Heterogeneous effects of B7-1 and B7-2 in the induction of both protective and therapeutic anti-tumor immunity against different mouse tumors

Experimental mouse tumors are classified as intrinsically immunogenic when, after a single injection into syngeneic mice as nonreplicating cell vaccines, they elicit a protective immune response against a subsequent lethal challenge. Tumors that do not retain this residual immunogenicity are defined as poorly immunogenic or nonimmunogenic. The expression of the B7-1 co-stimulatory molecule on immunogenic tumors can further increase their capacity to induce a T cell-dependent anti-tumor immunity, whereas it has limited effects on nonimmunogenic tumors. Recently, B7-2, a second molecule with an apparently similar co-stimulatory activity, has been cloned. In this report, we compare the efficiency of nonreplicating cells from one immunogenic and two nonimmunogenic mouse tumors transfected with B7-1 or B7-2 in the induction of protective and curative anti-tumor immunity. Immunogenic lymphoma cells expressing B7-1 or B7-2 are equally effective in both protecting against a subsequent challenge and curing established tumors. By contrast, nonimmunogenic adenocarcinoma and melanoma cells expressing B7-2 provide superior protective immunity, and only B7-2⁺ adenocarcinoma cells induce an efficient curative immunity. CD8⁺ and polymorphonuclear cells, but not CD4⁺ T cells, are critically involved in the rejection of the adenocarcinoma elicited by both B7-1⁺ and B7-2⁺ vaccines. These data indicate that B7-1 and B7-2 are not redundant co-stimulatory molecules and that, in these experimental models, B7-2 is superior to B7-1 in the induction of an efficient immunity when the immunogenicity of a tumor is a limiting factor.     

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.     

CD28 is not required for rejection of unmanipulated syngeneic and autologous tumors

To gain a better understanding of the requirement of CD28 co-stimulation in different types of T cell-dependent tumor rejection responses, we performed a series of syngeneic and autologous tumor rejection experiments on CD28 knockout mice. In a preimmunization-challenge model, virally-induced ALC lymphoma and methylcholanthrene-induced MC57X fibrosarcoma transplants were rejected similarly by syngeneic CD28 knockout and immunocompetent controls. ALC-specific cytotoxic T lymphocytes (CTL) and MC57X-specific tumor necrosis factor (TNF) release were induced in CD28 knockouts, although at a reduced level in the latter case. Secondly, the spontaneous regression of Moloney murine sarcoma virus (MMSV)-induced primary tumors in the autologous hosts occurred equally in CD28 knockouts and in immunocompetent control mice. A comparable virus-specific CTL response was generated in both, as revealed in cytolytic assays against RBL-5 targets. Thirdly, the spontaneous rejection of the B7-transfected EL-4 lymphoma by immunocompetent hosts was abrogated in CD28 knockout mice, since more than 82% CD28 knockouts developed tumors after inoculation with B7-transfected EL-4 cells. Our results therefore show that CD28 co-stimulatory molecules are not required for the rejection of unmanipulated syngeneic tumors in hyperimmunized hosts and the regression of MMSV-induced sarcoma in autochthonous hosts.     

Human Naive and Memory T-Helper Cells Display Distinct Adhesion Properties to ICAM-1, LFA-3 and B7 Molecules

In this paper the contribution of different accessory molecules to the adhesion of resting, naive and memory CD4⁺ T cells was examined utilizing a panel of CHO cell transfectants as model antigen-presenting cells (APCs). CD4⁺ T lymphocytes demonstrated strong adhesion to HLA-DR4 transfected CHO cells co-expressing B7, ICAM-I or LFA-3 molecules, suggesting that all three adhesion pathways is utilized by resting CD4⁺ cells. Monoclonal antibodies (MoAbs) against the corresponding receptors on T cells, e.g. anti-CD28, anti-LFA-1β and anti-CD2, inhibited completely T-cell adhesion to natural ligands expressed on transfected CHOcells. Pretreatment of CD4⁺ T cells with NKI-L16 MoAb, which interact with an activation epitope on LFA-loc chain, enhanced adhesion to ICAM-1 but not B7 or LFA-3 expressing CHO cells. Analysis of T helper-cell subsets revealed that memory T cells bound several fold stronger to ICAM-1 expressing transfectants compared to the CD4⁺ 45RA⁺ naive T cells, whereas adhesion to B7, LFA-3- and B7/LFA-3-expressing CHO cells was similar in both T-cell subsets. The kinetics of adhesion of naive and memory CD4⁺ T cells to ICAM-1 was rapid and similar in both subsets. The NKI-L16 MoAb multiplied several times ICAM-1-dependent adhesion in naive compared to memory cells, which enabled the naive cells to reach a similar adhesion level as memory cells. The results suggest that resting naive CD4⁺ T cells utilize preferentially the CD2/LFA-3 or CD28/B7 adhesion pathways upon adhesion to APCs, while memory CD4⁺ T cells utilize the CD2/LFA-3, CD28/B7 and LFA-l/ICAM-1 adhesion pathways. The NK.I-L16 MoAb-induced upregulation of adhesion involves an increased affinity of LFA-1 for its ligand and not a change in the number of LFA-1 molecules. This is compatible with a view that naive cells express a large number of inactive LFA-1 molecules, whereas memory cells express preferentially activated LFA-1 molecules. The inherent low number of active LFA-I molecules on naive CD4⁺ T cells may be important in keeping these cells in a resting state.     

