Inducible Deletion of CD28 Prior to Secondary Nippostrongylus brasiliensis Infection Impairs Worm Expulsion and Recall of Protective Memory CD4+ T Cell Responses

IL-13 driven Th2 immunity is indispensable for host protection against infection with the gastrointestinal nematode Nippostronglus brasiliensis. Disruption of CD28 mediated costimulation impairs development of adequate Th2 immunity, showing an importance for CD28 during the initiation of an immune response against this pathogen. In this study, we used global CD28(-/-) mice and a recently established mouse model that allows for inducible deletion of the cd28 gene by oral administration of tamoxifen (CD28(-/lox)Cre(+/-)+TM) to resolve the controversy surrounding the requirement of CD28 costimulation for recall of protective memory responses against pathogenic infections. Following primary infection with N. brasiliensis, CD28(-/-) mice had delayed expulsion of adult worms in the small intestine compared to wild-type C57BL/6 mice that cleared the infection by day 9 post-infection. Delayed expulsion was associated with reduced production of IL-13 and reduced serum levels of antigen specific IgG1 and total IgE. Interestingly, abrogation of CD28 costimulation in CD28(-/lox)Cre(+/-) mice by oral administration of tamoxifen prior to secondary infection with N. brasiliensis resulted in impaired worm expulsion, similarly to infected CD28(-/-) mice. This was associated with reduced production of the Th2 cytokines IL-13 and IL-4, diminished serum titres of antigen specific IgG1 and total IgE and a reduced CXCR5(+) T-FH cell population. Furthermore, total number of CD4(+) T cells and B220(+) B cells secreting Th1 and Th2 cytokines were significantly reduced in CD28(-/-) mice and tamoxifen treated CD28(-/lox)Cre(+/-) mice compared to C57BL/6 mice. Importantly, interfering with CD28 costimulatory signalling before re-infection impaired the recruitment and/or expansion of central and effector memory CD4(+) T cells and follicular B cells to the draining lymph node of tamoxifen treated CD28(-/lox)Cre(+/-) mice. Therefore, it can be concluded that CD28 costimulation is essential for conferring host protection during secondary N. brasiliensis infection. Author Summary CD28 is an important costimulatory molecule, involved in the activation of naive T cells, enhancing cytokine production, preventing T cell anergy and apoptosis. Furthermore, CD28 plays a crucial role in the organisation of secondary lymphoid tissue by assisting in the recruitment of T cells into the B cell follicles, thus promoting germinal center formation, isotype switching and B cell maturation. The requirement of CD28 costimulatory signalling during recall of memory responses against infections has remained controversial. Hence, here we utilised a mouse model that allowed for inducible deletion of the cd28 gene (CD28(-/lox)Cre(+/-)) by oral administration of tamoxifen to resolve this controversy. CD28(-/-) mice and mice given tamoxifen prior to secondary infection failed to expel adult N. brasiliensis worms. This was related to reduced production of the Th2 cytokines IL-13 and IL-4, diminished type 2 antibody titres, and a reduced number of memory CD4(+) T cells. In summary, CD28 is crucial for protection against N. brasiliensis secondary infection and plays a key role in the recruitment of T-FH cells, memory CD4(+) T cells and follicular B cells.