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.

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.

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.

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. 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.