Antigen presentation by interferon-γ-treated thyroid follicular cells inhibits interleukin-2 (IL-2) and supports IL-4 production by B7-dependent human T cells

The consequence of recognition of antigen on antigen-presenting cells that are induced to express major histocompatibility complex (MHC) class II molecules following an inflammatory process is still not clear. In this study, we have investigated the outcome of antigen presentation by epithelial cells and we have used as a model thyroid follicular cells (TFC) that are known to express MHC class II molecules in autoimmune thyroid diseases and acquire the capacity to present autoantigens to T cells infiltrating the thyroid gland. The result show that MHC class II-expressing TFC were unable to stimulate a primary T cell alloresponse, using CD4⁺ T cells from three HLA-mismatched responders. Phenotypic analysis showed that TFC, after incubation with interferon-γ, do not express the co-stimulatory molecules B7-1 (CD80) and -2 (CD86). Addition of murine DAP.3 cells expressing human B7-1 (DAP.3-B7) to cultures containing peripheral blood CD4⁺ T cells and DR1-expressing TFC led to a proliferative response, suggesting that the failure of TFC to stimulate a primary alloresponse was due to a lack of co-stimulation. Similarly, HLA-DR-restricted, influenza-specific T cell clones dependent on B7 for co-stimulation did not respond to peptide presented by TFC; again the lack of response could be overcome by co-culture of TFC with DAP.3-B7. Furthermore, recognition of antigen on TFC inhibited interleukin-2 (IL-2) production in the B7-dependent T cells. In contrast, in T helper type 0 (Th0) T cells, IL-4 release was not affected by TFC presentation. In addition, antigen presentation by TFC favored IL-4 production relative to IL-2 production by B7-indpendent Th0 clones. These results suggest that antigen presentation by MHC class II⁺ TFC may induce tolerance in autoreactive Th1 cells but may simultaneously favors a Th2 response in uncommitted T cells, and thereby support autoantibody production.     

Myoblasts fail to stimulate T cells but induce tolerance

Recent interest in myoblast transfer and in the use of myoblastsas vehicles in gene therapy has made it important to understandthe potential immunogenicity of allogeneic or neoantlgen-expresslngmyoblasts. Given the problems of producing a pure populationof myoblasts, In this study we used a tumour-derived musclecell line (TE671), with phenotyplc features of myoblasts, whichwe transfected to express HLA-DR1. However, this cell line wasunable to stimulate either established HLA-DR1-specific alloreactlveT cell clones or a primary alloresponse. Nor could it presenthaemagglutlnln peptide HA 306–324 to DR1-restricted, HA306–324-speciflc T cell clones or lines. Indeed, prelncubatlonwith DR1-expressing TE671 and HA 306–324 rendered suchT cells tolerant as Judged by their subsequent inability toproliferate in response to a DR1⁺ B cell line plus peptide HA306–324. These results imply that myoblasts do not providecostlmulatory signals, and are therefore unlikely to stimulateallospeclfic T cells following myoblasts transplantation orto initiate neoantlgen-speclfic immune responses following Invivo transfection.     

T cell activation by antigens on human melanoma cells co-stimulation by B7-1 is neither sufficient nor necessary to stimulate IL-2 secretion by melanoma-specific T cell clones in vitro

B7-1 expression, induced by transfection in poorly Immunogenicmurine tumours, was shown to elicit a T cell-mediated rejectionof these tumours and further active immunity against the nontransfectedtumour. We therefore asked to what level similarly induced expressionof B7 on human melanoma cells would affect the antigen-dependentresponses of tumour-specific T cell clones in vitro. Data presentedshow that B7-1 expression by melanoma lines: (i) significantlyinduced, or improved, an IL-2-dependent proliferative responseof such clones to the antigen; (ii) increased the amount ofIL-2 produced by two clones in response to the parental non-transfectedtumour cells;and (iii) increased the TNF responses of all theCD4⁺ clones. However, despite these clear co-stimulatory effectson antigen-induced responses of all T cell clones, which demonstratedaneffective interaction of the B7-1 transfected molecule withone or the other of its counter-receptors expressed on T cellclones, B7 co-stimulation did not correct the defect of IL-2secretion exhibited by many of these clones in response to invitro antigen presentation by melanoma cells. We further showthat defective IL-2 secretion in response to melanoma antigenswas not due to a T cell clone refractoriness induced by theculture, since one of these clones could be induced to secreteIL-2 by an antigen-expressing melanoma line, upon increasedlymphocyte function associated antigen-3expression induced bygene transfectlon. Together these data suggest that defectiveIL-2 secretion by many tumour-infiltrating lymphocytes clonesin response to antigen presentation by melanoma cells in vitrois not exclusively due to the inability of these cells to providean appropriate co-stimulation through the B7-1 molecule.     

