Anti-CD4-binding domain antibodies complexed with HIV type 1 glycoprotein 120 inhibit CD4+ T cell-proliferative responses to glycoprotein 120.

HIV-specific CD4+ helper T cell responses, particularly to the envelope glycoproteins, are usually weak or absent in the majority of HIV-seropositive individuals. Since antibodies, by their capacity to alter antigen uptake and processing, are known to have modulatory effects on CD4+ T cell responses, we investigated the effect of antibodies produced by HIV-infected individuals on the CD4+ T cell response to HIV-1 gp120. Proliferative responses of gp120-specific CD4+ T cells were inhibited in the presence of either serum immunoglobulin from HIV-infected individuals or human monoclonal antibodies specific for the CD4-binding domain (CD4bd) of gp120. Human monoclonal antibodies to other gp120 epitopes did not have the same effect. The anti-CD4bd antibodies complexed with gp120 suppressed T cell lines specific for varying gp120 epitopes but did not affect T cell proliferation to non-HIV antigens. Moreover, inhibition by the anti-CD4bd/gp120 complexes was observed regardless of the types of antigen-presenting cells used to stimulate the T cells. These results indicate that the presence of anti-CD4bd antibodies complexed with gp120 can strongly suppress CD4+ helper T responses to gp120.

Non-nominal HIV surveillance: preserving privacy while tracking an epidemic.

OBJECTIVE: To enhance HIV surveillance within a non-nominal provincial testing system. METHODS: Confirmatory HIV tests from a provincial laboratory were analyzed during 1995 and 1996. Enhancements included elimination of repeat positive tests for the same individual using automated matching of non-nominal identifiers and nurse call-back of health care providers, completion of missing information through call-back and connection of providers with resources for patient care. RESULTS: Forty-seven percent of 2,683 reactive HIV tests were identified as duplicates for the same individual, meaning that 1,401 people tested positive for the first time. From laboratory test data to enhanced unduplicated data after call-back, the proportion of tests for which risk and ethnic information was unknown dropped from 37% to 11% and from 64% to 18% respectively (p < 0.0001). CONCLUSIONS: Enhanced non-nominal surveillance for HIV is a practical means of marrying the needs of public health for epidemiological information and the rights of patients to privacy.

Antigen presenting cell function in HIV-1 infected patients.

Antigen presenting cell (APC) function is central to the activation of anti-viral cytotoxic T-cells and antibody production. In previous studies we have evaluated APC function in HIV-1 infected patients as the capacity to present peptide to a well defined panel of CD4 T-cell clones. We found that APC from HIV-1 infected patients were defective in the capacity to present peptide to CD4 T-cell clones. The APC defect uncovered by this method was present early in infection and worsened with increasing viral load, suggesting that it was an important determinant of progression and anti-viral efficacy. The CD4 T-cell clones were, however, found to vary in their susceptibility to the APC defect. CD4 T-cell clones that failed to respond to peptide presented by HIV + APC were 1000-fold more readily inhibited by anti-CD4 antibody than T-cell clones which consistently responded to APC from patients infected with HIV-1 (HIV + APC). An intermediate group of T-cell clones were also identified that only responded to peptide and HIV + APC from asymptomatic patients. These results suggested that the underlying mechanism for the APC defect was binding of T-cell CD4 by APC-associated gp120. In this paper we discuss the evidence to support this hypothesis.

Immunization with human immunodeficiency virus type 1 rgp120W61D in QS21/MPL adjuvant primes T cell proliferation and C-C chemokine production to multiple epitopes within variable and conserved domains of gp120W61D.

