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Melanoma is notoriously resistant to chemotherapy, but variable responses to biotherapies, including the IFNs and IL-2, provide intriguing avenues for further study. Systemic IL-2 treatment has provided significant clinical benefit in a minority of patients with metastatic melanoma, leading to long-term survival in a few cases. We hypothesize that one previously unidentified mechanism of effective IL-2 therapy is through direct upregulation of the tumor suppressor IL-24 in melanoma tumor cells resulting in growth suppression. In this study, five melanoma cell lines were treated with high dose recombinant human IL-2. Three (A375, WM1341, WM793) showed statistically significant increases in IL-24 protein; two (WM35, MeWo) remained negative for IL-24 message and protein. This increase was abolished by preincubating with anti-IL-2 antibody or blocking with antibodies against the IL-2 receptor chains. These IL-2 responsive melanoma cell lines expressed IL-2Rβ and IL-2Rγ mRNA. The IL-2Rβγ complex was functional, as measured by IL-2-induced signal transducers and activators of transcription activation as well as IL-15 signaling through its shared receptor complex. IL-24 upregulation was observed in response to either IL-2 or IL-15. Cell growth was significantly decreased by treatment of IL-24-positive cells with IL-2 or IL-15, whereas no effect was seen in negative cells. Incubating the IL-24 inducible-cells with anti-IL-24 antibody as well as transfecting with IL-24 small interfering RNA effectively reversed the growth suppression seen with IL-2. Thus, we have shown that one mechanism of clinically effective IL-2 therapy may be the direct action of IL-2 on a biologically distinct subset of melanoma cells leading to upregulation of the tumor suppressor IL-24.

Interleukin-24 (IL-24) is a novel tumor suppressor/cytokine gene expressed in normal human melanocytes but for which expression is nearly undetectable in metastatic melanoma. Overexpression of the IL-24 protein has been shown to inhibit tumor cell proliferation and induce apoptosis in many melanoma cell lines, and is now considered a tumor suppressor. Erlotinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has been widely studied for the treatment of human lung cancer and other solid tumors, but the erlotinib-targeted therapy has not been tested in melanoma. The objective of this study is to investigate the potency of erlotinib in suppressing the growth of human melanoma cells and whether IL-24 could enhance the antitumor activity of erlotinib. In cell viability and apoptosis assays, treatment with erlotinib dependently inhibited the growth of different melanoma cell lines and when combined with adenoviral vector-mediated IL-24 gene therapy, a significant increase in cell growth inhibition and apoptosis induction resulted (P<0.05). Immunoblot assay showed that the combination treatment of erlotinib and IL-24 considerably increased the cleavage of caspase-3 and caspase-9 and the expression of Apaf-1 protein in melanoma cells, inducing activation of the Apaf-1-dependent apoptotic pathways. Moreover, this combination treatment markedly inhibited phosphorylation of the EGFR, phosphatidylinositol-3 kinase, and Akt proteins, inactivating the Akt-dependent cell survival signaling pathway. These results show that a combination of IL-24-mediated molecular therapy and EGFR inhibitors such as erlotinib may be a promising treatment strategy for human melanoma and will serve as a basis for guiding the combination treatment designs in future preclinical and clinical trials.

Interleukin (IL)-24 is the protein product of melanoma differentiation-associated gene 7 (MDA-7). Originally identified as a tumor suppressor molecule, MDA-7 was renamed IL-24 and classified as a cytokine because of its chromosomal location in the IL-10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. We previously reported that IL-24 is expressed by cytokine-activated monocytes and T lymphocytes. Here, we show that IL-24 is expressed in keratinocytes during wound repair. Paraffin-embedded tissues prepared from human skin sampled at days 2, 6, and 10 after wounding were examined by immunohistochemistry for the expression of IL-24. Protein expression was detected in the keratinocyte population with maximum expression at days 2 and 6, and no expression by day 10 (four of four subjects). In vitro studies showed that cytokines involved in wound repair, most notably transforming growth factor alpha (TGFalpha), TGFbeta, IFNgamma, and IFNbeta, upregulated IL-24 protein expression in normal human epidermal keratinocytes (NHEKs). Examination of the function of IL-24 in both in vitro wound repair and migration assays demonstrated that IL-24 inhibits TGFalpha-induced proliferation and migration of NHEKs. These data support the hypothesis that IL-24 functions during an inflammatory response in the skin by inhibiting the proliferation and migration of keratinocytes.