Identification of cross-reactive T cell restriction epitopes located on the DR7 beta 1 and DR beta 4 molecules.

L cell fibroblasts transfected with HLA class II cDNA clones isolated from a cDNA library produced from a DR7 homozygous cell line were used as antigen-presenting cells (APC) for three HLA DR-restricted, diphtheria toxoid-specific T-cell clones in order to assess the antigen-presenting ability of the transfectants and to define the class II restriction of each clone. Class II-expressing transfectants are capable of presenting antigen to antigen-specific T-cell clones, although the transfectants are less efficient at antigen presentation than conventional APC. Paraformaldehyde fixation of transfectants prior to antigen pulsing abrogated antigen presentation, demonstrating that the transfectants require antigen processing. Antigen presentation by transfectants is completely inhibited by CD4-specific monoclonal antibodies (mAb) and one of four DR-specific mAb, whereas antigen presentation by conventional APC is only partially inhibited. Both the DR alpha:DR7 beta 1 and DR alpha:DR beta 4 (DR omega 53) molecules of the DR7 allotype serve as restriction elements for the diphtheria toxoid-specific T-cell clones. One clone is restricted by the DR7 beta 1 molecule, another clone by the DR beta 4(DR omega 53) molecule, and a third clone by a cross-reactive T cell epitope on DR7 beta 1 and DR beta 4(DR omega 53) molecules. The two DR beta 4(DR omega 53)-restricted clones react, however, differently with a panel of HLA-DR DR omega 53-positive human peripheral blood lymphocytes used as APC. Therefore the data presented here clearly document that the DR beta 4 (DR omega 53) chain may serve as restriction elements for DT-specific T-cell clones. They also provide the first evidence for functional cross-reactivity of the products of two different DR beta loci and in addition emphasize the high complexity of the supertypic HLA-DR omega 53 specificity.     

Comparison of cytokines and CD80 for enhancement of immunogenicity of cervical cancer cells

Tumor cells fail to activate specific cytotoxic T lymphocytes due to lack of costimulatory molecules e.g. CD80 (B7.1). We were able to render cervical carcinoma cells immunogenic by introduction of the CD80 gene into the tumor cells. In order to enhance the efficiency of T cell activation we investigated whether addition of interleukins would augment immunostimulation by CD80. To this end, allogeneic T cells were stimulated with CD80-expressing HeLa cells or CaSki cells in the absence or presence of IL-2, IL-7, IL-12, or combinations thereof. The proliferative response of the T cells was determined. CD80-transduced HeLa or CaSki cells induced a stronger proliferative response in allogeneic T cells than parental or mock transfected control cells. All three interleukins enhanced the proliferative response of allogeneic T cells to CD80-expressing tumor cells. IL-2 or IL-7 had stronger effects in expanding the T cells than IL-12. Combination of IL-2 and IL-7 resulted in best T cell expansion. The proliferating T cells were mainly CD8+ cells with MHC class I restricted and unrestricted cytotoxic activity. Stimulation with CD80 alone or in combination with IL-7 induced mainly cytotoxic T lymphocytes. IL-2, IL-12 or the combination of IL-2 and IL-7 induced natural killer cell-like activity and specific cytolytic activity against parental and CD80-positive tumor cells. Our data suggest that the expression of both CD80 and IL-2 plus IL-7 can enhance the efficacy of tumor vaccines.     

