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.

A Balanced Type 1/Type 2 Response Is Associated with Long-Term Nonprogressive Human Immunodeficiency Virus Type 1 Infection

“…PBMC were isolated by density gradient centrifugation and cultured in supplemented RPMI 1640 medium (22,23). All cell lines were cultured in 10% fetal calf serum-RPMI in 25-cm 2 flasks (Greiner Laboritechnik, Dursley, United Kingdom) and incubated at 37°C in 5% CO 2 .…”

“…. The HIV-1 recombinant antigens used were described previously (22). Cells were cultured with antigen or peptide at a final concentration of 10 g/ml.…”

“…PBMC (10 5 /well) were cultured with antigen, mitogen, or cytokine in round-bottomed microtiter plates (Greiner) in 10% AB plasma (200 l; Sigma) in RPMI. The antigens, mitogens, and cytokines used were described previously (22 (22,23). Results are expressed as the stimulation index and as the mean counts per minute (cpm) for triplicate cultures, with an error of the mean of Ͻ15%.…”

“…For cytokine assays, 50 l of supernatants from proliferative and bulk cultures was transferred to 96-well round-bottomed plates in triplicate for measurement of IL-2, IL-4, and IL-10 on the indicator cell lines CTLL-2 (European Collection of Animal Cell Cultures, Salisbury, United Kingdom), CT.h4S (a generous gift of W. Paul, Bethesda, Md. ), and D36 (Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany) as described previously (22,23,43). Briefly, CTLL-2 (10 3 cells/well), CT.h4S (5 ϫ 10 3 cells/well), or D36 (2 ϫ 10 3 cells/well in the presence of 16 U of mouse recombinant IL-4 per ml; R&D Sytems, Abingdon, United Kingdom) was added in 50 l to 50 l of supernatant to give a final volume of 100 l. After 24 h in culture, wells were pulsed with [methyl-3 H]thymidine, and cells were harvested as described above.…”

Uncoupled Anti-HIV and Immune-Enhancing Effects when Combining IFN-α and IL-7

“…However, the proinflammatory activities of TNF-␣ render it a poor candidate for therapies. In an in vivo study, HAART with IL-2 and GM-CSF reduced HIV replication to a greater extent than HAART and IL-2 (16). Clinical improvement from immunotherapy with IL-2 and GM-CSF, however, was reported in only one of three patients.…”