Recombinant TLR5 agonist CBLB502 promotes NK cell-mediated anti-CMV immunity in mice

Abstract

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.

Figure 1. Prophylactic rflagellin administration protected mice from lethal mCMV infection.

A. A total of 5 groups WT B6 mice were treated with 25 µg rflagellin/mouse i.p 96, 72, 48, 24 or 0 hours before a lethal dose (2×LD50, 1×10⁶ pfu/mouse i.p) of mCMV infection. Control mice were treated with PBS only 48 hours before the same lethal dose of mCMV infection. Infected mice were monitored every day to record for mortality. The % survival recorded until day 17 after mCMV infection is shown. The symbol “*” indicates the p value<0.05, Log Rank Test of groups rflagellin 48 hrs or 72 hrs vs PBS-treated group. B. The % weight loss measured on day 5 after mCMV infection. All mice receiving rflagellin at 0 hours after mCMV infection died before day 5 after mCMV infection and the % weight loss data of this group was not available. 5–10 mice were used per group. C. WT B6 mice were treated with a single dose of rflagellin 25 µg/mouse or 0.2 ml PBS i.p. 48 hours later mice were infected either with 0.5×10⁶, 1×10⁶ or 2.5×10⁶ mCMV pfu/mouse i.p. The % survival on day 28 after mCMV infection is presented. 10 mice were used per group. The % survival recorded until day 17 after mCMV infection is shown. The symbol “*” indicates the p value<0.05, Log Rank Test of while compared with the survival data of rflagellin-treated mice vs corresponding mCMV infection dose in PBS-treated mice. 5–10 mice were used per group. D. WT B6 mice were infected with 0.5×10⁶ mCMV pfu/mouse i.p. A single dose of rflagellin (25 µg/mouse) was injected i.p 24 or 48 hours after mCMV infection. Control mice were injected with 0.2 ml PBS i.p 24 hours after mCMV infection. Survival of mice after mCMV infection was monitored each day and % survived mice until 12 days after infection is presented. 6–8 mice were used per group.

Figure 2. Prophylactic rflagellin administration reduced liver mCMV load in WT B6 mice.

WT B6 and TLR5 KO B6 mice were given 25 µg rflagellin or 0.2 ml PBS i.p 48 hours before a sub-lethal dose (1×10⁵ pfu/mouse i.p) of mCMV. Mice were sacrificed on day 3 and 10 after mCMV infection and viral load per liver was determined as described in Materials and Methods. A. Virus titer in livers of rflagellin- and PBS-treated WT B6 mice. B. Virus titer in livers of rflagellin- and PBS-treated TLR5 KO B6 mice. The data are the representative of three similar experiments using 5 mice per group at each time point. C. WT B6 mice were given 25 µg rflagellin/mouse (rFlagellin 48 Hrs, closed circle) or 0.2 ml PBS (open circle) i.p 48 hours before or at the same time as (rFlagellin 0 Hr, closed triangle) a sub-lethal mCMV infection (1×10⁵ pfu/mouse i.p). Weights of individual mice were measured on day 0, 1, 2, 3, 4 and 8 days after mCMV infection. Percent weight changes per group of experimental mice are presented. D. Mice receiving rflagellin (rFlagellin 0 Hr) during mCMV infection became dehydrated and hunched and were sacrificed on day 3 after mCMV infection and liver viral load was determined. The data are the representative of two similar experiments using 5 mice per group. The symbols “*” and “**”represent the p values<0.05 and <0.005, respectively, Students t-Test.

Figure 3. NK cells are required to induce enhanced early anti-mCMV immunity in rflagellin-treated mice.

A). Experimental design of anti-asialo GM1 administration in WT B6 mice to deplete NK cells in vivo. Reconstituted anti-asialo GM1 in PBS and 0.5 ml was injected i.p to B6 mice on −4, −3 and −1 days of mCMV infection as described in Materials and Methods. Control WT B6 mice were injected with 0.5 ml PBS. 25 µg rflagellin was injected per mouse i.p 48 hours before mCMV infection in anti-asialo GM1-treated and or PBS treated WT B6 mice. All groups of treated mice were infected with a lethal dose (5×10⁵ pfu/mouse) of mCMV i.p on day 0. B). Survival data were recorded by observing mice every day or mice were euthanized having weight loss >25% following mCMV infection and percent survived mice of each group are presented. This experiments was performed once using 8 to 10 mice per group. The symbol “**”represents the p value<0.005, Log Rank Test (Kaplan-Meier estimator).

Figure 4. rFlagellin treatment increased NK cell activation in the absence and presence of mCMV infection.