Up-regulation of human epidermal Langerhans' cell B7-1 and B7-2 co-stimulatory molecules in vivo by solar-simulating irradiation

Ultraviolet (UV) radiation impairs cutaneous immune functions and induces antigen-specific tolerance both locally at the irradiated skin site, as well as at distant skin sites and systemically. It has been postulated that in the local model, altered Langerhans' cells (LC) provide tolerogenic signals, and studies in vitro have indicated that UV radiation may down-regulate the expression of co-stimulatory molecules on the surface of these cells. To examine the effect of UV radiation on LC co-stimulatory molecules in vivo, we irradiated human volunteers with erythematogenic doses of solar-simulating UV radiation (SSR), and analyzed the expression of cell surface markers in dermatome skin samples obtained 1–72 h post-irradiation. For flow cytometric analysis, epidermal cell (EC) suspensions were prepared and double labeled with monoclonal antibodies against CD1a or HLA-DR, and B7-1 (CD80), B7-2 (CD86), ICAM-1 (CD54), ICAM-3 (CD50), LFA-3 (CD58), E-cadherin, or integrin-β₄ (CD104). In unirradiated control skin samples, keratinocytes (KC) expressed high levels of E-cadherin. LC expressed high levels of both E-cadherin and ICAM-3, and low levels of B7-2, LFA-3, ICAM-1, and integrin-β₄. Following SSR, a triphasic reaction pattern was seen: an immediate, down-regulatory phase prevailing 2–6 h post-irradiation, when the number of DR⁺ and CD1a⁺ cells were temporarily reduced; a delayed, up-regulatory phase in which the number of LC was increased and the expression intensities of CD1a, HLA-DR, B7-1, and B7-2 were strongly up-regulated, maximally evident 12–24 h after irradiation, but no more seen at 48 h; and a late phase at 72 h, in which an influx of monocytes and a concomitant rise in DR⁺ cells was recorded. We conclude that to understand real-life cutaneous UV immunology, studies in vitro need to be complemented with studies in vivo. In the case of LC, the effects of erythematogenic UV radiation in vivo on human LC B7 co-stimulatory molecules include an up-regulatory stage.     

Lack of correlation between rejection of tumor cells co-expressing interleukin-2 and B7.1 and vaccine efficiency

Genetically modifying tumor cells to express a variety of cytokines such as interleukin-2 (IL-2) or the co-stimulatory molecule B7.1 leads to increased immunogenicity and reduced tumorigenicity of tumors in several models with T cells involved in the process. We have previously reported decreased tumorigenicity of the murine plasmacytoma J558L [major histocompatibility complex (MHC) class I⁺ and class II^?] expressing IL-2 or B7.1. When systemic immunity was analyzed, immunization with either J558-IL2 or J558-B7.1 cells generated moderate protection against unmodified J558L tumor cells, comparable to immunization with a tumor cells/adjuvant Corynebacterium parvum mixture. In this study, we asked whether the co-expression of IL-2 and B7.1 in tumor cells would augment vaccine potency, cytotoxic T lymphocyte (CTL) activity and protective immunity. Rejection of single IL-2 or B7.1 or co-transfected IL-2/B7.1 cells occurred in most syngeneic animals but not in T cell-deficient nude mice, thus confirming that T cells were required for tumor rejection. We knew from previous experiments that CD8⁺ T cells were responsible for rejection. Surprisingly, immunization with J558-IL2/B7.1 cells followed by challenge with parental J558L caused a reduction in systemic protection as compared to J558-B7.1 or J558-IL2 alone. We examined the mechanism underlying this unexpected result: 6 days after injection of J558-IL2/B7.1 cells, tumor were nearly completely destroyed and were almost devoid of CD8⁺ cells, while CD8⁺ cells were increased in both IL-2- and B7.1-transfected tumors. In addition, immunization with J558-IL2/B7.1 tumors had an adverse effect on the generation of CTL. Mice immunized with J558-B7.1 and to a lesser extent J558-IL2 cells mounted a CTL response against J558L cells while, in contrast, no CTL activity could be detected in mice immunized with J558-IL2/B7.1, thus showing a correlation between the absence of CTL activity and the lack of in vivo protection. We demonstrate that “hyperstimulation” of the immune response by genetically modified cancer vaccines can have adverse effects on tumor immunity, even though the mechanism is not yet completely understood.     