Purpose: Our goal was to perform studies on the specificity and antimelanoma mechanism of a novel bis-anthracycline, WP760. WP760 initially identified in the NCI 160 screen as anti-melanoma., Methods: The methyl thiazolyl tetrazolium reduction (MTT) assay was used to test tumor cell growth inhibition; confocal microscopy to view WP760 intracellular distribution; flow cytometry for cell-cycle arrest and apoptosis; and Western blotting was employed to identify and compare quantities and kinetics of cell growth related molecule levels., Results: WP760 induced G(2)/M-phase cell-cycle arrest and apoptosis in melanoma cell lines and short-term melanoma explants established from clinical specimens in a time and concentration dependent manner at nM concentrations. In contrast, effects on fibroblasts and A549 lung cancer cells required higher concentrations, suggesting that WP760 possesses selectivity for melanoma. Molecular studies indicated that WP760 induced p53 stabilization, checkpoint kinase 2 and p27(Kip1) protein upregulation, and activation of caspase-3. Endogenous nitric oxide (NO) production has been implicated in the chemoresistance of melanoma; WP760 caused inhibition of the inducible nitric oxide synthase (iNOS) protein as well as inhibition of phosphorylation of ERK, known to drive the iNOS pathway. Based on WP760 localization into mitochondria, and caspase-3 inhibitor block the killing of WP760, the intrinsic pathway of apoptosis appears to have been activated., Conclusions: Our results indicate that WP760 affects a critical and unique set of growth regulatory effects in melanoma, and is a promising candidate for further preclinical studies.

Interleukin-24 (IL-24), also known as melanoma differentiation-associated gene-7 (mda-7), is a member of the IL-10 family that exhibits both tumor suppressor and proinflammatory properties. We describe the purification of this novel dual-function tumor suppressor/cytokine from the supernatant of IL-24 gene-transfected HEK 293 cells and define the biochemical and functional properties of the soluble human IL-24 protein. Size exclusion chromatography demonstrates that an IL-24 macromolecular complex fractionates in a broad peak with a median of 110 kDa and comprises several IL-24 isoforms, identified by immunoblotting with anti-IL-24 polyclonal antibody after reducing SDS-PAGE analysis. IL-24 was found to associate with two serum components, albumin and C1q. Cation exchange purification results in the isolation of at least two N-linked glycosylated IL-24 dimers covalently associated via intermolecular disulfide bonds. These molecularly defined N-glycosylated IL-24 dimers elicited dose-dependent secretion of tumor necrosis factor-alpha (TNF-alpha) and IL-6 from human monocytes, as well as cytotoxicity to human melanoma cell lines. Thus, we demonstrated that the secreted, glycosylated, dimeric, human IL-24 is immunomodulatory to monocytes and exhibits tumor cell growth inhibition.

Interleukin-24 (IL-24) is a recently identified member of the IL-10 family of cytokines. It was originally identified as a tumor suppressor molecule, melanoma differentiation-associated gene 7, and then renamed IL-24 and classified as a cytokine, based on its chromosomal location in the IL-10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. Here, we correlate the kinetics of IL-24 mRNA and protein expression in human peripheral blood mononuclear cells (PBMC) stimulated by polyclonal activators phytohemagglutinin (PHA) and lipopolysaccharide (LPS) or by allogeneic major histocompatibility complex. PHA-stimulated PBMC express IL-24 mRNA, reaching peak levels at 8-12 h after stimulation. Protein expression, as measured by intracellular flow cytometry, followed the message, reaching maximum expression at 24 h. Subset analysis of mitogen-stimulated PBMC showed that IL-24 was expressed primarily in T cells and macrophages. Expression of IL-24 in mitogen-stimulated PBMC is the result of cytokine stimulation. Individual cytokines including IL-2, IL-7, IL-15, tumor necrosis factor alpha, granulocyte macrophage-colony stimulating factor, and IL-1beta stimulate the expression of IL-24 mRNA and protein, whereas interferons and T helper cell type 2 cytokines fail to induce substantial IL-24. When LPS- or PHA-stimulated cells were treated with Actinomycin D, IL-24 mRNA persisted at high levels over the 4-h course of treatment. These data strongly suggest that the expression of IL-24 in human PBMC results from cytokine stimulation and is regulated at the post-transcriptional level through stabilization of IL-24 mRNA.

