Frequency of gC1qR+CD4+ T cells increases during acute hepatitis C virus infection and remains elevated in patients with chronic infection

Abstract

CD4+ T cell responses are impaired in chronic HCV infection. To determine factor(s) involved in CD4+ T cell dysregulation, we examined the effect of extracellular core on the alteration of CD4+ T cell responses and the cell surface level of core-binding protein, gC1qR on CD4+ T cells from acute HCV patients with resolved and chronic infection. During the acute phase of infection, the frequency of gC1qR+CD4+ T cells increased in both resolved and chronic HCV infection compared to healthy controls. Notably, 6 months later, the frequency of gC1qR+CD4+ T cells maintained elevated in chronic patients compared to that in resolved patients. In addition, TCR stimulation increased the frequency of gC1qR+CD4+ T cells, resulting in core-induced inhibition of T cell responses in both resolved and chronic patients. These results suggest that HCV infection expands gC1qR+CD4+ T cells, which increase the susceptibility to core-mediated immune dysregulation and facilitate the establishment of HCV persistency.

Figure 1. Extracellular core inhibits proliferation of purified CD4⁺ T cells

A) PBMC and B) purified CD4⁺ T cells from representative healthy donors were stimulated with anti-CD3/CD28 antibodies in the presence or absence of core or control protein β-gal for 5 days. Proliferation was determined by incorporation of [³H]-thymidine after 18 hours of culture. * indicates p-value significance is p<0.05 C) Percent inhibition of proliferation of CD4⁺ T cells from 5 healthy donors. Mann-Whitney analysis. P-value significance is p<0.05. D) PBMC cell death was determined by staining cells with 7AAD after 4 days of culture with anti-CD3/CD28 antibodies in the presence or absence of core or control protein β-gal. D) CD4⁺ T cell death after HCV core-treatment or β-gal-treatment for 3 days was determined by staining with LIVE/DEAD dye. Representative of 5 healthy donors. PHA-treated cells acted as positive controls for cell death.

Figure 2. HCV patient serum core levels

Serum core levels of individuals that resolve infection and that fail to resolve infection determined at Month 6, including long-term chronic patients. Horizontal bar indicates median. Mann-Whitney analysis. P-value significance is p<0.05.

Figure 3. Differential expression of gC1qR on CD4⁺ T cells of individuals that resolve HCV infection and those that become chronically infected

A) Representative gC1qR staining of PBMC from a healthy donor, an acute-to-resolved patient (Month 0) and an acute-to-chronic patient (Month 0) after gating down to the CD4⁺CD3⁺ lymphocytes. B) Compiled relative gC1qR expression (%gC1qR⁺ -%FMO) on CD4⁺ T cells from healthy controls, acutely infected individuals (Month 0) and those same individuals roughly six months later (Month 6), at which time chronicity or resolution were determined. Horizontal bar indicates median. Mann-Whitney analysis. P-value significance is p<0.05.

Figure 4. Activation of cells increases the frequency of gC1qR⁺CD4⁺ T cells

A) Matched pair analysis of the frequency of gC1qR⁺CD4⁺ T cells before and after stimulation of PBMC from A) acute-to-resolved patients (n=8) and B) acute-to-chronic patients (n=9) at Month 0. Matched pair analysis of the frequency of gC1qR⁺CD4⁺ T cells before and after stimulation of PBMC from C) acute-to-resolved (n=6) patients and D) acute-to-chronic patients (n=7) at Month 6. Matched pair analysis of the frequency of gC1qR⁺CD4⁺ T cells, before and after stimulation of PBMC, from E) long-term chronic patients (n=10). Wilcoxon analysis. P-value significance is p<0.05.

Figure 5. Suppressive response to HCV core in acute-to-resolved and acute-to-chronic individuals

PBMC of acute-to-resolved and acute-to-chronic patients were stimulated with anti-CD3/CD28 antibodies for 5 days in the presence or absence of HCV core antigen or β-gal control protein. [³H]-thymidine was added to wells on the fifth day of culture and incubated for an additional 18 hours before cell harvest. A) Percent inhibition of proliferation of healthy donors and infected patients at time of enrollment (Month 0). Mann-Whitney analysis. P-value significance is p<0.05. B) Percent inhibition of proliferation of healthy donors and infected patients at Month 6. C) Percent inhibition of proliferation of healthy donors and long-term chronic patients. D) Matched pairs analysis of change in inhibition of proliferation from Month 0 to Month 6 in acute-to-resolved patients (n=7). Wilcoxon analysis. P-value significance is p<0.05. E) Matched pairs analysis of change in inhibition of proliferation from month 0 to month 6 in acute-to-chronic patients (n=9).