Emerging patterns of regulatory T cell function in tuberculosis.

Tuberculosis (TB) is one of the top 10 causes of mortality worldwide from a single infectious agent and has significant implications for global health. A major hurdle in the development of effective TB vaccines and therapies is the absence of defined immune-correlates of protection. In this context, the role of regulatory T cells (Treg ), which are essential for maintaining immune homeostasis, is even less understood. This review aims to address this knowledge gap by providing an overview of the emerging patterns of Treg function in TB. Increasing evidence from studies, both in animal models of infection and TB patients, points to the fact the role of Tregs in TB is dependent on disease stage. While Tregs might expand and delay the appearance of protective responses in the early stages of infection, their role in the chronic phase perhaps is to counter-regulate excessive inflammation. New data highlight that this important homeostatic role of Tregs in the chronic phase of TB may be compromised by the expansion of activated human leucocyte antigen D-related (HLA-DR)+ CD4+ suppression-resistant effector T cells. This review provides a comprehensive and critical analysis of the key features of Treg cells in TB; highlights the importance of a balanced immune response as being important in TB and discusses the importance of probing not just Treg frequency but also qualitative aspects of Treg function as part of a comprehensive search for novel TB treatments.

HIV-TB co-infection: mechanisms that drive reactivation of Mycobacterium tuberculosis in HIV infection.

HIV infection predisposes the host to tuberculosis by impairing the hosts' immune system principally by killing and altering CD4 T-cell function. How HIV infection disrupts CD4 T-cell function, which specifically compromises host immunity to Mycobacterium tuberculosis, is poorly understood and is a critical roadblock in developing better vaccine- or immune-based strategies to control and monitor TB in HIV-infected subjects. This review considers key pathways that are altered in HIV-infected subjects that impair anti-TB immunity.

WFDC1/ps20 is a novel innate immunomodulatory signature protein of human immunodeficiency virus (HIV)-permissive CD4+ CD45RO+ memory T cells that promotes infection by upregulating CD54 integrin expression and is elevated in HIV type 1 infection.

Understanding why human immunodeficiency virus (HIV) preferentially infects some CD4(+) CD45RO(+) memory T cells has implications for antiviral immunity and pathogenesis. We report that differential expression of a novel secreted factor, ps20, previously implicated in tissue remodeling, may underlie why some CD4 T cells are preferentially targeted. We show that (i) there is a significant positive correlation between endogenous ps20 mRNA in diverse CD4 T-cell populations and in vitro infection, (ii) a ps20(+) permissive cell can be made less permissive by antibody blockade- or small-interference RNA-mediated knockdown of endogenous ps20, and (iii) conversely, a ps20(low) cell can be more permissive by adding ps20 exogenously or engineering stable ps20 expression by retroviral transduction. ps20 expression is normally detectable in CD4 T cells after in vitro activation and interleukin-2 expansion, and such oligoclonal populations comprise ps20(positive) and ps20(low/negative) isogenic clones at an early differentiation stage (CD45RO(+)/CD25(+)/CD28(+)/CD57(-)). This pattern is altered in chronic HIV infection, where ex vivo CD4(+) CD45RO(+) T cells express elevated ps20. ps20 promoted HIV entry via fusion and augmented CD54 integrin expression; both of these effects were reversed by anti-ps20 antibody. We therefore propose ps20 to be a novel signature of HIV-permissive CD4 T cells that promotes infection in an autocrine and paracrine manner and that HIV has coopted a fundamental role of ps20 in promoting cell adhesion for its benefit. Disrupting the ps20 pathway may therefore provide a novel anti-HIV strategy.

Evidence for a role for galectin-1 in pre-mRNA splicing.

Galectins are a family of beta-galactoside-binding proteins that contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. The polypeptide of galectin-1 contains a single domain, the CRD. The polypeptide of galectin-3 has two domains, a carboxyl-terminal CRD fused onto a proline- and glycine-rich amino-terminal domain. In previous studies, we showed that galectin-3 is a required factor in the splicing of nuclear pre-mRNA, assayed in a cell-free system. We now document that (i) nuclear extracts derived from HeLa cells contain both galectins-1 and -3; (ii) depletion of both galectins from the nuclear extract either by lactose affinity adsorption or by double-antibody adsorption results in a concomitant loss of splicing activity; (iii) depletion of either galectin-1 or galectin-3 by specific antibody adsorption fails to remove all of the splicing activity, and the residual splicing activity is still saccharide inhibitable; (iv) either galectin-1 or galectin-3 alone is sufficient to reconstitute, at least partially, the splicing activity of nuclear extracts depleted of both galectins; and (v) although the carbohydrate recognition domain of galectin-3 (or galectin-1) is sufficient to restore splicing activity to a galectin-depleted nuclear extract, the concentration required for reconstitution is greater than that of the full-length galectin-3 polypeptide. Consistent with these functional results, double-immunofluorescence analyses show that within the nucleus, galectin-3 colocalizes with the speckled structures observed with splicing factor SC35. Similar results are also obtained with galectin-1, although in this case, there are areas of galectin-1 devoid of SC35 and vice versa. Thus, nuclear galectins exhibit functional redundancy in their splicing activity and partition, at least partially, in the nucleoplasm with another known splicing factor.

