Dimorphic HLA-B signal peptides differentially influence HLA-E- and natural killer cell-mediated cytolysis of HIV-1-infected target cells.

As a mechanism of self-protection, signal peptides cleaved from human leukocyte antigen (HLA) class I products bind to HLA-E before the complex interacts with the natural killer (NK) cell receptor CD94/NKG2A to inhibit NK-mediated cell lysis. Two types of the signal peptides differ in their position 2 (P2) anchor residue, with P2-methionine (P2-M) having higher HLA-E binding affinity than P2-threonine (P2-T). All HLA-A and HLA-C molecules carry P2-M, whereas HLA-B products have either P2-M or P2-T. Epidemiological evidence suggests that P2-M is unfavourable in the context of HIV-1 infection, being associated with accelerated acquisition of HIV-1 infection in two African cohorts. To begin elucidating the functional mechanism, we studied NK-mediated killing of CD4(+) T cells and monocyte-derived macrophages infected with two laboratory-adapted HIV-1 strains and two transmitted/founder (T/F) viruses. In the presence of target cells derived from individuals with the three HLA-B P2 genotypes (M/M, M/T and T/T), NK-mediated cytolysis was elevated consistently for P2-T in a dose-dependent manner for all cell and virus combinations tested (P = 0·008-0·03). Treatment of target cells with an anti-HLA-E monoclonal antibody restored NK-mediated cytolysis of cells expressing P2-M. Observations on cell lysis were also substantiated by measurements of HIV-1 p24 antigen in the culture supernatants. Overall, our experiments indicate that the anti-HIV-1 function mediated by NK cells is compromised by P2-M, corroborating the association of HLA-B genotype encoding P2-M with accelerated HIV-1 acquisition.

A significant number of human immunodeficiency virus epitope-specific cytotoxic T lymphocytes detected by tetramer binding do not produce gamma interferon.

Despite the seemingly important role of cytotoxic T-lymphocyte (CTL) responses in human immunodeficiency virus (HIV) disease pathogenesis, their measurement has relied on a variety of different techniques. We utilized three separate methodologies for the detection of CTLs in a cohort of HIV-infected individuals who were also human leukocyte antigen A2 (HLA-A2) positive. Among the different CTL assays, a correlation was seen only when the Gag epitope-specific HLA A*0201-restricted tetramer assay was compared with the ELISPOT assay performed after stimulation with the Gag epitope; however, this correlation was of borderline statistical significance. On average, the tetramer reagent detected a 10-fold-higher number of cells than were seen to produce gamma interferon by the ELISPOT assay. The implications of this CTL assay comparison and the possibility of phenotypic differences in HIV-specific CD8(+) T lymphocytes are discussed.

Cytokine profiles in seronegative volunteers immunized with a recombinant canarypox and gp120 prime-boost HIV-1 vaccine. NIAID AIDS Vaccine Evaluation Group.

OBJECTIVES: To study memory T cell proliferative responses and cytokine profiles induced in HIV-1 seronegative volunteers immunized with a live recombinant canarypox vector expressing HIV-1 antigens (ALVAC-HIV) and boosted with a recombinant gp120 subunit vaccine. DESIGN: HIV-specific T cell proliferative responses and cytokines were measured 2 weeks after vaccination. Cytokines secreted by T helper 1 cells (Th1) [interleukin (IL)-2 and interferon-gamma (IFN-gamma)] and T helper 2 (Th2) cells (IL-4, IL-5, IL-6, and IL-10) were assessed both at the mRNA and the protein level. METHODS: Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with HIV antigens. Subsequently, T cell proliferation was measured in a standard lymphoproliferation assay; secreted cytokines were measured using an enzyme-linked immunosorbent assay and upregulation of cytokine mRNA was measured using reverse transcriptase polymerase chain reaction. RESULTS: All individuals who had received ALVAC-HIV followed by the protein vaccine exhibited HIV-1-specific T cell proliferative responses. Moreover, the PBMC of all prime-boost vaccinated individuals produced detectable IFN-gamma and IL-10 in response to stimulation with HIV-1 envelope glycoprotein antigens; 83% also had detectable levels of IL-2 and IL-6, 71% had detectable levels of IL-4, and 86% had detectable levels of IL-5. CONCLUSIONS: These data indicate that this vaccination regimen was inducing both Th1- and Th2-type responses to HIV-1 envelope antigens. This prime-boost vaccination approach elicited T cell help for the generation of cytotoxic T lymphocyte responses as well as help for antibody production and so promises to generate a broad HIV-1-specific immune response.

Protective immunity to rotavirus shedding in the absence of interleukin-6: Th1 cells and immunoglobulin A develop normally.