Expression and co-stimulatory function of B7-2 on murine CD4+ T cells

Co-stimulatory signals mediated by the interaction of B7-1/B7-2 with CD28 are important for the activation of CD4⁺ T cells stimulated with antigen on antigen-presenting cells. There are controversies about the expression and function of B7-1/B7-2 on CD4⁺ T cells. The aim of this study was to analyze the expression of B7-1/B7-2 on naive and memory CD4⁺ T cells and the co-stimulatory function in the activation of naive CD4⁺ T cells stimulated by TCR ligation. Present results indicate that memory CD4⁺ T cells express B7-2 molecules on their surface, whereas naive CD4⁺ T cells do not. Neither memory nor naive CD4⁺ T cells expressed B7-1 molecule on their surface, although B7-1 mRNA was faintly expressed in memory T cells. B7-2 molecules expressed on memory T cells co-stimulated CD4⁺ naive T cells stimulated with plate-coated anti-CD3 to produce IL-2. Naive CD4⁺ T cells were shown to express B7-2 after co-stimulation with B7-2 and TCR ligation, because the naive T cells stimulated with anti-CD3 and B7-2CHO expressed B7-2 on their surface, although it remained to be studied whether the co-stimulation with B7-2 directly induced B7-2 expression on naive T cells. Our present results indicate that memory CD4⁺ T cells play some role in the activation of naive CD4⁺ T cells through the co-stimulation with B7-2 molecules.     

The threshold for autoimmune T cell killing is influenced by B7-1

The concept that naive CD4⁺ and CD8⁺ T cells require co-stimulatory signals for activation and proliferation is well documented. Less clear is the need for co-stimulation during the effector phase of the T cell response. Here we examined the influence of B7-1 (CD80) during the effector phase of an autoimmune response to pancreatic islets using transgenic mouse lines which expressed B7-1 in either all or only some of their β cells ( “confluent” or “patchy” RIP-B7-1 mice). Transgenic expression of B7-1 in normal mouse islets that co-expressed the pro-inflammatory cytokine, IL-2, resulted in early spontaneous autoimmunity. Islets with IL-2 and “confluent” B7-1 expression were destroyed whereas islets with IL-2 and “patchy” B7-1 expression showed selective killing of the B7-1⁺ β cells. Islet-reactive T cells, circulating in the RIP-B7-1/IL-2 mice, rejected syngeneic islet grafts, but only if these expressed B7-1. Introduction of the B7-1 transgene into the nonobese diabetic (NOD) genetic background likewise resulted in early spontaneous autoimmunity, but splenocytes from the diabetic animals could only transfer diabetes to NOD scid recipients that expressed B7-1 on their β cells. In both these transgenic models, therefore, islet destruction required continuous B7-1 expression by target β cells. Thus, although the normal repertoire contains T cells with potential islet reactivity, these T cells remain harmless because parenchymal cells like the β cell cannot normally express B7-1. Our results also have implications for tumor immunotherapy in that the ability of T cells to kill poorly immunogenic targets may be dependent upon B7-1 expression by the target cell itself.     

