Induction of cross clade reactive specific antibodies in mice by conjugates of HGP-30 (peptide analog of HIV-1(SF2) p17) and peptide segments of human beta-2-microglobulin or MHC II beta chain.

HGP-30, a 30 amino acid synthetic peptide homologous to a conserved region of HIV-1(SF2) p17 (aa86-115), has previously been shown to elicit both cellular and humoral immune responses when conjugated to KLH and adsorbed to alum. However, the free HGP-30 peptide is not immunogenic in animals. In order to improve the immunogenicity of HGP-30, peptide conjugates consisting of a modified HGP-30 sequence (m-HGP-30/aa82-111) and a peptide segment, residues 38-50, of the MHC I accessory molecule, human beta-2-microglobulin (beta-2-M), referred to as Peptide J, or a peptide from the MHC II beta chain (peptide G) were evaluated in mice. The effects of carriers and adjuvants on serum antibody titers, specificities to various HIV-1 clade peptides similar to HGP-30 and isotype patterns were examined. Peptides J or especially G conjugated to modified-HGP-30 (LEAPS 102 and LEAPS 101, respectively) generated comparable or better immune responses to modified HGP-30 than KLH conjugates as judged by the induction of: (1) similar antibody titers; (2) broader HIV clade antigen binding; and (3) antibody isotype response patterns indicative of a TH1 pathway (i.e. increased amounts of IgG2a and IgG2b antibodies). The ISA 51 and MPL(R)-SE adjuvants induced higher antibody responses than alum, with the ISA 51 being more potent. Immune responses to LEAPS 102, as compared to LEAPS 101, were weaker and slower to develop as determined by antibody titers and cross clade reactivity of the antibodies induced. Compared to KLH conjugates which induced significant anti-KLH antibody titers, minimal antibody responses were observed to peptide G, the more immunogenic conjugate, and peptide J. These results suggest that modified HGP-30 L.E.A.P.S. constructs may be useful as HIV vaccine candidates for preferential induction of TH1 directed cell mediated immune responses.

Immunologic approaches to the treatment of prostate cancer.

The presence of several organ-specific molecules that could serve as immunogens or targets of an immune attack, the nonessential nature of the prostate gland, the substantial failure rate after treatment of the primary tumor, and the lack of effective chemotherapy for metastatic disease make prostate cancer an ideal candidate for immunotherapy. This report reviews the current status of two novel approaches to the treatment of prostate cancer. The first is an effort to induce antitumor immunity by enriching the cytokine environment within the primary cancer by intraprostatic injection of Leukocyte Interleukin (Cel-Sci Corp, Vienna, VA), a mixture of natural cytokines that includes interleukin-1 beta (IL-1beta), IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha). The second approach uses OncoVax-P (Jenner Biotherapies, Inc, San Ramon, CA), a vaccine consisting of liposome-encapsulated recombinant prostate-specific antigen (PSA) and lipid A. When administered as an emulsion or in association with bacillus Calmette-Guérin (BCG)/cyclophosphamide or GM-CSF with or without IL-2/cyclophosphamide, immunologic tolerance is broken as evidenced by the generation of humoral and cellular immunity. Both of these approaches have been shown to be feasible and safe, and are now being tested in patients with less advanced disease to determine if manipulation of the immune system can favorably influence clinical outcome.

Leukocyte Interleukin, Inj. (LI) augmentation of natural killer cells and cytolytic T-lymphocytes.

