Primary cytomegalovirus phosphoprotein 65-specific CD8+ T-cell responses and T-bet levels predict immune control during early chronic infection in lung transplant recipients

Abstract

Background: Cytomegalovirus (CMV) remains an important pathogen in solid organ transplantation, particularly lung transplantation. Lung transplant recipients (LTRs) mismatched for CMV (donor positive/recipient negative [D(+)R(-)]) are at highest risk for active CMV infection and have increased mortality. However, the correlates of immune control during chronic CMV infection remain incompletely understood.

Methods: We prospectively studied 22 D(+)R(-) LTRs during primary CMV infection and into chronic infection. Immune responses during primary infection were analyzed for association with viral relapse during early chronic infection.

Results: Primary CMV infection was characterized by a striking induction of T-box transcription factor (T-bet) in CD8(+) T cells. CMV-specific effector CD8(+) T cells were found to be T-bet(+). After primary infection, 7 LTRs lacked immune control with relapsing viremia during early chronic infection. LTRs with relapsing viremia had poor induction of T-bet and low frequencies of phosphoprotein 65 (pp65)-specific CD8(+) effector T cells during primary infection. However, frequencies of IE1-specific CD8(+) effector T cells during primary infection were not associated with early relapsing viremia.

Conclusions: T-bet plays an important role in coordinating CD8(+) effector responses to CMV during primary infection. Moreover, CD8(+) T-bet induction and pp65-specific CD8(+) effector responses at the time of primary infection are important predictors of immune control of CMV during early chronic infection.

Figure 1.

Inadequate phosphoprotein 65 (pp65)–specific CD8⁺ effector responses during primary cytomegalovirus (CMV) infection are associated with relapsing viremia. A, Representative gating strategy for analysis of side scatter (SSC), forward scatter (FSC), LiveDead, CD3+ and CD8+ for peripheral blood mononuclear cells (PBMCs) and staining for interferon γ (IFN-γ) from 2 representative lung transplant recipients (LTRs). PBMCs were restimulated with either medium, pooled pp65 peptides, or staphylococcal enterotoxin B (SEB) and evaluated for CD3⁺CD8⁺ T cell production of IFN-γ with values indicating the frequency of CD3⁺CD8⁺ T cells producing IFN-γ. B, Comparison of pp65-specific and IE1-specific CD8⁺IFN-γ⁺ responses during primary CMV infection between 15 LTRs experiencing durable viral control and 7 LTRs experiencing relapsing viremia during early chronic infection. Bars represent median values, and P values were calculated by the Mann–Whitney–Wilcoxon test. C, Comparison of pp65-specific CD8⁺ and CD4⁺ production of IFN-γ between 15 controller and 7 relapser LTRs. Bars represent median values, and P values were calculated by the Mann–Whitney–Wilcoxon or Wilcoxon signed-rank test, as appropriate. Abbreviation: n.s., not significant.

Figure 2.

Massive induction of T-box transcription factor (T-bet) expression in CD8⁺ T cells with type 1 effector function caused by primary cytomegalovirus (CMV) infection. A, CD3⁺CD8⁺ T cells from a representative donor-positive/recipient-negative lung transplant recipient (LTR 50) stained for intracellular T-bet expression at a time point after lung transplantation but before primary CMV infection (pre-CMV) and then during primary CMV infection, with values indicating the frequency of CD3⁺CD8⁺ T cells expressing T-bet. B, Comparison of intracellular expression of T-bet between pre-CMV and primary CMV infection time points among 19 LTRs. Bars represent median values, and P value was calculated by the Wilcoxon signed-rank test. C, Peripheral blood mononuclear cells from a representative LTR (31) restimulated with medium, pooled phosphoprotein 65 (pp65) peptides, or staphylococcal enterotoxin B (SEB) during primary CMV infection and CD3⁺CD8⁺ T cells were evaluated for coexpression of intracellular T-bet and interferon γ (IFN-γ).

Figure 3.

Association of inadequate induction of T-box transcription factor (T-bet)⁺CD8⁺ T cells during primary cytomegalovirus (CMV) infection with relapsing viremia and correlation with phosphoprotein 65 (pp65)–specific CD8⁺interferon γ (IFN-γ)⁺ effector responses. A, Comparison of expression of T-bet in CD3⁺CD8⁺ T cells during primary infection between 15 lung transplant recipients (LTRs) with durable viral control and 7 LTRs with relapsing viremia. Bars represent median values, and P value was calculated with the Mann–Whitney–Wilcoxon test. B, Evaluation of the relationship between pp65-specific production of IFN-γ and T-bet expression in CD3⁺CD8⁺ T cells during primary CMV infection by scatterplot analysis; a best-fit nonlinear correlation (1-phase exponential association) was used. Correlation coefficients (r_s) and P value were calculated using the Spearman rank correlation test.

Figure 4.

Receiver operator characteristic (ROC) curves of T-box transcription factor (T-bet)⁺CD8⁺ and phosphoprotein 65 (pp65)–specific CD8⁺ interferon γ (IFN-γ)⁺ effector T cells for prediction of relapsing viremia during early chronic cytomegalovirus (CMV) infection. ROC curve (1 − specificity value vs sensitivity value) analyses, with calculation of the area under the curve (AUC), of the ability of the frequency of pp65-specific CD8⁺IFN-γ⁺ T cells (A) or T-bet⁺CD8⁺ T cells (B) during primary CMV infection to predict relapsing viremia during early chronic infection were performed. Indicated threshold values for each characteristic demonstrate optimal balance between sensitivity and specificity.