Mixed lymphocyte culture responses of mice. Genetic analysis of the responses to H-2Dd specificities.

Cellular responses in vitro to H-2D region histocompatibility antigens were demonstrated to be under the genetic control of two or three (P = 0.013) independently segregating loci. The H-2 region itself accounts for one of these loci, however, its activity appears to be dependent upon an association with other non-H-2-associated genetic information. The ability to stimulate a response and to respond to that stimulus are two separate genetic functions in certain MLR combinations. The stimuli in our studies were products of the H-2D region and cell donors must differ at that region in order for a response to occur. The control of the level of responses was determined by other genetic material. Differences at these "response loci" were not necessary for the induction of a proliferative response in the mixed lymphocyte cultures.

Major histocompatibility complex restriction of soluble helper molecules in T cell responses to altered self.

Evidence for major histocompatibility complex (MHC) restriction of soluble helper effects was observed in the generation of syngeneic killer T cells to trinitrophenyl-altered self. Ia-bearing T cells obscure the observation of such interactions, thus, must be removed to detect MHC restriction of nonspecific soluble helper factor supernates (HFS). Genetic mapping studies demonstrated that the strain producing HFS must be compatible in the H-21A region with the strain utilizing the helper molecules for optimal helper signals to be delivered.

Kidney transplants in mice. An analysis of the immune status of mice bearing long-term, H-2 incompatible transplants.

Kidney transplants between strains of mice which are incompatible at either the K or the D end of the H-2 complex usually function for prolonged periods supporting the lives of nephrectomized recipients. This occurs with no recipient treatment. With multiple H-2 and non-H-2 determined incompatibilities, transplants may be rejected but more slowly than skin grafts. In the strain combination studied most extensively in these experiments (B10.D2 to B6AF(1)) in which the incompatibility was confined to the K end of the H-2 region, about 70 percent of recipients survived for many weeks with normal blood urea nitrogen levels. Skin grafts between untreated members of these strains were rejected promptly (mean survival time of 13.5 +/- 1.1 days) as were kidney transplants to recipients of prior skin grafts. Donor strain skin grafts to recipients of kidney transplants after kidney transplantation enjoyed greatly prolonged survival whereas skin grafts from a third party (A.SW) were rejected normally. If kidney tissue was transferred in the form of free grafts without primary vascular union, it was rejected promptly leaving its recipient highly immunized. Cellular and humoral immunity to donor antigens declined over the first few weeks after transplantation, and the spleens of long-term recipients contained no "killer cells." Recipient lymphoid cells could mount active graft versus host reactions to donor strain antigens on transfer to neonatal mice. Nevertheless, they were distinctly less able to respond specifically by the production of killer cells to donor strain antigens after sensitization in vitro. No evidence that this defect was associated with the presence of suppressor cells was forthcoming from several types of in vivo and in vitro tests.

Antigen-specific soluble helper activity for murine major histocompatibility complex-encoded molecules. I. Kinetics of factor production after skin transplantation and genetic mapping of the H-2 region specificity.

Antigen-specific soluble helper molecules are produced during major histocompatibility complex-disparate allograft priming. Genetic mapping studies with appropriate recombinant and mutant lines of mice have defined the antigen specificities of the soluble helper molecules described here as being directed against the H-2Dd molecules. The production of antigen-specific helper molecules is a relatively early event after H-2Dd-region allograft priming. A later phase of factor production near the time of graft rejection also contains nonspecific helper factors and IL-2.

Immune cell functions in pancreatic cancer.