A serum free lymphokine preparation derived from human buffy-coat mononuclear cells [buffy coat interleukins (BC-IL)], also named Leukocyte Interleukin, Inj. (LI), trade name Multikine, containing glycosylated interleukin-2 (IL-2) among other interleukins, was tested in three head and neck cancer patients. They responded with tumor regressions associated with increased tumor infiltration of lymphocytes and tumor cell lysis indicating an LI Interleukin-2 induced tumor-specific immune response. To determine whether these responses elicited by LI were IL-2 driven, augmentation of natural killer cells (NKC) and cytolytic T cells (CTL), was tested both in vitro and in vivo. A single intraperitoneal (i.p.) injection of LI in adult BALB/c mice at doses of 3, 10, 30 and 100 of IL-2 equivalence International Units per mouse, led to significant (p < 0.01) augmentation of NKC cytotoxicity to YAC tumor cells. NKC cytotoxicity remained elevated for 7 days, peaking at 5 days post-treatment. Multiple treatments with LI did not increase NKC cytotoxicity above single injection, nor did it lead to NKC hyporesponsiveness. The most effective treatment routes leading to heightened NKC cytotoxicity were: intravenous(i.v.) > intraperitoneal (i.p.) > intramuscular (i.m.) > subcutaneous (sc). Significant (p < 0.05 to < 0.01) NKC cytotoxicity was achieved by all four routes. In vitro incubation of murine splenocytes with 30 and 100 International Units/ml (IU/ml) of IL-2 equivalent elevated NKC cytotoxicity significantly (p < 0.01) at all effector to target cell ratios tested and exceeded the response achieved with rhIFN gamma. NKC cytotoxicity of human peripheral blood lymphocytes (HPBL) against the K562 human tumor cell was also significantly elevated (p < 0.01) at the 30 and 100 IU/ml doses and (p < 0.05) at 3 and 10 IU/ml doses. Of particular interest was the significant increase of CTL response in HPBL generated by LI. Significant activity (p < 0.01) was achieved with levels of 10, 30 and 100 IU/ml at effector to target cell ratios as low as 6 to 1. These results indicate that the LI containing IL-2 led to the significant increase in NKC and CTL cytolytic activities. Relatively lower doses of LI were needed to attain equivalent cytolytic activities as achieved with rhIL-2 or rhIFN gamma.

Antigen-specific immunoregulation and autoimmune thyroiditis.

1. Large Thy1+, CD8+ immunoregulatory cells were found in the thymus of mice 9-16 days after injection of soluble mouse thyroglobulin (without adjuvant). 2. Similar immunoregulatory cells appeared in the spleen 14-21 days after antigen administration suggesting that they were generated in the thymus and later migrated to the spleen. 3. The immunoregulatory cells were antigen-specific and attached to cognate antigen. 4. The degree of inhibition by the immunoregulatory cells differed among various inbred strains of mice and were virtually absent from SJL mice. 5. The regulatory function of the cells was strain specific in vitro and in vivo, and was restricted by both H-2 and non-H-2 haplotype. 6. By panning on antigen-coated plates, immunoregulatory cells were isolated and cell lines and clones with inhibitory function established.

A non-cytotoxic suppressor of immunoglobulin synthesis and secretion by B cells of normal humans and patients with rheumatoid arthritis and systemic lupus erythematosus.

A factor secreted by thymocytes of immunized rabbits totally suppressed both the initiation of, and ongoing synthesis and secretion of, lectin (PWM)-induced synthesis of IgM and IgG immunoglobulins by the circulating B lymphocytes of normal humans, and of twenty consecutive patients with rheumatoid arthritis and twelve consecutive patients with systemic lupus erythematosus. The suppressor factor, referred to as human Ig synthesis/secretion suppressor factor or HISSF, is not HLA restricted in its activity and is not cytotoxic to the circulating human mononuclear cells (B cells, T cells, Null cells and monocytes). It was demonstrated that T cells precultured with HISSF were transformed into suppressor cells which, when added to fresh cultures of autologous B cells, suppressed the synthesis and secretion of IgM and IgG. On the basis of its suppressive and non-cytotoxic properties in vitro, HISSF may be an effective immunosuppressant in the treatment of patients with autoimmune diseases.

Cells involved in the immune response. XXXVII. Antigen-specific suppressor cells, capable of secreting an antigen-specific suppressor factor, migrate from the thymus to the spleen following primary immunization.

