T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells.

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.

Crosslinking of the T cell-specific accessory molecules CD7 and CD28 modulates T cell adhesion.

Regulated adhesion enables T cells to migrate through tissue and transiently interact with an endless succession of cells. Monoclonal antibody (mAb) engagement of the CD3/T cell receptor (TCR) complex results in a rapid and transient augmentation of the adhesion function of LFA-1 and VLA integrin molecules on human T cells. We show in this study that mAb crosslinking of the T cell-specific accessory molecules CD7 and CD28, or treatment with the Ca2+ ionophore A23187, results in the rapid induction of integrin-mediated adhesion to three distinct ligands: the extracellular matrix protein fibronectin, and the cell surface molecules ICAM-1 and VCAM-1. Like CD3 crosslinking, increased adhesion via CD7 and CD28 crosslinking appears to involve both protein kinase C (PKC) and cAMP-dependent protein kinases. In contrast, A23187 induction of adhesion is unaffected by PKC inhibitors. CD7 is preferentially expressed on naive T cells and is unique in being a potent inducer of naive T cell adhesion. Enhanced expression/function of adhesion-inducing molecules thus overcomes relative deficits in adhesion receptor expression.

Costimulation by purified intercellular adhesion molecule 1 and lymphocyte function-associated antigen 3 induces distinct proliferation, cytokine and cell surface antigen profiles in human "naive" and "memory" CD4+ T cells.

Activation of resting human CD4+ "naive" (CD45RA+CD45RO-) and "memory" (CD45RA-CD45RO+) T cells requires costimulatory signals in addition to engagement of the T cell receptor/CD3 complex (TCR/CD3). The adhesion pathways mediated by lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 (LFA-1/ICAM-1) and CD2/LFA-3 are capable of providing such costimulatory signals. Our work shows that these costimulatory adhesion pathways are critically involved in regulation of T cell differentiation/maturation. Evidence for subset-specific costimulatory requirements is demonstrated by the finding that only memory CD4+ T cells were costimulated by LFA-3, whereas both naive and memory CD4+ T cells were costimulated by ICAM-1. In addition, these costimulatory adhesion pathways regulated reciprocal cytokine secretion patterns for interleukin 5 (IL-5) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Repeated costimulation of CD4+ memory T cells with LFA-3 led to secretion of high levels of IL-5, while repeated costimulation with ICAM-1 induced high levels of secreted GM-CSF. Significant interferon gamma (IFN-gamma) production was observed with either of the costimulatory ligands. Extensive cell surface analysis of these in vitro cultures of peripheral blood derived memory CD4+ T cells, with monoclonal antibodies obtained from the 5th Leucocyte Typing Workshop, revealed differential expression of a singular antigen, CD60. This antigen was preferentially expressed on LFA-3-costimulated cells suggesting a positive correlation between CD60 expression and a T helper type 2-like cytokine profile. In conclusion, this report demonstrates a new functional role for costimulatory adhesion molecules in regulating differential cytokine secretion in human memory CD4+ T cells.

Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1.