Pancreatic cancer kills nearly 29,000 people in the United States annually-as many people as are diagnosed with the disease. Chemotherapeutic treatment is ineffective in halting progression of the disease. Yet, specific immunity to pancreatic tumor cells in subjects with pancreatic cancer has been demonstrated repeatedly during the last 24 years. Attempts to expand and enhance tumor-specific immunity with biotherapy, however, have not met with success. The question remains, "Why can't specific immunity regulate pancreatic cancer growth?" The idea that tumor cells have evolved protective mechanisms against immunity was raised years ago and has recently been revisited by a number of research laboratories. In pancreatic cancer, soluble factors produced by and for the protection of the tumor environment have been detected and are often distributed to the victim's circulatory system where they may effect a more generalized immunosuppression. Yet the nature of these soluble factors remains controversial, since some also serve as tumor antigens that are recognized by the same T cells that may become inactivated by them. Unless the problem of tumor-derived immunosuppressive products is addressed directly through basic and translational research studies, successful biotherapeutic treatment for pancreatic cancer may not be forthcoming.

Role of beta2 integrins in the prevention of apoptosis induction in chronic lymphocytic leukemia B cells.

Immunologically committed lymphocytes, especially mature, leukemic B cells, proliferate then accumulate without further cell division in chronic lymphocytic leukemia patients (CLL). These mature, leukemic B cells often produce autoantibodies. Under normal circumstances, immunologically committed lymphocytes that are autoreactive are deleted by a programmed cell death mechanism. In CLL cells, these mechanisms appear to be inhibited; therefore, cells accumulate rather than be destroyed. To understand the mechanism by which cell survival is selected over death in CLL cells, we studied the role of beta2 integrins and their ligands in the regulation of apoptosis. CLL cells were treated with monoclonal antibodies directed against beta2 integrins. Antibodies directed against the I-domain of the alpha chain of CD11b/CD18 inhibited apoptosis. The identity of the physiological ligand or counter-receptor for beta2 integrins that was required for the inhibition of apoptosis induction was sought. The ligand iC3b, but not ICAM-1 or fibrinogen, was identified as a ligand that could prevent apoptosis of CLL B cells. Free iC3b levels were elevated in CLL patients indicating that this ligand is available in vivowhere it may interact with beta2 integrins on CLL B cells and sustain their viability by preventing activation of the programmed cell death pathway. Leukemia (2000) 14, 34-39.

Gene expression in chronic lymphocytic leukemia B cells and changes during induction of apoptosis.

Our studies in chronic lymphocytic leukemia (CLL) are directed at understanding which signals maintain viability in vivo and become lost upon removal of leukemic cells from the body, such that they immediately begin to undergo apoptosis ex vivo. In this report, we examine changes in gene expression observed between freshly isolated CLL B cells and after maintenance in vitro with and without Fludara. We compare these effects with an Epstein-Barr virus (EBV)-transformed cell line treated similarly. Kinetic effects of drug treatment on apoptosis and cell division were examined with DNA laddering, radioisotopic labeling, and flow cytometry using the fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Reverse transcriptase polymerase chain reaction and hybridization blots of microarray cDNA analyses were performed to examine gene expression. We demonstrate that many genes, especially cyclin D1, were downregulated after culture of CLL cells. Anti-apoptotic genes BAG-1 and Akt2 were upregulated. The greatest positive effect with Fludara was the upregulation of JNK1. The EBV-transformed cell line was resistant to classic DNA laddering induced with Fludara. Although DNA synthesis was blocked, the EBV-transformed cell line had some ability to recover from treatment following drug washout. CLL cells express cell cycle regulatory genes that are specific for activated cells in the G(1)-S phase of the cell cycle. Growth regulatory signals are lost when the leukemic cells are isolated from the body. Fludara enhances kinetics of apoptosis and induces expression of a gene responsive to stress that regulates expression of a kinase involved in initiation of the apoptotic pathway.

Immunosuppressive activities of rat antisera raised against supernates from skin-graft-primed cells.

