Human MBP-specific T cells regulate IL-6 gene expression in astrocytes through cell-cell contacts and soluble factors.

One of the distinctive features of multiple sclerosis (MS) attacks is homing to the CNS of activated T cells able to orchestrate humoral and cell-based events, resulting in immune-mediated injury to myelin and oligodendrocytes. Of the complex interplay occurring between T cells and CNS constituents, we have examined some aspects of T-cell interactions with astrocytes, the major components of the glial cells. Specifically, we focused on the ability of T cells to regulate the gene expression of interleukin-6 (IL-6) in astrocytes, based on previous evidence showing the involvement of this cytokine in CNS disorders. We found that T-cell adhesion and T-cell soluble factors induce IL-6 gene expression in U251 astrocytes through distinct signaling pathways, respectively, resulting in the activation of NF-kappaB and IRF-1 transcription factors. In a search for effector molecules at the astrocyte surface, we found that alpha3beta1 integrins play a role in NF-kappaB activation induced by T-cell contact, whereas interferon-gamma (IFN-gamma) receptors dominate in IRF-1 induction brought about by T-cell-derived soluble factors. Similar phenomena were observed also in normal fetal astrocyte cultures. We therefore propose that through astrocyte induction, T cells may indirectly regulate the availability of a cytokine which is crucial in modulating fate and behavior of cell populations involved in the pathogenesis of MS inflammatory lesions.

Adhesion of immature and mature T cells induces in human thymic epithelial cells (TEC) activation of IL-6 gene trascription factors (NF-kappaB and NF-IL6) and IL-6 gene expression: role of alpha3beta1 and alpha6beta4 integrins.

T cell precursors homed to thymus develop in close contact with stromal cells. Among them, thymic epithelial cells (TEC) are known to exert dominant roles in their survival and functional shaping. Key molecules mediating TEC/thymocytes interactions include cytokines and growth factors secreted by the two cell types and adhesion receptors mediating cell contact. Signaling events triggered in thymocytes by adhesion to epithelial cells have been extensively investigated, whereas little is known on the opposite phenomenon. We have previously investigated this issue in a co-culture system composed of TEC cultures derived from human normal thymus and heterologous thymocytes. We demonstrated that thymocytes adhere to TEC involving beta1 and beta4 integrins and induce the clustering of alpha3beta1 and alpha6beta4 heterodimers at the TEC surface. In addition thymocyte adhesion was followed by activation of NF-kappaB and NF-IL6 gene transcription factors and enhanced IL-6 production. The two latter phenomena were reproduced by the cross-linking of the alpha3, alpha6, beta1 and beta4 integrins, thus implying that the alpha3beta1 and alpha6beta4 heterodimers can signal during thymocyte adhesion. We have extended our previous work investigating in the same experimental setting the inducing activity of non stimulated or activated policlonal or clonal mature T cells as representative of the more mature thymocyte subset. We found that adhesion of unstimulated T cell i) involved beta1, but not beta4 integrin functions at the surface ii) induced the clustering of alpha3beta1, but not alpha2beta1 heterodimers at the TEC surface and iii) up-regulated the nuclear binding activity of NF-kappaB transcription factor and the IL-6 secretion. We propose that alpha3beta1 and alpha6beta4 heterodimers are induced to cluster at the TEC surface recognizing yet unknown cellular ligands differentially expressed during T cell development.

Tumor necrosis factor alpha and human Schwann cells: signalling and phenotype modulation without cell death.

