The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain.

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.     

Identification of the antigenic epitopes in staphylococcal enterotoxins A and E and design of a superantigen for human cancer therapy

Monoclonal antibodies have a potential for cancer therapy that may be further improved by linking them to effector molecules such as superantigens. Tumor targeting of a superantigen leads to a powerful T cell attack against the tumour tissue. Encouraging results have been observed preclinically and in patients using the superantigen staphylococcal enterotoxin A, SEA. To further improve the concept, we have reduced the reactivity to antibodies against superantigens, which is found in all individuals. Using epitope mapping, antibody binding sites in SEA and SEE were found around their MHC class II binding sites. These epitopes were removed genetically and a large number of synthetic superantigens were produced in an iterative engineering procedure. Properties such as decreased binding to anti-SEA as well as higher selectivity to induce killing of tumour cells compared to MHC class II expressing cells, were sequentially improved. The lysine residues 79, 81, 83 and 84 are all part of major antigenic epitopes, Gln204, Lys74, Asp75 and Asn78 are important for optimal killing of tumour cells while Asp45 affects binding to MHC class II. The production properties were optimised by further engineering and a novel synthetic superantigen, SEA/E-120, was designed. It is recognised by approximately 15% of human anti-SEA antibodies and have more potent tumour cell killing properties than SEA. SEA/E-120 is likely to have a low toxicity due to its reduced capacity to mediate killing of MHC class II expressing cells. It is produced as a Fab fusion protein at approximately 35 mg/l in Escherichia coli.     

Targeting of superantigens

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on MHC class II antigens. In order to induce T lymphocytes to reject a tumor, we substituted the specificity of SEA for MHC class II molecules with specificity for tumor cells by combining SEA with a MAb recognizing colon carcinomas. Chemical conjugates or recombinant fusion proteins of the MAb C215 and SEA retained excellent antigen binding properties whereas the binding to MHC class II was markedly reduced. The hybrid proteins directed SEA responsive T cells to tumors with specificity determined by the specificity of the MAb. Significant tumor cell killing was obtained at picomolar concentrations of the hybrid proteins and was the result of direct cell mediated by cytotoxicity as well as production of tumoricidal cytokines by T cells. Targeting of superantigens represents a novel approach to specific immunomodulation and deserves further study as a potential therapy for malignant disease.     

Combined activation of murine lymphocytes with staphylococcal enterotoxin and interleukin-2 results in additive cytotoxic activity

This report demonstrates that in vitro activation of murine spleen cells with interleukin-2 (IL-2) or the bacterial superantigen staphylococcal enterotoxin A (SEA) results in different patterns of activation and function of cytotoxic cells. Lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity (ADCC) are mainly mediated by IL-2 activated natural killer (NK) cells. SEA is the most powerful T cell mitogen known so far and retarets cytotoxic T lymphocytes (CTL) to tumors expressing major histocompatibility complex (MHC) class II in staphylococcal-enterotoxin-dependent cellular cytotoxicity (SDCC). Culture of mouse spleen cells with SEA led to expansion and activation of T cells which demonstrated strong SDCC activity and some NK-like cytotoxicity after 5 days in culture. Cell sorting revealed that both CD8⁺ and CD4⁺ T cells mediated SDCC but the former were more effective. Phenotypic analysis showed that SEA preferentially stimulated and expanded T cells expressing T cell receptor V11, in particular CD8⁺ T cells. Combined activation with SEA and IL-2 resulted in simultaneous induction of T and NK cell cytotoxicity. Moreover, IL-2 had additive effects on SEA-induced SDCC. Combined treatment with SEA and IL-2 might therefore be an approach to induce maximal cytotoxicity against tumors and to recruit both T and NK cells in tumor therapy.     

CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex-peptide dimers

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.     

