Secondary replicative function of CD8+ T cells that had developed an effector phenotype

Abstract

Models of the differentiation of memory CD8+ T cells that replicate during secondary infections differ over whether such cells had acquired effector function during primary infections. We created a transgenic mouse line that permits mapping of the fate of granzyme B (gzmB)-expressing CD8+ T cells and their progeny by indelibly marking them with enhanced yellow fluorescent protein (EYFP). Virus-specific CD8+ T cells express gzmB within the first 2 days of a primary response to infection with influenza, without impairment of continued primary clonal expansion. On secondary infection, virus-specific CD8+ T cells that became EYFP+ during a primary infection clonally expand as well as all virus-specific CD8+ T cells. Thus, CD8+ T cells that have acquired an effector phenotype during primary infection may function as memory cells with replicative function.

Fig. 1

Requirements for expression of EYFP by cells from gzmBCreER^T2/ROSA26EYFP mice. (A) Naive CD8⁺ T cells from gzmBCreER^T2/ROSA26EYFP mice were stimulated for 6 days under gzmB-inducing (anti-CD3ε, IL-2, IFN-γ, and IL-7) or non-inducing conditions (anti-CD3ε, sCD70, IL-7, and antibodies to IL-2, CD25, and IFN-γ), in the presence or absence of 4-OHT and assessed for expression of gzmB and EYFP. (B) gzmBCreER^T2/ROSA26EYFP mice were infected intranasally with the HKx/31 strain of influenza and received tamoxifen or carrier alone on days 1-8 p.i. The lungs were assessed on day 10 p.i. for EYFP⁺ CD8⁺ T cells. (C) Splenic CD4⁺ and CD8⁺ T cells were evaluated for expression of EYFP and CD44 on days 10 and 100 p.i., and (D) splenic B cells, NK cells, dendritic cells, and myeloid cells were assessed for EYFP on day 10 p.i.

Fig 2

Clonal expansion by CD8⁺ T cells that had expressed gzmB during the first days of influenza infection. (A) gzmBCreER^T2/ROSA26EYFP mice were infected intranasally with the HKx/31 strain of influenza and were given tamoxifen 2 days prior to infection, on days 1 and 2 p.i., or on days 7 and 8 p.i.. On day 10 p.i. we determined the proportion (mean ± SEM) of D^b/NP-pentamer-binding CD8⁺ T cells that was EYFP⁺. (B) Influenza-infected mice that had received tamoxifen on days 1 and 2 were given BrdU on days 5-9 p.i., and EYFP⁺ CD8⁺ T cells from the lungs were assessed on day 10 p.i for incorporation of BrdU.

Fig 3

Expression of gzmB, CD62L, IL-7Rα, and CD25 by EYFP⁺ CD8⁺ T cells responding to influenza infection. (A) gzmBCreER^T2/ROSA26EYFP mice were infected intranasally with the HKx/31 strain of influenza and treated with tamoxifen on days 1-8 p.i. The EYFP⁺ CD8⁺ T cells from the lungs, MLNs and spleen were analyzed for intracellular gzmB, CD62L, and IL-7Rα at the peak of the primary response and during the memory phase (6 or 7 weeks p.i.). (B) Influenza-infected mice were given tamoxifen on days 1-4 p.i. and BrdU 12 hours prior to analysis on day 8 for BrdU incorporation and CD25 expression by EYFP⁺ CD8⁺ T cells from the MLNs.

Fig 4

Absence of impaired secondary clonal expansion by CD8⁺ T cells that had expressed gzmB during primary influenza infection. gzmBCreER^T2/ROSA26EYFP mice were intranasally infected with the HKx/31 strain of influenza and given tamoxifen on days 1-8 p.i. On days 10 and 49 p.i., CD8⁺ T cells from the lungs, MLNs, and spleens were assessed for the binding of D^b/NP pentamers and expression of EYFP⁺. The mice were challenged on day 49 p.i. with the PR8 strain of influenza in the absence of tamoxifen, and the same measurements were performed 7 days later. (A) The proportions (mean ± SEM) of D^b/NP-specific CD8⁺ T cells that were EYFP⁺ are shown. (B) The numbers of total (squares) and EYFP⁺ (crosses) D^b/NP-specific CD8⁺ T cells in the lungs, MLNs, and spleens of each mouse are shown.