Many ligands of adhesion molecules mediate costimulation of T cell activation. The generality of this emerging concept is best determined by using model systems which exploit physiologically relevant ligands. We developed such an "antigen-specific" model system for stimulation of resting CD4+ human T cells using the following purified ligands: (a) major histocompatibility complex class II plus the superantigen Staphylococcus enterotoxin A, to engage the T cell receptor (TCR); (b) adhesion proteins vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and endothelial leukocyte adhesion molecule 1 (ELAM-1), to provide potential cell surface costimulatory signals; and (c) recombinant interleukin 1 beta (rIL-1 beta)/rIL-6 as costimulatory cytokines. In this biochemically defined system, we find that resting CD4+ T cells require costimulation in order to respond to TCR engagement. This costimulation can be provided by VCAM-1 or ICAM-1; however adhesion alone is not sufficient since ELAM-1 mediates adhesion but not costimulation. The cytokines IL-1 beta and IL-6 by themselves cannot mediate costimulation, but augment the adhesion ligand-mediated costimulation. Direct comparison with the model of TCR/CD3 engagement by CD3 monoclonal antibody demonstrated comparable costimulatory requirements in both systems, thereby authenticating the commonly used CD3 model. The costimulation mediated by the activation-dependent interaction of the VLA-4 and LFA-1 integrins with their respective ligands VCAM-1 and ICAM-1 leads to increased IL-2R alpha (CD25) expression and proliferation in both CD45RA+ CD4+ and CD45RO+ CD4+ T cells. The integrins also regulate the secretion of IL-2, IL-4, and granulocyte/macrophage colony-stimulating factor. In contrast the activation-independent adhesion of CD4+ T cell to ELAM-1 molecules does not lead to T cell stimulation as measured by proliferation, IL-2R alpha expression, or cytokine release. These findings imply that adhesion per se is not sufficient for costimulation, but rather that the costimulation conferred by the VLA-4/VCAM-1 and LFA-1/ICAM-1 interactions reflects specialized accessory functions of these integrin pathways. The new finding that VLA-4/VCAM-1 mediates costimulation adds significance to observations that VCAM-1 is expressed on a unique set of potential antigen-presenting cells in vivo.

CD31 expressed on distinctive T cell subsets is a preferential amplifier of beta 1 integrin-mediated adhesion.

The CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]/endothelial cell adhesion molecule [endoCAM]) molecule expressed on leukocytes, platelets, and endothelial cells is postulated to mediate adhesion to endothelial cells and thereby function in immunity, inflammation, and wound healing. We report the following novel features of CD31 which suggests a role for it in adhesion amplification of unique T cell subsets: (a) engagement of CD31 induces the adhesive function of beta 1 and beta 2 integrins; (b) adhesion induction by CD31 immunoglobulin G (IgG) monoclonal antibodies (mAbs) is sensitive, requiring only bivalent mAb; (c) CD31 mAb induces adhesion rapidly, but it is transient; (d) unique subsets of CD4+ and CD8+ T cells express CD31, including all naive (CD45RA+) CD8 T cells; and (e) CD31 induction is selective, inducing adhesive function of beta 1 integrins, particularly very late antigen-4, more efficiently than the beta 2 integrin lymphocyte function-associated antigen-1. Conversely, CD3 is more effective in inducing beta 2-mediated adhesion. Taken together, these findings indicate that unique T cell subsets express CD31, and CD31 has the capacity to induce integrin-mediated adhesion of T cells in a sensitive and selective fashion. We propose that, in collaboration with other receptors/ligands, CD31 functions in an "adhesion cascade" by amplifying integrin-mediated adhesion of CD31+ T cells to other cells, particularly endothelial cells.

Four molecular pathways of T cell adhesion to endothelial cells: roles of LFA-1, VCAM-1, and ELAM-1 and changes in pathway hierarchy under different activation conditions.

T cell adhesion to endothelium is critical to lymphocyte recirculation and influx into sites of inflammation. We have systematically analyzed the role of four receptor/ligand interactions that mediate adhesion of peripheral human CD4+ T cells to cultured human umbilical vein endothelial cells (HUVEC): T cell LFA-1 binding to ICAM-1 and an alternative ligand ("ICAM-X"), T cell VLA-4 binding to VCAM-1, and T cell binding to ELAM-1. Contributions of these four pathways depend on the activation state of both the T cell and HUVEC, and the differentiation state of the T cell. ELAM-1 plays a significant role in mediating adhesion of resting CD4+ T cells to activated HUVEC. LFA-1 adhesion dominates with PMA-activated T cells but the strength and predominant LFA-1 ligand is determined by the activation state of the HUVEC; while ICAM-1 is the dominant ligand on IL-1-induced HUVEC, "ICAM-X" dominates binding to uninduced HUVEC. Adhesion via VLA-4 depends on induction of its ligand VCAM-1 on activated HUVEC; PMA activation of T cells augments VLA-4-mediated adhesion, both in the model of T/HUVEC binding and in a simplified model of T cell adhesion to VCAM-1-transfected L cells. Unlike LFA-1 and VLA-4, ELAM-1-mediated adhesion is not increased by T cell activation. Differential expression of adhesion molecules on CD4+ T cell subsets understood to be naive and memory cells also regulates T/HUVEC adhesion. Naive T cell adhesion to HUVEC is mediated predominantly by LFA-1 with little or no involvement of the VLA-4 and ELAM-1 pathways. In contrast, memory T cells bind better to HUVEC and utilize all four pathways. These studies demonstrate that there are at least four molecular pathways mediating T/HUVEC adhesion and that the dominance/hierarchy of these pathways varies dramatically with the activation state of the interacting cells and the differentiation state of the T cell.