Enhanced induction of human WT1-specific cytotoxic T lymphocytes with a 9-mer WT1 peptide modified at HLA-A*2402-binding residues

The Wilms' tumor gene WT1 is overexpressed in most types of leukemias and various kinds of solid tumors, including lung and breast cancer, and participates in leukemogenesis and tumorigenesis. WT1 protein has been reported to be a promising tumor antigen in mouse and human. In the present study, a single amino-acid substitution, M-->Y, was introduced into the first anchor motif at position 2 of the natural immunogenic HLA-A*2402-restricted 9-mer WT1 peptide (CMTWNQMNL; a.a. 235-243). This substitution increased the binding affinity of the 9-mer WT1 peptide to HLA-A*2402 molecules from 1.82 x 10(-5) to 6.40 x 10(-7) M. As expected from the increased binding affinity, the modified 9-mer WT1 peptide (CYTWNQMNL) elicited WT1-specific cytotoxic T lymphocytes (CTL) more effectively than the natural 9-mer WT1 peptide from peripheral blood mononuclear cells (PBMC) of HLA-A*2402-positive healthy volunteers. CTL induced by the modified 9-mer WT1 peptide killed the natural 9-mer WT1 peptide-pulsed CIR-A*2402 cells, primary leukemia cells with endogenous WT1 expression and lung cancer cell lines in a WT1-specific HLA-A*2402-restricted manner. These results showed that this modified 9-mer WT1 peptide was more immunogenic for the induction of WT1-specific CTL than the natural 9-mer WT1 peptide, and that CTL induced by the modified 9-mer WT1 peptide could effectively recognize and kill tumor cells with endogenous WT1 expression. Therefore, cancer immunotherapy using this modified 9-mer WT1 peptide should provide efficacious treatment for HLA-A*2402-positive patients with leukemias and solid tumors.     

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A

Summary Activation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5^–CD56⁺) as well as T (CD5⁺) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.     

In vitro biological activities of transmembrane superantigen staphylococcal enterotoxin A fusion protein

The bacterial superantigen staphylococcal enterotoxin A (SEA) stimulates T cells bearing certain TCR V domains when binding to MHC II molecules, and is a potent inducer of CTL activity and cytokine production. Antibody-targeted SEA such as C215 Fab-SEA and C242 Fab-SEA has been investigated for cancer therapy in recent years. We have previously reported significant tumor inhibition and prolonged survival time in tumor-bearing mice treated with a combination of both C215Fab-SEA and Ad IL-18 (Wang et al., Gene Therapy 8:542–550, 2001). In order to develop SEA as an universal biological preparation in cancer therapy, we first cloned a SEA gene from S. aureus (ATCC 13565) and a transmembrane (TM) sequence from a c-erb-b2 gene derived from human ovarian cancer cell line HO-8910, then generated a TM-SEA fusion gene by using the splice overlap extension method, and constructed the recombinant expression vector pET-28a-TM-SEA. Fusion protein TM-SEA was expressed in E. coli BL21(DE3)pLysS and purified by using the histidine tag in this vector. Purified TM-SEA spontaneously associated with cell membranes as detected by flow cytometry. TM-SEA stimulated the proliferation of both human PBLs and splenocytes derived from C57BL/6 (H-2b) mice in vitro. This study thus demonstrated a novel strategy for anchoring superantigen SEA onto the surfaces of tumor cells without any genetic manipulation.Abbreviations SEA staphylococcal enterotoxin A - TM transmembrane - NK cell natural killer cell - CTL cytotoxic T lymphocyte Drs W. Ma and H. Yu are joint corresponding authors for this article.     

Superantigen-based tumor therapy: in vivo activation of cytotoxic T cells

We have recently demonstrated that the superantigen staphylococcal enterotoxin A (SEA) targets in vitro activated cytotoxic T lymphocytes against tumor cells expressing major histocompatibility complex (MHC) class II antigens. In this report we analyze the use of SEA as an immunoactivator in vivo. Treatment of mice with SEA activated a fraction of CD3⁺ T cells apparently as a function of their T cell receptor V expression. SEA induced interleukin-2 receptor expression and proliferation in both CD4⁺ and CD8⁺ T cells. This proliferative response was dose-dependent (0.1 – 100 µg/mouse), peaked during day 1 after treatment and declined to background levels within 4 days. The cytotoxic response, measured as cytotoxicity to SEA-coated MHC class II⁺ target cells (staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity, SDCC), was maximal at a dosage of 1 µg SEA/mouse. The SDCC was confined to the CD8⁺ T cell compartment, peaked 2 days after treatment and declined to background levels within 4 days. A second injection of SEA on day 5 after the first SEA treatment resulted in SDCC function with kinetics and magnitude identical to that seen after one injection. These results pave the way for the use of SEA in the treatment of MHC class II⁺ tumors.     