Antigen-presenting human T cells and antigen-presenting B cells induce a similar cytokine profile in specific T cell clones

One of the factors that may influence the cytokine secretion profile of a T cell is the antigen-presenting cell (APC). Since activated human T cells have been described to express major histocompatibility complex (MHC) class II molecules as well as costimulatory molecules for T cell activation, like e.g. ICAM-1, LFA-3 and B7, they might play a role as APC and be involved in the regulation of T-T cell interactions. To define further the role of T cells as APC we tested their capacity to induce proliferation and cytokine production in peptide- or allospecific T cell clones and compared it with conventional APC, like B lymphoblasts (B-LCL) or HTLV-1 - transformed T cells, or with non-classical APC, like activated keratinocytes or eosinophils. CD4⁺, DP-restricted T cell clones specific for a tetanus toxin peptide (amino acids 947-967) and CD4⁺, DR-restricted allospecific Tcell clones produced interleukin (IL)-2, IL-4, tumor necrosis factor-α and interferon-γ (IFN-γ) after phorbol 12-myristate 13-acetate and ionomycin stimulation and a more restricted cytokine pattern after antigen stimulation. Dose-response curves revealed that the antigen-presenting capacity of activated, MHC class II⁺, B7⁺ T cells was comparable to the one of B-LCL. Both APC induced the same cytokine profile in the T cell clones despite a weaker proliferative response with T cells as APC. Suboptimal stimulations resulted in a lower IFN-γ/IL-4 ratio. Cytokine-treated, MHC class II⁺ keratinocytes and eosinophils differed in the expression of adhesion molecules and their capacity to restimulate T cell clones. The strongly ICAM-1-positive keratinocytes induced rather high cytokine levels. In contrast, eosinophils, which express only low densities of MHC class II and no or only low levels of adhesion molecules (B7, ICAM-1 and LFA3), provided a reduced signal resulting in a diminished IFN-γ/IL-4 ratio. We conclude that non-classical APC differ in their capacity to restimulate T cell clones, whereby the intensity of MHC class II and adhesion molecules (B7, ICAM-1) expressed seems to determine the efficacy of this presentation.     

Low-dose UVB radiation perturbs the functional expression of B7.1 and B7.2 co-stimulatory molecules on human Langerhans cells

In previous studies, we have shown that ultraviolet (UV) B radiation perturbs the APC function of Langerhans cells (LC) by interfering with as-yet unidentified co-stimulatory signals. Recently, B7.1 and B7.2 on APC were shown to deliver important co-stimulatory signals through interaction with their counterreceptors CD28 and CTLA-4 on T cells. To determine whether UVB affects the functional expression of B7.1 or B7.2 on LC, B7.1 and B7.2 expression was studied on human LC by multiparameter flow cytometry. Little, if any, B7.1 or B7.2 was detected on LC freshly isolated from skin. However, following 48 h of tissue culture, expression of both B7.1 and B7.2 were markedly up-regulated. To test whether these molecules were functional, primary mixed epidermal cell leukocyte reactions (MECLR) were performed. Blocking monoclonal antibody (mAb) to B7.1 or B7.2 both inhibited the MECLR, with anti-B7.2 being much more effective than anti-B7.1. UVB radiation dose-dependently (100–200 J/m²) suppressed the culture-induced up-regulation of B7.1 and B7.2 on LC. Since LC exposed to the same UVB flux (UVB-LC) failed to stimulate alloreactive T cells in a MECLR, we questioned whether this was related to their inability to provide B7 co-stimulation. Indeed, when effective B7-CD28 signaling was ascertained by adding submitogenic doses of exogenous anti-CD28 mAb to UVB-LC, the proliferative response of alloreactive T cells was restored. We conclude that the suppressive effects of low-dose UVB radiation on the APC function of LC are, at least in part, due to an inhibition of functional B7.1 and B7.2 expression.     