The aim of the study was to evaluate the biological response of human Schwann cells (SC) to tumor necrosis factor alpha (TNFalpha) in vitro and to the inflammatory milieu of chronic inflammatory demyelinating polyradiculoneuritis (CIDP). By immunocytochemical and functional assays, we found that SC expressed TNF receptors and that TNFalpha promoted in SC cultures transient activation of transcription factors NFkappaB and c-jun in the absence of apoptosis. In addition, TNFalpha significantly increased the proportion of non-myelin-forming SC expressing the p75 nerve growth factor receptor. Such phenotypic effect was dose-dependent and partially mediated by NFkappaB, as assessed by functional blockage with acetylsalicylic acid. We then extended our study to a human disease in which SC are exposed to TNFalpha. Increased signals for NFkappaB, but not c-jun, molecules were observed by immunohistochemistry on SC nuclei in nerve biopsies from patients with CIDP, as compared with controls. Irrespective of the presence of nerve inflammation, SC showed no evidence of apoptosis. Taken together, our results suggested that SC are potential targets of TNFalpha and that this cytokine exerted no cytotoxic effects either in vivo or in vitro. Rather, TNFalpha may influence the fate of SC by activating transcriptional pathways and modulating their phenotype.

Lymphoid adhesion promotes human thymic epithelial cell survival via NF-(kappa)B activation.

Inside the thymus, thymic epithelial cells and thymocytes show an interdependent relationship for their functional differentiation and development. As regards possible interdependency for their mutual survival, it is clear that lympho-epithelial adhesion can control the survival of developing thymocytes whereas the effects of lymphoid adhesion on epithelial cell survival have never been described. To address this issue, we performed co-cultures between normal human thymic epithelial cells (TEC) and a mature lymphoid T cell line (H9) or unfractionated thymocytes. TEC were induced to apoptosis by growth factor deprivation and the level of cell death was measured by flow cytometry. TEC stimulated by cell adhesion showed a significant reduced apoptosis when compared to the control and this phenomenon was associated with increased binding activity of NF-(kappa)B, as measured by gel shift analysis. The activation of NF-(kappa)B was necessary to promote survival, since its inhibition by acetyl salicylic acid prevented the promoting effect. The mAb-mediated crosslinking of (alpha)(3)(beta)(1) was considered as a potential inducer of TEC survival, since we have previously demonstrated that the engagement of this integrin was able to induce NF-(kappa)B activation in TEC. The crosslinking of (alpha)(3)(beta)(1), which clustered at the lympho-epithelial contact sites, partially reproduced the promoting activity of cell adhesion. These results highlight that lympho-epithelial adhesion can control the survival of thymic epithelial cells through an intracellular pathway which requires the activation of NF-(kappa)B and is triggered by integrins of the (beta)(1) family.

8B4/20, a private CD43 epitope on developing human thymocytes, is involved in thymocyte maturation.

The 8B4/20 Ag is a 120-kDa molecule whose expression on human thymocytes varies according to the differentiation stage: high density on immature CD3-/low thymocytes, reduced density on CD3medium and double-positive thymocytes, and absent on CD3high and single-positive thymocytes and on circulating T cells. In this paper we present immunological and biochemical evidence demonstrating that 8B4/20 Ag is a variant of CD43. We show that 8B4/20-expressing molecules, which are a subset of the CD43 molecules on thymocytes, are heterogeneous in charge, suggesting varying sialylation levels. The 8B4/20 epitope was mapped to the peripherally exposed N-terminal region of CD43, and the 8B4/20 antigenic determinant was characterized by requirement for the sialic acid exocyclic polyhydroxyl side chain, a feature shared with ligands of CD22. Altogether, 8B4/20-CD43 expression pattern and biochemical characteristics suggest its participation in carbohydrate-based interactions in the thymus. We therefore used specific Ab to mimic putative 8B4/20 interactions with natural ligand and examined the effect on isolated thymocytes. Treatment with 8B4/20 had no effect on in vitro apoptosis of isolated thymocytes. In contrast, 8B4/20 ligation enhanced the conversion of isolated thymocytes to differentiated phenotypes. Increased numbers were found in 8B4/20-treated cultures of CD3high and single-positive thymocytes and decreased numbers of CD3-/low and double-positive thymocytes, strongly suggesting that engagement of 8B4/20 delivers a positive signal that favors completion of the thymocyte maturation program. The ability of 8B4/20 mAb to drive thymocyte maturation in vitro suggests that CD43 molecules bearing the 8B4/20 epitope participate in early events of thymic selection.