The splenic mononuclear cells (MNC) of rabbits 7-14 and 30-48 days following primary intravenous immunization with sheep erythrocytes generated large numbers of antibody-secreting or plaque-forming cells (PFC) in secondary immune responses induced in vitro, whereas the splenic MNC obtained from rabbits 18-30 days following primary intravenous immunization generated poor secondary immune responses (few PFC) in vitro. However, these latter splenic MNC depleted of T cells consistently generated many PFC in the secondary immune response in vitro. Furthermore, the splenic MNC of rabbits thymectomized prior to day 3 following primary intravenous immunization also generated good secondary immune responses in vitro, irrespective of the time of killing post-immunization, whereas the splenic MNC of rabbits thymectomized after day 7 following primary immunization generated poor secondary immune responses in vitro. These results indicate that the depressed ability of the splenic MNC, obtained from rabbits killed between days 18 and 30 post-primary immunization, to generate significant secondary immune responses in vitro is due to suppressor T cells. The suppressor cells are referred to as immune spleen suppressor cells or ISSC. It was demonstrated that the suppression by the ISSC is antigen-specific and that the ISSC secrete an antigen-specific suppressor factor referred to as immune spleen suppressor factor or ISSF. It is concluded that the ISSC are generated in the thymus within a few days following primary immunization, that they migrate to and infiltrate the spleen between days 3 and 7 following primary immunization, and that they suppress or down-regulate further antibody synthesis via the secretion, locally of ISSF.

The induction of antigen-specific thymic regulatory cells in the mouse.

Regulatory T cells are known to play a role in the termination of the immune response. In this investigation, we bring evidence in support of the induction of antigen-specific regulatory cells in the thymus of the mouse. These thymus-derived regulatory cells depend upon injection with a high dose of antigen for their induction, appear in the thymus early after primary immunization (Days 7-12), are H-2- and strain-restricted in their action, and can by themselves suppress directly the secretion of antibodies from syngeneic antibody-forming cells in vitro. These regulatory cells, therefore, differ from previously reported murine suppressor T cells which exert their effect on antibody formation indirectly, generally by acting on helper T cells.

Cells involved in the immune response. XXXV. The antigen-specific antibody response in the rabbit is suppressed by thymocytes of allogeneic immunized rabbits (ITSC) and by the non-toxic suppressor factor (ITSF) secreted by these thymocytes.

The antigen-specific suppressor cells (ITSC) detected in the thymus of the rabbit 7 days post-iv immunization with sheep or horse erythrocytes (SRBC and HRBC, respectively), and the antigen-specific suppressor factor (ITSF) which the ITSC secrete in culture, inhibit the antigen-specific primary immune response in vivo when injected iv into SRBC and/or HRBC immunized rabbits on Days 0, 3, and 5 (ITSC) or daily on Days 0 to 5 (ITSF) post-primary immunization. The rabbits recover the ability to synthesize the specific antibodies following reimmunization by day 80 post-primary immunization. The primary immune response toward a non-cross-reacting antigen is not inhibited by the antigen-specific ITSC or ITSF. Neither the thymocytes of unimmunized rabbits nor the secretions of these cells in culture can suppress the primary immune response in vivo to either SRBC or HRBC. It must be emphasized that the suppression of the immune response by ITSC and ITSF in the rabbit is antigen-specific. ITSC and ITSF are not cytotoxic to rabbit lymphocytes in vitro. No gross or microscopic changes were detected in any of the lymphoid and nonlymphoid organs of rabbits sacrificed 2 days following 5 daily iv injections of large doses (10 ml) of ITSF. ITSF causes no adverse reaction in vivo since it did not induce morbidity in the rabbits during the 80 days observation period following its injection iv daily for 5 days commencing with the primary immunization.

Cells involved in the immune response. XXXVI. The thymic antigen-specific suppressor cell in the immunized rabbit is a T cell with receptors for FcG and the antigen and it acts, via a secreted suppressor factor, directly on the immune splenic AFC B cell to inhibit antibody secretion.

