Potential target sites for antiviral inhibitors of human immunodeficiency virus (HIV).

The rapid identification of anti-HIV compounds in the laboratory following the isolation of the causative virus in 1983 and their subsequent use in the clinic was not unexpected. Three decades of previous work had established a scientific basis for the evaluation of antiviral compounds. However, no antiviral yet discovered can cause total blockade of a virus replicating in a cell. The combination of properties of HIV including latency, antigenic and biochemical variation is unusual and the virus represents a daunting challenge for chemotherapy. But at least 90 antiviral compounds have been discovered, many inhibiting the virus reverse transcriptase. Other targets for inhibition are possible including viral regulatory gene products, viral protease and endonuclease enzymes but compounds for initial study will have to be found by random searching. X-ray crystallography of HIV proteins will shortly be possible, enabling the commencement of a more molecular specific search for inhibitors. Meanwhile, advantage can be taken of comparative nucleotide sequences of the HIV-1 and -2 genomes to test short oligonucleotides as potential inhibitors of mRNA transcription. The pol gene also has a zinc finger amino acid sequence suggesting that chelation chemotherapy may have a potential role. In the absence of HIV vaccines, and associated theoretical problems in their development, antiviral chemotherapy is expected to occupy a central role in combating the AIDS epidemic.

The killing of tumour cell targets coupled to tuberculin (PPD) by human and murine PPD-reactive T helper clones. I. PPD specificity of killing.

This paper reports on the characteristics of killing by a human and a murine tuberculin (PPD)-specific T helper clone of targets to which PPD was attached via the lectin concanavalin A (Con A). The killing was specific for PPD from M. tuberculosis; and targets coupled to Con A alone or to PPD from M. paratuberculosis were not killed. Target cells carrying Con A-PPD were more effectively lysed than PPD-pulsed cells. This form of lymphocyte killing, though highly significant, was inefficient. Maximum killing of PPD carrying targets was 30-40% at effector to target ratios of 20:1 and at 16 h. Cells carrying 2 x 10(6) molecules of PPD and less than 1.5 x 10(6) molecules Con A per cell were killed most efficiently. A major distinction between this helper T cell killing and that mediated by cytotoxic T cells was that both TH clones displayed bystander lysis and killed PPD uncoupled targets when these were cultured with syngeneic PPD-bound targets. This suggests that the mechanism of cytotoxicity may involve soluble mediators.

Analysis of human peripheral blood T lymphocytes proliferating in response to sensitizing antigens: effect of interleukin-2 (IL-2)-responsive bystander cells.

The contribution of interleukin-2 (IL-2)-responsive bystander cells to the proliferative responses of human peripheral blood T lymphocytes to antigens used for sensitization such as Purified Protein Derivative (PPD), Tetanus toxoid (T T) and Influenza virus was investigated. Marked proliferation of the unfractionated peripheral blood mononuclear cells (PBMC) was observed following stimulation with these antigens to which the individuals were known to have been sensitized previously. Depletion of large granular lymphocytes (LGL) from PBMC resulted in substantial reduction in the response of the lymphoid cells in proliferating to the antigens. Proliferation of the T4+T8- (helper)-enriched population, or T4+T8- subset depleted of any IL-2 receptor (IL-2R)-bearing lymphoid cells to these antigens was comparable to that of LGL-depleted PBMC cultures. Cell titration experiments of the blast cells generated from these cultures revealed that PBMC-derived population contained fewer antigen-reactive lymphocytes. These results, therefore, suggested that IL-2-responsive LGL through expansion affected the concentration of antigen-proliferating T cells in the antigen-stimulated PBMC cultures.

B cell growth factor (BCGF) secreting human T cell clones reactive to a soluble glycoprotein antigen, streptococcal antigen (SA).

