Induction of HIV-1-specific T cell responses by administration of cytokines in late-stage patients receiving highly active anti-retroviral therapy

Abstract

Highly active anti-retroviral therapy (HAART) is associated with reduction in the morbidity and mortality of patients with advanced HIV-1 disease. The ability of such treatment to improve immune responses against HIV-1 and opportunistic pathogens is variable and limited. Addition of cytokine immunotherapy to this treatment may improve immune responses. IL-2 with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered to HIV-1+ individuals receiving HAART with undetectable viral loads, and CD4 counts < 100 cells/microl. In one patient presenting with Mycobacterium avium complex (MAC) infection, we evaluated the effect of cytokine immunotherapy on lymphocyte phenotype; plasma viral load; proliferative responses to mitogens, recall and HIV-1 antigens; cytokine production and message in response to non-specific and specific stimuli; and natural killer (NK) cell activity. Proliferation assays were performed in two similar patients. Before cytokine immunotherapy the predominant CD8+ population was mainly CD28-. No proliferation or IL-2 production was seen in response to mitogens, recall or HIV-1 antigens; and no HIV-1 peptide-specific interferon-gamma (IFN-gamma)-secreting cells were present. Low levels of IL-4 were detected in response to antigens to which patients had been exposed, associated with up-regulated expression of costimulatory molecules influenced by IL-4. Following IL-2 administration, loss of IL-4 was associated with increased NK cell activity and HIV-1 peptide-specific and non-specific IFN-gamma-producing cells. Proliferative responses associated with IL-2 production and responsiveness were only seen after subsequent concomitant administration of GM-CSF with IL-2. These changes mirrored clinical improvement. An imbalance of lymphocyte subsets may account for immune unresponsiveness when receiving HAART. Restoration of responses following immunotherapy suggests a shift towards a lymphocyte profile with anti-pathogen activity.

Fig. 3

IL-2 increases natural killer (NK) activity and induces non-specific and HIV-1 peptide-specific IFN-γ-secreting cells. (a) Measurement of the NK-mediated cytotoxicity by patient's NK cells before (•) and after (▪) IL-2 immunotherapy, and by uninfected controls (open symbols); s.d. of the mean for each effector cell dilution was < 5%. (b) ELISPOT assay measuring IFN-γ release in response to phytohaemagglutinin (PHA) by peripheral blood mononuclear cells (PBMC) obtained before and after IL-2 immunotherapy. The data represent average values at each point and variation among duplicates was < 10%. (c) ELISPOT assays measuring IFN-γ release in response to HLA-B35-restricted peptides. Patient cells obtained before and after IL-2 immunotherapy, and HLA-B35 uninfected donor cells were stimulated with or without HLA-B35-restricted peptides: (i) HIV-RT: HPDIVIYQY; (ii) HIV-p17: NSSKVSQNY; (iii) Flu-M: ASCMGLIY; (iv) EBV-EBNA3A: YPLHEQHGM. Patient 3 HLA phenotype: A1/24, B35/61, Bw6 C4/1202 DR4/7 DRB4-53 DQB0601; uninfected donor HLA phenotype: A11/33 B7/35 Bw6 C4/1202 DR1/1303 DRB3-52 DRB4-53 DQ5/7.

Fig. 1

Two-colour flow cytometric analysis of the expression of CD28 and CD57 molecules on the predominant CD8⁺ lymphocytes (> 90% CD8⁺) reveals accumulation of the CD8⁺CD28⁻CD57⁺ anergic subset in advanced HIV-1 disease. Expression was assessed by direct immunofluorescence, using the FITC-conjugated anti-CD28 and PE-conjugated anti-CD57 MoAbs. A representative result is shown.

Fig. 2

Absence of proliferative responses to recombinant HIV-1 antigens, recall antigens, mitogens and IL-2 in patients with advanced HIV-1 disease on highly active anti-retroviral therapy (HAART) (upper panel). Peripheral blood mononuclear cells (PBMC) were incubated with the indicated stimuli for 6 days and ³H-thymidine incorporation was measured. Proliferation of uninfected control cells is shown for comparison (results from a representative control are shown; lower panel).

Fig. 4

Gel electrophoresis of reverse transcriptase-polymerase chain reaction (RT-PCR) products of IL-2, IL-4 and IL-10 mRNAs obtained from patient peripheral blood mononuclear cells (PBMC) after the IL-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) immunotherapy. Lanes 1 and 17, DNA ladder; lane 2, positive control; lane 3, negative control; lane 4, day 0; lanes 5–8, day 1–4 stimulation with recombinant p24; lanes 9–12, day 1–4 stimulation with Candida antigen; lanes 13–16, day 1–4 stimulation with IL-2.