CD40 can costimulate human memory T cells and favors IL-10 secretion

Optimal proliferation of T cells, although initiated via ligation of the CD3/TCR complex, requires additional costimulatory signals. While the most well-defined in vitro pathway of costimulation is via the B7 family of molecules, a large body of data clearly demonstrates an in vivo role for the CD40/CD154 pathway in transplantation, autoimmunity and allergy. We have examined the role of CD40 as an independent costimulatory molecule by generating a panel of transfected human fibroblasts expressing DR1 with either CD80, CD86 or CD40. Functional assays using allogeneic CD4(+) T cells as responders demonstrated that CD40 was capable of costimulating CD4(+) T cell proliferation particularly in the CD45RO subset. Costimulation by CD40 induced much higher levels of IL-10 than were induced by B7-expressing cells. On day 3 the dominant costimulation was provided by CD40, while by day 5 this was overshadowed by CD80 and CD86. Nonetheless, the provision of costimulation by CD40 was enough to expand an alloreactive T cell line. These results were confirmed in experiments using primary cultures of CD40(+)CD80/CD86(-) renal tubular epithelial cells as the stimulator population. Thus, CD40 is capable of functioning independently (in the absence of B7) as a costimulatory molecule both in terms of proliferation and cytokine release. The data have interesting implications concerning the consequences of antigen presentation by tissue parenchymal cells.     

Class-II HLA Restriction of Antigen-Specific Human T-Lymphocyte Clones

Blast-enriched suspensions of T cells primed for Chlamydia trachomatis antigen were cloned by a limiting dilution technique. The class-II HLA restriction of T-lymphocyte clones (TLC) was studied by using allogeneic antigen-presenting cells (APC) carrying foreign class-II HLA antigens. Most of the TLC were restricted by one or the other of the D/DR determinants of the T-cell donor; that is, they did not respond when antigen was presented by APC expressing foreign D/DR determinants. Furthermore, heterogeneity of the DR4-expressing molecule could be demonstrated by T-cell clones from one person; APC from family members expressing DR4 gave high proliferative responses, whereas no proliferation was observed with most APC from unrelated persons expressing DR4. This heterogeneity of DR4 was confirmed by mixed lymphocyte culture (MLC) experiments, indicating a close relationship between restriction epitopes and those that activate allogeneic T cells. Other clones seemed to be restricted by other class-II HLA determinants, most probably MT determinants of the T-cell donor. The restriction specificities were confirmed by subcloning experiments.     

Co-stimulation of murine CD4 T cell growth: cooperation between B7 and heat-stable antigen.

The B cell activation antigen B7/BB1 has been shown to co-stimulate growth of human T cells by binding the T cell molecule CD28. In mice, the heat-stable antigen (HSA) has also been shown to act as a co-stimulator for T cell growth. In this study, we have evaluated the contributions of B7 and HSA to the co-stimulatory activity of antigen-presenting cells (APC). Mouse B7 provides co-stimulatory activity for murine CD4 T cells in anti-CD3-induced proliferation. Human CTLA4Ig, a chimeric molecule comprising the extracellular region of CTLA-4 fused to an immunoglobulin C gamma fragment, binds to murine B7. We, therefore, use human CTLA4Ig and the hamster anti-HSA monoclonal antibody 20C9 to analyze the relative contributions of B7 and HSA to the co-stimulatory activity of murine spleen APC. Our data reveal that both murine B7 and HSA are expressed by dendritic cells and by low-density spleen B cells. Either CTLA4Ig alone or anti-HSA alone inhibited CD4 T cell proliferation to anti-CD3 by > 90%, while CTLA4Ig and anti-HSA together were far more efficient in inhibiting clonal expansion of CD4 T cells. These results demonstrate that functionally defined co-stimulation involves at least B7 and HSA and suggest that signals delivered by B7 and HSA synergize in promoting T cell growth.     

A radiosensitive APC activity dissociates IL-2 secretion and activation-induced cell death by autoreactive T cell hybridomas

T cell hybridomas were generated from a LEW rat T cell linespecific for the uveltogenic peptide bov-B1 of bovine retinalS-antigen. Using these autoreactive hybridomas, IL-2 productionand activation-induced cell death (AICD) were dissociated asoutcomes of activation. The self-reactive hybridomas secreteIL-2 and undergo AICD in response to antigen presented by non-irradiatedsyngeneic splenocytes, whereas antigen presentation by irradiatedsplenocytes induced only AICD. IL-2 production by a non-selfreactive hybridoma was unaffected by irradiation of the APC.Pretreatment of the APC with phorbol ester or lipopolysaccharideand IL-4 protected their ability to induce IL-2 secretion after-irradiation. Although the co-stimulation-blocking reagent CTLA-4-Igmimicked the effect of -irradiation by preventing IL-2 secretionbut not AICD, B7 expression on the APC was not radiosensitive,nor did co-stimulation, provided ‘in trans’ witha B7-expressing thirdparty cell, reconstitute antigen-specifichybridoma IL-2 secretion in response to irradiated APC. In summary,the data show that IL-2 secretion and AICD of a self-reactiveT cell hybridoma can be dissociated as consequences of TCR occupancyin the presence of a functional co-stimulatory signal. It isproposed that the signals producing these events are transducedthrough the TCR-CD3 complex alone and reflect the differentialoutcomes of high- and low-affinity interactions.     

