Effect of GM-CSF on the uNK cells and uMDSC from pregnant mice.

<p>A. CBA/CaJ female mice were mated with C57BL/6 allogeneic male mice. Uterine NK cells were flow sorted to obtain a NK1.1^- DX5^- DBA⁺ cell population that was incubated with GMCSF (20ng/ml) ± IL-2 (20ng/ml) for 5–7 days. Expression of uNK and CD11b⁺Ly6G^hiLy6C^lo and CD11b⁺Ly6G^loLy6C^hi cell populations were analyzed using a flow cytometer. GMCSF treatment resulted in no significant change in the NK1.1^- DX5^- DBA⁺ cell population from the CBA mice (p = 0.1164). However, an increase of the CD11b⁺Ly6G^loLy6C^hi population was observed (p = 0.0592). An inverse correlation was observed between the uNK cells and uMDSC in presence of both GMCSF and IL-2 treatment but was not statistically significant. B. C57BL/6 female mice were mated with allogeneic CBA/CaJ male mice. Uterine NK cells were flow sorted to obtain a NK1.1⁺ DX5^- DBA⁺ cell population that was incubated with GMCSF (20ng/ml) ± IL-2 (20ng/ml) for 5–7 days. Expression of uNK and CD11b⁺Ly6G^hiLy6C^lo and CD11b⁺Ly6G^loLy6C^hi cell populations were analyzed using a flow cytometer. GMCSF treatment resulted in a significant change in the NK1.1⁺ DX5^- DBA⁺ cell population from the C57BL/6 female mice (p = 0.0310). However, the change in the CD11b⁺Ly6G^loLy6C^hi population was not significant (p = 0.2410). The effect of GMCSF was more pronounced in the uNK cells from the C57BL/6 female mice and a significant inverse correlation between uNK and uMDSC was also observed (p = 0.0158). Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done by one-way analysis of variance (ANOVA) using Kruskal-Wallis test and two way ANOVA.</p

Effect of RMT3-23 treatment on expression of activation markers and inhibitory surface receptors on uNK cells.

<p>Treatment with RMT3-23 significantly upregulates the expression of CD69 on NK1.1^- DX5^- DBA⁺ cell population. A similar trend of increased expression was also observed in case of B220 and CD25 levels after treatment with RMT3-23 although the data were not statistically significant. RMT3-23 treatment also significantly down regulates Ly49C expression and up regulates KLRG1 and Ly49G2 on NK1.1^- DX5^- DBA⁺ cells. Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done using unpaired t test.</p

Expression of NK cell markers in the uNK and pNK population from non-pregnant and pregnant mice.

<p>Expression of NK1.1, DX5, DBA and NKp46 were determined on CD3^-CD122⁺ cells isolated from the uteri of non-pregnant (A) syngeneically mated pregnant (B) and allogeneically mated (C) pregnant CBA mice. Expression of NK1.1, DX5, DBA and NKp46 on CD3^-CD122⁺ cells from spleen of non-pregnant CBA mouse (D), syngeneically mated (E) and allogeneically mated (F) pregnant CBA mouse. Red-boxed areas denote uNK populations positive for NK1.1 and DBA in allogeneically mated pregnant CBA mouse (C) and DBA positive uNK population in syngeneically mated pregnant CBA mouse (B). These positive populations are absent in the splenic NK cell population in the same mouse. Data is representative of at least 3 experiments.</p

Expression of CD11b, Ly6C and Ly6G on uNK cells from CBA mice.

<p>Female CBA mice were allogeneically mated toC57BL/6 males and uterine lymphocytes were isolated between GD 10.5 to 12.5. Expression of CD11b, Ly6C and Ly6G was determined on the NK1.1^- DX5^-DBA⁺ cell population. Data are representative of at least 4 experiments.</p

Effect of RMT3-23 treatment on cytokine production by uNK cells.