Staphylococcal Enterotoxin A Induces Small Clusters of HLA-DR1 on B Cells

The superantigen SEA causes non-specific hyperactivation of T and B cells at low concentrations. Studies of mutants or soluble proteins suggest SEA is bivalent for its ligand, MHC class II. However, the interaction between these molecules on intact cells is unknown. On primary mouse B cells expressing the MHC class II allele HLA-DR1, measurements of Förster Resonance Energy Transfer between HLA-DR1 molecules on SEA-treated cells indicated specific clustering, not observed in untreated or monovalent superantigen treated cells. Tomographic visualization and electron microscopy of immunogold-labeled SEA-treated B cells revealed small clusters of surface HLA-DR1 (≤4 gold labels). These results present direct visual evidence of SEA-mediated clustering of MHC class II molecules on treated antigen presenting cells, and provide a new structural approach to addressing problems of this nature.     

Superantigen-induced apoptotic death of tumor cells is mediated by cytotoxic lymphocytes, cytokines, and nitric oxide.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a potent inducer of CTL activity and cytokine production in vivo. Protein A (PA) of Staphylococcal aureus has been found to have diverse biological response modifying properties and to possess antitumor, antitoxic and antiparasitic effects. In this study we examined the anti-tumor effect of these two superantigens used separately as well as in combination in mice carrying the Ehrlich ascites tumor. With combined treatment, DNA cell cycle analysis of tumor cells showed a significant (P < 0.05) percentage of tumor cell death. Levels of the soluble mediators TNF-alpha, IFN-gamma and IL-1 as well as NO were elevated. Additionally, CD4(+) and CD8(+) specific T cells in spleen, thymus and PBMC in tumor carrying mice were increased (P < 0.01). Our data altogether suggests that enhanced tumor cell death is caused by the increased CTL activity, cytokine and nitric oxide levels, in response to the combined effect of SEA + PA.     

Antitumor Activity of T Cells Generated from Lymph Nodes Draining the SEA-expressing Murine B16 Melanoma and Secondarily Activated with Dendritic Cells

The successful use of tumor-draining lymph nodes (TDLN) as a source of effector cells for cancer immunotherapy depends largely on the immunogenicity of the tumor drained by the lymph nodes as well as the methods for secondary in vitro T cell activation and expansion. We transferred the bacterial superantigen staphylococcal enterotoxin A (SEA) gene into B16 murine melanoma tumor cells, and used them to induce TDLN (SEA TDLN) in syngeneic hosts. Wild-type (wt) TDLN induced by parental B16 tumor was used as a control. In vitro, SEA TDLN cells proliferated more vigorously, produced more IFNγ and demonstrated higher CTL activity than wt TDLN cells when activated with anti-CD3/anti-CD28/IL-2. In vivo, SEA TDLN cells mediated tumor eradication more effectively than similarly activated wt TDLN cells (p<0.01). Furthermore, use of dendritic cells (DC) plus tumor antigen in vitro in addition to anti-CD3/anti-CD28/IL-2 stimulation further amplified the immune function and therapeutic efficacy of SEA TDLN cells. DC-stimulated SEA TDLN cells eliminated nearly 90% of the pulmonary metastasis in mice bearing established B16 melanoma micrometastases. These results indicate that enforced expression of superantigen SEA in poorly immunogenic tumor cells can enhance their immunogenicity as a vaccine in vivo. The combined use of genetically modified tumor cells as vaccine to induce TDLN followed by secondary stimulation using antigen-presenting cells and tumor antigen in a sequential immunization/activation procedure may represent a unique method to generate more potent effector T cells for adoptive immunotherapy of cancer.     

Tumor cells with B7.1 and transmembrane anchored staphylococcal enterotoxin A generate effective antitumor immunity

