Cytomegalovirus-Specific CD4 T Cells Are Cytolytic and Mediate Vaccine Protection

Abstract

CD4 T cells provide protection against cytomegalovirus (CMV) and other persistent viruses, and the ability to quantify and characterize epitope-specific responses is essential to gain a more precise understanding of their effector roles in this regard. Here, we report the first two I-A d -restricted CD4 T cell responses specific for mouse CMV (MCMV) epitopes and use a major histocompatibility complex class II (MHC-II) tetramer to characterize their phenotypes and functions. We demonstrate that MCMV-specific CD4 T cells can express high levels of granzyme B and kill target cells in an epitope-and organ-specific manner. In addition, CD4 T cell epitope vaccination of immunocompetent mice reduced MCMV replication in the same organs where CD4 cytotoxic T lymphocyte (CTL) activity was observed. Together, our studies show that MCMV epitope-specific CD4 T cells have the potential to mediate antiviral defense by multiple effector mechanisms in vivo. C ytomegalovirus (CMV)/human herpesvirus 5 (HHV-5) (the prototypic betaherpesvirus) infection is endemic in humans and wild mice and establishes a lifelong infection in the absence of acute disease in healthy hosts. Adaptive immunity is a critical component of CMV defenses, restricting primary infection and dampening reactivation, helping to maintain the largely benign host-virus equilibrium. However, if immunity is naive or compromised (e.g., in transplant recipients or congenital infection), CMV can cause serious disease (1, 2). In immunocompetent mice, CD8 T cells help to control acute CMV infection and establish latency (3, 4), and their adoptive transfer prevents disease in mice and humans with weakened immunity (5-8). Although much less well studied, CD4 T cells also contribute to defense against CMV. Their rapid expansion and numbers correlate with reduced disease in transplant and HIV patients (9-11), and correspondingly, delayed induction of CMV-specific CD4 T cells is associated with increased congenital infection (12) and prolonged viral shedding in infants (13). Notably, no protective correlates were seen with CMV-specific CD8 T cell responses in several of these studies. In mice, CD4 T cells are absolutely required to control mouse cytomegalovirus (MCMV) replication in the salivary glands-the key site of viral dissemination, where CD8 T cells can exert no control (14, 15)-and also contribute to immune control in several other organs. Adoptive transfer of MCMV-specific transgenic CD4 T cells provides some protection in immunocompromised mice (16), and cotransferring CMV-specific CD4 T cells reduces viral load and promotes "help" for CMV-specific CD8 T cell responses in patients receiving cellular immunotherapy (17).In addition to traditional helper functions, CD4 T cells can also mediate direct antiviral activity in some cases. In chronic human CMV (HCMV), Epstein-Barr virus (EBV), and HIV infections, CD4 T cells displaying a terminally differentiated effector phenotype and/or expressing canonical cytolytic molecules (e.g., granzymes and perforin) are pres...

Human Cytomegalovirus (HCMV)-Specific CD4 + T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

“…This assay convincingly shows that CD4 ϩ T cells can respond directly to CMV-infected cells probably using both the cytokine expression and cytotoxicity mechanisms observed in the polyfunctional flow cytometry experiments. The ability of CMV-specific CD4 ϩ T cells to control viral dissemination is in spite of the large array of immune evasion mechanisms encoded by the virus genome and confirms the observations made in vivo in the murine model (68) and previously in infected macrophages in in vitro models of HCMV infection (69). Further interrogation of the CMV-specific CD4 ϩ T cell response using this in vitro model will be able to determine whether the cytokines or cytotoxicity produced by CD4 ϩ T cells is more important in resolving CMV infection or reactivation in the host.…”

“…This problem has been addressed to an extent in the murine model with MCMV infection. A recent study using novel epitope class II-restricted tetramers in vivo observed direct killing of infected cells (68). With respect to HCMV infections, Sinzger et al have shown that IE1-specific CD4 ϩ T cell clones are able to produce IFN-␥ in response to stimulation by TB40/E-infected macrophages which have downregulated MHC class II expression (69).…”

Cytotoxic CD4 T Cells—Friend or Foe during Viral Infection?

“…The data presented in this study suggest that MCMV-specific CD4 T cells can kill target cells in an epitope- and organ-specific manner. Additionally, they show that vaccination with CD4 T cell epitopes leads to reduced viral replication in tissues where CD4 CTL are observed (136). …”

TGF-β receptor maintains CD4 T helper cell identity during chronic viral infections

“…Moreover, absence of TGF-β signaling did not greatly impact antiviral cytokine secretion by D b :GP 33-41 LCMV-specific CD8 T cells upon cognate peptide stimulation (Supplemental Figure 1D). We also found that TGF-β signaling only minimally suppressed the effector markers KLRG and Ly6C on virus-specific CD8 T cells, or granzyme B on IFN-γ-producing cells, after GP [33][34][35][36][37][38][39][40][41] peptide stimulation (Supplemental Figure 1, E-G). Overall, these data show that deletion of Tgfbr2 prior to infection only minimally affected CD8 T cell antiviral responses early after chronic LCMV infection.…”

“…Cytokine measurements. Secretion of IFN-γ, TNF-α, and IL-2 from LCMV-specific T cells was performed ex vivo using GP [31][32][33][34][35][36][37][38][39][40][41] or GP [67][68][69][70][71][72][73][74][75][76][77] peptide stimulation of splenocytes incubated in the presence of 1 μg/ ml brefeldin A (BFA; Sigma-Aldrich) for 5 hours at 37˚C before staining.…”