<p>Treatment with RMT3-23 significantly down regulated cytokine production by NK1.1^- DX5^- DBA⁺ cell population. A significant decrease in the population of IFN, IL-6, IL-10, VEGF and GMCSF producing NK1.1^- DX5^- DBA⁺ cells was observed after treatment with RMT3-23 in comparison to Control group, whereas IL-4 production was significantly increased. Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done using unpaired t test.</p

Effect of GM-CSF and IL-15 on the uNK cells from pregnant CBA mice.

<p>CBA/CaJ female mice were mated with C57BL/6 allogeneic male mice. Uterine lymphocytes were isolated from Control and RMT3-23 treated mice and cultured with IL-15, IL-15+IL-12, IL-12, IL-15+GMCSF and GMCSF only for 7days in vitro in a 37°C humidified CO2 incubator. At the end of the incubation period the percentage of 3 different cell populations were determined by flow cytometry. A. RMT3-23 treated uNK cells proliferated more in response to IL-12 (p = 0.0284). B. This effect was more pronounced in the uNK cells expressing the MDSC markers (p<0.0001).</p

A. Expression of TIM-3 and DBA on uNK cells during gestation.

<p>A steady and similar increase in the expression of both TIM-3 and DBA is observed between GD 7.5 and 12.5, the time period when the uNK cells are most abundant in the uterus. TIM-3 and DBA expression were determined on the CD3^- CD122⁺ NK1.1^- DX5^- cells isolated from the uterus of pregnant CBA mice allogeneically mated with C57BL/6 males (n = 8). Data are presented as mean± SEM and are representative of at least 3 experiments. Data show the percentage of CD3- CD122+ NK1.1- DX5- cells expressing DBA and TIM-3 on different GDs. Statistical analysis was performed using 2 way ANOVA. B. Effect of TIM-3 blockade on uNK cell population size. Percentage of CD3^- CD122⁺ NK1.1^- and DX5^- cell population in control and RMT3-23 treated pregnant CBA mice at GD 7.5, 10.5 and 12.5. There was no significant change in the number of uNK cells between control and treated groups. Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done by one-way analysis of variance (ANOVA) using Kruskal-Wallis test. C. Effect of TIM-3 blockade on uNK cell cytotoxicity. No significant change was observed in the cytotoxicity of uNK cells following treatment with RMT3-23. Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done using unpaired t test. D. Effect of TIM-3 blockade on Granzyme production by uNK cells. A small but significant increase in the production of Granzyme B was observed in the RMT3-23 treated uNK cells in comparison to control. Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done using unpaired t test.</p

GMCSF concentration in the placenta of CBA/CaJ mice at GD 12.5 following TIM-3 blockade.

<p>A significant decrease in the GMCSF concentration was observed in the placenta of RMT3-23 treated mice in comparison to control. Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done using unpaired t test.</p

Effect of TIM-3 blockade on uterine MDSC population.

<p>Treatment with RMT3-23 did not change the size of the MDSC population in the uteri of pregnant CBA mice (C) although a significant decrease in the proinflammatory S100A8 (p = 0.0003) (A) and A9 (p<0.0001) (B) proteins in the placentae was observed. No change was observed in the concentration of the S100A8/A9 proteins in the RMT3-23 treated mice sera (D). Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done using unpaired t test.</p

Effect of RMT3-23 treatment on the cytokine and chemokine milieu in the placenta and on the uNK cell population size of pregnant CBA mice between G.D 10.5 to 12.5 that were allogeneically mated to C57BL/6 males.

<p>A significant decrease in IL-6, IL-15 and IL-9 concentrations and a significant increase in MIP1α, IL-8/KC and IP-10 mRNA expression is observed in the placental homogenate from mice treated with RMT3-23 in comparison to the control group. Data are presented as mean± SEM. Statistical analysis was done using unpaired t test. However, there was no significant change in the number of uNK cells (CD3- CD122+ NK1.1- and DX5-) between control and treated groups following treatment with RMT3-23. Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done by one-way analysis of variance (ANOVA) using Kruskal-Wallis test.</p