Introduction: Sentinel lymph node (SLN) biopsy allows identification of the first lymph node into which a primary tumor drains. In breast cancer, identification of tumor cells in the SLNs is a predictor of the tumor's metastatic potential. In the present article, we tested the hypotheses that a positive immune response can occur in tumor-free SLNs and that the activation state of dendritic cells (DCs), the major antigen presenting cells within SLNs, predicts the immune status and metastatic potential of the tumor., Methods: Fifty paraffin-embedded SLN sections, 25 tumor-free and 25 tumor-containing, from patients with breast cancer were analyzed by immunohistochemistry to determine the immune maturation state of their DCs. In addition, 12 lymph nodes from noncancer-containing breasts were analyzed. Tissues were stained with antibodies against CD3, MHC class II, CD1a, CD83, IL-10, and IL-12. Mature DCs were defined by CD83 expression and immature DCs by CD1a expression., Results: We found a trend toward higher numbers of mature CD83-positive DCs in tumor-free SLNs than in tumor-containing SLNs (P = 0.07). In addition, tumor-free SLNs were more likely to contain cells expressing IL-10 (P = 0.02) and, to a lesser extent, IL-12 (P = 0.12). In contrast, when all SLNs, both tumor-free and tumor-containing, were compared with uninvolved lymph nodes, the numbers of mature and immature DCs were similar., Conclusions: Our results suggest tumor-free SLNs are immunologically competent and potentially a site of tumor-specific T-cell activation, as evidenced by the presence of greater numbers of mature DCs and cytokine-producing cells in tumor-free SLNs.

Acute rejection of renal allografts is mediated by infiltrating alloreactive T cells. The goals of this study were to correlate T cell proliferation with rejection and to determine whether T cell proliferation in the absence of rejection would predict future rejection episodes. Toward this, kidney biopsies (n=100) were cultured in the presence of interleukin-2. Cultures were examined at 4, 24, and 48 hr for T cell proliferation. A strong correlation was observed between T cell proliferation at any time point and rejection. There was not a significant correlation between T cell proliferation in biopsies with no rejection and the occurrence of a rejection episode within 2 months. However, T cell proliferation after 4 hr was a better predictor of the occurrence of rejection within 2 months compared with observations after 24 and 48 hr. Therefore, a subgroup of patients with unremarkable biopsies but T cell proliferation may be at risk for rejection and warrant closer observation and possible tailoring of immunosuppression.

Background: Significant levels of donor soluble human leukocyte antigen (HLA) class I (sHLA) are present in patients after transplants. We investigated the possibility that sHLA may inhibit cytolytic T lymphocyte (CTL) activity by inducing apoptosis of the CTL, thereby serving as a mechanism for specific tolerance., Methods: sHLA-A2 and A3 were isolated from the sera of liver transplant recipients by affinity chromatography. T cell bulk lines directed against HLA-A2 and HLA-A3 were generated by stimulation with HLA-A2, A3+ peripheral blood leukocytes and B-lymphoblastoid cells. Induction of T cell apoptosis by sHLA was analyzed by adding sHLA to allospecific CTL 4 or for 24 hr before flow cytometric analysis of propidium iodide and fluorescein isothiocyanate-conjugated annexin V stained cells. T cell receptor (TCR) engagement by sHLA was demonstrated using a monoclonal antibody specific for the TCR., Results: sHLA-A3 inhibited CTL activity of a HLA-A3 T cell line by 53%, whereas sHLA-A2 had no effect. sHLA-A3 also increased T cell death by 77% over the control, whereas sHLA-A2 had no significant effect. However, sHLA-A2 induced 21% apoptosis of an anti-HLA-A2 T cell line, whereas sHLA-A3 caused only 3% apoptosis. The antibody complexed form of sHLA was ineffective in the induction of apoptosis. Preincubation of the T cells with anti-T cell receptor monoclonal antibody protected the T cells from sHLA-induced apoptosis, indicating that sHLA-TCR engagement is necessary for this process to occur., Conclusion: TCR-mediated apoptosis of alloreactive CTL may serve as a mechanism by which sHLA can modulate the immune response.