Tumour necrosis factors (alpha, beta) induced by HIV-1 in peripheral blood mononuclear cells potentiate virus replication.

Cytokines such as tumour necrosis factor (TNF) can induce HIV-1 production in T-cell tumour lines. However, it is not known if the same occurs in freshly isolated mononuclear cells, nor is it known if the virus can itself regulate cellular cytokine production. In this paper we report that HIV-1 induces peripheral blood mononuclear cells (PBMC) and CD4+ T lymphocytes to secrete TNF alpha, TNF beta and interferon-gamma (IFN gamma), three cytokines having multifunctional activities and complex physiological roles. We also show that separate addition of exogenous recombinant (r) TNF alpha or rTNF beta or rIFN gamma increases HIV-1-induced syncytium formation in both PBMC and CD4+ cells by up to 10,000-fold, with TNF alpha being most potent in this regard. Finally, we show that syncytium formation induced by diverse HIV-1 isolates and LAV-2 is inhibited without the addition of exogenous r-cytokines by the respective anti-cytokine antibodies. Our study therefore demonstrates that efficient HIV replication in primary mononuclear cells is associated with the ability of the virus to induce TNF and IFN gamma secretion.

Evidence for a post-entry barrier to R5 HIV-1 infection of CD4 memory T cells.

BACKGROUND: HIV-1 strains R5 and X4 can infect CD4 memory T cells in vivo. Anti-CD3/28 stimulation induces beta-chemokines and CCR5 down-regulation and renders these cells resistant to R5 HIV-1 infection. Here we describe an additional cellular mechanism that blocks productive R5 HIV-1 infection of CD4 memory T cells. METHODS: Blood-derived CD4 memory T cells and CD4 T-cell clones were infected with primary R5 and X4 HIV-1 strains. Virus replication was correlated with CCR5 expression and beta-chemokine production. Virus entry and infectivity were measured by PCR for early and late products of HIV reverse transcription respectively. RESULTS: R5 strains were up to 1000-fold less infectious than X4 viruses for CD4 memory T cells. This resistance was independent of CCR5 levels and of the Delta-32 mutation and the CCR2-V64I/CCR5-59653T linked mutations. Blocking endogenous beta-chemokines relieved minimally this restriction. At the single cell level, CD4 memory cells were either permissive or non-permissive for R5 HIV-1 infection. R5 HIV titre was up to 10-fold lower than X4 virus titre even in a permissive clone. However, R5 viruses replicated as efficiently as X4 viruses in the permissive clone when neutralizing anti-beta chemokine antibodies were added. Non-permissive cells blocked a post-entry step of the virus life-cycle and expressed early but not late HIV transcripts. Neutralizing anti-beta chemokine antibodies promoted R5 virus replication marginally in the non-permissive clone. CONCLUSION: Some blood memory CD4 T cells retard R5 HIV-1 replication via endogenous beta-chemokines whereas others block productive R5 HIV-1 infection by an additional mechanism that interferes with a post-entry step of the virus life cycle. These natural barriers might contribute to lower pathogenicity of R5 HIV-1 strains for CD4 memory T cells than X4 viruses that emerge late in disease.

HIV infection alters the production of both type 1 and 2 cytokines but does not induce a polarized type 1 or 2 state.

