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. 2016 Feb 15;196(4):1768-79.
doi: 10.4049/jimmunol.1501588. Epub 2016 Jan 13.

HIV-1 Env Glycoprotein Phenotype along with Immune Activation Determines CD4 T Cell Loss in HIV Patients

Anjali Joshi  1 , Melina Sedano  1 , Bethany Beauchamp  2 , Erin B Punke  1 , Zuber D Mulla  3 , Armando Meza  4 , Ogechika K Alozie  4 , Debabrata Mukherjee  4 , Himanshu Garg  5
Affiliations

HIV-1 Env Glycoprotein Phenotype along with Immune Activation Determines CD4 T Cell Loss in HIV Patients

Anjali Joshi et al. J Immunol. .

Abstract

The mechanism behind the selective depletion of CD4(+) cells in HIV infections remains undetermined. Although HIV selectively infects CD4(+) cells, the relatively few infected cells in vivo cannot account for the extent of CD4(+) T cell depletion, suggesting indirect or bystander mechanisms. The role of virus replication, Env glycoprotein phenotype, and immune activation (IA) in this bystander phenomenon remains controversial. Using samples derived from HIV-infected patients, we demonstrate that, although IA in both CD4(+) and CD8(+) subsets correlates with CD4 decline, apoptosis in CD4(+) and not CD8(+) cells is associated with disease progression. Because HIV-1 Env glycoprotein has been implicated in bystander apoptosis, we cloned full-length Envs from plasma of viremic patients and tested their apoptosis-inducing potential (AIP). Interestingly, AIP of HIV-1 Env glycoproteins were found to correlate inversely with CD4:CD8 ratios, suggesting a role of Env phenotype in disease progression. In vitro mitogenic stimulation of PBMCs resulted in upregulation of IA markers but failed to alter the CD4:CD8 ratio. However, coculture of normal PBMCs with Env-expressing cells resulted in selective CD4 loss that was significantly enhanced by IA. Our study demonstrates that AIP of HIV-1 Env and IA collectively determine CD4 loss in HIV infection.