We investigated whether interleukin-6 (IL-6) was required for the development of immunoglobulin A (IgA)- and T-helper 1 (Th1)-associated protective immune responses to rotavirus by using adult IL-6-deficient mice [BALB/c and (C57BL/6 x O1a)F(2) backgrounds]. Naive IL-6(-) mice had normal frequencies of IgA plasma cells in the gastrointestinal tract. Consistent with this, total levels of IgA in fecal extracts, saliva, and sera were unaltered. In specific response to oral infection with rhesus rotavirus, IL-6(-) and IL-6(+) mice exhibited efficient Th1-type gamma interferon responses in Peyer's patches with high levels of serum IgG2a and intestinal IgA. Although there was an increase in Th2-type IL-4 in CD4(+) T cells from IL-6(-) mice following restimulation with rotavirus antigen in the presence of irradiated antigen-presenting cells, unfractionated Peyer's patch cells failed to produce a significant increase in IL-4. Moreover, virus-specific IgG1 in serum was not significantly increased in IL-6(-) mice in comparison with IL-6(+) mice. Following oral inoculation with murine rotavirus, IL-6(-) and IL-6(+) mice mediated clearance of rotavirus and mounted a strong IgA response. When IL-6(-) and IL-6(+) mice [(C57BL/6 x O1a)F(2) background] were orally inoculated with rhesus rotavirus and later challenged with murine rotavirus, all of the mice maintained high levels of IgA in feces and were protected against reinfection. Thus, IL-6 failed to provide unique functions in the development of IgA-secreting B cells and in the establishment of Th1-associated protective immunity against rotavirus infection in adult mice.

[Not Available].

Oral delivery of vaccines results in these being taken up by specialised microfold epithelial cells covering Peyer's patches of the gastrointestinal tract, therefore stimulating regulatory T cells and surface IgA positive (sIgA+) B cells. T helper cells can be divided into 2 subsets, type 1 (T(H)1) and type 2 (T(H)2), according to their function and the cytokines they secrete. T(H)1 cytokines such as interleukin (IL)-2, interferon-gamma and tumour necrosis factor-beta (TNFbeta) elicit activation of T cells and macrophages, whereas T(H)2 cytokines such as IL-4, IL-5, IL-6 and IL-10 favour mucosal and parenteral B cell responses. Therefore, T(H)2-type T cells are of particular interest for mucosal responses, since they help in the differentiation of sIgA+ B cells into IgA-producing plasma cells. As a result, one can take advantage of the fact that different forms of antigen delivery systems generally influence the outcome of an immune response, and use those that best induce mucosal responses. An example of this would be orally administered live attenuated Salmonella versus the oral administration of cholera toxin. The first induces a dominant T(H)1-type response, and the second a T(H)2-type response. Thus these 2 delivery systems can be exploited in order to elicit the desired immune response depending on the protective response required. Alternatively, encapsulating antigens into polyglycolide microspheres or liposomes, or incorporating them into immune-stimulating complexes, has facilitated the delivery of antigens which otherwise do not result in an immune response when given orally. Much progress is being made in the construction of attenuated viral and bacterial vectors for the delivery of antigen to mucosal sites. For example, poliovirus has been used as a vector to deliver both rotavirus and HIV antigens. Bacterial vectors and attenuated mutant bacteria for use in vaccines have also been extensively researched. Examples of these include Salmonella typhi mutants, Vibrio cholerae, Shigella species, Helicobacter pylori and Campylobacter jejuni. In addition, new approaches are being developed to induce responses at mucosal surfaces such as the gastrointestinal, respiratory and genitourinary tracts. These include the use of adjuvants that stimulate mucosal responses such as cholera toxin and Escherichia coli heat labile toxin, as well as the coexpression of cytokine genes with antigenic proteins on live vectors to drive the immune response so that mucosal responses are favoured. Furthermore, nucleic acid vaccines and the potential use of transgenic plants are new technologies that are contributing to our ability to induce responses at mucosal surfaces.

Quantitation of antigen-specific immune responses in human immunodeficiency virus (HIV)-infected individuals by limiting dilution analysis.

The lymphocyte proliferative response to recall antigens is lost following HIV infection. We sought to devise a means by which the functional immune status of persons in the early stages of HIV infection could be monitored quantitatively. The response to tetanus toxoid was examined in 45 HIV-infected individuals and 11 controls using conventional lymphocyte proliferative assays concurrently with limiting dilution analysis utilizing the secretion of interleukin-2 as the measure of a response. Our data show that the limiting dilution analysis detects tetanus toxoid-reactive T cells in 80% of those tested, as compared to only 44% by proliferation. However, the frequency of tetanus-reactive T cells in HIV-infected individuals (median frequency = 1/59,156) is decreased five-fold as compared to seronegative controls (median frequency = 1/11,599). Longitudinal studies demonstrated a time-dependent decrease in the frequency of tetanus-specific T cell responses in the HIV-infected individuals. Thus, the limiting dilution analysis is a quantitative approach for detecting antigen-specific T cells in HIV-infected individuals, and may be used to monitor changes in T cell function in HIV infection.