Thymocyte contact or monoclonal antibody-mediated clustering of 3beta1 or 6beta4 integrins activate interleukin-6 (IL-6) transcription factors (NF-kappaB and NF-IL6) and IL-6 production in human thymic epithelial cells.

T-cell precursors develop within the thymus in contact with multiple supportive elements, among which thymic epithelial cells (TEC) are known to exert a dominant role in their homing, survival, and functional differentiation. All these functions are supported by cell-cell contacts and cytokine release. Signaling events triggered in lymphoid cells by adhesion to TEC are well characterized, but little is known about the opposite phenomenon. To address this issue, we derived cultures of TEC from human normal thymus. TEC monolayers were cocultured with thymocytes and immunostained with monoclonal antibodies (MoAbs) to integrin (2, 3, 4, and 6) and beta (beta1 and beta4) chains. Optical and confocal analysis showed that integrins were polarized on TEC at discrete surface locations: 6beta4 lined the basal surface of TEC monolayers, whereas 3beta1 was found mostly at TEC-TEC contacts; it is noteworthy that both 3beta1 and 6beta4 became highly enriched also at the boundaries with adherent thymocytes. Functional studies performed with MoAbs anti-beta1 and -beta4 integrins showed that beta1, and, to a much lower extent, beta4 heterodimers are involved in the TEC-thymocyte adhesion. Thymocyte contact or MoAb-mediated ligation of 3, 6, beta1, and beta4 integrins was investigated as a potential inducer of intracellular signaling in TEC. Thymocyte adhesion or cross-linking of MoAbs bound to integrins clustered at the TEC/thymocyte contact sites led to activation of interleukin-6 (IL-6) gene transcription factors, namely NF-IL6 serine phosphorylation and NF-kappaB nuclear targeting, as well as to increased IL-6 secretion. We propose that integrin clustering occurring during TEC-thymocyte contacts modulates in TEC the gene expression of a cytokine involved in thymocyte growth and functional differentiation.

HTLV type IIIB infection of human thymic epithelial cells: viral expression correlates with the induction of NF-kappa B-binding activity in cells activated by cell adhesion.

Productive infection by the LAV strain has been demonstrated in T cell precursors at different stages of intrathymic development, while viral replication in thymic epithelial cells is still controversial. In this article we show that epithelial cell cultures derived from the medullary component of normal thymus are infectable by HTLV-IIIB virus through cell-free and lymphoid-mediated transmission. Free virus inoculum results in the integration of proviral copies undergoing poor replication, whereas lymphoid-mediated transmission leads to substantial viral expression and the production of viral progeny able to secondary infect lymphoid cells. Interleukin 6 production and phenotype changes (increased expression of MHC class I and ICAM-1) were induced in TE cells by contact with free virus or by adhesion to infected lymphoid cells. By contrast, NF-kappa B-binding activity on the HIV-1 LTR kappa B enhancer element was upregulated only by contact with infected lymphoid cells, but not with virus. The viral replication observed in TE cells after lymphoid-mediated transmission correlates with the upregulation of NF-kappa B-binding activity. Interleukin 6 increased production and phenotype changes and increased NF-kappa B-binding activity were also induced by adhesion to uninfected lymphoid cells, demonstrating that lymphoepithelial cell contacts can activate TE cells. These results demonstrate that thymic epithelial cells are permissive to HIV infection and that viral replication in this cell lineage can be modulated by intracellular signals delivered by adhesive contacts.

Induction of CIITA and modification of in vivo HLA-DR promoter occupancy in normal thymic epithelial cells treated with IFN-gamma: similarities and distinctions with respect to HLA-DR-constitutive B cells.