Human immunodeficiency virus type 1 (HIV-1) gp120W61D-specific T cell lines (TCL) were generated from an HIV-1-seronegative volunteer who received rgp120W61D in QS21/MPL adjuvant with emulsion. TCL were challenged with pools of consecutive, overlapping peptides spanning the gp120W61D sequence and then with the individual peptides of the immunostimulatory pool. T cell epitopes were found within both variable and conserved domains, and there was no evidence of a single immunodominant epitope. The two most frequently recognized peptides were located in the C1 domain and in the C-terminal region of the V3 loop. Several TCL were shown to recognize multiple peptides from nonoverlapping regions. Peptides from both conserved and variable domains were capable of inducing MIP-1alpha, MIP-1beta, and RANTES production. When tested against the equivalent peptide from the HIV-1IIIB sequence, however, TCL were able to tolerate only minor conserved changes in the amino acid sequence.

Dendritic cells from HIV-1-infected patients naturally express HIV-1 gp120 V3 loop-derived peptide ligands.

Little is known of the peptide ligands expressed in vivo on antigen-presenting cells (APC) or of the APC lineages involved. In this study we have addressed this question using HLA-DRbeta1*0101-restricted CD4 T cell clones (TLC) specific for a synthetic peptide based on the HIV-1 gp120 V3 loop consensus sequence for the Clade B isolates predominantly found in European and North American patients. These TLC were found to respond, in a dose-dependent manner, to freshly isolated HIV-infected patient APC in the absence of exogenously added peptides. Further APC purification showed that the naturally expressed peptide ligands were present in both the APC lineages shown to be infected with the virus and were most strongly detectable on purified blood dendritic cells. Peptides based on consensus sequences of viruses isolated from one of the patients over the period when naturally expressed peptide ligands could be detected were all found to stimulate TLC proliferation. These studies, therefore, show that peptide ligands derived from natural infection are detectable on APC lineages, particularly on dendritic cells which play an important role in the immune response to viruses. Even small differences in sequence between the vaccine isolate and the natural infection, if they occur in the key residues of protective T cell epitopes, could therefore have a profound effect on the efficacy of vaccines against viruses with high rates of mutation.

Tandem peptide epitopes facilitate CD4-dependent activation of T cell clones.

Peptides that consist of two tandemly repeated epitopes joined by a flexible linker have an increased affinity for class II molecules and are more potent at inducing proliferation of T cell clones than monomeric epitopes. The increase in potency of peptides with two epitopes for individual T cell clones is proportional to the relative CD4 dependence of the clones. We show that epitope dimers activate T cell clones that respond sub-optimally to monomeric epitopes presented by APC from HIV-infected donors. We hypothesize that HIV+ APC normally fail to stimulate the clones because virally encoded gp 120 sequesters CD4 from the activation complex, but epitope dimers overcome this effect because they are better able to recruit CD4. The alpha beta heterodimer of human class II (HLA-DR1) is further ordered as a dimer of heterodimers (superdimer) at least in its crystal form. Since class II molecules have an open-ended antigen binding groove, the superdimer is theoretically permissive of stable binding of two peptide epitopes linked in tandem. Our data support a role for the MHC class II dimer of heterodimers in amplifying the proliferative response of T cells to antigen by dint of the superdimers having a higher affinity for CD4 than the nominal class II alpha beta heterodimers.

CD4+ T-cell recognition of diverse clade B HIV-1 isolates.

OBJECTIVES: To evaluate the effect of sequence variation within the gp120 V3 loop on CD4 T-cell recognition. DESIGN: CD4 T-cell clones were generated using synthetic peptides to circumvent the difficulties of using polyclonal T-cell responses. Peptides based on other HIV isolates were then used to determined the influence of single and multiple sequence differences. RESULTS: Three of the panels of T-lymphocyte clones (TLC), which were all specific to diverse HIV-1 clade B gp120 V3-loop peptides differing in a limited number of residues, had heterogeneous patterns of response to peptides differing in length and sequence indicating that they recognized distinct but overlapping epitopes. The panels of TLC also differed in the extent to which they tolerated sequence differences between cell-culture-adapted or primary HIV-1 isolates. One panel responded to peptides based on several clade B and one clade D isolate. In contrast, two panels, generated from two different donors using the same peptide, only responded to a limited number of clade B isolates, whereas another only recognized HIV-1BRU. Two of the panels were also stimulated by peptides based on clinical isolates from one patient with some sequence changes enhancing T-cell recognition. CONCLUSIONS: These data are consistent with highly diverse CD4 T-cell recognition of the HIV-1 gp120 V3 loop, which is influenced by the sequence differences within the T-cell epitopic region and has implications for the pathogenesis and design of vaccines against HIV-1.