B7-1-dependent co-stimulation results in qualitatively and quantitatively different responses by CD4+ and CD8+ T cells

To characterize better the co-stimulatory activity of native B7-1 in the absence of other receptor/ligand interactions that might contribute to the response, B7-1 was purified by monoclonal antibody (mAb) affinity chromatography. Immobilization of purified B7-1 with anti-T cell receptor (TCR) mAb on cell-sized latex microspheres provided an effective stimulus for activation of both CD4⁺ and CD8⁺ T cells as measured by proliferation, development of effector function, and changes in motility and adhesion. The CD4⁺ T cell response was prolonged and resulted in efficient interleukin-2 production and clonal expansion. In contrast, CD8⁺ responses were transient. Proliferation and clonal expansion peaked on days 3 and 4, coincident with maximal expression of lytic effector function, and the cells then died. These results demonstrate that B7-1 mediated co-stimulation is sufficient for the induction of effector function in both helper and cytotoxic T cell precursors, but suggest that B7-1 co-stimulation is not sufficient to sustain helper-independent CD8⁺ CTL responses. When the dose responses of CD4⁺ and CD8⁺ T cells to B7-1 were compared, CD8⁺ T cells were found to require higher densities of B7-1 to attain an equivalent level of activation, suggesting that the level of expression of B7-1 by APC may influence the development of helper or CTL responses. Finally, in contrast to results obtained by others with B7-1 transfectants, purified B7-1 did not provide co-stimulation when presented on a surface separate from the TCR stimulus.     

NKG2D ligation relieves 2B4‐mediated NK‐cell self‐tolerance in mice

Along with MHC class I (MHCI), 2B4 provides nonredundant NK‐cell inhibition in mice. The immunoregulatory role of 2B4 has been increasingly appreciated in models of tumor and viral infection, however, the interactions among 2B4, MHCI, and other activating NK‐cell receptors remain uncertain. Here, we dissect the influence of two distinct inhibitory pathways in modulating NK‐cell‐mediated control of tumors expressing strong activating ligands, including RAE‐1γ. In vitro cytotoxicity and in vivo peritoneal clearance assays using MHCI⁺CD48⁺ (RMA‐neo), MHCI⁺CD48⁺RAE‐1γ (RMA‐RAE‐1γ), MHCI^?CD48⁺ (RMA‐S‐neo), and MHCI^?CD48⁺RAE‐1γ (RMA‐S‐RAE‐1γ) tumor lines demonstrated that NKG2D activation supersedes the inhibitory effect of both 2B4‐ and MHCI‐mediated immune‐tolerance systems. Furthermore, 2B4KO mice subcutaneously challenged with RMA‐neo and RMA‐S‐neo exhibited reduced tumor growth and significantly prolonged survival compared with WT mice, implying that 2B4 is constitutively engaged in the NK‐cell tolerance mechanism in vivo. Nevertheless, the inhibitory effect of 2B4 is significantly attenuated when NK cells encountered highly stressed tumor cells expressing RAE‐1γ, resulting in an immune response shift toward NK‐cell activation and tumor regression. Therefore, our data highlight the importance of the 2B4‐mediated inhibitory system as an alternate self‐tolerance mechanism, whose role can be modulated by the strength of activating receptor signaling within the tumor microenvironment.     

A B7-1-transfected human melanoma line stimulates proliferation and cytotoxicity of autologous and allogeneic lymphocytes

B7 co-stimulation is necessary to activate resting T cells upon antigen recognition by the T cell receptor. To see whether expression of B7 may render human melanoma cells able to stimulate T cells, a cloned melanoma line (Me1B6), which did not express B7-1, was transfected with the human B7-1 gene. In proliferation assays, B7-1 transfected cells (Me1B6/B7) showed greater stimulatory activity of allogeneic and autologous peripheral blood lymphocytes (PBL) compared to parental, non-transfected tumor cells. This effect was also seen when allogeneic CD8⁺ and CD4⁺ subpopulations were used as effectors. In these studies, activation of lymphocytes was B7-1-dependent and HLA classes I and II mediated. The higher proliferation correlated with an increased lytic activity by PBL stimulated with B7-1⁺ tumor cells against the untransfected Me1B6. Furthermore, PBL from a metastatic melanoma patient stimulated by Me1B6/B7 developed an higher lytic activity not only against Me1B6 but also against their autologous, B7-1^? tumor. Finally, after Me1B6/B7 stimulation, PBL released interleukin (IL)-2 and interferon-γ, but not IL-4, suggesting a Th1-mediated response. These data support the use of B7-1 transfected melanoma cells in the therapeutic vaccination of melanoma patients.     