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Figures

Figure 1
Figure 1. Immune activation in CD8+ T cells correlates better with CD4+ T cell decline than CD4 immune activation
PBMCs purified from HIV+ patients or healthy controls were stained for CD3, CD4, CD8, CD38 and HLADR expression followed by flow cytometry analysis. CD4:CD8 ratios were correlated with (A) CD4+ counts (B) immune activation in CD8+ cells (C) immune activation in CD4+ cells in HIV-infected patients. Comparison of (D) CD4+CD38+HLADR+ and (E) CD8+CD38+HLADR+ T cells in HIV-infected versus healthy controls. (F) The percentage of activated CD38+HLADR+ cells is significantly higher (p<0.01) in the CD8+ versus the CD4+ T cell population in HIV-infected individuals. Comparison of (G) CD4+CD38+ (H) CD4+HLADR+ (I) CD8+CD38+ and (J) CD8+HLADR+ cells in HIV-infected versus heathy controls.
Figure 2
Figure 2. Immune activation correlates with plasma viremia
Correlation analysis between log viremia and (A) CD4+CD38+HLADR+ and (B) CD8+CD38+HLADR+ cells in HIV-infected patients. (C) Comparison of viral load in patients on HAART versus no therapy. Comparison of percent (D) CD4+CD38+HLADR+ and (E) CD8+CD38+HLADR+ cells in normal healthy controls, HIV-infected non-viremic and HIV-infected viremic patients. (F) Comparison of CD4:CD8 ratios in normal healthy controls, HIV-infected non-viremic and HIV-infected viremic patients. (G) CD4:CD8 ratios in patients on HAART versus no therapy.
Figure 3
Figure 3. CD4+ but not CD8+ T cell apoptosis correlates with CD4 decline
PBMCs purified from HIV+ patients or healthy controls were stained for various cell surface markers along with CaspACE FITC-VAD-FMK. Correlation analysis of CD4:CD8 ratios with (A) CD4+ apoptosis (B) CD8+ apoptosis in PBMCs derived from HIV-infected individuals. Analysis of (C) percent CD4+ apoptosis (D) percent CD8+ apoptosis in HIV-infected versus normal healthy controls. Comparison of (E) percent CD4+ apoptosis and (F) percent CD8+ apoptosis in normal uninfected controls, HIV-infected non-viremic and HIV-infected viremic patients. Correlation analysis of (G) CD4+ activation versus CD4 apoptosis and (H) CD8+ activation versus CD8 apoptosis in HIV infected viremic patients.
Figure 4
Figure 4. Apoptosis inducing potential of Primary Envs correlates with CD4 decline
(A) HeLa cells transfected with the cloned primary Env constructs were co-cultured with SupT-R5-H6 cells. 24 hours post co-culture, apoptosis was determined using the caspase glo 3/7 assay. YU-2 Env was used as a positive controls and pcDNA 3.1+ empty vector as the negative control. Percent apoptosis was determined after normalizing data to YU-2 control as 100% and pcDNA 3.1 as 0%. (B) Infectivity of the cloned primary Env constructs. Virus stocks were prepared by co-transfecting 293T cells with the pNL-Luc backbone along with the indicated Env constructs. Equal RT cpm of culture supernatants were used to infect TZM-bl cells in the presence of AMD-3100 (4μM) or Maraviroc (2μM) and luciferase activity determined. Infectivity is normalized to YU-2 Env as 100%. (C) Correlation of apoptosis inducing potential of the primary Env constructs with percent infectivity. (D) The co-culture assay described in part A above was conducted with selected primary Envs in the presence of media, AMD-3100 (4μM), Maraviroc (2μM) or T-20 (8μM) added at the time of co-culture. (E) YU-2 or Lai Env expressing HeLa cells were co-cultured with SupT-R5-H6 cells in the presence of indicated inhibitors and apoptosis was determined using the caspase glo 3/7 assay. (F) Phylogenetic analysis of the primary HIV Env clones used in the study. A phylogenetic tree showing Env relatedness was derived using the DNA Star software.
Figure 5
Figure 5. CD4 loss in vivo is a combination of Apoptosis Inducing Potential (AIP) of Envs and immune activation
(A) Correlation analysis of AIP of Envs with CD4:CD8 ratios shows an inverse correlation between the two phenomenon. (B) Correlation of CD4:CD8 ratios with CD4+ immune activation. (C) Correlation analysis between plasma viremia and CD4:CD8 ratios. (D) Correlation analysis between patient plasma viremia and CD4+ immune activation.
Figure 6
Figure 6. Immune activation per se does not alter the CD4:CD8 ratios
Lymphocytes obtained from HIV negative healthy donors were cultured in RPMI-1640 medium supplemented with 20% FBS and phytoheamagglutinin at 2.5 μg/ml and IL-2 at 10U/ml for 48h. Cells were then stained for immune activation markers as described in Figure 1. Increase in percentage of CD38+HLADR+ cells in the (A) CD4+ (B) CD8+ population after 48h of activation. (C) No significant alteration in the CD4:CD8 ratio after 48h of immune activation in vitro.
Figure 7
Figure 7. Immune activation enhances Env-mediated bystander apoptosis in PBMCs
Unstimulated PBMCs from heathy donors were co-cultured with HeLa cells expressing either (A) Lai Env or (B) YU-2 Env along with a vector control. Apoptosis was determined 24h later via caspase 3 activity measurement (*=p<0.05). (C) Fold increase in caspase 3 activity in resting versus PHA+IL-2 activated PBMCs when co-cultured with Lai Env expressing HeLa cells. (D) PBMCs derived from healthy donors NR010, NR015 and NR016 were either left unstimulated or activated with PHA and IL-2. These PBMCs were then co-cultured with HeLa cells expressing Lai Env, YU-2 Env or vector control followed by measurement of caspase 3 activity in cultures after 24 hrs. (E) Experiment in part D was repeated to determine the CD4:CD8 ratios by flow cytometry after 24 hrs of co-culture. The CD4:CD8 ratio was determined after normalizing to control pCDNA3.1 transfected cells. (F) PBMCs derived from healthy donors NR004, NR006 and NR016 were activated and then co-cultured with Lai Env, YU-2 Env or pCDNA3.1 control vector expressing HeLa cells. In some cultures, T-20 was added at the time of co-culture. CD4:CD8 ratios were determined after staining for surface CD4 and CD8 expression. (G) Percent CD4 apoptosis was determined via surface CD4 and CaspACE FITC-VAD-FMK staining followed by flow cytometry.
Figure 8
Figure 8
Model proposing how virus replication, immune activation and Env-mediated apoptosis would result in progressive loss of CD4+ cells during HIV disease progression leading to AIDS.
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References

    1. d’Ettorre G, Paiardini M, Ceccarelli G, Silvestri G, Vullo V. HIV-associated immune activation: from bench to bedside. AIDS Res Hum Retroviruses. 2011;27:355–364. - PubMed
    1. Buzon MJ, Massanella M, Llibre JM, Esteve A, Dahl V, Puertas MC, Gatell JM, Domingo P, Paredes R, Sharkey M, Palmer S, Stevenson M, Clotet B, Blanco J, Martinez-Picado J. HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nat Med. 2010;16:460–465. - PubMed
    1. Li G, Cheng M, Nunoya J, Cheng L, Guo H, Yu H, Liu YJ, Su L, Zhang L. Plasmacytoid dendritic cells suppress HIV-1 replication but contribute to HIV-1 induced immunopathogenesis in humanized mice. PLoS pathogens. 2014;10:e1004291. - PMC - PubMed
    1. Douek D. HIV disease progression: immune activation, microbes, and a leaky gut. Topics in HIV medicine : a publication of the International AIDS Society, USA. 2007;15:114–117. - PubMed
    1. Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, Kazzaz Z, Bornstein E, Lambotte O, Altmann D, Blazar BR, Rodriguez B, Teixeira-Johnson L, Landay A, Martin JN, Hecht FM, Picker LJ, Lederman MM, Deeks SG, Douek DC. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med. 2006;12:1365–1371. - PubMed

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