An early antigen-presenting cell defect in HIV-1-infected patients correlates with CD4 dependency in human T-cell clones.

We have used a defined panel of nine HIV peptide-specific T-cell clones (TLC) generated from a healthy volunteer to evaluate the antigen-presenting cell (APC) function of human immunodeficiency virus-1 (HIV- 1)-infected patients. Peripheral blood mononuclear cells (PBMC) from HLA-matched seropositive and uninfected volunteers were compared for their capacity to present peptide to TLC specific for the V3 loop of HIV- 1 envelope glycoprotein gp120, influenza haemagglutinin or the mycobacterial 19,000 MW antigen APC from uninfected volunteers (HIV- APC) invariably presented peptides to all TLC with comparable efficiency. In contrast using APC from HIV- 1-infected subjects (HIV+ APC) three patterns of responsiveness were observed. The first group of TLC was not stimulated by HIV+ APC even early in infection. The second responded to all APC comparably. The third and intermediate group, responded to APC from some clinically asymptomatic, but not acquired immune deficiency syndrome (AIDS), patients. The two additional TLC, derived from other donors and with specificity for non-HIV peptides, showed similar variation in response to HIV+ APC. The different patterns of response to HIV APC did not correlate with the fine specificity or cytokine phenotypes of the TLC. Neither was the defect due to decreased levels of expression of APC molecules involved in delivering the first or second signal required for T-cell activation APC mixing experiments showed no evidence of APC-derived inhibitory factor. Furthermore, the defect was independent of T cells or their products and was equally expressed in monocytes and dendritic cells. Instead, responsiveness was inversely related to the degree of CD4 dependency suggesting that the underlying mechanism was a CD4 APC-associated gp120 interaction. The early appearance of this defect in HIV- 1 infection co-incident with the loss of recall responses is consistent with a role for APC dysfunction in pathogenesis.

The effect of a single amino acid substitution within the V3 loop of HIV-1 gp120 on HLA-DR1-restricted CD4 T-cell recognition.

Viral variation has been proposed to play a role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1) infection, and is an important consideration in vaccine design. During the course of an infection, isolates with sequence changes in CD8 T-cell and B-cell epitopes arise. To determine whether sequence variation within the V3 loop of HIV-1 gp120 affects HLA-DR beta 1*0101-restricted CD4 T-cell recognition, we have generated CD4 T-cell clones (TLC) specific to gp120 V3 loop peptides. Four HLA-DR beta 1*0101-restricted groups of TLC were defined by distinct patterns of responses to a panel of peptides, consistent with a highly diverse T-cell repertoire recognizing the 30 amino acid stretch (296-326) of the gp120 V3 loop. Nevertheless, a single residue change at position 311 was found to abolish the recognition of two of the four groups of TLC. This was not due to an effect of the residue at 311 on binding to major histocompatibility complex (MHC), because: (1) irrespective of the residue at 311, peptides competed well with the influenza haemagglutinin peptide 307-319 for binding to cell-bound DR1; and (2) R311-specific TLC were also HLA DR beta 1*0101 restricted. Instead, the substitution of arginine for serine at position 311 blocked the interaction of the peptide with the T-cell receptor. Thus, despite the diversity of the T-cell response to the V3 loop of HIV-1, a single amino acid change can have a considerable influence on the responding T-cell population. As residue 311 is one of the most variable of the V3 loop residues, these results suggest that CD4 recognition can also exert pressure on viral variation consistent with a role for these cells in antiviral immunity.