LFA-3, CD44, and CD45: physiologic triggers of human monocyte TNF and IL-1 release.

The monocyte-derived cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), are central regulators of the immune response, but the physiologic stimuli for their release remain largely undefined. Engagement of three monocyte glycoproteins, LFA-3, CD44, and CD45, by specific monoclonal antibodies immobilized on plastic induced TNF-alpha and IL-1 beta release. In addition, TNF-alpha was released when monocyte LFA-3 bound immobilized, purified CD2, which is its physiologic receptor. Thus, a receptor-ligand interaction that mediates cell-cell adhesion can transmit the necessary signals for the release of monokines.

Roles of multiple accessory molecules in T-cell activation.

Accessory molecules expressed on T cells can mediate adhesion between T cells and other cells, or the extracellular matrix. The same T-cell accessory molecules participate in a dialogue with their ligands (counter-receptors) on the antigen-presenting cells, and elicit signals that determine the specifics of activation and subsequent differentiation of the T cells and antigen-presenting cells.

O6-alkylguanine-DNA alkyltransferase in cutaneous T-cell lymphoma: implications for treatment with alkylating agents.

Mycosis fungoides is a low-grade cutaneous T-cell lymphoma. Early treatment often involves the use of topical chemotherapy such as mechlorethamine or carmustine although single-agent oral chemotherapy with alkylators is common for advanced disease. Recently, in a Phase I study of the new alkylating agent temozolomide, two mycosis fungoides patients experienced a complete response. The mechanism of resistance to alkylating drugs such as temozolomide is thought to be due to the presence in tumor cells of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT). The protein mediates a reaction with the O6-position of guanine in DNA, removing the lesion and leaving guanine intact. We, therefore, examined the levels of AGT in CD4+ T lymphocytes obtained by negative antibody selection from the blood of noncancerous individuals and mycosis fungoides patients, and in paraffin-embedded sections from mycosis fungoides patch, plaque, or tumor lesions and cells from involved lymph nodes. AGT protein levels were measured by quantitative immunofluorescence microscopy using a monoclonal antibody against human AGT. Using this approach, the mean level of our positive control (AGT-expressing cells) was 84,807 molecules/nucleus; values below 5,000 molecules/nucleus are considered very low. The mean AGT level in CD4+ T lymphocytes from noncancerous and cancerous individuals was 18,618 (n = 12) and 8,593 (n = 5), respectively. The mean fraction of outliers in circulating CD4+ T lymphocytes from mycosis fungoides patients was statistically significantly lower than T cells in lymph nodes. AGT molecules/nucleus from lymph node biopsies from 8 of 10 patients showed low (< 10,000 molecules/nucleus) or undetectable levels (n = 5) of AGT. The mean AGT level from samples of mycosis fungoides patch/plaque and tumor was also low at 221 (n = 4) and 2,363 (n = 6), respectively. Surprisingly, Hut78, a mycosis fungoides T-cell lymphoma cell line, was positive for AGT activity (median: 77,700 molecules/nucleus), and Hut102--another mycosis fungoides cell line--was low (median: 5,990 molecules/nucleus). Because AGT is a primary means of cell resistance to alkylating agents, the low level of AGT in neoplastic T lymphocytes from patients with mycosis fungoides suggests that treatment with alkylating agents producing O6-alkylguanine adducts, such as carmustine or temozolomide, may produce improved clinical outcomes.

