Age-related resistance of C57BL/6 mice to Cryptococcus neoformans is dependent on maturation of NKT cells

Abstract

Conflicting results have been reported regarding the ability of C57BL/6 mice to clear infections due to Cryptococcus neoformans. Examination of the various experimental protocols used suggested that C57BL/6 mice might develop the ability to resist infection as they mature. We analyzed the ability of C57BL/6 mice of different ages to respond to immunization with cryptococcal antigen or to clear a cryptococcal infection. Mice were immunized with a soluble cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund's adjuvant (CneF-CFA). Delayed-type hypersensitivity (DTH) reactions elicited by the immunization were significantly stronger in 15-week-old C57BL/6 mice than in 7-week-old mice. Analysis of cryptococcal CFU 8 weeks following intratracheal infection of 7-week-old mice or 15-week-old mice revealed a relative inability of the younger animals to control the infection. Six-week-old immunized and infected mice cleared cryptococci from brain, spleen, and liver in a manner similar to that of immunized and infected 15-week-old mice. However, the older mice cleared cryptococci much more efficiently from the lungs. The possible role for NKT cells was determined by passive transfer of thymocytes from 10-week-old mice (containing mature NKT cells) or 2-week-old mice (containing immature NKT cells) to 6-week-old mice. The 10-week-old thymocytes significantly enhanced the ability of the mice to develop a DTH response after immunization with CneF-CFA, while animals treated with 2-week-old thymocytes did not improve their DTH response after immunization. The cells in the 10-week-old thymocyte population responsible for improvement of DTH responses were identified as being NK1.1 positive.

FIG. 1.

DTH responses of 7-week-old and 15-week-old B6 mice immunized with saline-CFA or CneF-CFA 7 days prior to footpad testing. Data represent the mean increase (expressed in inches) ± the standard error of the mean of footpad swelling from five animals per group. P values were determined using Student's t test. Footpad swelling in CneF-injected footpads of mice that were immunized with CneF-CFA was significantly greater than in the CneF-injected footpads of mice immunized with saline-CFA (P < 0.001).

FIG. 2.

Cryptococcal CFU from brain, spleen, liver, and lungs of 6-week-old and 15-week-old B6 mice infected with 10⁵ C. neoformans cryptococci intravenously. Mice were infected 7 days after immunization, and CFU were determined 7 days following infection. Numbers of CFU represent the mean ± standard error of the mean of total organ CFU obtained from five individual mice per group. P values were determined using Student's t test.

FIG. 3.

DTH responses of 6-week-old B6 mice treated intravenously with 0.5 ml of saline, 10⁸ thymocytes harvested from 2-week-old B6 mice, or 10⁸ thymocytes harvested from 10-week-old B6 mice just prior to immunization with CneF-CFA or saline-CFA. Mice were footpad tested 7 days after immunization. Data represent the mean increase (expressed in inches) ± the standard error of the mean in footpad swelling from five animals per group. P values were determined using ANOVA followed by the Bonferroni posttest. Footpad swelling in CneF-injected footpads of mice that were immunized with CneF-CFA was significantly greater than in the CneF-injected footpads of mice treated with saline-CFA (P < 0.001 for all groups). NS, not significant.

FIG. 4.

DTH responses of 6-week-old B6 mice that were treated with 0.5 ml of saline, 10⁸ thymocytes harvested from 10-week-old B6 mice, or 3 × 10⁷ thymocytes harvested from 10-week-old B6 mice and depleted of NK1.1-positive cells or treated with 3 × 10⁷ thymocytes harvested from 10-week-old mice and treated with isotype control antibody. Data represent the mean increase (expressed in inches) ± the standard error of the mean in footpad swelling from five animals per group. P values were determined using ANOVA with the Bonferroni posttest. Footpad swelling in CneF-injected footpads of mice that were immunized with CneF-CFA was significantly greater than in the CneF-injected footpads of mice treated with saline-CFA (P < 0.001 for all groups).