Rat antisera raised against supernates derived from draining lymph node cells of skin-graft-primed mice exhibit a number of immunosuppressive effects in vitro and in vivo. The skin graft-induced, cell-free-supernates had been demonstrated to contain a number of helper activities that led to an antigen-specific induction of cytolytic T lymphocytes and/or to the induction of interleukin-2 synthesis. The rat antisera administered to skin graft recipients resulted in prolongation of major-histocompatibility-complex-incompatible skin graft survival. The rat antisera appear to have a specificity for the inhibition of T cell responses in vitro, although binding to B and T cells was apparent. The responses of unprimed cells to T cell mitogens and alloantigens are blocked, whereas B cell responses to the lipopolysaccharide mitogen are not blocked by the antisera. The generation of cytolytic T lymphocytes and the cytolytic functions of such cells are both blocked by the rat antisera. The inhibition of the differentiation pathway in cells cultured continuously with the antisera was overcome only through the addition of conditioned medium obtained from stimulated concanavalin A rat spleen cells, as opposed to mouse cell conditioned media. The rat antisera do not appear to block T cell responses via the IL-2 receptor, and were found to be substantially less effective against activated and proliferating T cells. These rat antisera have allowed us to further examine the pathways involved in T cell responses.

Heterogeneity of type-II interleukin-1 receptors. Heterogeneity of B-cell interleukin-1 binding created by dimerization of type-II interleukin-1 receptors.

The binding of IL-1 alpha and IL-1 beta to two human lymphoblastoid B-cell lines, Raji and RPMI 1788, was compared with binding to the murine T-cell line, EL4. Dramatic differences in IL-1 binding were observed. Both human B-cell lines bound much less IL-1 alpha than IL-1 beta, expressed 5-10 times more receptors per cell for IL-1 beta than did the EL4 cell line, and demonstrated a large difference in the ability of IL-1 alpha to compete with IL-1 beta for binding. The B-cell lines demonstrated a low number of high-affinity IL-1 alpha receptors and a large number of IL-1 alpha receptors with a much lower affinity. Inhibition studies demonstrated that only IL-1 beta could compete for the binding of radiolabeled IL-1 beta to the B-cell IL-1R. Furthermore, SDS-PAGE analyses of lysates of the B-cell lines that had been affinity cross-linked with 125I-IL-1 alpha revealed two bands corresponding to IL-1R structures of 60 and 110 kD. These results coupled with a nonequilibrium binding study suggested a dimerization of a common type-II IL-1R polypeptide, the dimer being responsible for the high-affinity IL-1 alpha-binding site of the B-cell lines.

Normal and aberrant expression of cytokines in neoplastic cells from chronic lymphocytic leukemias.

Molecular expression of cytokines and cytokine receptors associated with B-cell growth and differentiation was examined in cells from B-cell chronic lymphocytic leukemia patients using the PCR. These studies were undertaken in order to determine whether a particular cytokine could be associated with leukemic transformation in this disease. The precursor-lymphoid and pro-B, pre-B-cell growth factor, interleukin-7, was found to be expressed in 30 of 30 patients, whereas, it was not expressed in normal donor peripheral blood lymphocytes (0 of 8) or in purified B-cell subsets from normal individuals. IL-1 beta and IL-2 receptors, on the other hand, were expressed by B cells from both normal and B-CLL patients. Other cytokine and cytokine receptors examined were not consistently expressed by all donors. Thus IL-7 was found to be the only cytokine tested that was expressed in CLL cells and not in normal cells.

Selective inhibitors of 5-lipoxygenase reduce CML blast cell proliferation and induce limited differentiation and apoptosis.

Inhibitors of the arachidonic acid metabolizing enzyme, 5-lipoxygenase, reduce the rate of proliferation of chronic myelogenous leukemia blast cells. The inhibitory agents studied were ETYA, A63162 and SC41661A. These reagents induced differentiation of cultured chronic myelogenous leukemia cells from blast to promyelocytic morphology. Promyelocytic cells then underwent apoptosis, which was identified by nuclear and cytoplasmic morphological features and by DNA laddering. Proliferation of monoblastoid U937 and myelomonocytic HL60 cell lines, known to contain 5-lipoxygenase and synthesize leukotrienes, was reduced by these inhibitors. U937 cells cultured with ETYA, A63162 or SC41661A for 48 h exhibited apoptosis as assessed by DNA laddering and morphology. Characteristic ultrastructural changes of apoptosis were seen at 120 h. MK886, an inhibitor of 5-lipoxygenase with a mechanism of action distinct from oxidation/reduction reagents, at 20-40 microM also inhibited CML and U937 cell proliferation and induced apoptosis, as shown by DNA laddering and ultrastructure.