Background: The tissue specificity of a cytolytic T lymphocyte is determined by the MHC class I bound peptide it recognizes. We have developed an allorestricted human CTL clone, DBS 1.5, that recognizes an epitope found on HLA-A3+ kidney epithelial cells but not on HLA identical B-lymphoblastoid cells. The peptide recognized by this clone has been isolated from HPLC separated, acid eluted peptides from purified HLA class I molecules from HLA-A3+ kidney tissue. This peptide shares no sequence homology with any known protein., Methods: To confirm the tissue specificity of the HLA-A3 restricted clone and the peptide it recognizes we have transfected the gene for HLA-A3 into a number of tumor cell lines both human and murine not expressing this antigen. The resulting transfected lines, confirmed by immunofluorescent staining, were used as targets to determine if expression of HLA-A3 alone was sufficient to allow recognition and lysis by the HLA-A3 restricted T cell clone., Results: The HLA-A3 restricted T cell clone recognized HLA-A3 when expressed on human kidney epithelial cells and to a lesser extent on human lung epithelium and human epidermal cells. Of the tumor lines transfected with HLA-A3 only the human kidney tumor cell line was lysed at a level equal to the original kidney epithelial cell used to develop the clone., Conclusion: These results confirm that this allorestricted human CTL clone is tissue specific recognizing a peptide found in human epithelial tissue that must be presented in the context of HLA-A3 for recognition.

Background: The presentation of donor MHC class II-derived peptides by host antigen-presenting cells in the context of self-MHC class II molecules has been suggested as a mechanism for the chronic rejection of kidney and heart allografts. The aim of this study was to determine whether indirect allorecognition of HLA class I-derived peptides occurred in lung transplant (LTx) recipients and whether it correlated with the development of bronchiolitis obliterans syndrome (BOS)., Methods: Peripheral blood mononuclear cells from LTx recipients were cultured with synthetic peptides corresponding to the hypervariable regions of the mismatched HLA class I antigens of the donor. Proliferation and precursor frequency (PF) of allopeptide reactive T cells were determined by the incorporation of [3H]thymidine into DNA and limiting dilution analysis., Results: Peripheral blood leukocytes of LTx recipients with BOS mismatched for HLA class I molecules showed a proliferative response three- to fourfold higher than those observed in mismatched recipients without BOS and in normal control individuals (P<0.001). Similarly, the PF of allopeptide-reactive T cell was 3- to 24-fold higher in recipients with BOS compared with recipients without BOS (P<0.05) as well as normal control individuals (P<0.03). The T cell PF to donor-specific allopeptides, as well as irrelevant allopeptides, was not significantly different in LTx recipients without BOS and normal control individuals., Conclusions: These data suggest that T cells from LTx recipients are sensitized to mismatched HLA class I antigens. The sensitization was significantly higher in LTx recipients with BOS compared with LTx recipients without BOS. Strategies to block T-cell responses generated by indirect allorecognition after lung transplantation may provide a means for the prevention or treatment of BOS in LTx recipients.

