To the Editor:
Current guidelines recommend initiation of antiretroviral therapy (ART) when a HIV patient has <200 CD4 T cells/μL. This is supported by many studies. For example, the probability of developing AIDS or dying while receiving ART decreases incrementally in patients with nadir CD4 T-cell counts of <50, 50-99, 100-199, 200-349 and >350 cells/μL.1 Moreover, patients in these categories achieved progressively higher CD4 T-cell counts on ART. Intuitively, it seems likely that poor prognosis of patients who survived a protracted period with AIDS reflects a higher burden of opportunistic pathogens and immunological defects that persist despite a virological response to ART. For example, the resolution of CD4 T-cell activation and restoration of lymphoproliferative responses to tetanus toxoid were less effective in patients with baseline CD4 T-cell counts below 160 cells/μL, when compared with patients treated at 160-350 CD4 T cells/μL.2 Similarly, nadir CD4 T-cell counts below 200 cells/μL correlated with poor responses to Candida and HIV p24.3 We described persistently poor lymphoproliferation and interferon-γ (IFN-γ) responses to antigens from Candida or cytomegalovirus (CMV) in patients who began ART with <100 CD4 T cells/μL.4 However, no studies have investigated the effects of “lower” nadir CD4 T-cell counts on immune function in patients who have a stable virological response to treatment. We address this retrospectively in patients with nadir CD4 T-cell counts of 0-60 cells/μL. Patients had a stable virological response to therapy when sampled 1-9 years later.
Briefly, adult HIV-infected patients receiving ART (triple therapy including a protease inhibitor or a nonnucleoside reverse transcriptase inhibitor) were selected from the HIV database of the Department of Clinical Immunology and Immunogenetics, Royal Perth Hospital, on the basis of nadir CD4 T-cell counts below 60 cells/μL, an increase of greater than 4-fold or to >200 CD4 T cells/μL, and a sustained virological response to treatment (<50 copies/mL HIV RNA in plasma over at least 6 months). Nadir CD4 T-cell counts were defined as the lowest values in our database. Our program was approved by the ethics committee of Royal Perth Hospital.
To assess IFN-γ responses, peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradients and cryopreserved in liquid nitrogen. Nitrocellulose-backed microtiter plates (Millipore, Bedford, MA) were coated with anti-IFNγ (15 μg/mL; Mabtech, Stockholm, Sweden) in 0.1 M bicarbonate buffer (pH 9.5). PBMC and CMV crude antigen (strain AD169), anti-CD3 (Mabtech; 10 ng/mL), Candida soluble antigen,4 or K562 cells(target:effector ratio = 1:10) was added for 18-20 hours (37°C, 5% CO2). Spots were detected with biotinylated anti-IFNγ antibody (2 hours), streptavidin-horseradish peroxidase conjugate (Genzyme, Cambridge, MA; 1 hour), and TMB substrate. Assays of PBMCs depleted of CD4 and CD8 T cells using magnetic beads (Miltenyi Biotech, North Ryde, NSW, Australia) showed that only CD4 T cells produce IFN-γ after CMV stimulation whereas both CD4 and CD8 T cells respond to Candida antigen and anti-CD3 (data not shown).
IFN-γ responses were related to nadir CD4 T-cell counts using linear mixed models in SPlus 7.0 (Insightful Corp, Seattle, WA) to accommodate repeated measurements on some individuals and using square root of ELISpot counts (per 200,000 cells) to better approximate normality and constant variance. P <0.05 was accepted as a significant difference.
PBMCs had been archived on up to 3 occasions from 39 patients who had begun treatment with ≤60 CD4 T cells/μL and achieved a sustained virological response. Three batches of samples from 14 to 25 patients had been selected from this archive for previous studies,5-7 respectively. The median (range) durations of treatment in the 3 studies were 69 (12-83), 103 (38-111) and 82 (40-107) months. CD4 T-cell counts were 384 (120-786), 601 (208-1152) and 598 (209-1225) cells/μL at the time of study.