Cytokines involved in the generation of cytolytic effector T lymphocytes.

The differentiation of precursor, antigen-competent, T cells into effective helper and/or cytolytic cells involves a number of different steps that are signaled by soluble molecules termed cytokines. We demonstrate here that subsets of T cells, distinguished on the basis of their expression of cell-surface markers, CD4 and CD8, receive distinct signals for differentiation. The precursor T cells of a T-cell subset that is MHC class II-restricted are readily activated by signals provided by rIL-1, rIL-2, rIL-3, or IFN-gamma. The precursor T cells of the MHC class I-restricted T-cell subset, on the other hand, are not readily activated by signals provided by rIL-1, rIL-3, or IFN-gamma. Recombinant IL-2 apparently functions in a nonrestricted manner, in that it can provide growth signals to both MHC class I- and class II-restricted T cells.

Genetic mapping of an H-2-associated antigen expressed on regulatory T cells.

T lymphocytes that serve regulatory functions in the generation of effector T cells to alloantigen and to altered syngeneic cells express a distinguishing H-2-associated cell surface marker that we have termed Ha. Genetic mapping studies with the B10.A(2R), B10.A(4R), B10.A(5R), and LG/Ckc lines of mice have delineated the locus determining the expression of Ha to the right of H-2K and left of H-2IB or within the I-A region. Thus these regulatory T lymphocytes express Ia antigens.

Cellular responses to murine alloantigens of the major histocompatibility complex: the role of cell subpopulations that express different quantities of H-2 associated antigenic markers.

Subsets of lymphoid cells that function in the initiation and differentiation of cell-mediated responses to H-2-coded alloantigens were defined with an antiserum raised between congenic resistant lines of mice that differed for a limited number of components of the H-2 complex. Only those cells that express "Ia markers" can stimulate responses to H-2K, D and/or I region antigens in mixed lymphoid cell cultures, even though all lymphoid cells apparently express the H-2K and H-2D-coded private antigens. Ia markers, therefore, serve to distinguish the subset of cells which includes as its members the stimulating cells. The Ia marker(s) is expressed on the cell membrane of at least one of the two T cell subsets that collaborate in the development of T effector cells to H-2-associated alloantigens, i.e. precursors and helpers. The cells remaining after lysis with our antiserum plus complement no longer can respond in the MLR. Syngeneic non-T cells cannot reconstitute the response. Most activated and proliferating cells express the Ia determinant(s). A proportion of T effector or killer cells, however, does not express the Ia markers. We suggest, therefore, that the MLR-responsive cell in normal lymph nodes is an "activated" cell and the "Ia markers" are involved in the differentiation of T precursor to T effector cells. The end stage T effector probably is devoid of the Ia marker.

Interleukin-1 receptor-like proteins synthesized by translation, in vitro, of immunopurified polysomal messenger RNA.