Thyrotropin-receptor and thyroid peroxidase-specific T cell clones and their cytokine profile in autoimmune thyroid disease.

We studied the cytokine profile and the immune responses to thyroid antigens of specific T cell clones (TCC) isolated from patients with Hashimoto's thyroiditis (HT) and Graves' disease (GD). Antigen-specific TCC were reactive to thyroid peroxidase (TPO), thyroglobulin (Tg) or human recombinant TSH-receptor extracellular domain (TSH-R), and/or their respective peptides. Of the 43 clones derived from HT patients, 65% were reactive to TPO, and 59% of the 32 clones derived from GD patients were reactive to TSH-R. TPO epitopes 100-119 and 625-644 were recognized by 75% of HT-derived clones, whereas TSH-R epitopes 158-176, 207-222, and 343-362/357-376 were recognized by 85% of GD-derived TCC. The TCC were classified according to their cytokine profile into T helper cell (Th)0 [secreting interleukin (IL)-4, IL-5, interferon (IFN)-gamma], Th1 (secreting IFN-gamma) and Th2 (secreting IL-4 and/or IL-5). Tumor necrosis factor-beta and IL-10 were produced by all subsets. The specific TCC were predominantly Th1-like cells in HT, and were Th0- and Th1-like cells in GD. Fifty three percent of Th0 clones were derived from GD patients and were reactive to TSH-R, whereas 50% of Th1 clones were derived from HT patients and were reactive to TPO or Tg. Most Th2 clones (82%) were reactive to TPO and were established from peripheral blood. All these clones produced IL-5, and 64% produced IL-4 and IL-10. Interestingly, IFN-gamma was highly produced by TPO- or Tg-specific clones established from HT thyroid tissue. These results confirm at the clonal level our previous studies regarding T cell epitopes on TPO and TSH-R molecules and support the concept that immunodominant T cell epitopes are located on amino acid residues 100-119 and 625-644 of TPO in HT and amino acid residues 158-176, 207-222 and 343-362/357-376 of TSH-R in GD. Our studies also demonstrate that thyroid-specific T cells can be classified into Th0, Th1, and Th2 subsets. TPO- or Tg-specific clones with Th1 phenotype appear to be involved in the pathogenesis of HT, mediating thyroid tissue destruction, whereas TSH-R clones with Th0 phenotype may induce thyroid-stimulating autoantibodies in GD.

Programmed cell death signaling via cell-surface expression of a single-chain antibody transgene.

BACKGROUND: The monoclonal antibody, 5H7, is specific for a monomorphic determinant on the a3 domain of human class I MHC (A, B, C). Immobilized 5H7 delivers programmed cell death (PCD) signals to human lymphoid tumor cells as well as peripheral blood mononuclear cells. METHODS: The potential clinical utility of 5H7 was addressed by design of a single-chain variable antibody (scFv), termed 5H7scFv, which was coupled to glycophosphotidylinostitol (GPI), thereby providing membrane expression of the 5H7 idiotype (5H7scFv-GPI). Membrane expression of 5H7scFv-GPI conferred PCD-inducing properties to cells that do not normally have the capability to process and express whole antibody molecules. The initial construction was undertaken in a bacterial expression system, and appropriate protein folding was determined by binding to class I MHC-expressing cells. RESULTS: 5H7scFv-GPI-transfected Chinese hamster ovary cells demonstrated reconstitution of the 5H7 idiotype and binding to soluble HLA-A2. Cross-linking of class I MHC, via membrane expression of the scFv, provided effective PCD signaling in B and T lymphocyte tumor cells. Peripheral blood mononuclear cells were susceptible to 5H7scFv-GPI-induced PCD, and augmentation of PCD signals was noted with anti-CD3 and anti-CD28 preactivation. Responder cells demonstrated typical histologic features of PCD and Annexin V-fluorescein isothiocyanate binding. CONCLUSIONS: Cell surface anchorage of scFv thus provides effective delivery of immune modulatory signals, which may be manipulated for various therapeutic strategies.