A panel of human T cell clones bearing exclusively the helper (T4) phenotype and showing reactivities to a soluble glycoprotein antigen (185,000 dalton Mol. Wt. Streptococcal antigen, SA) is described. Two of these clones namely, SA 1.53 and SA 1.82, are found to co-produce B cell growth factor (BCGF) and interferon-gamma (IFN-gamma) in the absence of interleukin 2 (IL2) upon stimulation with phytohaemagglutinin (PHA) or the specific antigen in the presence of irradiated autologous antigen-presenting cells (APC). Secretion of the lymphokines is genetically restricted in part by DR molecules that are expressed on the cloned cells and APC. Produced BCGF is differentiated from the BCGF-promoting property of IFN-gamma in that only IFN-gamma activity, but not BCGF activity is removed and inhibited by anti-IFN-gamma antibodies. Exogenous IL2 induces secretion of BCGF and IFN-gamma of the cloned cells, an observation which involves interaction of IL2 with IL2 receptors. An analysis of the proliferative responses to antigen of the T cell clones shows that BCGF-producing clones, unlike those that secrete IL2, fail to proliferate significantly to specific antigen restimulation.

Differential regulation of interleukin 2 (IL-2) receptor expression on antigen-specific human T cell clones by IL-2.

A panel of human T cell clones bearing exclusively the T4 (helper) phenotype and demonstrating specificity to a well-characterized soluble glycoprotein antigen (185,000 dalton streptococcal antigen, SA) is described. After having been cultured in exogenous interleukin 2 (IL-2) for 7 days in the absence of the specific antigen, two of the clones, namely SA1.4 and SA1.23, show stronger proliferative responses to the ligand as compared to the other T4 clones. Analysis of both the high- and low-affinity IL-2 receptor (IL-2R) levels reveals that IL-2 mediates differential regulation of high affinity IL-2R expression on these antigen-deprived cloned cells. Higher levels of surface expression of the IL-2 binding sites on SA1.4 and SA1.23 as compared to the other clones are observed throughout the 7-day culture period that these lymphocytes are maintained in exogenous IL-2. All the cloned cells appear to have returned to their "unstimulated states" as noted by their stable low expressions of Tac antigen and high affinity IL-2R. The unstimulated states of SA1.4 and SA1.23 are represented by higher levels of high affinity IL-2R expression. Under the condition in which the cloned cells are exposed to a decreasing concentration of IL-2, SA1.4 and SA1.23 are found to secrete a greater amount of IFN-gamma. The present results therefore suggest that a control mechanism involving the "mutual amplification" of IL-2 and IFN-gamma regulates the differential expression of high affinity IL-2R on antigen-specific T4 clones.

In vivo activated lymphoid cells (IVALC) affect the cloning efficiency of human T lymphocytes reactive to a soluble antigen, purified protein derivative.

Peripheral blood mononuclear cells (PBMC) of normal individuals were found to contain a proportion (4-9%) of in vivo activated lymphoid cells (IVALC). These IVALC were characterized by their expression of interleukin 2 (IL-2) receptors, and by the ability to proliferate in the presence of exogenous IL-2. There was a good correlation between the proportion of IVALC in different cell populations and the level of cell proliferation to IL-2. It was found that IVALC isolated from autologous PBMC of Bacillus Calmette-Guerin (BCG)-immunized individuals contained no significant proportion of purified protein derivative (PPD)-reactive lymphocytes. The addition of IVALC markedly enhanced proliferative responses of the autologous T4+T8-IL-2 receptor-negative cell cultures to antigen stimulation. An increased proportion of activated (IL-2 receptor-positive) lymphocytes was generated in PBMC as compared to autologous T4+T8-IL-2 receptor negative cell cultures after stimulation with PPD. Limiting dilution analysis showed that IL-2 responsive IVALC through expansion markedly affected the cloning efficiency of antigen-proliferating T cells of autologous PPD-stimulated PBMC cultures. Only 1 out of every 11-25 blast cells generated in the PBMC cultures could establish itself as a growing colony based on determinations in six BCG-positive individuals. By using a T4+T8- population depleted of IVALC to generate PPD-reactive lymphocytes, a three- to four-fold increase in the cloning efficiency of antigen-specific cells was obtained.

Immuno-modulation of human lymphocyte function by desferroxamine (Desferal).