In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantage that MHC class II induction is studied in a "physiologic" cell lineage that, as a result of this expression within the thymus, is thought to participate to the selection and maturation of the T cells. It was found that the MHC class II gene expression was associated with the de novo transcription of the gene encoding the CIITA trans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II DRA promoter and corresponding binding factors was analyzed by in vivo DNAse I footprint. It was found that treatment with IFN-gamma induces changes in the occupancy of the DRA gene regulatory sequences by nuclear factors. The resulting occupancy displays strong similarities with the one observed in the MHC class II-constitutive B cells, represented by both the Burkitt lymphoma line Raji and normal tonsil- derived B cells. However, some peculiar differences were observed between the TEC, either IFN-gamma-induced or not, and the constitutive B cells. These results suggest that both common mechanisms, such as the one mediated by the CIITA trans-activator, and distinct tissue-specific constraints contribute to the transcriptional control of constitutive and IFN-gamma-induced MHC class II gene expression.

HIV-1 spreads from lymphocytes to normal human keratinocytes suitable for autologous and allogenic transplantation.

Normal human keratinocytes can reconstitute in vitro cohesive sheets of epithelium suitable for grafting onto patients. Despite the widespread use of autografts and allografts, no data are yet available on productive infection by human immunodeficiency virus (HIV-1) of human keratinocytes. To address this point, we challenged keratinocytes at the second passage of culture with HTLV-IIIB virus by cell-free and cell-mediated inoculum. Viral entry was not achieved by cell-free inoculum, thus demonstrating that cultured keratinocytes do not provide the membrane requirements for viral binding and/or internalization. By contrast, the cell-mediated inoculum overcame specific receptor constraints, leading to viral integration and productive infection. The p24gag viral protein was transiently released in the culture supernatant, although at low level. The viral progeny produced by infected keratinocytes was rescued and amplified by co-culture experiments performed with the HIV-1 high sensitive CEM-SS human T-cell line. Viral integration, p24gag production, and secondary transmission to lymphoid cells was further confirmed with keratinocytes infected at the fourth passage of culture. Taken together, our results demonstrate that cultured keratinocytes can be infected by HTLV-IIIB virus, which can be maintained in semi-latent form for several passages after inoculum and rescued to full replication by a proper target. The in vitro demonstration of lympho-epithelial HIV-1 spreadings warns against the use of inappropriately screened biopsies for the preparation of skin grafts.

Susceptibility of human-mouse T cell hybrids to HIV-productive infection.

Interspecies human x mouse cell hybrids were used to investigate the genetic basis of human permissivity to HTLV-IIIB infection. T cell hybrids between the mouse BW 51.47 T lymphoma line and normal, PHA-IL-2 activated, human peripheral mononuclear cells (PBMCs) were generated. These hybrids preferentially segregated human chromosomes, as assessed by phenotype and karyotype analysis. Viral integration occurred only in those hybrids expressing CD4+ at the cell surface. However, infectious progeny production was demonstrated only in two of the three CD4+ hybrids tested. By segregation analysis, we could correlate the absence of human chromosomes 1, 3, and 9 with the lack of infectious viral progeny.

A novel integrin involved in thymocyte-thymic epithelial cell interactions.

Thymocytes differentiate in the thymic microenvironment into immunocompetent T cell through the interaction with a variety of accessory cells, including thymic epithelial cells (TEC). TEC plays an important role in the selection process presenting self antigens in association with major histocompatibility complex (MHC) molecules to the maturing T cells. The T cell receptor recognizes the self antigen-MHC complex, but other surface molecules help stabilize this interaction. Thus, the CD2/LFA-3 and LFA-1/intercellular adhesion molecule 1 pairs have been shown to participate in the binding between lymphoid cells and TEC. Here we describe an integrin of the very late activation antigen subfamily composed by the known beta 1 chain and by a novel alpha chain. This adhesion molecule is expressed on the surface of medullary TEC and is involved in the adhesion between TEC and thymocytes, but not peripheral blood T lymphocytes.

Establishment of an EBV-positive lymphoblastoid cell line that grows as a lymphoma in nude mice and expresses membrane CD2 molecules.