The effect of lipoylation on CD4 T-cell recognition of the 19,000 MW Mycobacterium tuberculosis antigen.

The mechanisms contributing to the dominant and degenerate recognition of the N-terminal region of the Mycobacterium tuberculosis 19,000 MW protein have been investigated. Using polyclonal and cloned T cells it was found that the apparently promiscuous response to the N-terminal peptide was due to the presence of multiple epitopes recognized in the context of different HLA determinants. This finding did not, however, explain the concentration of T-cell recognition on this part of the molecule. The 19,000 MW antigen is a lipoprotein, which raised the possibility that presence of a lipidation motif preceded by a signal peptide influenced the processing and presentation of the protein. Modified peptides covalently attached to lipid moieties were, therefore, tested on both polyclonal and cloned T cells. It was found that whilst lipoylation enhanced polyclonal T-cell recognition, the effect on cloned T cells was variable and depended on their epitope specificity and restricting HLA determinants. This suggested that whilst lipoylation may enhance some aspect of signalling for polyclonal T cells it does not affect the presentation of peptide to T-cell clones. The explanation for the immunodominance of the N-terminal region may, therefore, lie in some aspect of its processing.

Antigenic specificity and subset analysis of T cells isolated from the bronchoalveolar lavage and pleural effusion of patients with lung disease.

Cellular infiltrates of bronchoalveolar lavage (BAL) and pleural effusion from patients with tuberculosis (TB) and lung cancer were characterized for the presence of different T cell subsets by phenotypic analysis. The specificity of the T cells for mycobacterial antigens was then compared for the two disease compartments. The composition of T cell subsets within the BAL, in contrast to pleural effusion cells (PEC), revealed evidence of sequestration of CD8+ cells. BAL T cells were found to be a predominantly CD29+ DR+ memory population of activated cells. Although polyclonal populations of BAL T cells proliferated poorly to Mycobacterium tuberculosis antigens, mycobacterial antigen-reactive monoclonal T cell populations could be derived from the alveolar compartment. Two clones were shown to recognize the 65-kD heat shock protein of mycobacteria, and one of these clones recognized a conserved sequence of the molecule. Several BAL-derived clones, responding to a mycobacterial soluble extract, did not, however, recognize purified mycobacterial antigens, previously identified as highly stimulatory for PEC-derived T cells. T cell clones, derived from PEC of two TB patients, responded to the 38-kD and 71-kD, as well as the 65-kD mycobacterial antigens. Examination of the activation requirements of BAL-derived T cell clones, specific for mycobacterial antigens, revealed that exogenous IL-2 was necessary for the T cells to sustain proliferation. This was in contrast to the mycobacterial antigen-reactive T cells cloned from PEC. These results suggest that T cell populations with distinct antigen specificities and activation requirements are present in BAL and PEC.

Stress-induced modulation of antigen-presenting cell function.

The effect of two means of inducing a stress response, heat and oxygen radicals, on the ability of an HLA-DR1 B-cell line to stimulate DR1-restricted and anti-DR1 auto- and alloreactive T-cell clones has been examined. Both forms of stress enhanced the ability of B cells to stimulate auto- and alloreactive T-cell clones and to present peptide to an influenza-virus specific T-cell clone. Furthermore, the ability of the B-cell line to present whole influenza virus was augmented by heat stress. The stress-induced enhancement of T-cell responses coincided with a modest increase in the cell-surface expression of major histocompatibility class II products. This was, however, insufficient to account for the observed functional effects. In contrast to these effects, presentation of whole antigen was inhibited by the oxygen radical intermediate, hydrogen peroxide (peroxide), in a dose-dependent manner. When analysed by SDS-PAGE, it was found that whilst overall protein synthesis decreased following both types of stress, increased synthesis of heat-shock proteins (HSP), and in particular the 70,000 MW HSP, was only evident following heat stress. The absence of an increase in the synthesis of HSP 70, in the antigen-presenting cells (APC) following the uptake of UV-treated influenza virus, however, implied that HSP 70 induction was not necessary for the presentation of whole antigen. The effects of peroxide stress appeared to be qualitatively different in several respects. First, peroxide treatment did not cause the induction of any stress proteins; second, peroxide abolished the presentation of whole antigen. In addition, heat stress of APC was unable to protect from the adverse effects of peroxide treatment, in that cells treated sequentially with heat, followed by peroxide, were unable to present whole influenza virus. In order to determine the stage of antigen presentation at which peroxide was causing inhibition, APC were treated at varying time-points after pulsing with antigen. The kinetics of the peroxide effect paralleled those of aldehyde fixation. Taking these results together it would appear that peroxide interferes with some aspects of the antigen-processing pathway.