Desferroxamine inhibits lectin-stimulated proliferation of human lymphocytes. Inhibition is not due to toxic effect of the drug as it could be completely reversed by iron. It is found that the drug impairs the expression of both the total and high-affinity interleukin-2 (IL-2)-binding receptors on lymphoid cells in response to the mitogen phytohaemagglutinin. IL-2 production by mitogen-stimulated T4+T8- and T8+T4- populations is also markedly reduced by desferroxamine treatment. Decreased secretion of macrophage inhibition factor and macrophage activation factor is also observed as the result of the lymphocytes of the helper/delayed hypersensitivity subset being exposed to the drug. Collectively, our results suggest that desferroxamine interferes with the early activation events of human lymphokine-producing lymphocytes.

An antigen-specific T8+ human clone of cells with a nonspecific augmenting function on the T4 cell-B cell helper interaction.

We isolated a T8+ T3+ Ia+ clone of cells from the peripheral blood mononuclear cells of a healthy subject. The clone was expanded and maintained with autologous feeder cells, interleukin 2, and a streptococcal antigen. The T8+ clone of cells responded specifically to the streptococcal antigen, in the absence of accessory cells, and released a soluble factor. Both the cloned cells and the corresponding soluble factor expressed augmenting helper but not suppressor activity. The augmenting helper activity for B cell antibody synthesis was demonstrable only in the presence of autologous T4 cells. Although stimulation of the T8+ cloned cells was antigen-specific, the resulting soluble factor elicited nonspecific antibody synthesis in the presence of T4 and B cells. The T8+ cloned cell-derived factor was adsorbed by B cells but not by T4 cells. Preliminary studies suggest that the factor has the properties of a B cell growth factor. We suggest that the T8+ population consists of functionally heterogeneous cell subsets, some that have suppressor function and others that augment the T4+ helper-inducer activity in B cell antibody synthesis.

Clonal analysis of T-cell responses to herpes simplex virus: isolation, characterization and antiviral properties of an antigen-specific helper T-cell clone.

A herpes simplex virus (HSV)-specific long-term T-cell clone has been established from the draining lymph node cells of BALB/c mice; the cells required repeated in vitro restimulation with UV-irradiated virus. The established T-cell clone expresses the Thy-1 and Lyt-1+2,3- surface antigens. For optimal proliferation of the cloned cells, both the presence of specific antigen and an exogenous source of T-cell growth factor are required. The proliferative response of the cloned T cells was found to be virus-specific but it did not distinguish between HSV-1 and HSV-2. Adoptive cell transfer of the cloned T cells helped primed B cells to produce anti-herpes antibodies: the response was antigen-specific and cell dose-dependent. The clone failed to produce a significant DTH reaction in vivo, but did produce high levels of macrophage-activating factor. Furthermore, the T-cell clone could protect from HSV infection, as measured by a reduction in local virus growth, and by enhanced survival following the challenge of mice with a lethal dose of virus. The mechanism(s) whereby this clone protects in vivo is discussed.

Studies in the enhancement of tumour immunity by coupling strong antigens to tumour cells ('heterogenization of tumours'). Helper T cell clones against PPD help other T cells mount anti-tumour responses to PPD-coupled tumour cells.

PPD-reactive T cell clones have been used to analyse the nature of T lymphocytes that are involved in the 'heterogenization' of tumour cells. This is a phenomenon where coupling tumour cells to a strong antigen (in this case PPD) causes an enhanced immune response to tumour-specific antigens to be elicited providing that the host shows T cell immunity to the strong antigen (in this case is BCG positive). Clones of T cells with the Lyt1+2- phenotype which were unable to mediate delayed-type hypersensitivity but which provided efficient help to hapten-primed B cells were found to potentiate anti-tumour immunity in BCG-negative syngeneic mice when immunized with Con-A-PPD coupled, X-irradiated MC6A tumour cells. There therefore appears to be a mechanism whereby a helper T cell response to one antigen can provide help for the generation of a T cell response to a linked antigen which is analogous to the well-known phenomenon of help to hapten primed B cells. Furthermore the clones of T cells that help B cells the best are those that give maximal augmentation of T cell immunity.

Automated rosetting assay for cell surface antigens and autorosetting lymphocytes.