The immunogenic potential of human fetal retinal pigment epithelium and its relation to transplantation.

PURPOSE: To perform a quantitative analysis of the expression of major histocompatibility molecules (MHC classes I and II) and costimulatory molecules by human fetal retinal pigment epithelial (HFRPE) cells and to evaluate their potential role in providing costimulatory signals for the activation of human T cells in vitro. METHODS: Pure HFRPE cells were isolated and cultured. The ability of HFRPE cells to express MHC class I and II and costimulatory molecules before and after incubation with interferon (IFN)-gamma was quantitatively analyzed by flow cytometry. The potential of HFRPE cells to activate human T cells was assessed in three different lymphocyte activation models. In the first model, anti-CD3 (OKT3)-coated beads were used to provide the T-cell receptor (TcR) signal. In the second model, the allogenic potential of HFRPE cells was assessed, and in the third assay a potent superantigen (SEA) was used to provide the TcR signal. T-cell activation was determined by cell proliferation, measured by [3H]-thymidine incorporation. RESULTS: The cultured HFRPE cells expressed low levels of MHC class I and ICAM-1 molecules. After incubation with IFN-gamma, the expression of MHC class I and ICAM-1 molecules was further upregulated, and the expression of MHC class II and VCAM-1 molecules was induced. The expression of the costimulatory molecules B7-1 and B7-2 was not observed in normal or activated HFRPE cells. In the first T-cell activation model, neither normal nor IFN-gamma-activated HFRPE cells could provide T-cell costimulation for anti-CD3 (OKT3)-coated beads. However, the autologous peripheral blood mononuclear cells (PBMCs; used here as the source of antigen-presenting cells) could provide costimulation for the T cells, inducing their proliferation. In the second T-cell activation assay, normal or IFN-gamma-activated HFRPE cells could not stimulate an alloresponse from the T cells, but they could induce a significant alloimmune T-cell response in the presence of PBMCs. In the third T-cell activation assay, the IFN-gamma-activated HFRPE cells were able to provide T-cell costimulation for the SEA-mediated activation. CONCLUSIONS: In these in vitro experiments, the IFN-gamma-activated HFRPE cells stimulated only the T cells with the potent superantigen SEA. In the absence of antigen-presenting cells, the HFRPE cells did not provide T-cell costimulation in an anti-CD3 mAb-coated bead system or induce significant alloimmune response. These results suggest that in transplantation between donors and recipients with different MHC molecules, the direct MHC peptide presentation by HFRPE cells may not induce a significant allospecific immune response. Nevertheless, an allospecific immune response could occur as a consequence of the indirect presentation, to the host T cells by the host antigen-presenting cells, of the HFRPE cells' derived MHC alloantigens.

Human fetal retinal pigment epithelial cells induce apoptosis in the T-cell line Jurkat.