Following i.v. immunization of the normal outbred rabbit with sheep (SRBC) or horse (HRBC) erythrocytes, antigen-specific suppressor cells are generated in the thymus capable of inhibiting the generation of haemolytic plaques by the autologous or allogeneic splenic antibody-forming cells (AFC) in the plaque-forming cell (PFC) assay. These suppressor cells secrete an antigen-specific suppressor factor in short-term (4-24 hr) culture in vitro. The suppressor cells are not detected in the thymus prior to Day 4, exhibit peak activity between Days 5 and 11 post-immunization, and decline slowly thereafter. Suppressor cells can no longer be detected in the thymus by Day 60 postimmunization. Suppressor cells are not detected in any of the other lymphoid organs of the immunized rabbit nor in any lymphoid organ in the unimmunized rabbit. The thymic suppressor cell is a T cell with surface receptors for the antigen (SRBC or HRBC) and for FcG. On the other hand, the AFC B cells generated in the spleen of the immunized rabbit possess cell-surface receptors for only the antigen and not for FcG. Both the suppressor cells and the secreted suppressor factor act directly on the AFC B lymphocytes to inhibit the generation of antigen-specific haemolytic plaques in the PFC assay.

Cells involved in the immune response. XXXIV: Suppressor cells in the thymus of the immunized rabbit capable of secreting a factor which can suppress the secretion of antibodies from antibody-forming cells in vitro.

Outbred rabbits were immunized intravenously with 10(9) sheep erythrocytes (SRBC) and/or horse erythrocytes (HRBC) and sacrificed 1 to 60 days later. At the height of the immune response (Day 7 postimmunization), antibody-forming cells identified by their ability to form hemolytic plaques in the plaque-forming cell (PFC) assay were detected only in the spleen. The splenic PFCs were totally inhibited if they were first incubated with the autologous thymus cells (immune thymus suppressor cells or ITSCs) for 4 hr prior to assaying for PFCs. Incubation of the spleen mononuclear cells with cells of any of the other lymphoid organs of the immunized rabbit for up to 6 hr did not inhibit the PFCs to any significant degree. There is no MHC restriction in the suppressor activity of the ITSCs since they could totally suppress the PFCs of autologous and allogeneic 7-day immune spleen cells. Neither thymus cells nor cells of any of the other lymphoid organs of the unimmunized rabbit could inhibit allogeneic 7-day immune splenic PFCs. The ITSCs from rabbits immunized with SRBC could be isolated by rosetting with the immunizing antigen, SRBC, but not with the non-cross-reacting antigens HRBC, human erythrocytes (HuRBC), and rabbit erythrocytes (RRBC), and vice versa, thus demonstrating the antigenic specificity of the suppressor cells. The thymus cells, but not the cells of any of the other lymphoid organs of the SRBC-immunized rabbit, secreted a factor referred to as immune thymus suppressor factor (ITSF) during incubation for 4 hr at 37 degrees C which could, by itself, totally suppress the splenic PFC. ITSF could be absorbed out of solution by incubation with the immunizing antigen, SRBC, but not with the non-cross-reacting antigens HRBC, HuRBC, and RRBC. Similarly, the ITSF secreted by the thymus cells of the HRBC-immunized rabbit could be absorbed out of solution by incubation with the immunizing antigen, HRBC, but not with the SRBC, HuRBC, and RRBC. ITSF is therefore antigen specific as is its parent ITSC. The cells of the lymphoid organs of unimmunized rabbits did not secrete a suppressor factor during incubation in vitro. ITSCs capable of totally inhibiting the PFCs were detected in the immunized rabbits by Day 5 post-primary-immunization; the ITSCs were capable of secreting ITSF which could totally inhibit the PFCs by Day 7 post-primary-immunization. Although the ITSCs were detected in a suppressive state up to Day 40 post-primary-immunization, they lost their capacity to secrete ITSF by Day 21 post-primary-immunization.(ABSTRACT TRUNCATED AT 400 WORDS)