CD4+ T cell restoration and control of hepatitis C virus replication after childbirth

Abstract

Chronic hepatitis C virus (HCV) infection is characterized by persistent high-level viremia and defective cellular immunity, including a lack of functional HCV-specific CD4+ T cells. We previously described an exceptional period of viral control that occurs in some chronically infected women after childbirth. Here, we investigated whether reduced HCV replication after pregnancy is associated with recovery of CD4+ T cell immunity. Class II tetramer analysis revealed significantly greater frequencies of circulating HCV-specific CD4+ T cells at 3 months postpartum in women with concurrent declines in viremia compared with those with stable viremia. These HCV-specific CD4+ T cells had an effector-memory phenotype. Inhibitory coreceptor expression on these cells corresponded to the degree of viral control. Circulating CD4+ T cells produced IL-2 and IFN-γ after HCV antigen stimulation, demonstrating Th1 functionality. These data provide direct evidence that the profound loss of HCV-specific CD4+ T cell help that results in chronic infection is reversible following pregnancy, and this recovery of CD4+ T cells is associated with at least transient control of persistent viral replication.

Keywords: Hepatitis; Immunology; Infectious disease; T cells.

Figure 1. Function of HCV-specific CD4⁺ T cells in women with and without postpartum viral control.

(A) Plasma HCV RNA levels at the third trimester (T3) and 3 months postpartum (3PP) for 10 women with (controllers) and 22 women without (noncontrollers) postpartum viral load reductions of at least 1 log₁₀ IU/mL. (B) Example HCV-specific CD4⁺ T cell cytokine responses of 1 controller and 1 noncontroller assessed by intracellular cytokine stain following PBMC stimulation with 3 separate peptide pools spanning HCV NS3-NS4. (C) Background-subtracted frequencies of HCV-specific cytokine-producing CD4⁺ T cells at T3 and 3PP for 10 controllers (left) and 22 noncontrollers (right) (Wilcoxon matched-pairs signed rank test). (D) Pearson’s correlation of changes in viral load and HCV-specific IL-2⁺IFN-γ⁺ CD4⁺ T cell frequencies from T3 to 3PP. (E) HCV-specific CD4⁺ T cell IL-2⁺IFN-γ⁺ coproduction of controllers and noncontrollers at T3, 3PP, 6PP, and 12PP (Mann-Whitney U test). Horizontal lines indicate median values. *P < 0.05; **P < 0.01.

Figure 2. HCV-specific CD4⁺ T cell frequencies by class II tetramer staining.

(A) Examples of tetramer-positive populations among total CD3⁺ T cells (left) or CD4⁺CD8^–CD3⁺ T cells (right) for 2 controllers and 2 noncontrollers with matched HLA class II tetramers. (B) Frequencies of tetramer-positive CD4⁺ T cells per CD4⁺CD8^–CD3⁺ T cells at T3 and 3PP. Up to 4 tetramers were pooled for each subject based on HLA-type and tetramer availability. Frequencies were compared for 6 controllers and 5 noncontrollers at 3PP using the Mann-Whitney U test. Horizontal lines indicate median values. (C) Spearman’s correlation of change in viral load from T3 to 3PP with the frequency of tetramer-positive CD4⁺ T cells at 3PP. *P < 0.05; **P < 0.01.

Figure 3. Tetramer-positive CD4⁺ T cell phenotypes of 6 women with postpartum viral control.

(A) The viral load (column 1), tetramer-positive CD4⁺ T cell frequency (column 2), and phenotypic marker expression of tetramer-positive (black) and bulk (gray) CD4⁺ T cells (columns 3–5). All phenotypic analyses were performed at the 3PP time point, when tetramer-positive frequencies were highest. Relevant HLA alleles for class II tetramer studies are listed in the right column. (B) Spearman’s correlation of the change in viral load from T3 to 3PP with the frequency of tetramer-positive CD4⁺ T cells expressing PD-1 (left), CTLA-4 (middle), and PD-1 but not CTLA-4 (right) at 3PP.

Figure 4. Function of CD4⁺ T cells targeting tetramer epitopes in 3 women with robust postpartum viral control.

(A) Production of IL-2, IFN-γ, and TNF-α measured by intracellular cytokine staining following challenge of PBMCs with peptides corresponding to the epitopes used in class II tetramer studies (see Supplemental Table 2 for list of tetramers for each subject). (B) Polyfunctionality of CD4⁺ T cell responses. Pie charts represent populations of CD4⁺ T cells secreting a single cytokine (either IL-2, IFN-γ, or TNF-α; white pie slices), 2 cytokines (medium gray pie slices), or all 3 cytokines (dark gray pie slices). Arcs represent production patterns of the individual cytokines: IL-2 (inner black arcs), IFN-γ (middle medium gray arcs), and TNF-α (outer light gray arcs).