PURPOSE: To investigate the mechanism(s) involved in human fetal retinal pigment epithelium (HFRPE)-mediated T-cell death. METHODS: Pure HFRPE cells were isolated and cultured. Normal and interferon (IFN)-gamma-activated HFRPE from early and late in vitro passages were incubated with cells from the human T-cell leukemia line Jurkat (Jkt). Cultures were pulsed with [3H]-thymidine to measure Jkt cell proliferation. Jkt cells were evaluated for apoptosis either by staining with an ethidium bromide/acridine orange mixture (AO/EB) or with Annexin V-phycoerythrin. The role of Fas ligand (FasL) molecule in HFRPE-mediated apoptosis was assessed by using a mutant Jkt cell line (DD3), which is deficient in Fas-mediated signaling. The involvement of the antiapoptotic human gene bcl-xL was determined by using Jkt cells that were stably transfected with bcl-x(L). The role of cell- cell contact in the induction of apoptosis was evaluated in a transwell system in the presence or absence of neutralizing antibodies against IFN-gamma and tumor necrosis factor (TNF)-alpha. RESULTS: HFRPE cells inhibited the proliferation of Jkt cells by inducing apoptosis through a FasL-independent pathway. Passaging and IFN-gamma activation strengthened the inhibitory effect of HFRPE cells on the proliferation of Jkt cells. At lower HFRPE passages (P2), bcl-alphaL, overexpression rescued the HFRPE cell-mediated apoptosis. The separation of the cells by the transwell system did not affect the HFRPE cell-mediated suppression. This suppressive effect was not mediated by the secretion of IFN-gamma or TNF-alpha molecules. CONCLUSIONS: HFRPE cells suppressed the proliferation of Jkt cells by inducing apoptosis. HFRPE cells induced a stronger inhibitory effect on Jkt cells at higher in vitro passages. The HFRPE-induced apoptosis was not mediated through the FasL/Fas pathway or through the secretion of the apoptosis-inducing cytokines IFN-gamma and TNF-alpha. The bcl-xL gene may play a role in preventing HFRPE cell-induced apoptosis in Jkt cells. These combined results suggest that the HFRPE-mediated suppression of primary T cells may also be mediated by the induction of apoptosis. Therefore, the retinal pigment epithelium may play a role in the induction of immune privilege in the subretinal space.

Human fetal retinal pigment epithelium-induced cell cycle arrest, loss of mitochondrial membrane potential and apoptosis.

PURPOSE: To investigate the mechanism of action of the soluble immune suppressive product secreted by human fetal retinal pigment epithelial (HFRPE) cells in a model system using the human T-cell line Jurkat (Jkt). METHODS: Pure HFRPE cells were isolated and cultured. The supernatants of both nonactivated and IFN-gamma-activated HFRPE cells were isolated. Cells from the human T-cell line Jkt were incubated either in standard culture medium or in the supernatant isolated from HFRPE cells. In the first assay Jkt cell proliferation was measured by [(3)H]thymidine incorporation. In the second assay Jkt cell apoptosis was examined for annexin V staining by flow cytometry. In the third assay Jkt cell division was evaluated with carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye. In the last assay the mitochondrial transmembrane potential of Jkt cells was measured with the cationic lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)]. In all the assays the effect of supernatants isolated from both nonactivated and IFN-gamma-activated HFRPE cells were compared with standard culture medium. The involvement of antiapoptotic human gene bcl-x(L:)was determined by using a Jkt cell line that was stably transfected with bcl-x(L:). RESULTS: The supernatant isolated from HFRPE cells significantly suppressed the cell division in Jkt cells and induced apoptosis. These effects were stronger when the supernatant was isolated from IFN-gamma-activated HFRPE cells. The apoptosis pathway induced by the secreted product of HFRPE cells involved the early disruption of mitochondrial transmembrane potential. Although the overexpression of bcl-x(L) gene rescued the Jkt cells from supernatant-induced apoptosis, it could not restore the proliferation of Jkt cells. CONCLUSIONS: These data suggest that HFRPE cells secrete a product that initiates an early cell cycle arrest in the human T-cell line Jkt, which is followed by the activation of an apoptotic pathway that involves the loss of mitochondrial membrane potential. The latter could be prevented by bcl-x(L) overexpression. Also these data suggest that the HFRPE-induced T-cell apoptosis may play a significant role in maintaining the immune privilege in the subretinal space.

Fine specificity of human HLA-B7-specific cytotoxic T-lymphocyte clones. I. Identification of HLA-B7 subtypes and histotopes of the HLA-B7 cross-reacting group.