Background: The lung epithelium is among the first donor tissues encountered by the lung allograft recipient's immune system. The purpose of this study was to determine whether lung epithelium was recognized by T lymphocytes that are isolated from bronchoalveolar lavage fluid of lung allograft recipients during periods of acute rejection., Methods: Lymphocytes isolated from 45 bronchoalveolar lavage samples (from 41 lung transplant recipients) served as effector cells in standard cell-mediated cytolytic assays with several cell lines as targets: BEAS-2B (an immortalized airway epithelial cell line); B-lymphoblastoid cell lines; and K562 (a natural killer-sensitive cell line). Cytotoxic T-lymphocyte activity of bronchoalveolar lavage lymphocytes was correlated with pathologic status., Results: During acute rejection alone (ie, without concomitant cytomegalovirus infection), mean lysis of the airway epithelial target was significantly greater, compared with during no rejection, when these targets expressed donor-specific HLA class I antigens (P =.007). Lysis of donor class I-matched B-lymphoblastoid cell line targets during rejection was not significantly different from lysis during no-rejection periods (P =.18). Mean lysis of K562, a natural killer cell target, did not differ between acute rejection (without concomitant cytomegalovirus infection) and no rejection (P =.30). During cytomegalovirus infection (without concomitant acute rejection), there was no difference in mean lysis of airway epithelial cells, B-lymphoblastoid cell lines, or K562 targets compared with during no cytomegalovirus infection, whereas during acute rejection, compared with cytomegalovirus infection without rejection, there was a significant increase in mean lysis of the airway epithelial target when it expressed donor-specific HLA antigens (P =.01)., Conclusions: Donor HLA class I-specific cytotoxic T-lymphocyte activity directed at airway epithelial cells was demonstrated in bronchoalveolar lavage lymphocytes from lung transplant recipients. Lysis of these targets was significantly higher during episodes of acute rejection.

Allograft rejection is mediated by both CD4+ and CD8+ T cells. The lytic function of the classic CD8+ cytolytic T lymphocytes (CTL) occurs through recognition of allogeneic major histocompatibility complex (MHC) class I on the surface of the graft. CD4+ CTL recognize MHC class II through a direct recognition pathway or an indirect pathway where MHC peptides are presented in the context of self MHC class II. Lytic CD4+ cells may destroy graft tissue or, we hypothesize, the indirect CD4+ T cell may down regulate CD8+ CTL by recognition of donor MHC peptides presented by self MHC class II expressed on CD8+ T cells. To define the role of CD4+ CTL in allograft outcome we used a CD4+ CTL that is MHC class II restricted, recognizing human leucocyte antigen (HLA)-A1 and HLA-B8 peptides in the context of HLA-DR4. This line (MDSxA1/B8) will lyse DR4+ B lymphoblastoid cells (LCL) pulsed with HLA-A1/B8 peptides (amino acids 60-84 of the alpha1 domain of the MHC class I molecule). These T cells will also lyse peptide-pulsed antigen-specific T cell clones, both CD4+ and CD8+, that express HLA-DR4. These clones must process and present the MHC class I peptides for recognition and lysis to occur. These results suggest a possible mechanism to explain allograft tolerance. Lytic CD4+ T cells, that recognize donor HLA peptides through an indirect antigen presentation pathway, down-regulate donor-specific CTL through peptide-specific lysis resulting in graft tolerance.

Acute rejection, mediated by T lymphocytes recognizing donor MHC class I and II, is a major factor influencing renal transplant survival. To define the specificity of these effector cells we examined cytolytic activity of graft infiltrating T lymphocytes (GIL) from renal biopsies of individuals undergoing acute cellular rejection. The majority of these cells recognized MHC class I on both donor kidney epithelial cells (KCL) and B-lymphoblastoid cells (LCL) suggesting these T cells recognized peptides from various tissues. However, cold target inhibition experiments demonstrated a significant proportion of GIL T cells were tissue specific. We reported previously that kidney specific CTL can be isolated from biopsies of kidney allografts undergoing acute cellular rejection. Here we extend that observation showing we were able to isolate tissue specific CTL from two additional biopsies. Greater than 10% of the clones isolated (4 of 36 and 5 of 37) from these biopsies were CTL recognizing donor KCL but not LCL targets suggesting that peptides, recognized in the context of donor MHC, were tissue specific. Repeated isolation of significant numbers of tissue specific CTL suggests these T cells play a role in allograft rejection and may be important effector cells mediating rejection in HLA matched transplants.