Staphylococcus enterotoxin A (SEA) stimulates T cells bearing certain TCR beta-chain variable regions, when bound to MHC-II molecules, and is a potent inducer of CTL activity and cytokines production. To decrease toxicity of SEA to the normal MHC-II(+) cells and to localize the immune response induced by SEA to the tumor site, my colleague previously genetically fused SEA with B7.1 transmembrane region (named as SEAtm) to make SEA express on the surface of tumor cells and tumor cells modified with SEAtm could induce efficient antitumor immunity in vitro. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger antitumor immune responses than single-modified vaccines. In this study, we modified the tumor cell vaccine with B7.1 and SEAtm to improve efficiency in the application of SEA. First, SEAtm gene was subcloned from recombinant plasmid pLXSNSEP by PCR and murine B7.1 gene was cloned from splenocytes derived from C57BL/6 mice by RT-PCR. Then, the eukaryotic co-expression vector of SEA and murine B7.1 gene was constructed and named as pcDNA-BIS. B16 cell lines stably expressing SEA and/or B7.1 were established by screening with G418 after transfection and inactivated for the preparation of tumor cell vaccines to treat mice bearing established B16 tumors. The results indicated that the dual-modified tumor cell vaccine B16/B7.1+SEAtm (B16-BIS) elicited significantly stronger antitumor immune responses in vivo when compared with the single-modified tumor cell vaccines B16/B7.1 (B16-B7.1) and B16/SEAtm (B16-SEAtm), and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine with superantigen and co-stimulatory molecule.     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). II. Genuine class II specificity of Lyt-2+ CTL clones

Class II-specific allogeneic cytolytic T lymphocytes (CTL) consist of two types of cells, i.e., Lyt-2+L3T4- and Lyt-2-L3T4 T cells. The Lyt-2+L3T4- class II-specific CTL population constitutes a conspicuous exception to the general correlation observed between the class of major histocompatibility complex antigen recognized and the type of accessory molecules expressed by T cells. In order to examine the specificity of such an exceptional T cell population, CTL clones were established by limiting dilution of a bulk CTL line developed in an I region incompatible combination of mouse strains, B10.QBR anti-B10.MBR. These CTL lines showed single genetic specificity indicating their clonal nature with respect to CTL activities. Lyt-2+L3T4- (2+4-), Lyt-2-L3T4+ (2-4+) and Lyt-2-L3T4- (2-4-) clones were obtained. Among many CTL clones showing a spectrum of genetic specificities, 2+4- and 2-4+ clones with apparent I-Ak-specificity, were studied further and four lines of evidence confirmed their class II specificity: 1) genes encoding the target antigen for these CTL clones were mapped within the I-A subregion by simple genetics; 2) an I-Ak-specific monoclonal antibody readily blocked specific cytolysis by these clones; 3) the clones failed to react with cells expressing mutated I-Ak antigens; and 4) a B cell tumor transfected with alpha- and beta-chain genes of I-Ak was specifically lysed by these CTL clones. These data therefore establish the existence of Lyt-2+ CTL with genuine class II specificity. All 2-4+ CTL were sensitive to the blocking effect of an antibody to L3T4, whereas none of the 2+4- class II-specific CTL were sensitive to blocking by an anti-Lyt-2 antibody, indicating that class II-specific CTL with "wrong phenotype" is not dependent on the function of the accessory molecule. Besides true class II-specific CTL clones, 2+4- clones with a spectrum of genetic specificities were obtained, including clones recognizing a combination of an I-Ak product and the Kb molecule. Two 2-4- clones were also specific for the combination of Kb + I-Ak. These clones most likely recognize an allogeneic class II antigen in the context of a class I antigen and therefore would more appropriately be included in the class I-restricted T cell population.     

The CD8 T cell coreceptor exhibits disproportionate biological activity at extremely low binding affinities

T lymphocytes recognize peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells. Recognition specificity is determined by the alphabeta T cell receptor (TCR). The T lymphocyte surface glycoproteins CD8 and CD4 enhance T cell antigen recognition by binding to MHC class I and class II molecules, respectively. Biophysical measurements have determined that equilibrium binding of the TCR with natural agonist peptide-MHC (pMHC) complexes occurs with KD values of 1-50 microm. The pMHCI/CD8 and pMHCII/CD4 interactions are significantly weaker than this (KD >100 microm), and the relative roles of TCR/pMHC and pMHC/coreceptor affinity in T cell activation remain controversial. Here, we engineer mutations in the MHCI heavy chain and beta2-microglobulin that further reduce or abolish the pMHCI/CD8 interaction to probe the significance of pMHC/coreceptor affinity in T cell activation. We demonstrate that the pMHCI/CD8 coreceptor interaction retains the vast majority of its biological activity at affinities that are reduced by over 15-fold (KD > 2 mm). In contrast to previous reports, we observe that the weak interaction between HLA A68 and CD8, which falls within this spectrum of reduced affinities, retains substantial functional activity. These findings are discussed in the context of current concepts of coreceptor dependence and the mechanism by which TCR coreceptors facilitate T cell activation.     