Interleukin-1 (IL-1) receptors can be solubilized from murine cell surfaces and immunoprecipitated with a xenogeneic rat antiserum raised in this laboratory. We demonstrated first that this antiserum contains antibodies directed against IL-1 receptors. We have now successfully used this antiserum as a reagent to immunopurify polysomes along with their messenger RNA from a murine leukemic cell line known to express relatively high levels of IL-1 receptors. The immunoselected mRNA was translated into proteins in vitro. The translation products contained an IL-1 binding protein which could specifically bind to immobilized IL-1 but not to other immobilized ligands such as interleukin-2 or tumor necrosis factor-alpha. The translation products which bound to IL-1 could be acid-eluted from the immobilized ligand, and the proteins released could still specifically bind to IL-1 in a receptor-ligand binding reaction. The eluted IL-1 binding proteins, as well as soluble receptor-ligand complexes derived from them, could also be immunoprecipitated with the xenogeneic rat antiserum. The xenogeneic rat antiserum could, furthermore, immunoprecipitate the IL-1 binding proteins from the translated products before ligand was added. The residual translated products no longer interacted with IL-1. We conclude that our antiserum contains antibodies that recognize determinants expressed on the following proteins: on nascent chains of IL-1 binding proteins; on soluble translated IL-1 binding proteins; on soluble complexes of IL-1 binding proteins that had been cross-linked with IL-1 ligand; and on cell surface-associated IL-1 receptors. The translated and unprocessed IL-1 binding proteins have a molecular mass of approximately 52,000-56,000 daltons.

Xenogeneic antiserum to soluble products from activated lymphoid cells inhibits interleukin 1-mediated functions in the helper pathway of cytolytic-effector-cell differentiation.

We have produced an antiserum that inhibits interleukin 1-mediated functions in immune responses. Skin graft-induced helper factor-containing supernatant (SgHFS) was used an immunogen in rats. The resultant antiserum was immunosuppressive of T-cell functions both in vivo and in vitro. We have further studied the effects of this antiserum on cell surface molecules that are involved in the generation of cytolytic effector T cells. Rat anti-SgHFS inhibited the differentiation of precytolytic effector cells in mixed lymphocyte cultures by blocking the helper-cell pathway. Both the level and the kinetics of interleukin 2 production were affected as the duration of rat anti-SgHFS pretreatment was increased. Interleukin 1 production after 24 hr in culture was unaffected. Monokines, including partially purified interleukin 1, actively compete with the rat anti-SgHFS by activating helper cells and thus circumvent suppression. Rat anti-SgHFS inhibits interleukin-1-mediated functions by a time-dependent active process. Thus, the target cell surface molecule(s) affected by the rat anti-SgHFS are associated with interleukin 1 function and may be the interleukin 1 receptor.

A role for helper cell subpopulations in a genetically controlled cytolytic T lymphocyte response to the H-2Db antigen.

The primary cytolytic T lymphocyte (CTL) response to the H-2Db antigen in several strain combinations appears to be under genetic control. Our studies were undertaken to determine the mechanisms involved in responders that were absent from the nonresponders and that led to CTL generation. In these studies, H-2Dk-anti-H-2Db combinations served as CTL responders, and H-2Dd-anti-H-2Db combinations served as CTL nonresponders. The nonresponsiveness of the H-2Dd-anti-H-2Db strain combinations appeared to be due to the inability of the Db antigen to activate a helper cell subpopulation. The activation of this helper cell subpopulation, as demonstrated in the H-2Dk-anti-H-2Db response, resulted in the production of factors that led to the induction of CTL differentiation. Antigen-specific pre-CTL for Db were present in the nonresponders as well as in the responders. Interleukin 2 (IL 2)-producing cells were also present in both nonresponders and responders, because all strain combinations tested in this system produced detectable levels of IL 2. Additional analysis of these data suggested that different determinants on the H-2Db antigen were recognized by distinct populations of cells. The activation of pre-CTL and IL 2-producing cells by the recognition of determinants on the H-2Db antigen was not sufficient for the generation of effector CTL, as demonstrated in the H-2Dd-anti-H-2Db response. We suggest that determinants on the H-2Db antigen that are distinct from those recognized by pre-CTL and IL 2-producing cells are recognized by a helper cell subpopulation in the H-2Dk strains, and that activation of this subpopulation is required for the production of factors that mediate CTL differentiation. This is the first description of the role that CTL differentiation factors play in a genetically controlled response.