The cytolytic activity of T lymphocytes infiltrating renal allografts from recipients undergoing episodes of acute cellular rejection was studied. These T-cell populations, composed of both CD4+ and CD8+ cells, demonstrated significant cytolytic activity against both donor-derived KCLs and B-LCLs. In five of 21 biopsy-derived lines greater cytolytic activity was measured against donor KCLs compared to donor B-LCLs, suggesting the presence of kidney antigen-specific, MHC-restricted clones. Clones developed by stimulation with donor B-LCLs lysed both donor B-LCLs and KCLs while clones developed on donor KCLs as stimulator cells showed tissue specificity. Three of 26 clones recognized tissue-specific antigens in the context of donor MHC class I antigens lysing donor KCLs but not B-LCLs. These data demonstrate that a subpopulation of T cells recognizing kidney-specific antigens are present in biopsies of renal allograft recipients undergoing acute cellular rejection. This subpopulation of tissue-specific cytotoxic T lymphocytes may prove to contribute significantly to the pathology of allograft rejection.

The molecular nature of tissue-specific Ags involved in MHC-restricted CTL responses is as yet undefined. To determine the specificity of these peptides, their function, and their possible relationship to allograft rejection, we have utilized human kidney-specific CD8+ CTL clones to screen reversed-phase HPLC (RP-HPLC)-separated self peptides presented by allo-class I molecules. One of these clones is HLA-A3-restricted and the other HLA-B62-restricted, lysing human kidney cell lines but not MHC identical B lymphoblastoid cells which express the appropriate HLA molecules. We have identified a biologically active RP-HPLC fraction containing self peptides eluted from affinity-purified MHC molecules from HLA-A3+ kidney. This peptide is not expressed in HLA-A3+ spleen. Similarly, a HLA-B62-associated peptide fraction was identified in kidney but not in spleen using the HLA-B62-restricted CTL clone. Sequence analysis of the biologically active fraction from HLA-A3 kidney revealed multiple peptides. Because of the ambiguity of the peptide sequence, a mixed peptide library corresponding to this sequence was synthesized that included the HLA-A3 binding motif. The biologically active peptide library was RP-HPLC fractionated and the fraction containing HLA-A3-restricted CTL activity was sequenced. The resulting sequence of the alloreactive HLA-A3-restricted peptide epitope is GPPGVTIVK. By using this unique strategy, we describe the successful isolation and sequencing of an antigenic peptide that is recognized by a human alloreactive kidney-specific CTL clone.

Severe combined immunodeficient (SCID) mice have become a promising tool for the development of models of human immunologic process. We report the development of a reproducible technique for engrafting SCID mice with human PBL (hu-PBL-SCID). Our results show that a booster injection of anti-CD3 antibody stimulated human lymphocytes given 2 days after the initial injection of lymphocytes will improve the efficiency of chimera establishment to 86.7% (13 out of 15). There was also good correlation among detection of human Ig, human CD3+ cells, and human DNA by polymerase chain reaction amplification in the circulation of hu-PBL-SCID mice. Questions remain concerning the immune function of the human lymphoid cells in the SCID mouse. In this study, we analyzed the ability of human T cells in SCID mice to reject human islet allografts transplanted under the kidney capsule. Human islet allograft function assessed by human C-peptide levels demonstrated failure of islet allografts within 21 days after transplantation in hu-PBL-SCID. In contrast, human islets grafted in unreconstituted SCID mice continued to function for greater than 60 days. Recovered human T cells from rejected islets of hu-PBL-SCID mice displayed specific cytolytic activity against HLA class I-matched islets, while the recovered cells from spleen of hu-PBL-SCID mice showed minimal specific cytotoxicity against islets. These results suggest that graft-infiltrating lymphocytes were activated by the engrafted islets within the hu-PBL-SCID, causing the eventual rejection of the human islet allograft. Thus, engraftment of the anti-CD3 antibody-primed human PBL results in a mouse-human chimera with a functionally competent human immune system that is capable of rejecting a human islet allograft.