Changes in the strength of co-stimulation through the B7/CD28 pathway alter functional T cell responses to altered peptide ligands

T cells require a TCR and a co-stimulatory signal for activation. We have examined the effect of the strength of TCR and co-stimulatory signals on proliferation and production of cytokines by differentiated T cell clones. The TCR signal was varied using antigen dose and altered peptide ligands. The co-stimulatory signal was varied by using as antigen-presenting cells, Chinese hamster ovary cell transfectants that express different levels of the B7-1 molecule with similar levels of MHC class II. Our results show that the level of co-stimulation has a profound effect on the response to an antigen, and that a strong co-stimulatory signal can convert a weak agonist into a full agonist and an agonist into a superagonist. Antigenicity is not absolute but a function of the strengths of the TCR and co-stimulatory signals. Increasing the strength of co-stimulation can lower antigen concentration required for maximal proliferative responses by T cell clones by 5 log. These results show that the level of expression of co-stimulatory molecules will profoundly regulate T cell clonal expansion and effector functions.     

Alloantigen-specific T-cell anergy induced by human keratinocytes is abrogated upon loss of cell-cell contact

The activation of primary human T cells largely depends on the expression of both major histocompatibility complex (MHC) class II and B7 molecules on antigen-presenting cells (APC), whereas APC expressing HLA class II but not B7 antigens are expected to induce anergy. According to this concept, interferon-γ (IFN-γ)-activated keratinocytes (KC) expressing HLA class II but not B7 costimulatory antigens should be able to induce anergy. However, in terms of anergy versus activation contradicting data have been published on the outcome of interaction between T cells and human KC. In addition, it has been shown that human KC can express a B7-like molecule with unknown function, whereas MHC expression may be functionally impaired. To evaluate this item we transfected the human A431 KC cell line with B7-1 coding sequences and up-regulated HLA-DR by treatment with IFN-γ, yielding A431^DR,B7-1 cells. Irradiated A431^DR,B7-1 cells were found to be capable of inducing vigorous proliferative primary T-cell responses in resting allogeneic T cells, whereas A431^DR cells could induce proliferation only when interleukin-2 (IL-2) was added. These data indicate that KC can present alloantigens, and that lack of costimulatory molecules on KC is the main reason why these cells cannot induce primary T-cell responses. Surprisingly, however, no evidence could be obtained of stable anergy induction by A431^DR cells, as T cells contacted with A431^DR cells and then transferred to A431^DR,B7-1 cells clearly demonstrated alloresponsiveness. T-cell non-responsiveness was maintained only when T cells remained in contact with A431^DR cells. These data indicate that, despite expression of HLA class II in the absence of B7 costimulatory molecules, human KC cannot induce stable anergy but rather induce short-term anergy in primary resting T cells.     

Similar co-stimulation requirements of CD4+ and CD8+ primary T helper cells: role of IL-1 and IL-6 in inducing IL-2 secretion and subsequent proliferation.

