates dendritic cells, which in turn promote the proliferation and differentiation of CD4(+) T cells. We now unexpectedly demonstrate the presence of TSLP receptors on activated human CD4(+) T cells. Strikingly, whereas freshly isolated peripheral blood human T cells show little if any response to TSLP, TCR stimulation allows a potent response to this cytokine. Moreover, TSLP increases the sensitivity of human CD4(+) T cells to low doses of IL-2, augmenting responsiveness of these cells to TCR engagement. Our results establish that human CD4(+) T cells are direct targets for TSLP.