An automated procedure, using a Coulter counter, is described for enumerating rosette-forming lymphocytes in 2 rosetting systems in mice, detecting antibodies to cell surface antigens, and the interaction of autologous erythrocytes with thymocytes (autorosetting). The procedure gives results comparable with determinations of rosettes by light microscopy. The procedure not only estimates rosetting percentages, but can be used to titrate anti-lymphocyte antibodies, to detect autorosette inhibition factor in serum and to assay cell surface antigens in detergent lysates of spleen cells.

Anti-self receptors. V. Properties of a mouse serum factor that blocks autorosetting receptors on lymphocytes.

Murine lymphocytes spontaneously bind autologous and allogeneic erythrocytes via receptors that primarily recognize self H-2L molecules on the erythrocyte surface. Normal mouse serum contains a factor, termed autorosette inhibition factor (AIF), that very effectively blocks autorosette formation. This paper describes experiments that determine the origin and nature of serum AIF. It was found that AIF lacks strain and species specificity, serum from several mammalian and non-mammalian species inhibiting the autorosetting of BALB/c thymocytes. However, mouse strains differed in the levels of AIF in their serum. Furthermore, AIF appears to directly interact with autorosetting receptors on lymphocytes as thymocytes from the BALB/c-H-2dm2 mutant strain, which lack autorosetting receptors, were unable to absorb the factor. Several lines of experimental evidence indicated that AIF is secreted by a population of short-lived, radiosensitive macrophages (or monocytes). Firstly, in vivo administration of the anti-macrophage agents carrageenan and silica profoundly depressed AIF levels in serum. Secondly, in vitro culturing of different lymphoid cells revealed that AIF is secreted by an adherent population of peritoneal cells. Thirdly, total body irradiation experiments demonstrated that AIF production is dependent upon a radiosensitive cell that is bone marrow derived. Finally, AIF was purified to homogeneity from mouse plasma and shown to be a single polypeptide chain with a molecular weight of 84,000.

Anti-self receptors. IV. H-2-restricted receptors on thymocytes recognize carbohydrate structures on target cells.

Rosetting between thymocytes and autologous erythrocytes in mediated by receptors on thymocytes that primarily recognize self H-2L molecules on erythrocytes. This paper describes preliminary attempts to chemically characterize the receptor and acceptor molecules involved in this H-2-restricted interaction. On the basis of sugar inhibition studies and the sensitivity of the receptors to protease and glycosidase treatments it appears that a protein receptor on thymocytes recognizes the carbohydrate portion of a glycoprotein on erythrocytes. Furthermore, the thymocyte receptor appears to recognize terminal D-galactose, D-mannose and sialic acid residues on a branched-chain carbohydrate structure on erythrocytes, with mouse strains of different H-2 haplotype expressing carbohydrate structures that differ in the linkage of these three terminal sugars. These findings indicate that H-2-restricted carbohydrate-protein interactions can occur between cells, a conclusion with important theoretical implications.

Anti-self receptors. III. Lack of allelic exclusion and thymic epithelium dependence of H-2L-region-restricted receptors on lymphocytes.

Murine thymocytes and peripheral lymphocytes bind autologous erythrocytes via H-2L-region-restricted receptors. After inhibiting autorosetting with different erythrocyte sonicates the specificity of these anti-self receptors was examined in F1 hybrid and chimaeric mice. Most F1 lymphocytes simultaneously expressed receptors against both parental haplotypes. Furthermore, analysis of lymphocytes from allogeneic and semi-allogeneic chimaeras clearly demonstrated that radioresistant elements in the recipient thymus did not modify the haplotype specificity of the receptors on donor-derived lymphocytes.

Anti-self receptors. II. Demonstration of H-2L region-restricted receptors on subpopulations of peripheral T and B lymphocytes.