Three HLA-B7-specific cytotoxic T lymphocyte (CTL) clones are described. Their fine specificity is examined by extensive panel and family studies. Four CTL-defined subtypes of HLA-B7 could be found: HLA-B7.1, -B7.2, -B7.3, and -B7.4. Biochemical evidence was obtained in one-dimension isoelectric focusing for only two subtypes. The estimated subtype frequencies in the Caucasian population are presented. Furthermore, a linkage disequilibrium between HLA-A29 and HLA-B7.3 is suggested. The CTL clones exerted distinct "extra-reactivities", i.e., lysis of some non-B7 target cells. These extra reactivities could be assigned to the cross-reacting group of HLA-B7.

An improved biochemical method for the analysis of HLA-class I antigens. Definition of new HLA-class I subtypes.

A simple method is described for the biochemical analysis of HLA class I antigens. It is a modification of a previously published procedure for one-dimensional isoelectric focusing (1D-IEF), giving improved resolution and offering larger sample capacity. One million viable cells suffice for analysis, and no more than 25 muCi of radioisotope (35S-methionine) are required. The usefulness of the method is illustrated by the characterization of a total of four biochemically distinct subtypes of HLA-B27, three subtypes of HLA-A24, two subtypes of HLA-A11, three subtypes of HLA-A2, two subtypes of HLA-Bw60, two subtypes of HLA-Bw62, and four subtypes of HLA-B35 in a panel of 24 cells selected for the expression of HLA-B27. We envision that this technique will allow a rigorous classification of HLA-A,B antigens into novel subtypes. Populations of distinct ethnic origin are especially of interest in this regard. This technique might also be used as an additional criterion for the official classification of HLA-A,B antigens.

Identification of new B27 subtypes (B27C and B27D) prevalent in oriental populations.

With the aid of alloreactive cytotoxic T lymphocytes, three subtypes of HLA-B27 can be defined: B27W, B27K, and B27C (non-B27W and non-B27K). The B27C subtype can be distinguished by 1D-IEF, is not recognized by B27W- or B27K-restricted influenza-A-specific cytotoxic T lymphocytes, and is prevalent in Oriental populations. Preliminary data indicate that the various B27 subtypes (W, K, C) are in different linkage disequilibria with HLA-C antigens. A fourth subtype of B27 (non-B27W and non-B27K), designated as B27D, was detected by 1D-IEF. (See accompanying paper by Neefjes et al.) The finding of four B27 subtypes indicates that the serologically defined B27 antigen comprises a family of various, strongly cross-reactive class-I molecules.

Small intestinal submucosa induces loss of mitochondrial integrity and caspase-dependent apoptosis in human T cells.

Porcine small intestinal submucosa (SIS) is a cell-free biomaterial used in humans for wound healing and as scaffold material for constructive remodeling of damaged or missing tissue. We have previously shown that SIS contains a factor that suppresses human helper T cell subset differentiation and expansion by inducing programmed cell death. Our aims here were to identify in detail the processes involved in SIS-induced T cell apoptosis and to perform the first characterization of the apoptosis-inducing factor present in SIS. In in vitro experiments, we utilized human T cell lines, Jurkat and CEM, to identify the processes involved in SIS-induced T cell apoptosis. Two types of sterile SIS material were used: hydrated sheets and rehydrated clinical-grade sheets. We found that SIS-mediated apoptosis as detected by induction of membrane annexin V staining involved the loss of mitochondrial membrane potential and was dependent on caspase activation. We eliminated transforming growth factor beta (TGF-beta), Fas ligand (FasL), and galectin family members as factors in SIS-mediated T cell apoptosis. We further established that processes required to prepare SIS for clinical use, freeze-drying, and gas sterilization destroyed the apoptosis-inducing factor. SIS contains a factor that induces loss of mitochondrial integrity and caspase-dependent apoptosis in human T cells. This factor is destroyed by freeze-drying and gas sterilization and is not TGF-beta, FasL, or a galectin family member. Normal T cell homeostasis in gut-associated tissues may be regulated in part by this unknown factor.