Primary murine CD4+ and CD8+ T helper (Th) cells provide help for various immune responses by secreting lymphokines which activate effector cells. The purpose of the present study was to investigate the co-stimulatory signals that, together with T cell receptor (TCR) cross-linking, induce phenotypically distinct primary Th cells to secrete IL-2 and proliferate. We isolated highly purified populations of primary CD4+ or CD8+ T cells and stimulated them in vitro with platebound anti-CD3 mAb. TCR cross-linking by anti-CD3 mAb induced both IL-2 receptor expression and responsiveness to exogenous IL-2, but was not sufficient to induce either IL-2 secretion or T cell proliferation. Rather, for both CD4+ and CD8+ primary Th cells, IL-2 secretion and proliferation required both TCR cross-linking and antigen presenting cell (APC)-derived co-stimulatory signals. Based on G-10 adherence and sensitivity to gamma-irradiation, the APC populations able to induce primary CD4+ Th cells and primary CD8+ Th cells to secrete IL-2 were indistinguishable. In addition, we found that either IL-1 or IL-6 could replace the requirement for APC-derived co-stimulatory signals for IL-2 secretion and proliferation by both primary CD4+ Th cells and primary CD8+ Th cells. Thus, the present study has examined and compared the co-stimulatory requirements of rigorously purified subsets of IL-2-secreting primary CD4+ and primary CD8+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low expression of CD40 and B7 on macrophages infiltrating UV-exposed human skin; role in IL-2Rα− T cell activation

After UV exposure of skin, epidermal Langerhans cells (LC) are depleted, whereas CD11b⁺ CD36⁺ CD1a⁻ monocytes/macrophages (UV-Mϕ) infiltrate. Different immunological outcomes in vivo are mediated by LC (sensitization) and UV-Mϕ (tolerance) which may be related to the distinct T cell activation states that these antigen-presenting cells (APC) induce. We previously demonstrated that CD4⁺ T lymphocytes activated by UV-Mϕ are, in contrast to LC-activated T cells, IL-2Rα deficient, and we hypothesize that this differential T cell activation is related to differences in co-stimulatory molecules between UV-Mϕ and LC. Using four-color flow cytometry, we found a reduced capacity to up-regulate expression of the important co-stimulatory molecules CD40, B7-1 and B7-2 by UV-Mϕ relative to LC. This alteration in co-stimulatory molecule expression was selective, because UV-Mϕ express equal levels of ICAM-1 and ICAM-3, and increased levels of LFA-1, relative to LC. After bidirectional signaling with T cells during alloantigen presentation, UV-Mϕ still exhibited less CD40 and B7-1 than LC. Addition of IFN-γ induced CD40 and B7-1 expression on UV-Mϕ and restored IL-2Rα expression on UV-Mϕ-activated T cells but had no effect on IL-2Rα on resting or LC-activated T cells. The restoration of IL-2Rα expression on UV-Mϕ-activated T cells by IFN-γ was inhibited (67 %, p = 0.005) by addition of neutralizing anti-CD40. Therefore, differences in co-stimulatory molecule expression, in particular CD40, on UV-Mϕ and LC are critical in determining the distinct T cell activation induced by these APC.     

Alloreactive T cell recognition of the HLA-DR beta N-terminal polymorphic region

T cell alloreactivity, originally discovered as a tissue transplantation effect, is believed to be a manifestation of the normal major histocompatibility complex (MHC) restriction of antigen presentation by accessory cells to T cells. The molecular features of the human class II-MHC proteins (HLA) which are recognized by alloreactive T cells are not at present understood, although they are clearly related to the polymorphic nature of the MHC proteins. Human CD4+ T cell clones were selected by response to the HLA-DR2 peptides beta 1-15 or beta 51-65, in an MHC-restricted manner. In addition, these clones respond to cell lines expressing the DR2 haplotype, without the requirement for accessory antigen presenting cells. DR2 beta peptide 1-15 blocks the T cell alloresponse and polymorphic residues are shown to stimulate the peptide-specific response of these clones. Thus, the polymorphic residues contained within the DR beta sequence 1-15 are demonstrated to be directly recognized by alloreactive T cells.     

