CD127 expression, exhaustion status and antigen specific proliferation predict sustained virologic response to IFN in HCV/HIV co-infected individuals

Abstract

Hepatitis C virus (HCV) infection is a major cause of morbidity and mortality in the HIV co-infected population. Interferon-alpha (IFN-α) remains a major component of anti-HCV therapy despite its deleterious effects on the immune system. Furthermore, IFN-α was recently shown to diminish the size of the latent HIV reservoir. The objectives of this study were to monitor the impact of IFN-α on T cell phenotype and proliferation of HIV and HCV-specific T cells during IFN therapy, and to identify immune markers that can predict the response to IFN in HICV/HIV co-infected patients. We performed longitudinal analyses of T cell numbers, phenotype and function in co-infected patients undergoing IFN-α therapy with different outcomes including IFN-α non-responders (NR) (n = 9) and patients who achieved sustained virologic response (SVR) (n = 19). We examined the expression of activation (CD38, HLA-DR), functional (CD127) and exhaustion markers (PD1, Tim-3, CD160 and CD244) on total CD4 and CD8 T cells before, during and after therapy. In addition, we examined the HIV- and HCV-specific proliferative responses against HIV-p24 and HCV-NS3 proteins. Frequencies of CD127+ CD4 T cells were higher in SVR than in NR patients at baseline. An increase in CD127 expression on CD8 T cells was observed after IFN-α therapy in all patients. In addition, CD8 T cells from NR patients expressed a higher exhaustion status at baseline. Finally, SVR patients exhibited higher proliferative response against both HIV and HCV antigens at baseline. Altogether, SVR correlated with higher expression of CD127, lower T cell exhaustion status and better HIV and HCV proliferative responses at baseline. Such factors might be used as non-invasive methods to predict the success of IFN-based therapies in co-infected individuals.

Figure 1. Sustained virologic response to IFN-α therapy correlates with baseline expression of CD127 and Tim-3 on CD4 T cells.

Expression of the memory marker CD127 and the indicated activation or exhaustion molecules on CD4 T cells was monitored longitudinally in NR (n = 7) and SVR (n = 12) HCV/HIV co-infected patients before, during and after IFN-α therapy. (A) Representative flow cytometry plots for one patient from each group NR (top panel) and SVR (bottom panel) demonstrating expression of the different markers on total CD4 T cells (gated on CD8^negCD3⁺ lymphocytes). (B) Expression of the memory marker CD127; (C-G) CD4 activation/exhaustion was measured by the absence of CD127 and the expression of the activation/exhaustion markers HLA-DR, PD-1, CD160, CD244 and Tim-3. P-values were calculated using a two-tailed Mann Whitney U test to compare NR to SVR patients. Wilcoxon signed rank test was used to perform longitudinal statistical analysis. Open symbols represent patients of group A and closed symbols represent patients of group B. (* p<0.05).

Figure 2. Sustained virologic response to IFN-α therapy correlates with higher baseline and on treatment expression of CD127 while non-response correlates with increased CD8 T cell activation.

Resting and activated CD8 T cells were defined as CD127⁺CD38^neg and HLA-DR⁺CD38⁺ CD8⁺CD3⁺ lymphocytes, respectively, and monitored longitudinally in NR (n = 8) and SVR (n = 14) HCV/HIV co-infected patients before, during and after IFN-α therapy. (A) Representative flow cytometry plots for one patient from each group NR or SVR at baseline. (B) Longitudinal expression of CD127 on CD38^neg CD8 T cells. (C) Longitudinal activation of CD8 T cells measured as percent of HLA-DR⁺CD38⁺ CD8 T cells. P-values were calculated using a two-tailed Mann Whitney U test to compare NR to SVR patients. Wilcoxon signed rank test was used to perform longitudinal statistical analysis. Open symbols represent patients of group A and closed symbols represent patients of group B. (* p<0.05, *** p<0.001).

Figure 3. Non response to IFN-α therapy correlates with increased CD8 T cell exhaustion at baseline.

Expression of the indicated activation/exhaustion molecules on CD8 T cells was monitored longitudinally in NR (n = 8) and SVR (n = 10) HCV/HIV co-infected patients before, during and after HCV therapy. (A) Representative flow cytometry plots for one patient from each group NR (top panel) and SVR (bottom panel) demonstrating expression of the different markers on total CD8 T cells (gated on viable CD8⁺CD3⁺ lymphocytes). (B-E) CD8 activation/exhaustion was measured by the absence of CD127 and the expression of the activation/exhaustion markers HLA-DR, PD-1, CD160, CD244 and Tim-3. P-values were calculated using a two-tailed Mann Whitney U test. Open symbols represent patients of group A and closed symbols represent patients of group B. (* p<0.05, ** p<0.01, *** p<0.001).

Figure 4. Sustained virologic response to IFN-α therapy correlates with higher HIV- and HCV-specific proliferation of CD4 and CD8 T cells at baseline.

Proliferation of HIV- and HCV-specific CD4 and CD8 T cells in response to HIV P24 (A, B) and HCV NS3 (C, D) antigens was measured in NR (n = 9) and SVR (n = 11) HCV/HIV co-infected patients before, during and after IFN-α therapy. Briefly, patient PBMCs were labelled with CFSE and stimulated with 1 ug/ml of the indicated antigens for 6 days then stained as described in Materials and Methods. Proliferating antigen-specific T cells were identified by gating on viable CFSE^lowCD4⁺CD3⁺ (A, C) or CFSE^lowCD8⁺CD3⁺ lymphocytes (B, D). Stimulation Index (SI) was calculated using the following formula: % CFSE^low (antigen stimulated)/% CFSE^low (unstimulated). P-values were calculated using a two-tailed Mann Whitney U test to compare NR to SVR patients. Wilcoxon signed rank test was used to perform longitudinal statistical analysis. Open symbols represent patients of group A and closed symbols represent patients of group B. (* p<0.05, ** p<0.01, *** p<0.001).