Strain and gender dependent effects of a systemic PD-1 knockout in mice.

Background

PD-1 is a negative immunoregulatory coreceptor on activated T and B cells that maintains self-tolerance. In cancer therapy, immune checkpoint inhibitors (ICI) are used to activate the immune system in order to supress tumor growth. However, an increased number of autoimmune side effects have been reported in many patients. Therefore, we examined the effect of a PD-1 knockout in a mouse strain very susceptible for the induction of many experimental autoimmune diseases (A/J) compared to one known to be mostly resistant (BL/6), as well as gender specific differences.

 

Methods

PD-1 deficient (pd-1^-/-), PD-1 heterozygote (pd-1^+/-) and PD-1 wildtype (pd-1^+/+) A/J and BL/6 mice were observed on their survival rate in a period of 0-15 weeks. At the age of 4-6 weeks, echocardiography was performed. Mice at the same age were sacrificed and heart and blood samples were collected. The hearts were examined for signs of inflammation and fibrosis, using hematoxylin eosin (HE) and acid fuchsin orange G (Afog) staining. HsTnT levels as well as auto-antibody titer against Troponin I were measured in serum. For the hsTnT analysis, a treshold of 100 pg/mL was set due to test sensitivity reasons.

Results
A/J pd-1^-/- mice showed significantly decreased survival rates in comparison to pd-1^+/- and pd-1^+/+ mice (χ²(2)=54,20, p<0.0001, Fig.1A) although BL/6 pd-1^-/- mice did not (χ²(1)=82,80. p<0.0001, Fig.1B).

 

Fig.1: Kaplan-Meier curves of mice from age 0 to 15 weeks. Comparison of A) pd-1^-/-, pd-1^+/-, pd-1^+/+ A/J mice and B) pd-1^-/- A/J and BL/6 mice.

 

Female pd-1^-/- A/J mice showed increased hsTnT levels (1661±1206) in comparison to pd-1^+/- (51.61±13.36, p<0.01), pd-1^+/+ (56.30±7.66, p<0.01). Regarding the strains pd-1^-/- female A/J mice showed significantly higher levels than female pd-1^-/- (22.30±13.80, p<0.01) and pd-1^+/- BL/6 mice (20.10±8.20, p<0.01). Even though elevated hsTnT levels were observed in female BL/6 pd-1^+/+ mice (65.03±1.96) compared to pd-1^+/- (p>0.05) and pd-1^-/- animals (p<0.05), all levels were under the treshold (Fig.2A).

In males, hsTnT levels were significantly increased in pd-1^-/- (278.1±142.7) than pd-1^+/- A/J mice (50.61±11.07, p<0.05). Significantly higher hsTnT levels were measured in pd-1^+/+ (219.7±159.4) than pd-1^+/- (30.24±7.67, p<0.05) and pd-1^-/- BL/6 mice (13.38±13.38, p<0.05). Also, pd-1^-/- A/J mice showed significantly higher levels than pd-1^-/- (p<0.01) and pd-1^+/- BL/6 mice (p<0.001). Compared to pd-1^-/- BL/6 mice, hsTnT levels of pd-1^+/+ A/J mice were also elevated (p<0.05), however still remained under the threshold (Fig.2B).

 

Fig.2: Serum hsTnT levels of pd-1^-/-, pd-1^+/-, pd-1^+/+ mice. A) female- and B) male A/J vs. BL/6 mice.

Conclusion

In A/J mice susceptible to autoimmunity, PD-1 deficiency led to a decreased survival rate, elevated hsTnT levels, production of anti-TnI-antibodies and inflammation of the myocardium. Regarding gender-specific differences, the effects were stronger in female A/J mice. In contrast, pd-1^-/- BL/6 mice, mostly known to be more resistant against autoimmunity, showed no signs of myocardial inflammation. Our results indicate, that the effects of PD-1 depend on both, the genetic background and gender. Therefore, further studies should elaborate these differences in order to investigate the underlying pathomechanisms as well as possible targets for the treatment of ICI caused autoimmunity.