Transcription factors and MAPK signaling in CD56 subset.

<p>EOMES and T-bet control cytotoxic function of effector T cells. (A) Majority of CD56+ CD8 T cells expressed T-bet and/or EOMES and were double positive for these transcription factors, whereas a significant proportion of CD56- CD8 T cells were double negative for EOMES and T-bet. The representative plots show the trends presend in CD56+ or CD56- subsets from all patient groups irrespective of disease status (B) NVS group had highest frequency of T-bet expressing CD8 T cells whereas there was no significant difference in EOMES positive CD8 T cells among different cohorts of HIV infected patients. (C) Overall, the CD56+ fraction of CD8 T lymphocytes had higher levels of Erk phosphoryation upon PMA+inomycin stimulation compared with the CD56- CD8 T lymphocytes when samples from all groups were combined for analysis. CD56+ or CD56- CD8 T lymphocytes showed similar levels of Erk phosphorylation in all infected and control groups.</p

IL-15 restores CD56 on CD8 T cells from HIV infected patients.

<p>(A) Most CD8 T cells and almost all CD56 + CD8 T cells express the common gamma chain (γ_c) (or CD132). Representative plots (from left ND, PD, NVS, VIR) show there was no significant downregulation of this receptor on CD8 T lymphocytes from any infected group (B) Culture of PMBC in IL-15 resulted in a significant upregulation of CD56 on CD8 T lymphocytes at day 12 relative to start of culture in infected and uninfected samples.</p

Preserved CD56+ CD8 T lymphocytes in elite controllers.

<p>(A, B) Higher expression of CD56 on CD8dim T compared with CD8bright T lymphocytes. (C) Representative samples shows CD56+ CD8 T lymphocytes are mostly effector memory type whereas (D) CD56- CD8 T lymphocytes have a significant subset of naïve and central memory and type cells. (E) Both CD56+ and CD56- subset of CD8 T cells have similar frequency of HIV tetramer binding cells. Cells from an HLAB5701 HIV infected individual were stained with TW10 tetramer. Cells are gated on CD8+ CD3+ lymphocytes. (F) Significantly elevated CD8 T cell frequencies in both ART treated and low viremia (VIR) groups but not in elite controllers (NVS). (G) The frequency of CD56+ CD8+ T lymphocytes is significantly reduced in HIV+ ART treated group compared with uninfected controls. NVS (elite controllers) did not show this defect and have normal levels of CD56 expression on CD8 T cells. (H) Analysis of CD8bri (H) or CD8dim (I) populations of T cells show that NVS group has superior levels of CD56 subset among both populations whereas ART group has lowest levels of CD8bri cells expressing CD56. VIR group has high frequency of CD56 expressing CD8bri but not CD8dim cells when compared with ART patients. For F-I, Kruksal-Wallis test followed by Dunn's test for multiple correction was used to analyze the four groups.</p

Demographic and Clinical Characteristics of Patients and Control groups.

a<p>HIV uninfected samples are blood from commercial source</p>b<p>Data are for patients who received antiretrovirals, patients with natural virus suppression (NVS) and chronic low viremia (VIR) and are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088884#pone.0088884-Hardy1" target="_blank">[50]</a>.</p

Exhausted CD56+ CD8 T lymphocytes in treated HIV infection.

<p>The expression of Tim-3 on T cells identified exhausted T lymphocytes. (A) A higher proportion of CD56+ CD8 T cells express Tim-3 compared to the CD56- subset. (B) Cumulative data from all individuals irrespective of disease status shows significantly higher Tim-3 expression by CD56+ CD8 T cells (C) The proportion of CD56+CD8+ T cells that are exhausted as judged by their Tim-3 expression were significantly higher in HIV+ART but not in elite controllers when compared to uninfected group. (D) The levels of Tim-3 on CD56- CD8 T cells is comparable in all HIV- and HIV+ groups.</p

Elite controllers have higher levels of perforin/GranzymeB+CD56+ CD8 T lymphocytes.

<p>Intracellular expression of preformed perforin (A) and Granzyme B (B) is significantly higher on gated CD8 T lymphocytes from elite controllers and low viremia groups compared to uninfected controls or HIV+ART subjects. The level of CD56 expression on perforin (C) or Granzyme B (D) expressing CD8 T lymphocytes was significantly reduced in HIV+ ART treated and low viremia groups but not in elite controllers. Results in panels E-G show perforin upregulation in response to stimulation with HIV gag peptides measured with DG48 clone of anti-perforin antibody. (E) Both elite controller and low viremia group have significantly higher production of perforin upregulation in response to stimulation compared with ART treated individuals. (F). Representative sample from each infected group showing majority of CD56+ CD8 T cells upregulated perforin in response to stimulation while a minority of CD56- CD8 T cells upregulate perforin. Cells are gated on CD8 T lymphocytes. (G) Combined results from all infected groups show proportion of CD56 fraction of CD8 T cells upregulating perforin in response to stimulation with HIV peptides is significantly higher than proportion of CD56- CD8 T cell that upregulate perforin. Significantly greater proportions of CD56+ CD8 T cells degranulate (H) as well as produce IFNγ (I) in response to stimulation with HIV gag peptides. Results in G, H and I show show comparison of CD56+ and CD56- subsets irrespective of the patient's disease status.</p

Association of SNPs rs1801274 (FCGR2A) and rs396991 (FCGR3A) with HIV status.

<p>Significant differences related to HIV status are shown. For FCGR3A, carriage of V allele was associated with increased risk of HIV seropositivity. Among HIV sero-positive cases, there was a significant association between VV genotype (i.e., lack of allele F) and the progressor phenotype. For the FCGR2A∶FCGR3A extended haplotype, the RR∶FF genotype was associated with disease progression.</p><p>Fisher's exact test (2-tailed) p values and Odds ratios are listed where significant.</p><p>*None of the NVS donors carried the F allele, so odds ratio were not calculated.</p><p>#Include Progressors and NVS.</p><p>**Not significant.</p

Study populations demographic.

<p>Abbreviations: AA, African Americans; NVS, Natural Virus Suppressors; M, Male; F, Female; HAART, Highly Active Antiretroviral Therapy.</p><p>The populations were mostly African Americans (AA), except for a small percentage of progressors and controls, which was composed of Caucasians and Asians. HIV progressors contained a higher proportion of males and this reflects our HIV clinic populations.</p

Genotypic and allelic frequencies of SNP rs1801274 (FCGR2A) and rs396991 (FCGR3A) in HIV negative controls, NVS and HIV Progressors.

<p>Abbreviations: SNP, Single Nucleotide Polymorphism; NVS, Natural Virus Suppressors.</p><p>FCGR genotypic variants (FCGR2A H131R and FCGR3A F158V) were studied in HIV progressors, NVS as well as HIV negative controls. Genotypic and allelic frequencies of rs396991 and rs1801274 are shown. The genotypic frequencies of the HIV sero-negative patients are in Hardy-Weinberg equillibrium.</p

Validation of differentially expressed genes identified by DNA Microarray analysis.

<p>Expression of genes related and unrelated to B cells are down regulated with Rituximab (RTX) treatment. As shown in Fig A, expression of B cell related genes CD19, CD27, CD24, and CD20 were significantly lower after RTX treatment than at baseline (p<0.0001). Fig 2B shows expression of non-B cell genes CD182, CCR2, and TWEAKR were all down regulated after RTX treatment than at baseline (p<0.0001). Fig 2C shows expression of IFIGs including total IFIG (p<0.0001), IFIT3 (p = 0.0003), IFI44 (p<0.0001), IFIT1 (p<0.0001) that were down-regulated after RTX treatment.</p