Analysis of human T-cell epitopes in the 19,000 MW antigen of Mycobacterium tuberculosis: influence of HLA-DR.

The potential number of T-cell epitopes in the 19,000 molecular weight (MW) antigen has been investigated using overlapping peptides which comprise the complete sequence. Sixteen potential epitopes could be deduced from the responses to these peptides by polyclonal T cells derived from 22 antigen-responsive donors. The majority of epitopes were not predicted by either of the major paradigms, the Rothbard motif and the amphipathic helix. A hierarchy of epitopes was indicated by the responses, which ranged from strong and frequent in the N-terminal region, to moderate or weak elsewhere. Some epitopes were restricted by single HLA-DR determinants, or families of determinants sharing structural features in common, whilst the two N-terminal peptides were recognized by donors with a diversity of DR types. The high degree of T-cell recognition of the N-terminal region may be of relevance to the design of a sub-unit vaccine capable of priming T cells against Mycobacterium tuberculosis.

Heat shock proteins: an autoprotective mechanism for inflammatory cells?

Heat shock/stress proteins are synthesized in all cell types under a variety of stressful conditions. Stress associated with ongoing inflammation relates, at least in part, to toxic products locally generated by cells accumulating in the inflamed tissue and organ. These products include oxygen free radicals, cytokines, proteases, chemotactic factors and, in the particular case of the eosinophil, toxic basic proteins. The heat shock response in inflammatory cells appears to be specifically regulated by their own proinflammatory products (oxygen free radicals, cytokines) generated during physiological functions such as phagocytosis, during differentiation, or in certain pathological states such as inflammatory lung diseases. We suggest that in human monocytes-macrophages heat shock proteins belong to the autoprotective equipment against oxidative stress.

The generation and use of human T cell clones.

In this review a number of uses of human T cell clones have been discussed. Before considering T cell cloning, however, it is worth bearing in mind that there are certain disadvantages to this approach to T cell immunity, not the least of which is that these cells, adapted as they are for in vitro growth, may be unrepresentative of the normal T cell, in terms of both specificity, and function. In addition, cloning is sufficiently difficult for it to be undertaken only where monoclonal populations are essential to the desired aim. Nevertheless, the range of uses discussed, and the fact that many have had a fundamental impact on our understanding of immune mechanisms, not only as mediated by T cells, but also of the intracellular mechanisms of antigen-presentation, the nature and mode of action of the cytokines, as well as the cell surface molecules and cascade of signals that orchestrate T cell activation, indicate the importance of T cell cloning. In the future, it is probable that the use of T cell clones with defined receptor usage will improve our understanding of the mechanisms underlying disease pathogenesis, and thus aid both the prevention and treatment of disease. In addition, the T cell receptor structure will, no doubt, be elucidated, leading to a further quantum leap in our understanding of T cell immune mechanisms, as well as suggesting other avenues for exploration. In all these areas there is no doubt that the methodology of T cell cloning will continue to make a fundamental contribution.

Functional evidence for the recognition of endogenous peptides by autoreactive T cell clones.