We describe a human lymphoblastoid cell line (LCL), called ZS, that originated spontaneously from the cultures of gamma-irradiated (50 Gy) peripheral-blood mononuclear cells of a normal donor. When injected subcutaneously in sublethally irradiated, splenectomized and anti-asialo-GM1-treated nude mice, ZS cells invaded the lymph nodes, that appeared 10 to 50-fold enlarged in all of the mice tested. Furthermore, ZS cells expressed a typical T-cell surface structure, the CD2 molecule, detectable by a variety of different anti-CD2 monoclonal antibodies (MAbs). However, other T-cell markers were not found, with the possible exception of a truncated messenger of the beta chain of the T-cell receptor and ZS cells could be identified as B cells since they (i) expressed a battery of markers of the resting and activated B cells, (ii) displayed a monoclonal rearrangement of the IgH chain locus and (iii) synthesized IgM K molecules. The Epstein-Barr virus (EBV) genome was detected in ZS cells in approximately ten copies per cell by DNA hybridization techniques. Furthermore, the cells were positive for EBV nuclear antigens (EBNA). Western blotting analysis of EBV encoded antigens demonstrated clear differences with those present in the B 95.8 virus-producer cell line, indicating that ZS cells were not infected by EBV in vitro and that they already harbored the virus in vivo. ZS cells formed colonies in vitro with a high cloning efficiency and displayed chromosomal abnormalities in all of the mitoses (karyotype 47, xy, +13, -14, 8p+, 21p+, +m). In spite of these malignant features, ZS cells expressed the full range of EBV latent proteins as usually do "normal" LCSs and did not have any of the chromosomal abnormalities that juxtapose the c-myc oncogene to one of the genes coding for immunoglobulin molecules.

Assessment of the potential malignancy of Epstein-Barr virus (EBV)-infected B cells in AIDS-related disorders.

Spontaneous lymphoblastoid cell lines (LCLs) were established from the peripheral blood of ten human immunodeficiency virus (HIV)-seropositive patients in order to investigate whether or not a progression of the cells toward a malignant state could be traced. The LCLs studied displayed no differences in their surface phenotype, karyotype, or tumorigenicity in nude mice when compared with a wide panel of control LCLs. However, four of the ten LCLs derived from HIV-seropositive patients formed colonies in agar with a cloning efficiency (0.1 to 0.9%) that was much lower than that of a control neoplastic B cell line (50%). Some sublines that were derived form the agar colonies expressed new activation markers (CD10 and Bac-1) but did not produce tumors in nude mice or display chromosomal abnormalities. These sublines might comprise cells that have progressed toward a more transformed state.

Studies on the oncogenic potential of Epstein-Barr-virus (EBV)-infected B cells in AIDS-related disorders.

Spontaneous lymphoblastoid cell lines (LCLs) were established from the peripheral blood of 10 human immunodeficiency virus (HIV)-seropositive patients in order to investigate whether or not progression of the cells towards a malignant state could be traced. The LCLs studied displayed no differences in their surface phenotype, karyotype, and tumorigenicity in nude mice as compared with a wide panel of control LCLs. Furthermore, no c-myc rearrangement could be detected in any of the LCLs. However, 4 of the 10 LCLs derived from HIV-seropositive patients formed colonies in agar with a cloning efficiency of 0.1-0.9%. This percentage was much lower than that of a control neoplastic B cell line (50%), but consistently higher than that observed for a battery of spontaneous LCLs. The cells of a number of sublines that were derived from the agar colonies expressed new activation markers (CD10 and Bac-1) but did not induce tumors in nude mice or display chromosomal abnormalities. These sublines might comprise cells that have progressed towards a more markedly transformed state.

Selective inhibition of interleukin 2 gene function following thymocyte antigen/major histocompatibility complex receptor crosslinking: possible thymic selection mechanism.

