CD127 expression and regulation are altered in the memory CD8 T cells of HIV-infected patients--reversal by highly active anti-retroviral therapy (HAART)

Abstract

HIV infection activates abnormally the immune system and the chronic phase is accompanied by marked alterations in the CD8 compartment. The expression of CD127 (IL-7R alpha chain) by memory CD8 T lymphocytes in HIV-infected patients is analysed and reported. The memory CD8 T cell subset was characterized by expression of CD45RA and CD27 markers, and CD127 cell surface expression was measured ex vivo by four-colour flow cytometry. HIV infection was associated with a fall in the proportion of CD127(+) cells among memory CD8 lymphocytes that resulted in a higher CD127(-) CD45RA(-)CD27(+) CD8 T cell count in HIV-infected patients. Diminished CD127 cell surface expression [mean fluorescence intensity (MFI)] by positive cells was also observed in this subset. The data suggest that these defects were reversed by highly active anti-retroviral therapy (HAART). The regulation of CD127 expression was also studied in vitro. Down-regulation of CD127 by interleukin (IL)-7 was observed in memory CD8 lymphocytes from healthy donors and HAART patients. Expression of CD127 by memory CD8 lymphocytes cultured in the absence of IL-7 confirmed that IL-7R regulation is altered in viraemic patients. Under the same experimental conditions, memory CD8 lymphocytes from HAART patients were shown to express CD127 at levels comparable to cells from healthy individuals. Altered CD127 cell surface expression and defective CD127 regulation in the memory CD8 T lymphocytes of HIV-infected patients are potential mechanisms by which these cells may be impeded in their physiological response to endogenous IL-7 stimulatory signals. Our data suggest that these defects are reversed during the immune reconstitution that follows HAART.

Fig. 1

Ex vivo expression of CD127 by memory CD8 lymphocyte taken from viraemic and highly active anti-retroviral therapy (HAART)-treated HIV-infected patients. Peripheral blood mononucear cells (PBMC) were incubated with antibody mixtures containing anti-CD8-APC (clone DK25, IgG1k, Dako Cytomation A/S, Glostrup, Denmark), anti-CD27-FITC (clone M-T27, IgG1k, Dako Cytomation S/A), anti-CD45RA Cy5 (clone HI 100, IgG2b, Pharmingen, San Diego, CA, USA) and anti-CD127-RPE (R34·34, IgG1, Immunotech, Marseille, France). The binding of anti-CD127 monoclonal antibodies (mAb) R34·34 to interleukin (IL)-7R alpha was not exposed to competition from IL-7 under our experimental conditions. After 30 min at 4°C, the cells were washed and fixed in phosphate-buffered saline (PBS) paraformaldehyde (1%). An isotype control was performed for each reagent. Staining analyses were performed on a fluorescence-activated cell sorter (FACSCalibur) flow cytometer running cellquest version 3·1 software (Becton Dickinson, Mountain View, CA, USA). Data were expressed as medians and interquartile ranges. Statistical analyses were performed by the Mann–Whitney U-test. The main comparisons were performed between healthy donors and viraemic patients, and between viraemic and HAART patients. (a) CD127 expression by CD8⁺ CD27⁺ lymphocytes. Naive cells (CD45RA⁺) and memory cells (CD45RA⁻). Results are shown for one representative individual in each group. CD45RA labelling delineated naive cells (CD45RA⁺) from memory CD8 lymphocytes (CD45RA⁻). (b) CD45RA and CD27 were used to delineate naive (CD45RA⁺ CD27⁺), memory (CD45RA⁻ CD27⁺), effector (CD45RA⁻ CD27⁻) and terminally differentiated effector lymphocytes (CD45RA⁺ CD27⁻). Proportion of CD127⁺ lymphocytes [mean fluorescence intensity (MFI) > 50] in the four CD8 lymphocyte subsets in healthy individuals, viraemic patients and HAART patients; n = 10 for each group. (c) Absolute counts per cubic millimetre for total CD127⁻ lymphocytes (MFI < 50) in naive, memory, effector and terminally differentiated CD8 lymphocytes from each study group. □, Healthy donors; ▪ viraemic patients; , HAART patients. *P < 0·05 when compared to healthy donors; **P < 0·05 when compared to untreated viraemic patients.

Fig. 2

Regulation of CD127 expression in memory CD8 lymphocytes taken from viraemic and highly active anti-retroviral therapy (HAART)-treated HIV-infected patients. Peripheral blood mononucear cells (PBMC), prepared as above, were cultured in the absence or presence of interleukin (IL)-7 (10 ng/ml). IL-7 was kindly provided by Cytheris SA (Vanves, France). Cultures (2 × 10⁵ cells/200 microlitre) were performed in 96-well U-bottomed microtitre plates. The medium consisted of RPMI-1640 supplemented with 0·5% human AB serum, 2 mM glutamine, 10 mM HEPES and 50 mM 2-mercaptoethanol. On the days indicated the cells were stained and analysed as described in the legend to Fig. 1. Data were expressed as median [mean fluorescence intensity (MFI)] of all cells in each CD8 lymphocyte subset. For the sake of clarity the figure does not include the interquartile ranges. Statistical analyses were performed by the Wilcoxon matched-pairs signed-ranks test. (a) CD127 down-regulation in the presence of IL-7 was monitored on day 3 and on day 6 was found to be significant when compared to CD127 expression on day 0, and this for all experimental conditions used (P< 0·05). Dotted lines represent the level of CD127 expression on day 0. (b) CD127 over-expression was measured on day 3 in the absence of IL-7. The P-values obtained between day 3 and day 0 are indicated. IL-7 plasma levels were measured in each group by enzyme-linked immunoassay (Quantikine HS kit, RD System, Paris, France): viraemic group [16·2 (13·6–17·2) pg/ml], HAART group [11·0 (8.9–12.8) pg/ml] and healthy donors [1·4 (0·9–2·1) pg/ml]. *P < 0·05 when compared to day 0.