Cytokine modulation of costimulatory molecules on human fetal retinal pigment epithelial cells.

PURPOSE: Experiments were performed to evaluate the effect of various pro- and anti-inflammatory cytokines on the human fetal retinal pigment epithelium's (HFRPE) expression of major histocompatibility complex (MHC) and costimulatory molecules. METHODS: Pure cultures of HFRPE cells were isolated. HFRPE cells were incubated in the presence of Interferon-gamma (IFN-gamma), IFN-beta, Tumor Necrosis Factor-alpha (TNF-alpha), Interleukin-1beta (IL-1beta), Tumor Growth Factor-beta (TGF-beta), and a combination of IFN-gamma and TGF-beta (pre-incubation and simultaneously incubated). The expression of MHC class I and class II, Intercellular cell adhesion molecule (ICAM-1), B7-1 (CD80), and B7-2 (CD86) molecules was quantitatively analyzed by flow cytometry. RESULTS: The cultured HFRPE cells expressed high levels of MHC class I and low levels of MHC class II and ICAM-1 molecules. After culture with the above mentioned cytokines, IFN-gamma up-regulated the HFRPE's expression of MHC class II and ICAM-1. IFN-beta and IL-beta1 only up-regulated the expression of ICAM-1. TGF-beta was unable to suppress the up-regulatory effect of IFN-gamma in HFRPE cells (pre-incubated and simultaneously incubated). The other cytokines did not have any significant effect on HFRPE's expression of MHC I and II or the selected costimulatory molecules. CONCLUSIONS: Our findings indicate that TGF-beta cannot suppress up-regulating effects of IFNgamma- on HFRPE's expression of MHC and costimulatory molecules. Overall, the weak or lack of expression of costimulatory molecules after stimulation with various cytokines further confirms that HFRPE cells are weak antigen presenting cells.

Human fetal retinal pigment epithelial cells induce apoptosis in allogenic T-cells in a Fas ligand and PGE2 independent pathway.

PURPOSE: To evaluate the inhibitory effect of human fetal retinal pigment epithelium (HFRPE) on the activation of human T-cells. METHODS: Pure cultures of HFRPE cells were incubated with purified human T-cells in three different activation assays: 1) allogenic peripheral blood mononuclear cells; 2) OKT3 coated beads in the presence of accessory cells; and 3) stimulation with phorbol ester and phytohemagglutinin. RESULTS: HFRPE cells suppressed the activation of T-cells in all three assays. The mechanism of HFRPE mediated T-cell suppression was apoptosis. The role of Fas ligand(FasL)/Fas-mediated T-cell suppression was excluded, since FasL protein or mRNA could not be detected on HFRPE cells with flow cytometry and by reverse transcriptase polymerase chain reaction, respectively. Additionally, the inhibitory effect of HFRPE cells could not be blocked by anti-Fas ligand or antagonistic anti-Fas antibodies. Moreover, HFRPE cells suppressed the proliferation of anti-CD3 mAb mediated T-cell proliferation of murine splenocytes isolated from lpr mice. The inhibitory effect of HFRPE cells was not PGE2 mediated, since indomethacin could not restore the T-cell activation. Although the HFRPE mediated T-cell apoptosis was cell-cell contact independent, it was not induced by secretion of TNF-alpha, TGF-beta, or IL-10. The ratio between HFRPE and T-cells had a major impact on the HFRPE's inhibitory effect. CONCLUSIONS: HFRPE cells suppressed the activation of human T-cells by induction of T-cell apoptosis through a process that involves the secretion of soluble factors. The HFRPE mediated T-cell suppression was dependent on the ratio between HFRPE and T-cells. This undefined pathway of T-cell apoptosis may play a role in the maintenance of immune privilege in the subretinal space and may reduce the severity of the immune response after HFRPE transplantation.