Considerable evidence now exists to support the notion that the 50-kDa sheep erythrocyte binding protein, T11, represents an essential cell surface component of a human T-cell-lineage activation pathway. Furthermore, it is known that the human T-cell antigen-major histocompatibility complex (MHC) receptor complex T3-Ti is capable of regulating cell growth mediated by the T11 structure. Here we show that, within the T3+ thymocyte compartment, T3-Ti crosslinking rapidly inhibits T11-initiated interleukin 2 (IL-2) gene transcription and translation. This inhibition is restricted to the IL-2 gene (IL2) as transcription of both the IL-2 receptor gene (IL2R) and the Ti beta-chain gene (TCRB) are not affected (human gene designations are in parentheses). Perhaps more importantly, T3-Ti-mediated IL-2 inhibition of this type is not operational in peripheral T lymphocytes. The results imply that the majority of T3+ thymocytes are functionally distinct from peripheral T lymphocytes despite their T3+ phenotype and may possess a unique endogenous regulatory component for suppressing IL-2 gene activity. Moreover, since IL-2 is likely rate-limiting for growth within the thymus, the findings provide one plausible mechanism for thymic selection--namely, T3-Ti crosslinking of thymocytes upon interaction with self-major histocompatibility complex inhibits clonal expansion of high-affinity autoreactive cells.

Functional and molecular aspects of human T lymphocyte activation via T3-Ti and T11 pathways.

Two pathways of human lymphocyte activation are known to exist on T-lineage cells. The first involves the T-lymphocyte receptor for antigen (T3-Ti) which operates in conjunction with gene products of the MHC complex and is a molecular complex composed of 5 polypeptide chains. Both the 49KD alpha and 43KD beta chains are immunoglobulin-like and thus contain variable domains responsible for ligand binding. In contrast, the 20-25KD T3 gamma, delta and epsilon chains are monomorphic structures presumably involved in transmembrane signalling. The alpha and beta subunits are disulfide bonded to each other and held in noncovalent association with the T3 chains. The second pathway involves the 50KD T11 sheep erythrocyte binding protein. The T11 pathway is operational during early intrathymic ontogeny, prior to T3-Ti receptor expression. Under physiologic conditions, T3-Ti and T11 pathways appear to function in series with T11, representing a more "nuclear proximal" structure. However, each pathway, independently of the other, can activate the phosphoinositol cascade and lead to elevation in cytosolic free calcium. The latter is critical for transcriptional activation of the endogenous IL-2 gene. The ability of the T3-Ti complex to regulate T11-mediated activation is discussed with reference to its possible role in thymic selection.

Selective inhibition of interleukin 2 gene function following thymocyte antigen/major histocompatibility complex receptor crosslinking: possible thymic selection mechanism.

Considerable evidence now exists to support the notion that the 50-kDa sheep erythrocyte-binding protein, T11, represents an essential cell surface component of a human T-cell lineage activation pathway. Furthermore, it is known that the human T3-Ti T-cell antigen/major histocompatibility complex receptor complex is capable of regulating cell growth mediated by the T11 structure. Here we show that, within the T3+ thymocyte compartment, T3-Ti crosslinking rapidly inhibits T11-initiated interleukin 2 (IL-2) gene transcription and translation. This inhibition is restricted to the IL-2 gene (IL2) as transcription of both the IL-2-receptor gene (IL2R) and the Ti beta-chain gene (TCRB) are not affected (human gene designations are in parentheses). Perhaps more importantly, T3-Ti-mediated IL-2 inhibition of this type is not operational in peripheral T lymphocytes. The results imply that the majority of T3+ thymocytes are functionally distinct from peripheral T lymphocytes despite their T3+ phenotype and must possess a unique endogenous regulatory component for suppressing IL-2 gene activity. Moreover, since IL-2 is likely rate-limiting for growth within the thymus, the findings provide one plausible mechanism for thymic selection--namely, T3-Ti crosslinking of thymocytes upon interaction with self-major histocompatibility complex inhibits clonal expansion of high-affinity autoreactive cells.