Transgenic mice expressing the human insulin gene do not produce insulin-specific antibody after injection of human insulin. Nevertheless, they have some peripheral T cells that proliferate to human insulin in vitro. To investigate the nature of these T cells, human insulin-specific T cell hybridomas were produced from transgenic and nontransgenic mice. Transgenic hybridomas required more insulin to achieve maximum responses and they produced lower levels of lymphokines than nontransgenic hybridomas. The majority of nontransgenic hybridomas recognized only human and pork insulin whereas transgenic hybridomas recognized beef, sheep, and/or horse insulin in addition to human and pork insulin. The TCR expressed by transgenic and nontransgenic hybridomas were determined by Northern analysis. Both types of hybridomas used several different V alpha and V beta gene families and no favored association between V alpha and V beta gene usage was detected in either type. V beta 1 was used by 7 of 16 nontransgenic hybridomas but only by 1 of 16 transgenic hybridomas. V beta 6 receptors were predominantly expressed by the transgenic hybridomas and all V beta 6-bearing hybridomas recognized beef as well as human insulin. The differences in Ag reactivity and TCR gene usage suggest that V beta 1-bearing human insulin-reactive T cells were clonally deleted or inactivated in the transgenic animal. Other clones, representing a minor subpopulation in nontransgenic mice, were recovered from transgenic mice.

We have examined mechanisms of tolerance to circulating self-proteins in mice that are transgenic for human insulin. Normal, nontransgenic mice develop serum antibody responses when injected with human insulin in CFA; syngeneic transgenic mice do not. B cell responsiveness was assessed by immunizing with human insulin coupled to a T-independent Ag, Brucella abortus. No differences were found in the numbers of insulin-specific splenic plaque-forming cells between transgenic and nontransgenic mice suggesting that insulin-specific B cells are not tolerant in transgenic mice. Similarly, APC from transgenic and nontransgenic mice display no differences in their ability to process and present human insulin to human insulin-specific T cells in vitro. However, marked differences were detected between transgenic and nontransgenic T cells. Lymph node T cells from transgenic mice primed with human insulin provided no detectable helper activity for secondary antibody responses to human insulin whereas, lymph node T cells from nontransgenic mice did. Nevertheless, lymph node T cells from transgenic mice developed significant proliferative responses to human insulin. Lymph node T cells obtained from transgenic and nontransgenic mice were fused to BW5147 and human insulin-specific T cell hybridomas were generated. The fact that human insulin-specific T cell hybridomas were obtained from the transgenic mice suggests that these T cells were not clonally deleted. In addition, APC from transgenic mice did not stimulate human insulin-specific hybridomas from normal mice in the absence of exogenous insulin. We suggest that T cells specific for human insulin are not deleted in the thymus of transgenic mice because APC in the thymus do not bear the requisite levels of endogenous human insulin/Ia complexes. Therefore, we conclude that tolerance in the transgenic mice is preserved by peripheral mechanisms.

Toxic-shock syndrome toxin-1 (TSST-1), a nonspecific T lymphocyte mitogen, was shown to cause the suppression of immunoglobulin-secreting cells (ISCs) generated in pokeweed mitogen (PWM)-stimulated cultures of human peripheral blood mononuclear cells (PBMs). Greater than 90% suppression of the PWM-stimulated ISC response was achieved when 2 X 10(6) PBMs were cultured in the presence of 1 microgram of TSST-1. The nonspecific suppression induced by the toxin was dose related; amounts of TSST-1 as low as 1.0 ng/2 X 10(6) cultured cells caused significant suppression of the ISC response. TSST-1-induced suppression was due to the activation of a population of cells that secreted a soluble factor that mediated the resulting suppression. PBMs were activated by a 24-hr preincubation of 2 X 10(6) cells in the presence of 1 microgram of TSST-1 followed by repeated washing to remove residual toxin. These cells, when co-cultured with unstimulated PBMs and PWM, suppressed the normal ISC response. The supernatant factor, active at volumes of 10 microliters/2 X 10(6) PBMs, was generated by harvesting the supernatant fluid of TSST-1-activated cells. The results of these in vitro experiments confirm that TSST-1 is an immunosuppressing agent that affects human lymphocyte function and, in such a capacity, may play a role in the development of toxic shock syndrome in humans.