OBJECTIVE: To test the T-helper (TH)1/TH2 cytokine paradigm in HIV infection. DESIGN AND METHODS: Cytokine profiles in two separate studies of HIV patients and controls are presented: (i) a longitudinal study of HIV patients with CD4 counts > 500 x 10(6)/l tested at three timepoints compared with controls; (ii) a blinded cross-sectional study of controls and patients with high (> 500 x 10(6)/l) and low (< 500 x 10(6)/l) CD4 counts. Peripheral blood mononuclear cells (PBMC) from patients and controls were tested for the production of two type 1 [interleukin (IL)-2, interferon (IFN)-gamma] and two type 2 (IL-4, IL-10) cytokines by enzyme-linked immunosorbent assay. Both spontaneous and mitogen-induced cytokine production was measured. RESULTS: HIV infection was noted to have the following effects on cytokine production: (i) it led to the in vivo activation of type 2 cytokines in a small group of individuals with high CD4 numbers characterized by the spontaneous release of IL-4 and IL-10. Longitudinal data showed high spontaneous IL-4 and IL-10 to be a consistent feature of the patient group (at each timepoint some patients were high producers) but to be variable in a given individual; (ii) HIV infection impaired the ability of PBMC to respond to stimuli (selected for their ability to optimally induce each cytokine) in terms of IL-2, IL-4 and IL-10 production in patients with both high and low CD4 cell counts; and (iii) conversely, HIV infection led to an overproduction of IFN-gamma in patients with high CD4 counts; patients with low CD4 produced normal levels of IFN-gamma. CONCLUSIONS: Our observations did not suggest polarization of the type 1/type 2 cytokine profile in HIV patients. Instead, the data suggested more complex changes to type 1/type 2 cytokine patterns in HIV infection than originally proposed by the TH1/TH2 dichotomy.

Immunization of human HIV-seronegative volunteers with recombinant p17/p24:Ty virus-like particles elicits HIV-1 p24-specific cellular and humoral immune responses.

OBJECTIVE: To evaluate the immune response to HIV-1 p24 generated in vivo by p17/p24:Ty virus-like particles (p17/p24:Ty-VLP) by examining the lymphoproliferative and antibody (Ab) responses to HIV-1 p24, as well as Gag-specific cytotoxic T lymphocytes (CTL), in HIV-seronegative volunteers immunized with hybrid p17/p24:Ty-VLP. DESIGN AND METHODS: Sixteen HIV-seronegative volunteers were immunized with p17/p24:Ty-VLP at two dose levels (100 or 500 micrograms) and monitored for the following 48 weeks for production of anti-p24 and anti-p17 Ab, in vitro lymphoproliferative responses to HIV-1 p24 and p17, and in vitro CTL responses to HIV-1 Gag. RESULTS: Twelve out of the 16 volunteers had significant p24-specific proliferative responses, with volunteers on the higher dose schedule exhibiting earlier proliferative responses than those on the lower dose schedule. Proliferative responses in both volunteer groups were similar in overall magnitude but appeared at different times during the immunization schedule. Anti-p24 Ab were detected in six out of the nine individuals in the lower dose group and in five out of the seven in the higher dose group. There was a good correlation between the presence of p24-specific Ab and the detection of lymphoproliferative responses to the p24 protein in peripheral blood mononuclear cells isolated from the same individuals. Anti-p17 Ab were detected in five volunteers. No Gag-specific CTL responses were detected. CONCLUSION: We conclude that hybrid HIV-1 p17/p24:Ty-VLP are capable of inducing both cellular and humoral immunity to HIV-1 Gag p17 and p24 components and are worthy of further study as a potential HIV immunotherapeutic.

The related cytokines interleukin-13 and interleukin-4 are distinguished by differential production and differential effects on T lymphocytes.

We have compared the production of the related cytokines IL-13 and IL-4 by T lymphocytes, and the effects of the two cytokines on these cells. IL-13 and IL-4 production differ in a number of respects. IL-13 is produced at higher levels than IL-4 by activated T lymphocytes, and its accumulation in the culture medium can be more prolonged, corresponding partly to differential mRNA accumulation and partly to a preferential depletion of IL-4 from the culture medium. Certain inducing combinations such as PMA and anti-CD28, stimulate high levels of IL-13 and IL-13 mRNA, but little or no IL-4 or IL-4 mRNA. The ratio of IL-13 to IL-4, both at protein and mRNA levels, is higher in CD8+ lymphocyte than in CD4+ lymphocyte populations. Although after in vitro polarization of peripheral blood lymphocytes leading to type 1 and type 2 populations, IL-13 is made principally by cells of a type 2 phenotype, as is IL-4; it can also be produced by type 1 CD4+ and CD8+ T lymphocyte clones making large amounts of IFN-gamma and very little IL-4. IL-13 and IL-4 exert different effects on T lymphocyte functions. IL-13 does not significantly inhibit the IL-2-induced T lymphocyte production of IFN-gamma, RANTES, MIP-1 alpha or MIP-1 beta, nor that of perforin mRNA, as does IL-4. We have also been unable to demonstrate STAT6 activation by IL-13 on T lymphocytes purified in a number of ways, despite strong activation of STAT6 by IL-4 in these cells. This is contrary to some previous reports, but is consistent with the notion that the majority of T lymphocytes lack functional IL-13 receptors. A higher and more prolonged T lymphocyte production of IL-13 than that of IL-4 may thus be permissible because IL-13 does not inhibit T-cell functions. Conversely, sustained IL-13 production may be partly due to the absence of receptor-mediated depletion of this cytokine.