Direct evidence for a functional role of HLA-DRB1 and -DRB3 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T lymphocytes

The contribution of the HLA-DRB1, -B3, and -B5 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T cells isolated from an atopic individual (HLA-DRw12, DR7; DRw52b) with perennial rhinitis was investigated. Using a panel of histocompatible and histoincompatible accessory cells, the restriction specificity obtained for a long term T cell suggested that a component of the dust mite reactive repertoire recognized antigen in association with DRB3 gene products. Oligonucleotide DNA typing of the presenting cell panel demonstrated a correlation between the DRw52b allele and T cell responsiveness. Murine fibroblasts expressing DRw52b, but not DRw52a or -c molecules, presented antigen to both the T cell line and cloned T cells (DE26) derived from the line, indicating that the supertypic specificity DRw52b was able to restrict recognition of dust mite antigens. Additional T cell clones (DE9 and DE41) also isolated from the line were restricted by the products of the B1 gene locus (DRw12B1) as determined by murine fibroblasts transfected with the appropriate HLA-DR genes. Clone DE9 was degenerate in its restriction specificity, also recognizing dust mite presented by accessory cells expressing the DR2 subtypes. Presentation by fibroblasts transfected with DRw12B1, DR2Dw2B5 genes and EBV-transformed B cell lines expressing DR2DW21B1 and -B5 indicated that the functional site restricting recognition may be associated with residues 70 and 71 of the DR beta chain helical wall of the antigen combining site. Furthermore, we have recently demonstrated that both T cell clones DE9 and DE26 induce allergen dependent IgE synthesis in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells

Summary: Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC), At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR). whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal I to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal I alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-l can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and [CAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.     

Specificity of alloactivated human T lymphocyte clones in secondary proliferation, cell-mediated lympholysis and interleukin-2 release

Alloreactive cytolytic T cell clones were generated from mixed leucocyte reactions (MLRs) between unrelated individuals. Two clones exhibited proliferative responses specific for class I HLA antigens B21 and B17. They were also found to be cytolytic toward cells bearing these HLA-B antigens as measured in cell-mediated lympholysis (CML) assays. Four clones exhibited primed lymphocyte test (PLT) and CML activity specific for various class II HLA antigens, namely DR1, DR5, DQw3 and DRw53. For each of these six clones, the CML specificity was identical to the PLT specificity. Both class I and class II specific clones released interleukin-2 (IL-2) upon restimulation with irradiated cells carrying the relevant HLA specificity. This stimulator-induced IL-2 release showed the same specificity pattern as that observed in the PLT assay. Monoclonal antibody (mAb) inhibition studies on alloreactive T cell clones showed similar inhibition profiles of PLT, CML and IL-2 release assays. These findings suggest that cytolytic activity, secondary proliferation and IL-2 release by alloactivated T cells may be induced by the same HLA encoded determinant.     

A co-stimulatory role for CD28 in the activation of CD4+ T lymphocytes by staphylococcal enterotoxin B.

In this study we investigated the differential effect of the co-stimulatory receptor ligand molecules CD2/LFA-3, LFA-1/ICAM-1, and CD28/B7 on microbial superantigen mediated activation of CD4+ T cells. Highly purified CD4+ T cells, depleted of antigen presenting cells (APCs), do not proliferate in response to the superantigen, staphylococcal enterotoxin B (SEB). However, CD4+ T cells do respond to SEB in the presence of the LFA-3, ICAM-1, and B7 positive erythroleukemic cell line K562, murine L cells, human B7 transfected L cells or CD28 mAb. The K562 plus SEB induced response can be inhibited by combinations of mAbs to CD2 and LFA-1, and to LFA-3, ICAM-1, and B7. Addition of CD28 mAb to the CD2 and LFA-1 inhibited cultures could restore the response. Furthermore, soluble CD28 mAb alone is able to synergize with SEB to induce a proliferative CD4+ T cell response. CD4+ T cells depleted of APCs could also be activated by a pool of four mAbs directed to the V beta 5, V beta 6, V beta 8, and V beta 12 region of the TCR when a co-stimulatory signal was provided by the CD28 mAb, while the V beta mAbs alone or in combination are unable to activate CD4+ T cells in the absence of APCs. In contrast, addition of soluble mAbs to CD2 and LFA-1 molecules failed to co-stimulate SEB activated CD4+ T lymphocytes. The kinetics of the different modes of activation are distinct. SEB induced proliferation is most efficient in the presence of autologous APCs with maximal proliferation at a log4 lower SEB concentration than when CD28 mAbs were used. SEB plus K562 activation peaks on day 7, while SEB plus CD28 mAb induced proliferative responses do not peak until day 9. Thus, superantigen mediated activation of CD4+ T cells requires co-stimulatory signals, among which CD28 has distinct and unique effects.