The fine specificity of two human T cell clones responding to autologous HLA-DR1 expressing antigen-presenting cells (APC) in the absence of nominal antigen has been investigated using Epstein-Barr virus-transformed B cells (BCL) of known DR beta 1 domain sequence. It was found that responsiveness was markedly affected by changes in a limited number of residues in this domain. Substitution of the DR1 beta sequence at one residue, position 74, even conservatively, was found to be particularly significant. Located on the beta 1 domain alpha-helix, this residue is predicted to point into the antigen-binding groove and is therefore unlikely to make contact with the T cell receptor. This finding suggests that these T cells are specific for a bound endogenous peptide within the autologous major histocompatibility (MHC) binding groove. The autospecific T cell clones also responded to murine L cell transfectants expressing DR alpha DR1 beta as well as to transfectants expressing the mouse/human hybrid MHC molecule I-E alpha DR1 beta but not to the reciprocal combination DR alpha I-E beta, thus confirming the importance of the beta 1 domain to T cell recognition. In contrast to the autocytotoxicity observed with BCL, cytolysis of the murine L cells expressing the HLA-DR1 molecule was slight and only found at high effector-target ratios. In addition, although fixation enhanced the recognition of BCL, capacity of the murine L cells bearing the HLA-DR1 molecule to stimulate T cell clone proliferation was markedly reduced by aldehyde fixation. When taken together, these results suggest that the endogenous peptides recognized by these autoreactive T cells are of human origin.

Prediction and identification of an HLA-DR-restricted T cell determinant in the 19-kDa protein of Mycobacterium tuberculosis.

An allele-specific motif has been identified in the sequence of several peptides which are recognized by T cells in association with HLA-DR1. In order to test the predictive values of such a motif we analyzed the 19-kDa antigen from Mycobacterium tuberculosis and identified a sequence containing a pattern characteristic of DR1 restriction. Peripheral blood mononuclear leukocytes from every DR1 and 4 individual tested responded to the corresponding synthetic peptide. Nine other donors, constituting seven different DR alleles, failed to recognize this sequence. Recognition of the peptide in association with DR1 and DR4 was confirmed using T cell clones and transfected murine L cell lines expressing DR molecules.

Mapping of T cell epitopes using recombinant antigens and synthetic peptides.

Two complementary approaches were used to determine the epitope specificity of clonal and polyclonal human T lymphocytes reactive with the 65-kd antigen of Mycobacterium leprae. A recombinant DNA sublibrary constructed from portions of the 65-kd gene was used to map T cell determinants within amino acid sequences 101-146 and 409-526. Independently, potential T cell epitopes within the protein were predicted based on an empirical analysis of specific patterns in the amino acid sequence. Of six peptides that were predicted and subsequently synthesised, two (112-132 and 437-459) were shown to contain human T cell epitopes. This corroborated and refined the results obtained using the recombinant DNA sublibrary. Both of these regions are identical in M. leprae and M. tuberculosis and are distinct from the known B cell epitopes of the 65-kd protein. This combination of recombinant DNA technology and peptide chemistry may prove valuable in analysis of the cellular immune response to infectious agents.

T-cell activation by anti-idiotypic antibody: evidence for the internal image.

Human lymphoproliferative responses to a rabbit anti-idiotypic antibody (anti-Id TB71) and the corresponding mycobacterial protein antigen [38,000 molecular weight (MW)] have been investigated in a number of donors. It was found that responsiveness to anti-Id TB71 correlated with responder and non-responder (four subjects each) status to the 38,000 MW antigen. Furthermore, the induction of T-cell proliferation by both the 38,000 MW antigen and the anti-Id TB71 was dependent on accessory cells. When taken together with the concordance between the 38,000 MW antigen and anti-Id responsiveness, this implies that the 38,000 MW antigen and anti-Id TB71 stimulate related, or at least partially overlapping, repertoires of T cells. This was confirmed by the finding that cloned T cells reactive with the 38,000 MW antigen also proliferated in response to the anti-Id TB71. These observations are readily explained if the anti-idiotypic antibody contains an internal image of, and can therefore mimic, the antigen.