Activities of Murine Peripheral Blood Lymphocytes Provide Immune Correlates That Predict Francisella tularensis Vaccine Efficacy

Abstract

We previously identified potential correlates of vaccine-induced protection against Francisella tularensis using murine splenocytes and further demonstrated that the relative levels of gene expression varied significantly between tissues. In contrast to splenocytes, peripheral blood leukocytes (PBLs) represent a means to bridge vaccine efficacy in animal models to that in humans. Here we take advantage of this easily accessible source of immune cells to investigate cell-mediated immune responses against tularemia, whose sporadic incidence makes clinical trials of vaccines difficult. Using PBLs from mice vaccinated with F. tularensis Live Vaccine Strain (LVS) and related attenuated strains, we combined the control of in vitro Francisella replication within macrophages with gene expression analyses. The in vitro functions of PBLs, particularly the control of intramacrophage LVS replication, reflected the hierarchy of in vivo protection conferred by LVS-derived vaccines. Moreover, several genes previously identified by the evaluation of splenocytes were also found to be differentially expressed in immune PBLs. In addition, more extensive screening identified additional potential correlates of protection. Finally, expression of selected genes in mouse PBLs obtained shortly after vaccination, without ex vivo restimulation, was different among vaccine groups, suggesting a potential tool to monitor efficacious vaccine-induced immune responses against F. tularensis. Our studies demonstrate that murine PBLs can be used productively to identify potential correlates of protection against F. tularensis and to expand and refine a comprehensive set of protective correlates.

FIG 1

Expression of genes that are potential correlates of protection is upregulated in mouse PBLs after vaccination with LVS. PBLs (A and C) and splenocytes (B and D) obtained from naive mice or from mice immunized with LVS were used to purify total RNA at the indicated time points after vaccination (A and B) or after vaccination and lethal challenge with LVS (C and D). Semiquantitative analyses of gene expression were performed using selected sets of primers/probes, chosen from among those that best reflected the hierarchy of in vivo vaccine efficacy (16, 19). The values shown are the median fold changes of expression of the indicated genes in immunized mice compared to their levels of expression in naive mice calculated from three independent experiments.

FIG 2

PBL populations differ slightly from splenocyte populations. Single-cell preparations obtained from naive mouse spleens and PBLs were stained with a panel of fluorescent antibodies to cell surface markers and with a fluorescent viability dye. After exclusion of fragments and aggregates by side scatter A (SSC-A) versus forward scatter A (FSC-A) and FSC-W versus FSC-A, cells were initially analyzed for viable leukocytes (live CD45⁺ leukocytes). Subpopulations were then quantified. B and T cells were identified as B220⁺ CD19⁺ T-cell receptor β (TCRβ) negative (TCRβ⁻) and TCRβ positive (TCRβ⁺) B220⁻ CD19⁻, respectively. T cells were further discriminated according to CD4 and CD8a markers. The remaining non-B and non-T cells were analyzed using NK1.1, CD11b, CD11c, and Gr-1 markers. NK1.1⁺ cells represent both natural killer cells (NK1.1⁺ TCRβ⁻) and NK T cells (NK1.1⁺ TCRβ⁺). CD11b⁺ cells include neutrophils (CD11b⁺ Gr-1⁺), dendritic cells (CD11c⁺ CD11b⁺ Gr-1⁻), and macrophages (for spleens) or monocytes (for PBLs) (CD11b⁺ Gr-1⁻ CD11c⁻). Shown are the percentage of leukocytes identified using naive cells from five experiments; error bars indicate standard deviations.

FIG 3

In vitro functions correlate with the numbers of PBLs added to cocultures. (A) Decreasing numbers of PBLs derived from LVS-vaccinated mice were added to a constant number of LVS-infected macrophages. After 3 days of coculture, BMMϕ were washed, lysed, and plated to evaluate the recovery of intracellular bacteria. The values shown are the mean numbers of CFU per milliliter ± SD for viable bacteria from triplicate independent experiments. (B and C) Supernatants were collected and separated from cells for analyses of IFN-γ by ELISA (B) and NO by the Griess reaction (C). The concentrations of each were calculated using standard curves as references. The values shown are the mean concentration ± standard deviation from triplicate independent experiments of similar designs and outcomes. Mac, macrophages.

FIG 4

PBLs from mice vaccinated with LVS-related vaccines exhibit a hierarchy of control of intramacrophage LVS growth comparable to that of splenocytes. BMMϕ from C57BL/6J mice were infected with LVS (macrophages alone [Macs]) and cocultured with PBLs (A) or splenocytes (B) obtained from naive or vaccinated C57BL/6J mice, as indicated. After 2 days of coculture, BMMϕ were washed, lysed, and plated to evaluate the recovery of intracellular bacteria. The values shown are the mean numbers of CFU of viable bacteria per milliliter ± SD for triplicate samples. The results shown are from one representative of five independent experiments of similar designs and outcomes. Brackets indicate a significant difference (P < 0.05) between the recoveries of the bacteria in cocultures. There were no significant differences in the recovery of bacteria from cocultures between LVS-immune cells and LVS-G-immune cells (B).

FIG 5

The in vitro production of cytokines and chemokines by PBLs from vaccinated mice exhibits differential expression patterns. Supernatants derived from PBLs cocultured for 2 days were collected and separated from cells for analyses of selected cytokines and chemokines by use of a protein array quantification system. The values shown are the mean concentration ± standard deviation for quadruplicate samples. The results shown are from one representative of five independent experiments of similar designs. Proteins with higher or lower concentrations are shown in panels A and B, respectively (note the different scales in each panel). Brackets indicate significant differences (P < 0.05) between the amounts of proteins produced in the cocultures.

FIG 6

Expression of genes that are potential correlates of protection is differentially upregulated in PBLs after vaccination with LVS-derived vaccines. PBLs obtained from naive mice or from mice immunized with LVS-derived vaccines were used to purify total RNA 2 days (A) and 15 days (B) after vaccination. Semiquantitative analyses of gene expression were performed using selected sets of primers/probes chosen from among those that best reflected the hierarchy of in vivo vaccine efficacy in splenocytes (16) or PBLs (Table 1). The values shown are the fold changes in the levels of expression of the indicated genes compared to their levels of expression in PBLs from naive mice calculated from one representative of three independent experiments. Gzmb, granzyme B.