Subpopulations of murine spleen, lymph node, and bone marrow cells can bind autologous erythrocytes. The specificity of this interaction was investigated and it was found that these lymphoid cells, like thymocytes, primarily recognize self-H-2L antigens on red cells. Three experimental approaches were used to reach this conclusion: i) The inhibition of autorosetting with erythrocyte sonicates from different H-2 congenic and recombinant mouse strains; ii) the specific blocking of autorosette-inhibition with anti-H-2L antibodies, and iii) the analysis of H-2L mutant mice. The inhibition studies also demonstrated that extrathymic lymphocytes, like thymocytes, carry receptors that can distinguish between b, q and s haplotypes but cannot differentiate between the H-2L molecules expressed by d and k haplotypes. It was found that subpopulations of both T and B lymphocytes autorosette via H-2L restricted receptors. In fact, the majority (80%) of autorosetting cells in spleen were B lymphocytes. Furthermore, the H-2L restricted receptors on B lymphocytes were distinct from surface Ig and could develop in athymic (nude) mice. These findings imply that H-2 restricted receptors on lymphocytes play a much more fundamental and comples role in the immune system than simply directing the interaction of cytotoxic T lymphocytes with target cells.

Major histocompatibility gene complex (MHC)-coded determinants on antigen-specific suppressor factor for delayed-type hypersensitivity and surface phenotypes of cells producing the factor.

Antigen-specific suppressor factor for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) was obtained by incubating in vitro spleen cells from CBA mice (H-2k) injected intravenously 3 days previously with 1 x 10(9) SRBC. The suppressor factor was characterized for major histocompatibility gene complex (MHC)-coded antigenic determinants by passing the factor through immunosorbents coupled with appropriate alloantisera. The suppressor factor was absorbed by anti-H-2k, anti-Iak and anti-I-Jk immunosorbents but was not retained by anti-Ias, anti-I-Js, anti-I-Ak, anti-I-E/Ck or anti-H-2Kk immunosorbents. In addition, the factor bound to an immunosorbent coupled with rabbit antibodies against carbohydrate-defined Ia antigens. Furthermore, the suppressive activity that was absorbed was quantitatively recovered in the acid eluates from the immunosorbents. Treatment of the spleen cells with anti-Lyt-1.1 antiserum and complement completely abrogated their ability to elaborate the suppressor factor in vitro. In contrast, treatment with anti-Lyt-2.1 or anti-Iak antiserum and complement had no effect. Thus, it appears that the suppressor factor for DTH to SRBC bears I-J subregion-coded determinants, and its production is dependent on cells which have the Lyt-1+,2- and Ia- phenotype.

Anti-self receptors. I. Direct detection of H-2L region-restricted receptors on murine thymocytes.

A high proportion (20--50%) of murine thymocytes form rosettes with either syngeneic or allogeneic erythrocytes. The specificity of this interaction was investigated by measuring the ability of different erythrocyte sonicates to inhibit rosette formation. With erythrocyte sonicates from recombinant mouse strains it was demonstrated that rosetting with syngeneic erythrocytes was mediated by H-2L and/or H-2D region-restricted receptors. The specificity of autorosetting was directly mapped to the H-2L region by the inability of erythrocyte sonicates from the BALB/c-H-2dm2 mutant, an H-2L-deletion mutant, to inhibit the rosetting of wild-type (BALB/c) thymocytes. The B10,2D2-H-2dm1 mutant, which has substantially modified H-2L and H-2D antigens, supported this conclusion. Furthermore, anti-H-2L sera were able to specifically block the inhibition of rosetting by erythrocyte sonicates. The above procedures clearly implicated the H-2L region in the thymocyte rosetting of d and k haplotypes. With the s haplotype the rosetting receptor was mapped to the H-2L/H-2D region, whereas with the b and q haplotypes rosetting was only mapped to the D end of the H-2 complex. This study also suggested complete cross-reaction between the thymocyte receptors carried by the k and d haplotypes, whereas the receptors of b, q, and s haplotypes were haplotype specific. In addition, the inhibition assay indicated that the rosetting of thymocytes with allogeneic and xenogeneic (rat) erythrocytes was mediated by a receptor primarily directed against self-H-2L. Finally, the critical role played by the H-2L region in this rosetting phenomenon was demonstrated by the inability of thymocytes from the H-2L-deletion mutant (H-2dm2) to rosette with syngeneic, allogeneic (rat) erythrocytes.