There were significant differences in the intercepts of the 3 lines relating CD4 T-cell IFNγ responses to CMV to nadir CD4 T-cell count (P < 0.001). This may reflect use of separate batches of CMV antigen. After adjusting for the differences in intercept, the slopes of the lines were similar (P = 0.9). The common slope was significantly positive (P = 0.04), so responses to CMV correlated directly with nadir CD4 T-cell counts even though all the patients had experienced extreme immunodeficiency (Fig. 1A). This was not seen with responses to Candida (P = 0.3, Fig. 1B) or anti-CD3 (P = 0.4, Fig. 1C). These agents stimulate CD4 and CD8 T cells. However, there was a significant "inverse" relationship between the IFNγ responses to the natural killer (NK) cell target (K562 cells) and nadir CD4 T-cell counts (P = 0.03, Fig. 1D). The rates of change in NK responses were similar in the 2 studies (P = 0.7), but the levels for study 1 were higher (P = 0.01).
The results associate low nadir CD4 T-cell counts with low IFNγ responses of CD4 T cells stimulated with CMV antigen and high NK IFNγ responses, within a cohort of patients who all began therapy with ≤60 CD4 T-cell/μL. This is the first study ever to dissect the responses among patients with nadir CD4 T-cell counts in this range. The findings are reproducible when assessed in separate studies with different batches of CMV antigen.
As the first group of HIV patients to receive ART become older, it is important to capture data defining the recovery of their immunological capacity under “optimal” circumstances. Here we describe patients treated for up to 9 years with stable virological responses. Many had experienced monotherapy, and protracted episodes of immunodeficiency before triple therapy became available in 1996-1997. Hence, the cohort was effectively selected by survival pre-ART and response to ART at the time of study. These factors may favor patients with a genetic profile that impacts upon the immune responses measured. For example, the association between extreme immunodeficiency before ART and persistently high NK IFNγ responses (Fig. 1D) may correlate with evidence from the same clinic population that patients who experienced CMV as an AIDS-defining illness had high numbers of activating KIR genes.8 These findings suggest a genetic capacity to exploit an adaptive mechanism that has a lifelong footprint. Longitudinal studies are warranted as samples become available.
This is publication 2007-51 (Clinical Immunology & Immunogenetics, Royal Perth Hospital).
Patricia Price, PhD*†
Sonia Fernandez, PhD†
Dino B. A. Tan, BSc(Hons)†
Ian R. James, PhD‡
Niamh M. Keane, PhD†‡
Martyn A. French, MD*†
*Clinical Immunology and Immunogenetics
Royal Perth Hospital
†School of Pathology and Laboratory Medicine
University of Western Australia
‡Centre for Clinical Immunology and Biomedical Statistics
Murdoch University and Royal Perth Hospital
Western Australia, Australia
Supported by the National Health and Medical Research Council of Australia.
1. Battegay M, Nuesch R, Hirschel B, et al. Immunological recovery and antiretroviral therapy in HIV-1 infection. Lancet Infect Dis
2. Lange CG, Valdez H, Medvik K, et al. CD4+
T-lymphocyte nadir and the effect of highly active antiretroviral therapy on phenotypic and functional immune restoration in HIV-1 infection. Clin Immunol
3. Siddique MA, Hartman KE, Dragileva E, et al. Low CD4+
T cell nadir is an independent predictor of lower HIV-specific immune responses in chronically HIV-1-infected subjects receiving highly active antiretroviral therapy. J Infect Dis
4. Burgess K, Price P, James IR, et al. Interferon-gamma responses to Candida recover slowly or remain low in immunodeficient HIV patients responding to ART. J Clin Immunol
5. Fernandez S, Price P, McKinnon EJ, et al. Low CD4+
T-cell counts in HIV patients receiving effective antiretroviral therapy are associated with CD4+
T-cell activation and senescence but not with lower effector memory T-cell function. Clin Immunol
6. Fernandez S, Stone S, Price P, et al. Effector CD4+
T-cell responses in HIV patients responding to ART may be limited by low proportions of plasmacytoid dendritic cells rather than poor production of accessory cytokines. Clin Immunol
. 2008; [Epub ahead of print].
7. Price P, Keane NM, Lee S, et al. A T2 cytokine environment may not limit T1 responses in human immunodeficiency virus patients with a favourable response to antiretroviral therapy. Immunology
8. Price P, Witt C, Santis DD, et al. Killer immunoglobulin-like receptor genotype may distinguish immunodeficient HIV-infected patients resistant to immune restoration diseases associated with herpes virus infections. J Acquir Immune Defic Syndr