The killing of tumour cell targets coupled to tuberculin (PPD) by human and murine PPD-reactive T helper clones. II. Major histocompatibility complex restriction of killing.

This paper takes up the major histocompatibility complex (MHC) restriction of killing by a murine and a human tuberculin (PPD)-specific T helper clone of PPD-Con A bound targets. In the previous paper we demonstrated that the specificity of killing of such targets was directed against PPD and not the lectin. This paper provides further evidence to suggest that the PPD-specific clones recognize PPD on PPD-Con A-bound cells though the T cell antigen receptor complex, since the killing was restricted by MHC class II products. Using a range of syngeneic, allogeneic, and semi-syngeneic targets we have shown the fine specificity of the restricting element to be one of the two alleles of the DR region (DR 2) for the human clone, and to be the I-A subregion for the murine clone. Binding studies with radiolabelled class II antibodies were performed to see whether killing efficiency was dependent on the number of class II products expressed. The findings showed that the human B-EBV targets express 2-3 x 10(6) molecules per cell, while the susceptible murine tumours, the Abelson line and the 6A tumour, only expressed 600-800 binding sites per cell. Target cell susceptibility appeared to be linked to the number of class II molecules expressed; thus the syngeneic murine MBL-2 tumour expressing 200-300 binding sites per cell was not killed and the lysis of the 6A and Abelson tumours could be enhanced by doubling the number of class II binding sites by incubating cells with Con A-conditioned medium. However, maximum lysis did not exceed 30-40%, suggesting that class II expression alone did not govern killing.

Migration inhibition factor secreting human T-cell lines reactive to PPD: a study of their antigen specificity, MHC restriction and the use of Epstein-Barr virus-transformed B-cell lines as requirement for antigen-presenting cells.

Two PPD-reactive T-cell lines and two clones derived from them have been characterized. The lines were maintained for a period of 10-12 weeks in I1-2 containing medium. The clones were derived from the uncloned lines by the limiting dilution method and maintained in culture for 12 weeks. The cloning efficiency was 1%. Both the cloned and the uncloned lines were highly reactive to tuberculin in a proliferation assay and produced migration inhibition factors following antigenic stimulation. Both these functions were dependent on the addition of antigen-presenting cells and genetically regulated by Class II molecules of the MHC. Each uncloned line and the clones derived from them were restricted by just one of the DR alleles of the autologous host. An analysis of cell types involved in antigen presentation showed that macrophages and Epstein-Barr virus-transformed B-cell lines induced both proliferation and MIF secretion in the T-cell lines and clones cultured with PPD. Phenotypic studies indicate that the cells are Sheep E+, OKT4+, OKT8- and HLA-DR+.

Raising antibodies by coupling peptides to PPD and immunizing BCG-sensitized animals.

The use of PPD (purified protein derivative of tuberculin) as a carrier has several significant advantages. It provides very powerful T cell help and it gives rise to virtually no antibody response against itself. This is particularly useful if it is intended to go on to make monoclonal antibodies, where the presence of a large amount of anti-carrier antibody is a nuisance! Furthermore, unlike most comparably powerful adjuvant systems, it can be used in man. PPD coupling has been used to raise antibodies to haptens and to raise T cell responses to tumour cells. It is here reported that small peptides coupled to PPD will give rise to good titres of anti-peptide antibody. For peptides that contain no cysteine, coupling has been achieved by attaching succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) to the alpha-amino group of the peptide and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to the PPD and allowing an uncleavable bond to form between them. Data on immunization with the leucotactic nonapeptide of the alpha chain of the complement component C3 and with some oncogene-related peptides have been obtained.

HIV-1 infection of human CD4+ T cells in vitro. Differential induction of apoptosis in these cells.

