A Comprehensive Ex Vivo Functional Analysis of Human NKT Cells Reveals Production of MIP1-α and MIP1-β, a Lack of IL-17, and a Th1-Bias in Males

NKT cells contribute to the modulation of immune responses and are believed to be important in the pathogenesis of autoimmune and infectious diseases, as well as cancer. Variations in the composite NKT cytokine response may determine individual disease susceptibility or severity. Due to low frequencies in peripheral blood, knowledge of the breadth of ex vivo human NKT cell functions has been limited. To bridge this gap, we studied highly purified NKT cells from PBMC of healthy donors and assessed the production of 27 effector functions using sensitive Elispot and multiplex bead assays. We found the ex vivo human NKT cell response is predominantly comprised of the chemokines MIP1-α, and MIP1-β as well as the Th1 cytokines IFN-γ and TNF-α. Although lower in magnitude, there was also significant production of IL-2, IL-4, and perforin after mitogen stimulation. Surprisingly, little/no IL-5, IL-6, IL-10, or IL-13 was detected, and no subjects' NKT cells produced IL-17. Comparison of the NKT functional profiles between age-matched male and female subjects revealed similar IL-4 responses, but higher frequencies of cells producing IFN-γ and MIP1-α, from males. There were no gender differences in the circulating NKT subset distribution. These findings implicate chemokines as a major mechanism by which NKT cells control responses in humans. In addition, the panoply of Th2 and Th17 cytokine secretion by NKT cells from healthy donors may not be as pronounced as previously believed. NKT cells may therefore contribute to the gender bias found in many diseases

Stimulation of PBMC and Monocyte-derived-Macrophages via Toll-Like Receptor (TLRs) Activates Innate Immune Pathways in HIV-Infected Patients on Virally-Suppressive Combination Antiretroviral Therapy (cART)

In HIV-infected cART-treated patients, immune activation and microbial translocation persist and associate with inadequate CD4 recovery and morbidity/mortality. We analyzed whether alterations in the TLR pathway could be responsible for the immune hyper-activation seen in these patients.PBMC/MDM of 28 HIV+ untreated and 35 cART treated patients with HIV-RNA<40cp/mL (20 Full Responders: CD4≥350; 15 Immunological Non Responders:CD4<350) as well as of 16 healthy controls were stimulated with a panel of TLR agonists. We measured: CD4/CD8/CD14/CD38/HLA-DR/Ki67/AnnexV/CD69/TLR4/8 (Flow Cytometry); PBMC expression of 84 TLR pathway genes (qPCR); PBMC/MDM cytokine release (Multiplex); plasma LPS/sCD14 (LAL/ELISA). PBMC/MDM from cART patients responded weakly to LPS stimulation but released high amounts of pro-inflammatory cytokines. MDM from these patients were characterized by a reduced expression of HLA-DR+MDM and failed to expand activated HLA-DR+CD38+ T-lymphocytes. PBMC/MDM from cART patients responded more robustly to ssRNA stimulation; this resulted in a significant expansion of activated CD38+CD8 and the release of amounts of pro-inflammatory cytokines comparable to those seen in untreated viremic patients. Despite greater constitutive TLR pathway gene expression, PBMC from Immunological Non Responders seemed to up-regulate only type I IFN genes following TLR stimulation, whereas PBMC from Full Responders showed a broader response. Systemic exposure to microbial antigens drives immune activation during cART by triggering TLRs. Bacterial stimulation modifies MDM function/pro-inflammatory profile in cART patients without affecting T-lymphocytes; this suggests translocating bacteria as selective stimulus to chronic innate activation during cART. High constitutive TLR activation is seen in patients lacking CD4 recovery, suggesting an exhausted immune milieu, anergic to further antigen encounters

Similar distribution of NKT cell subsets between sexes.

<p>Eight-color flow cytometry was performed on PBMC samples from the same blood draw as the sorted NKT cell populations to compare (A) the NKT total cell frequencies as a per cent of lymphocytes and (B) the CD4+ CD8+ (Double Positive, or DP), CD4+CD8− cells (CD4 SP), CD4− CD8+ (CD8 SP), CD69+; CD161+, and CD56+ NKT cell subsets between male (white boxes) and female (grey boxes) subject groups. No significant differences were found. Data is shown as box and whisker plots, with the center line noting the median, the boxes noting the 25–75 percentile, and the lines representing the range for each group.</p

Donor information with sorting purity.

<p>Subjects are listed in descending NKT cell frequency. Sort purities are expressed as percent of CD3+ T cells.</p

Th1 bias of NKT cells from male donors.

<p>The ex vivo effector functions of NKT cells from male and female subject groups were compared. (A) The gender differences (n = 6/group) in the number of cytokine producing cells detected via elispot; statistically significant differences between groups are noted. White and grey boxes denote the male and female groups, respectively. Data is shown as box and whisker plots, with the center line noting the median, the boxes noting the 25–75 percentile, and the lines representing the range for each group. The ratio of Th1 cytokines (B) and non-Th1 cytokines (C) to IL-4 from pure NKT cell populations measured by the luminex assay. In (B) and (C), each circle represents one donor, with the line noting the mean for each group.</p

Total frequencies of NKT cells exerting different effector functions <i>ex vivo</i> from healthy donors.

<p>The data is expressed as box and whisker plots, with the median for all subjects shown as the center line, the box representing the 25–75 percentile, and the lines showing the range of the data. Statistically significant differences between unstimulated (white boxes) and stimulated (grey boxes) wells (p<0.05) are noted with an asterisk. Eight subjects were included in this analysis.</p

Measurement of the <i>ex vivo</i> effector fuctions of purified NKT cells by elispot assays.

<p>Determination of the frequencies of NKT cells exerting 14 different effector functions via elispot assays. (A) Representative images of elispot wells with either sorted NKT cells with no stimulation, sorted NKT cells with PMA and Ionomycin, or total PBMC with PMA and Ionomycin (positive controls). (B) NKT cell dilution results from two donors. Each symbol represents one effector function.</p