The role of B7-CD28 co-stimulation in tumor rejection

The role of B7 co-stimulatory signaling in in vivo tumor rejection remains incompletely characterized. In particular, the relative competence of B7-1 (CD80) and B7-2 (CD86) to provide effective co- stimulus is not well defined, and the identification of the T cell co- stimulatory receptor that mediates B7 co-stimulation in tumor rejection has not been addressed. These issues were studied by assessing rejection of B7-negative or B7-transfected tumor cells in CD28- expressing or CD28-deficient hosts. B7-negative EL4 tumor cells grew progressively in normal syngeneic C57BUL6 (B6) mice. In contrast EL4 cells transfected with either full length B7-1 or full length B7-2 were rejected, indicating that both B7-1 and B7-2 are competent to mediate rejection of EL4 tumor cells. Expression of truncated B7-1 or B7-2 products, with complete deletion of cytoplasmic domains, was as effective as expression of full length B7-1 or B7-2 in mediating rejection. In contrast to the rejection of B7-transfected EL4 cells observed in CD28-expressing syngeneic hosts, B7-1- and B7-2-positive EL4 cells as well as control EL4 cells grew progressively in CD28- deficient mice, demonstrating the requirement for host expression of CD28 in B7-mediated tumor rejection. These results indicate that interaction of host CD28 with co-stimulatory extracellular B7-1 or B7-2 ligands expressed on tumor cells can play a necessary role in mediating tumor rejection.      

Mechanism of enhanced antigen presentation by B cells activated with anti-μ plus interferon-γ: Role of B7-2 in the activation of naive and memory CD4+ T cells

B cells activated with anti-γ antibody plus interferon (IFN)-γ exerted strong antigen presentation activity for T cell proliferation. The enhanced antigen presentation function was shown to be due to the increase in B7-2 expression. When B cells were stimulated with anti-μ, expression of MHC major histocompatibility complex class II, heat-stable antigen (HSA), ICAM-1 and B7-2 was increased. The presence of IFN-γ further augmented the expression of B7-2 on anti-μ-stimulated B cells. B7-1 was not expressed on B cells under these conditions. The participation of B7-2 in the elicitation of the proliferative response of T cells was confirmed by the inclusion of anti-B7-2 antibody in cultures. The enhanced expression of either HSA or ICAM-1 was shown not to play a major role in the increased B cell antigen presentation capacity. The major T cell population responding to this activated B cell antigen presentation was shown to be CD44^low naive CD4⁺ T cells, whereas CD45RB^low memory CD4⁺ T cells responded only weakly. The difference in proliferative responses between naive and memory CD4⁺ T cells was explained by the different efficiency in IL-2 production of these cell populations in response to antigen presentation by B cells activated by anti-μ plus IFN-γ. These results suggest that IFN-γ plays an important role in recruitment of naive T cells for an immune response.     

Tumor cells cotransfected with interleukin-7 and B7.1 genes induce CD25 and CD28 on tumor-infiltrating T lymphocytes and are strong vaccines

Interleukin-7 (IL-7) and the membrane molecule B7 are both able to provide proliferation and activation signals for T cells. However, tumor cells transfected to express either molecule alone are not reliably rejected in syngeneic hosts or are not sufficiently immunogenic to serve as potent tumor vaccines. Since IL-7 and B7 have shown synergistically to induce activation and proliferation of T cells in vitro, we have expressed B7.1 by means of a retrovirus in the mammary adenocarcinoma TS/A which arose spontaneously in a BALB/c mouse and in the plasmacytoma J558L and their IL-7-transfected sublines to improve vaccine efficacy. Expression of IL-7 or B7.1 alone in tumor cells decreased tumorigenicity, but nevertheless tumors grew in a substantial number of mice. In contrast, IL-7/B7.1 cotransfected cells did not grow as tumor in a single case. This inhibition of tumor growth was completely T cell dependent, because TS/A-IL-7/B7.1 cells retained their full tumorigenic potential in T cell-deficient mice. Analysis of tumor-infiltrating T lymphocytes revealed increased numbers of T cells in B7, IL-7 and IL-7/B7 transfected compared to parental tumors. In IL-7/B7 transfected tumors, T cell numbers were not further increased compared to that in singlegene-transfected tumors. However, T cells in B7 and IL-7 transfected tumors differed phenotypically with respect to activation markers. In B7 transfected tumors, T cells were predominantly CD28⁺ and CD25⁺, while in IL-7-transfected tumors, T cells were mainly CD28^? and CD25⁺. In IL-7/B7 cotransfected tumors, the majority of T cells was CD28⁺ and CD25⁺. Thus, IL-7 and B7 induced an anti-tumor immune response by complementary T cell directed pathways in a cooperative fashion. Importantly, immunization of mice with the transfected cells and subsequent contralateral challenge with parental tumor cells showed that IL-7/B7 co-expressing cells induced the most strongly protective immunity, which is superior to that induced by single-gene transfectants and to the adjuvant Corynebacterium parvum. Vaccine efficacy was abrogated when irradiated cells were used for vaccination. Together, our results show that IL-7 and B7.1 transfected tumor cells induce strong T cell activation and tumor immunity.     

