Multifunctional CD4 Cells Expressing Gamma Interferon and Perforin Mediate Protection against Lethal Influenza Virus Infection

Abstract

CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H1N1 influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in the draining lymph node (DLN) and gamma interferon (IFN-γ) and IL-10 in the lung. IFN-γ-deficient CD4 cells produced larger amounts of IL-17 and similar levels of TNF-α, IL-10, and IL-2 compared to wild-type (WT) CD4 cells. Both WT and IFN-γ −/− CD4 cells exhibit influenza virus-specific cytotoxicity; however, IFN-γ-deficient CD4 cells did not promote recovery after lethal infection as effectively as WT CD4 cells. PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung but not the DLN. These cells expressed granzyme B (GrB) and required perforin to lyse peptide-pulsed targets. Lethally infected mice given influenza virus-specific CD4 cells deficient in perforin showed greater weight loss and a slower time to recovery than mice given WT influenza virus-specific CD4 cells. Taken together, these data strengthen the concept that CD4 T cell effectors are broadly multifunctional with direct roles in promoting protection against lethal influenza virus infection.

Intraepithelial T-Cell Cytotoxicity, Induced Bronchus-Associated Lymphoid Tissue, and Proliferation of Pneumocytes in Experimental Mouse Models of Influenza

“…Although classically CD8 T-cells are considered cytotoxic cells, there is increasing evidence for CD4 cytotoxic T-cells (7,8,27). Naïve CD4 T-cells do not contribute to viral clearance during primary influenza infection, but memory CD4 T-cells provide potent secondary immunity (8).…”

“…In the sixth model (6), Tregs were depleted by injecting an antibody to CD25 (PC61) on days -3 and -1 before secondary infection (12) and then challenged with 60,000 (8). During secondary influenza virus infection, both tetramer-positive and tetramer-negative Tregs accumulated in the lungs after infection.…”

“…Naïve CD4 T-cells do not contribute to viral clearance during primary influenza infection, but memory CD4 T-cells provide potent secondary immunity (8). To better document how protective memory CD4 Tcells contribute to protective immune responses against influenza, and how they might also impact the regulation of repair processes after viral clearance, two sets of experiments were done: transplantation of CD4 memory T-cells to unprimed wild mice and then transplantation to SCID mice.…”

“…Unprimed WT mice received either naïve or memory CD4 T-cells specific for an epitope of the influenza virus before challenge with a lethal dose of 10,000 EID 50 (7,8). The slides were prepared 2 days after infection to determine early inflammation driven by memory CD4 T-cells.…”

Memory CD4+T-cell–mediated protection depends on secondary effectors that are distinct from and superior to primary effectors

“…The transfer of 1°effectors to unprimed mice can protect against lethal challenge (8)(9)(10), and studies demonstrating memory CD4 + T-cell protection in mice deficient for CD8 + T and B cells suggest that an important helper-independent protective contribution of memory CD4 + T cells may be mediated directly by the 2°e ffectors (11)(12)(13). In addition, IAV-specific 1°effector (7,10) and memory (14,15) CD4 + T cells isolated from the lung and from secondary lymphoid organs (SLO) display distinct functional and phenotypic characteristics. Whether 2°effectors comprise a similarly heterogeneous population is unknown.…”