Variation of expression of histocompatibility antigens on tumor cells: absence of H-2Kk-gene products from a gross-virus-induced leukemia in BALB.K

The antigenic profile of the K-GV tumor of BALB.K origin, induced by Gross virus and maintained in vitro and in vivo, was investigated by serological and immunochemical methods and techniques of cell-mediated immunity. The H-2K^k-gene products were absent by several criteria: (1) monoclonal antibody and conventional alloantisera directed against the H-2K^k antigenic specificities were nonreactive by direct testing and by absorptions. (2) H-2K^k products could not be precipitated from glycoprotein or protein extracts of the radiolabeled K-GV tumor. (3) Cytotoxic effectors against H-2K^k produced by sensitization in vitro and in vivo failed to kill K-GV target cells. (4) The tumor could neither stimulate BALB.B congenic mice to produce cytotoxic effectors nor specific cytotoxic antibody against H-2K^k-gene products. In contrast, the H-2D^k antigen was readily detectable by all these criteria. These findings therefore describe a tumor which has selectively lost the H-2K-gene products. The K-GV tumor was able to generate Gross-virus-specific CTL, but had greatly reduced susceptibility to lysis by Gross-virus-specific CTL generated by H-2K expressing AKR (H-2 ^k) tumors. These findings have important implications for the associative recognition of tumor antigens and the immune surveillance of virally induced tumors.Abbreviations used in this paper MHS major histocompatibility system - LcH Lens culinaris hemagglutinin - SDS sodium dodecyl sulfate - CTL cytotoxic T lymphocytes - GCSA Gross-virus-induced cell-surface antigen - MuLV murine leukemia virus     

Polyclonal CTL responses observed in melanoma patients vaccinated with dendritic cells pulsed with a MAGE-3.A1 peptide

Vaccination with mature, monocyte-derived dendritic cells (DC) pulsed with the MAGE-3(168-176) peptide, which is presented by HLA-A1, has been reported to induce tumor regressions and CTL in some advanced stage IV melanoma patients. We present here a precise evaluation of the level of some of these anti-MAGE-3.A1 CTL responses and an analysis of their clonal diversity. Blood lymphocytes were stimulated with the MAGE-3.A1 peptide under limiting dilution conditions and assayed with an A1/MAGE-3 tetramer. This was followed by the cloning of the tetramer-positive cells and by TCR sequence analysis of the CTL clones that lysed targets expressing MAGE-3.A1. We also used direct ex vivo tetramer staining of CD8 cells, sorting, and cloning of the positive cells. In three patients who showed regression of some of their metastases after vaccination, CTL responses were observed with frequencies ranging from 7 x 10(-6) to 9 x 10(-4) of CD8(+) blood T lymphocytes, representing an increase of 20- to 400-fold of the frequencies found before immunization. A fourth patient showed neither tumor regression nor an anti-MAGE-3.A1 CTL response. In each of the responses, several CTL clones were amplified. This polyclonality contrasts with the monoclonality of the CTL responses observed in patients vaccinated with MAGE-3.A1 peptide or with an ALVAC recombinant virus coding for this antigenic peptide.     

Role of dendritic cells in the immune response induced by mouse mammary tumor virus superantigen.

After mouse mammary tumor virus (MMTV) infection, B lymphocytes present a superantigen (Sag) and receive help from the unlimited number of CD4(+) T cells expressing Sag-specific T-cell receptor Vbeta elements. The infected B cells divide and differentiate, similarly to what occurs in classical B-cell responses. The amplification of Sag-reactive T cells can be considered a primary immune response. Since B cells are usually not efficient in the activation of naive T cells, we addressed the question of whether professional antigen-presenting cells such as dendritic cells (DCs) are responsible for T-cell priming. We show here, using MMTV(SIM), a viral isolate which requires major histocompatibility complex class II I-E expression to induce a strong Sag response in vivo, that transgenic mice expressing I-E exclusively on DCs (I-EalphaDC tg) reveal a strong Sag response. This Sag response was dependent on the presence of B cells, as indicated by the absence of stimulation in I-EalphaDC tg mice lacking B cells (I-EalphaDC tg muMT(-/-)), even if these B cells lack I-E expression. Furthermore, the involvement of either residual transgene expression by B cells or transfer of I-E from DCs to B cells was excluded by the use of mixed bone marrow chimeras. Our results indicate that after priming by DCs in the context of I-E, the MMTV(SIM) Sag can be recognized on the surface of B cells in the context of I-A. The most likely physiological relevance of the lowering of the antigen threshold required for T-cell/B-cell collaboration after DC priming is to allow B cells with a low affinity for antigen to receive T-cell help in a primary immune response.     