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection

In a rat model of corneal transplantation, Fischer 344 (RT1^lv1) rats received orthotopic corneal isografts or Wistar-Furth (RT1^u) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8⁺ than CD4⁺ cells was found at days 1–3 following rejection, whereas there was a higher proportion of CD4⁺ cells at days 5–8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1–2, 4 and 7–10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4⁺ > CD8⁺ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1–2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes—from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4⁺ cells was found in both aqueous and graft at later times.     

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.     

CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro

The interaction of CD28 and its ligands is critical for antigen-inducedT cell activation. Recent studies have demonstrated the existenceof at least two members of the B7 receptor family. In this report,the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2)were compared to CD28 ligation by mAb. We demonstrate that thekinetics of induction of T cell proliferation after anti-CD3stimulation was similar regardless of the form of co-stimulation.Similarly, B7-1 and B7-2 could both maintain long-term expansionof CD4 cells. The co-stimulatory effects of both B7-1 and B7-2were dependent on CD28 cross-linking, based on complete inhibitionof proliferation by CD28 antibody Fab fragments. Co-stimulationwith B7-1 and B7-2 induced high levels of cytokine secretionby resting T cells, and the effects of B7-1 and B7-2 could notbe distinguished. This conclusion is based on analysis of theinitial activation of CD28⁺ T cells. as well as T cell subpopulationsconsisting of CD4⁺ and CD8⁺ T cells. Both B7-1 and B7-2 couldelicit IL-4 secretion from CD4⁺ T cells while anti-CD28 antibodyinduced substantially less IL-4 secretion. Furthermore, bothB7-1 and B7-2 could stimulate high levels of IFN- and IL-4 fromCD4⁺CD45RO⁺ cells, while neither B7 receptor could co-stimulateIFN- and IL-4 secretion from CD4⁺CD45RA⁺ T cells. B7-1 and B7-2could, however, co-stimulate CD4⁺CD45RA⁺ T cells to secreteIL-2. By contrast, when previously activated T cells were tested,re-stimulation of CD4⁺ T cell blasts with B7-1 or B7-2 resultedin higher secretion of IL-4 and IL-5 than anti-CD28, while re-stimulationwith anti-CD28 antibody maintained a higher level of secretionof IL-2 and IFN- than B7-1 or B7-2. These observations may haveimportant implications because they suggest that the mannerof CD28 ligation can be a critical determinant in the developmentof cytokine secretion that corresponds to T_h1- and T_h2-likepatterns of differentiation. Together these observations suggestthat there are no Intrinsic differences between B7-1 and B7-2in their ability to co-stimulate the populations of cells thatwe have tested.     

Paralysis of B7 co-stimulation through the effect of viral IL-10 on T cells as a mechanism of local tolerance induction

The Epstein-Barr virus (EBV) encodes an open reading frame with significant homology to the cellular IL-10 gene. This viral IL-10 (vIL-10) might enable EBV to evade antiviral T cells. We employed transfectants of a murine tumor cell line (P815) to investigate whether vIL-10 interferes with the first (antigenic) or second (co-stimulatory) signal of T cell activation. Untransfected P815 cells caused tumors in syngeneic DBA/2 mice after s.c. inoculation. In contrast, transfectants that provided either a strong antigenic stimulus (P815-K^b cells) or a strong co-stimulatory signal (P815-B7 cells) were rejected. Injection of double-transfected P815 cells expressing K^b and secreting high levels of vIL-10 (P815-K^b -vIL-10) did not result in tumor growth. We then investigated whether vIL-10 could paralyse co-stimulation by B7 under the same conditions. Therefore P815-B7 cells were mixed with vIL-10-secreting P815-K^b cells and co-injected into DBA/2 animals. Most of these mice developed a tumor. Explanted tumor cells expressed the B7 molecule but not the K^b antigen. These observations in vivo were mirrored by experiments in vitro: vIL-10 could induce T cell tolerance towards P815-B7 cells but not P815-K^b cells. Taken together our results suggest that vIL-10 acts directly on T cells to inhibit co-stimulatory signals mediated via B7 receptors such as CD28 or CTLA-4.