The H9 and CEM CD4+ T cell lines were infected with HIV-1 (NY5/LAV-1 isolate) and monitored for losses in cell viability, syncytium formation, and internucleosomal DNA cleavage (a marker for apoptosis). H9 cells were found to undergo cell death via apoptosis as a result of HIVNY5 infection, but this effect was not apparent in CEM cell cultures. The differential effects of HIV-1NY5 in terms of its apoptosis-inducing properties correlated with the relative abilities of H9 and CEM cells in supporting replication of this HIV-1 isolate, since infected CEM cell cultures produced 10-fold lower levels of HIV-1 p24 protein, and very few of these cells stained positive for cell-associated p24 by comparison with H9 cell cultures infected at the same multiplicity of infection. Furthermore, a different HIV-1 isolate (RF), which replicated equally efficiently in both H9 and CEM cells, produced similar levels of apoptosis in these cultures. HIV-1NY5 was also found to be capable of inducing apoptosis in purified peripheral blood CD4+ T cells as well as inhibiting anti-CD3-driven proliferation of these cells. In contrast, incubation of purified CD8+ T cells with HIV-1NY5 under similar conditions produced no cytopathic effects. Substantial levels of apoptosis were also recorded in HIV-1NY5-infected PHA blasts cell cultures. Soluble rHIV-1IIIB type CHO-derived gp120 was found to mimic the effects of HIV in terms of inhibition of anti-CD3/TCR mAb-induced proliferation of T cells, but apoptosis was not detected in gp120-treated T cell cultures whether cross-linked or used in conjunction with anti-CD3 mAb or not. We conclude therefore that both HIV-1NY5 and HIV-1RF isolates have the capacity to directly trigger apoptotic cell death in CD4+ T cells and that this appears to be at least partly associated with the efficiency of virus replication in these cells.

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.

The effects of complement activation by cobra venom factor on the migration of T and B lymphocytes into rat thoracic duct lymph.

Experiments were done to see whether C3 or C3-split products are involved in lymphocyte recirculation, with particular reference to B lymphocytes which have C3b receptors. Rats were injected with cobra venom factor (CVF), and the output of subclasses of lymphocytes was measured in thoracic duct lymph in hourly collections during the subsequent 24 h. During the period of acute C3 activation which lasted for 2-8 h, the output of lymphocytes decreased by 47%, but returned to normal at later times, when C3 levels were reduced to less than 20% normal. There was no effect on the output of C3b receptor lymphocytes, and this receptor was not blocked probably because initial C3 levels in lymph were only 13% of blood levels, so that only small amounts of C3b were generated in lymph. When these lymphocytes were labelled and injected i.v. they migrated with the slow rate which is characteristic of normal B lymphocytes. The main effect of CVF was to reduce the output of T lymphocytes by 58% during the phase of acute C3 activation. When normal thoracic duct lymphocytes were labelled and injected, their rate of reappearance in thoracic duct lymph was only reduced during this phase. It was concluded that recirculation of lymphocytes is not C3 dependent, and that insufficient C3b is generated in lymphoid tissues to block C3b receptors on B lymphocytes during periods of rapid C3 activation. However the migratory rate of T lymphocytes through these tissues is reduced during this period, and it is suggested that this may be due to an effect of C3 split products on macrophages which lie along T-lymphocyte traffic routes.

The heterogenization of tumour cells with tuberculin. I. The coupling of tuberculin to cell surfaces using con-A as ligand.

The introduction of an antigenic determinant strongly recognized by T cells on to a cell enhances the immune response to weak cellular antigens. Tuberculin (PPD) is a particularly suitable antigenic determinant for this purpose since it behaves in many ways as a 'T-cell hapten'. It has been found that direct chemical coupling of PPD to cell surfaces damages their antigenicity, but that this can be circumvented by coupling PPD to the lectin Concanavalin A and using this as the ligand for binding PPD to the cell. Techniques for preparing Con-A/PPD using little glutaraldehyde or SPDP as cross-linking agents are described.

The heterogenization of tumour cells with tuberculin. II. Studies of the antigenicity of tuberculin-heterogenized murine tumour cells in syngeneic BCG positive and BCG negative mice.

The possibility of enhancing the antigenicity of tumour cells by chemically attaching purified protein derivative (PPD) of tubercle bacillus as an immunogenic carrier determinant into the tumour cell surface has been explored in these studies. It was observed that multiple immunizations with PPD-coupled tumour cells did potentiate a marked anti-rumour response even to tumours that were very weakly antigenic. Moreover, such immunizations could be used to retard the growth of tumours in previously unimmunized animals. An attempt has been made to elucidate the important factors involved when PPD heterogenized tumour cells are used for immunization.