Bim-mediated deletion of antigen-specific CD8 T cells in patients unable to control HBV infection

Abstract

HBV-specific CD8(+) T cells are critical for a successful immune response to HBV infection. They are markedly diminished in number in patients who fail to control the virus, but the mechanisms resulting in their depletion remain ill defined. Here, we dissected the defective HBV-specific CD8(+) T cell response associated with chronic HBV infection by gene expression profiling. We found that HBV-specific CD8(+) T cells from patients with different clinical outcomes could be distinguished by their patterns of gene expression. Microarray analysis revealed that overlapping clusters of functionally related apoptotic genes were upregulated in HBV-specific CD8(+) T cells from patients with chronic compared with resolved infection. Further analysis confirmed that levels of the proapoptotic protein Bcl2-interacting mediator (Bim) were upregulated in HBV-specific CD8(+) T cells from patients with chronic HBV infection. Blocking Bim-mediated apoptosis enhanced recovery of HBV-specific CD8(+) T cells both in culture and directly ex vivo. Consistent with evidence that Bim mediates apoptosis of CD8(+) T cells expressing low levels of CD127 (IL-7R), the few surviving HBV-specific CD8(+) T cells were CD127(hi )and had elevated levels of the antiapoptotic protein Mcl1, suggesting they were amenable to IL-7-mediated rescue from apoptosis. We therefore postulate that Bim-mediated attrition of HBV-specific CD8(+) T cells contributes to the inability of these cell populations to persist and control viral replication.

Figure 1. cDNA microarray data of HBV-specific CD8⁺ T cells from resolved and CHB patients.

(A) Envelope-specific CD8⁺ T cells were purified by flow cytometric sorting of a short-term PBMC line from a chronic patient (middle and left plots, respectively). mRNA extracted from the purified cells was then profiled by dual-color cDNA microarray technology (right). (B) TreeView analysis of average linkage hierarchically clustered (with self-organized mapping) gene expression data. The top dendrogram represents the similarity between individual arrayed samples (vertical plane) based on the global gene expression profile; a yellow line segregates the main clusters: chronic (C) and resolved (R). The blue box highlights a section of the heat map where a group of genes exhibited marked upregulation in branch C compared with R (listed with original and current unigene references on the right; genes in red participate in apoptosis; genes overlapping with the SAM short list are indicated by asterisks). (C) SAM plot illustrating the most significant differentially regulated genes (false discovery rate, 1.3%) between the group with chronic and that with resolved HBV infection. (D) cDNA array data of 5 highly significant apoptosis-related genes. Error bars indicate mean ± SD.

Figure 2. Bim expression is increased at the protein level in HBV-specific CD8⁺ T cells from patients with CHB.

(A) Representative example of Bim expression in HBV-specific CD8⁺ T cells from resolved and CHB patients (left and middle) and the cumulative data (right). Bim expression in resolved responses was similar to background levels with an isotype control. (B) Representative example of Bim expression in total CD8⁺ T cells (T) and HBV-specific CD8⁺ T cells (H) in a patient with CHB (left and middle) and cumulative data (right). (C) Correlation between viral load and Bim expression in HBV-specific CD8⁺ T responses in CHB patients (left) and relative levels of Bim expression in resolved (R) and CHB patients segregated according to eAg status (right). (D) Representative example of Bim expression directly ex vivo in HBV-specific CD8⁺ T cells in resolved and CHB individuals (left and middle) and cumulative data (right). (E) Bim expression directly ex vivo in tetramer-positive HBV-specific CD8⁺ T cell responses from resolved and CHB patients. Shown are examples of tetramer and Bim staining (left and middle) and summary data for all responses (right). Significance testing of all cumulative data by Mann-Whitney test. (F) Bim expression directly ex vivo in HBV-specific CD8⁺ T cells quantified over the course of acute HBV infection (using overlapping peptides in HLA-A2^– patient R17 and using HLA-A2/HBV tetramers in 2 HLA-A2⁺ patients, R7 and R13). Bim levels in HBV-specific CD8⁺ T cells are plotted against HBV DNA, with serology and ALT in the acute and resolved (right of dotted line) phases indicated below. Error bars indicate mean ± SD.

Figure 3. Rescue of in vitro–cultured HBV-specific CD8⁺ T cells derived from individuals with CHB.

Representative flow cytometry plots and cumulative data (below) showing the effect of pancaspase inhibition on the detection of envelope and core-specific CD8⁺ T cells (A and B, respectively) in 10-day peptide-stimulated cultures of PBMCs from individuals with chronic infection. Differences in responses with and without caspase inhibition were calculated with the paired Student’s t test (P < 0.0001). (C) Representative plots of the detection of HBV-specific CD8⁺ T cells in short-term lines of PBMCs from an individual with chronic infection utilizing pools of overlapping peptides corresponding to the HBV precore/core (PreC/C), X, envelope (Ep1 and Ep2), and polymerase (Pp1–4) proteins with and without caspase inhibition. (D) Influenza A–specific CD8⁺ T cells detected in short-term lines from individuals with chronic infection ± caspase inhibition. (E) HBV-specific CD8⁺ T responses detected in short-term lines from resolved individuals with and without caspase inhibition. (F) HBV-specific CD8⁺ T cell rescue following specific inhibition of proapoptotic Bax in short-term lines from patients with chronic infection.

Figure 4. Direct ex vivo rescue of HBV-specific CD8⁺ T cells from patients with CHB.

Representative flow cytometry plots and cumulative data (below) indicating direct ex vivo frequencies of HBV-specific CD8⁺ T cells in patients with chronic infection as detected following stimulation with viral peptide with and without treatment with the pancaspase inhibitor zVAD-fmk.

Figure 5. Mcl1 and CD127 expression of HBV-specific CD8⁺ T cells.

(A) Intracellular staining for Mcl1 in core and envelope-specific CD8⁺ T cells expanded in vitro from individuals with chronic and resolved infection (left) with cumulative data (right). (B) Summary of Mcl1 levels in HBV-specific (H), total (T), and influenza-specific (I) CD8⁺ T cells from individuals with persistent HBV infection. (C) Correlation between viral load and the level of Mcl1 expression in HBV-specific CD8⁺ T responses from individuals with chronic infection (left) and relative levels of Mcl1 expression in resolved and persistently infected patients segregated according to eAg status (right). (D) Intracellular stain for Mcl1 in CD127^hi populations of HBV-specific and total CD8⁺ T cells. Error bars indicate mean ± SD.