The binding of iodinated cholera and Escherichia coli (LT) enterotoxins to Y1 mouse adrenal cells was studied by using saturation analysis (Scatchard). Each toxin bound to Y1 cells with similar affinity [KA = (1.5--2.0) x 10(9)M-1], but there appeared to be twice as many receptor sites per cell for E. coli toxin (approximately 4 x 10(5). Despite the increased binding of E. coli toxin, Y1 cells respond sooner to, and to smaller concentrations of, cholera toxin. The binding of each toxin was inhibited competitively by both toxins, although twice as much E. coli toxin was required to inhibit 50% of the binding of cholera toxin as was needed for either homologous inhibition or the inhibition of E. coli toxin binding by cholera toxin. The B subunits of both toxins were equally effective in competing for the binding of both iodinated toxins. Whereas the A subunits of both toxins had little or no effect on the binding of E. coli toxin, they consistently inhibited 20--40% of the binding of cholera toxin to cells. These results suggest that there are receptor loci on cells for the A subunit and that conformational differences exist between the two toxins that might explain the greater sensitivity of Y1 cells to cholera toxin. A model is suggested in which cholera toxin exhibits a greater degree of multivalent ligand binding than does the E coli toxin, resulting in a more favorable situation for apposition of the A subunit to its receptor or for its insertion into the membrane.

Toxic-shock-syndrome toxin 1 (TSST 1) produced by Staphylococcus aureus induced in vitro proliferation of lymphocytes isolated from rabbit spleens, murine spleens, and both human peripheral blood and cord blood. This mitogenic response was nonspecific in all three systems. In the mouse and human systems, proliferation depended upon the presence of macrophages in the responding lymphocyte population. Inability to remove macrophages from rabbit splenocyte suspensions made it impossible to determine the contribution of this cell in rabbit splenocyte proliferation. Kinetic analysis of TSST 1-induced mitogenicity showed that proliferation of lymphocytes was maximal between days 4 and 6 in each of the three systems examined. Sensitivity to TSST 1 was similar in each system, with maximal proliferation achieved at TSST 1 doses as low as 0.1 ng/5 X 10(5) murine splenocytes or 2 X 10(5) rabbit splenocytes, and 0.01 ng/3 X 10(5) human mononuclear cells from human peripheral blood. Study of separated populations of mouse splenocytes and mononuclear cells from human peripheral blood showed that the TSST 1-induced proliferative response resided solely in the T cell populations.

The use of the nontoxic B subunits of cholera and Escherichia coli enterotoxins in vitro and in vivo led to a decrease in toxin binding to target cells and a decrease in toxin-induced effects (i.e., morphological effects, adenylate cyclase activation, and fluid secretion). The reduction in toxin binding involves a process of down-regulation of cellular receptors for the toxin and not toxin occupancy of receptors. The extent of inhibition was dependent on the amount of B subunit used and on the duration of time after its use. Thus, in vivo exposure to a single bolus of B subunit was sufficient to block toxin binding and activity for up to 18 h. Because the B subunit binds extensively to the esophagus and the stomach, peroral administration will require a preparation that allows the subunit to reach the small bowel in a protected form. Our data provide a rationale for using B subunit therapy for short-term protection against the effects of enterotoxins, before the development of an immune response.

Toxic-shock-syndrome toxin-1 (TSST-1), produced by Staphylococcus aureus and associated with toxic shock syndrome, functions in vitro as both a lymphoproliferative and immunosuppressive protein for human peripheral blood mononuclear cells (PBMs). We analyzed TSST-1-target cell interactions by receptor-ligand binding analyses. In competitive binding experiments, 2 X 10(5) human PBMs or purified cell populations were incubated in the presence of small amounts of (5-50 ng) of 125I-labeled TSST-1 and increasing amounts of unlabeled TSST-1 (25-10,000 ng). Data were analyzed by the method of Scatchard. Toxin-specific receptors were shown to exist on T lymphocytes within the PBM population. T4+ cells had 27.5 X 10(6) receptors per cell, and T8+ cells had 9 X 10(6) receptors per cell. T4+ and T8+ receptors had dissociation constants of 2.58 X 10(-8) M and 1.8 X 10(-8) M, respectively. These studies confirm earlier work showing that TSST-1 causes the functional activation of a population of T lymphocytes involved in suppression of immunoglobulin responses.