Protection against metastasis by immunization with an allogeneic lymphocyte antigen

Q5 antigens are expressed on the surface of various experimental murine tumor cells. They share partially common antigenicity with Qa-2 alloantigens expressed on normal lymphocytes. For that reason we tested the immunoprotection by anti-Qa-2 immunization of mice against a Q5+ tumor. Nerve fibrosarcoma (NSFA) tumor, which specifically develops metastasis in the lung, has been reported to be poorly immunogenic. However, expression of the Q5 antigen was evident on the surface of NFSA cells. After immunizing (C3H/He x B6.K1)F1 (Qa-2-) mice with B6 (Qa-2+) lymphocytes, the protection against the proliferation of the semi-syngeneic NFSA tumor was examined First, immunization of normal mice induced resistance to NFSA cell transplants. Second, when the tumor cells were transplanted to the hind foot of a mouse and the resulting tumor was removed by amputating the leg, the mice were protected against the development of lung metastasis after immunization by intraperitoneal inoculation of B6 cells 3 days after tumor removal. Immunization with attenuated NFSA cells in this system failed to protect the mice from lung metastasis. On the other hand, inoculation of the mice with B6 cells without removal of the original tumor on the foot showed little effect on the progression of the tumor. Thus, cytotoxic T lymphocytes (CTL), which seemed to be present in an inactive form in the mice from which the tumor had not been removed, were induced in the mice after the removal of the major tumor followed by immunization with B6 lymphocytes. The induction of CTL by the immunization was suppressed in mice bearing large tumors. Cells stimulated by the tumor antigen seemed to be involved in the suppression. It was also shown that the Q5 antigen is the direct recognition target of the CTL since the activity of Q5-specific CTL clones in lysing tumor cells was inhibited by a monoclonal antibody specific for the Q5 antigen. In contrast to immunization with attenuated tumor cells, our novel allogeneic lymphocyte immunization procedure offers high CTL activation, by-passing the induction of T cell unresponsiveness.     

Stimulation of human naive and memory T helper cells with bacterial superantigen. Naive CD4+45RA+ T cells require a costimulatory signal mediated through the LFA-1/ICAM-1 pathway.

The role of the accessory molecule ICAM-1 in activation of subpopulations of human T cells was examined using the bacterial superantigen staphylococcal enterotoxin A (SEA) as a MHC class II and TCR-dependent polyclonal T cell activator. Human T cells responded with different sensitivity to SEA when presented on mouse accessory cells expressing a human transfected MHC class II gene product. Mouse L cells cotransfected with both MHC class II (DR2A or DR7) and ICAM-1-stimulated T cells at 100-fold lower concentrations of SEA as compared to the single transfected cells. mAb reacting with the CD11a, CD18, or ICAM-1 molecules efficiently inhibited T cell activation with the cotransfected HLA-DR2A/ICAM-1 cell but did not influence T cell activation with the HLA-DR2A single transfected cell. Analysis of the ICAM-1 requirement on CD4+ memory (CD4+45RO+) and naive (CD4+45RA+) T cells revealed that CD4+45RA+ naive Th cells were hyporesponsive to SEA-induced activation with the HLA-DR2A single transfectant. However, cotransfection of ICAM-1 enabled these cells to respond to low doses of SEA implicating that they are more dependent on accessory molecules than the CD4+45RO+ cells. rICAM-1 immobilized on a plastic surface, was able to strongly costimulate SEA-induced T cell activation with the HLA-DR2A single transfectant, suggesting that costimulatory signals mediated to the T cells through LFA-1 can be delivered physically separated from the TCR signal. CD4+45RO+ memory and CD4+45RA+ naive Th cells apparently differ in their capacities to be activated by SEA bound to HLA-DR. Although the TCR molecule densities are similar in these two subsets, costimulation with ICAM-1 is required for activation of the CD4+45RA+, but not the CD4+45RO+ T cell subset at 1 to 10,000 ng/ml concentrations of SEA. This observation indicates different activation thresholds of naive and memory Th cells when triggering the TCR over a wide dose interval of superantigen.