Splenocytes were harvested from rflagellin- and PBS-treated WT B6 and TLR5 KO B6 mice on day 0 and 3 after mCMV infection (1×10⁵ pfu/mouse i.p). A and B. Nucleated cells per spleen were determined from WT B6 and TLR5 KO B6 mice. C–J. Numbers of: CD3-NK1.1+ NK cells (C and D); KLRG2+ NK cells (E and F); ICOS-1+ NK cells (G and H); and CD69+ NK cells (I and J) per spleen were measured from WT B6 and TLR5 KO B6 mice. K–P. Numbers of: CD11b+ NK cells (K); CD122+ NK cells (L); 2B4+ NK cells (M); Ly49G2+ NK cells (N); Ly49C/H+ NK cells (O); and Ly49D+ NK cells (P) per spleen were determined from rflagellin- and PBS-treated WT B6 mice on day 0 and 3 after mCMV infection. The “*” and “**” represent p values<0.05 and <0.005, respectively, Students t-test. Q. mRNAs were harvested from the TLR5-transfected and TLR5-ve Null cells and FACS-sorted splenic CD3-NK1.1+ NK cells from naïve WT B6 mice as described in Materials and Methods. The cDNA bands specific for TLR5 and GAPDH were measured by RT-PCR and were visualized by ethidium bromide staining. Lane 1 = TLR5 expressing cells, Lane 2 = FACS sorted NK cells, and Lane 3 = TLR5-ve Null cells.

Figure 5. rFlagellin increased NK cell lytic activity in the presence and absence of mCMV infection.

Splenocytes were harvested on days 0 and 3 after mCMV infection and NK cell lytic activity was measured using standard 4-hour ⁵¹Cr-release assay by Yac-1 target cells as described in Materials and Methods. A and B. The % cell lytic activity of NK cells of rflagellin- and PBS-treated WT B6 mice on days 0 and 3 after infection. C and D. The % cell lytic activity by NK cells harvested from rflagellin- and PBS-treated TLR5 KO B6 mice on day 0 and 3 post infection. E. Kinetics of NK cells lytic activity from the splenocytes harvested from rflagellin and PBS-treated WT B6 mice on day 0, 1, 2, 3 and 8 after mCMV infection. The data shown in A–D are representative of three independent experiments and data shown in E are from one experiment. 5 mice were used per group per time point. The “*” indicates p value<0.05, Student's T-test.

Figure 6. rFlagellin enhanced NK cells cytotoxicity by increasing CD107a degranulation in NK cell.

Splenocytes were harvested from rflagellin- and PBS-treated mice on days 0 (48 hours after rflagellin treatment) and 3 after mCMV infection (1×10⁵ pfu/mouse i.p). Flowcytometric analysis for CD107a expressed by NK cells was performed by both regular surface stainings as described in Materials and Method. A. The representative FACS data of CD3-NK1.1+ NK cells expressing CD107a in the spleen of rflagellin- and PBS-treated mice determined by regular surface staining. B. The absolute numbers of CD107a+NK cells per spleen were determined from rflagellin- and PBS-treated mice on day 0 (48 hours after rflagellin treatment) and 3 after mCMV infection by using the flowcytometric data. 5 mice were used per group per time point. The “*” and “**” represent the p values<0.05 and <0.005, respectively, Student's T-test.

Figure 7. rFlagellin treatment enhanced maturation and increased expression of Ly49H on NK cells after mCMV infection.

Harvested splenocytes on day 3 after mCMV infection from rflagellin- and PBS-treated control mice were stained with mAbs to NK1.1, CD27, CD11b along with Ly49H as described in Materials and Methods. A. FACS plots of % CD3-NK1.1+ NK cells of lymphocyte-gated populations. B. % Ly49H expressed by NK cells. C. CD11b−CD27−(DN), CD11b−CD27+, CD11b+CD27+ (DP) and CD11b+CD27− NK cell populations. D. Ly49H+ NK cells of 4 subsets gated populations described in C. E. The total numbers of Ly49H+ NK cells and all 4 subsets of NK cells (as described in D) per spleen expressed Ly49H on day 3 after mCMV infection. The “*” represents p value<0.05, Student's T-test. 5 mice were used per group.

Figure 8. The effect of rflagellin treatment on cytokine production in the presence and absence of mCMV infection.

Splenocytes harvested from rflagellin- and PBS-treated control mice on day 0, 1, 2, 3 and 8 after mCMV infection were stimulated with PMA ionomycin for 4 hours at 37°C as described in Materials and Methods. Cells were stained for intracellular expression of IFN-γ and granzyme B along with NK cell surface markers. A and B represent the numbers of IFN-γ producing NK cells per spleen in the absence and presence of PMA-ionomycin stimulation. C and D represent the numbers of granzyme B producing NK cells per spleen in the absence and presence of PMA ionomycin stimulation. E and F represent the numbers of IFN-γ and granzyme B producing NK cells per spleen in the absence and presence of PMA ionomycin stimulation, harvested on day 0 after mCMV infection. G. Serum IFN-α on day 0, 2 and 3 after mCMV infection determined by Luminex assay. H and I represent serum cytokines determined on day 0 and 3 after mCMV infection, determined by Luminex assay. 5 mice were used per group in each time point. The “*” and “**” represent p values<0.05 and <0.005, respectively, Student's T-test.