key: cord-0252885-czqsd1up authors: Mouat, Isobel C.; Shanina, Iryna; Horwitz, Marc S. title: Age-associated B cells are long-lasting effectors that restrain reactivation of latent γHV68 date: 2022-04-21 journal: bioRxiv DOI: 10.1101/2021.12.29.474434 sha: 0c61ebed0461386172efb22a4cda25624791c738 doc_id: 252885 cord_uid: czqsd1up Age-associated B cells (ABCs; CD19+CD11c+T-bet+) are a unique population that are increased in an array of viral infections, though their role during latent infection is largely unexplored. Here, we use murine gammaherpesvirus 68 (γHV68) to demonstrate that ABCs remain elevated long-term during latent infection and express IFNγ and TNF. Using a strain of γHV68 that is cleared following acute infection, we show that ABCs persist in the absence of latent virus, though their expression of IFNγ and TNF is decreased. With a fluorescent virus we demonstrate that ABCs are infected with γHV68 at similar rates to other previously activated B cells. We find that mice without ABCs display defects in anti-viral IgG2a/c antibodies and are more susceptible to γHV68 reactivation when challenged with heterologous infection. Together, these results indicate that ABCs are a persistent effector subset during latent viral infection that restrains γHV68 reactivation. . e n c o d e s a p a c k a g i n g p r o t e i n t h a t a s s i s t s i n m o v i n g t h e n e w l y 212 r e p l i c a t e d v i r a l g e n o m e s t o t h e p a c k a g i n g m o t o r , w h e r e t h e y a r e l o a d e d i n t o c a p s i d s 3 7 . O m i c e t o c o n t r o l γ H V 6 8 l a t e n c y ; p r e v i o u s f i n d i n g s i n d i c a t e t h a t v a r i o u s c e l l t y p e s 234 i n f e c t e d b y γHV68 m a y d i s p l a y d i f f e r e n t i a l s u s c e p t i b i l i t y t o r e a c t i v a t i o n 2 3 , 4 0 . . γHV68, F o r a n a l y s i s o f e x t r a c e l l u l a r a n d i n t r a c e l l u l a r a n t i g e n s , 2 m i l l i o n c e l l s p e r s p l e e n s a m p l e o r a l l 399 c e l l s c o l l e c t e d p e r b l o o d s a m p l e w e r e s t a i n e d w i t h a p p r o p r i a t e a n t i b o d i e s . P r i o r t o s t a i n i T N F P e -C y 7 M P 6 -X T 2 2 1 : 1 0 0 B i o l e g e n d I L -1 7 A P E D a z z l e 5 9 4 T C 1 1 -1 8 H 1 0 . 1 1 : 1 0 0 B i o l e g e n d C D 4 5 A l e x a f l u o r 7 0 0 3 0 -F 1 1 1 : 2 0 0 T h e r m o F i s h e r S c i e n t i f i c C D 4 5 A P C -e F l u o r 7 8 0 3 0 -F 1 1 1 : 2 0 0 T h e r m o F i s h e r S c i e n t i f i c C D 4 S B 6 4 5 R M 4 -5 1 : C D 8 T c e l l s w e r e i d e n t i f i e d b y s t a i n i n g w i t h a t e t r a m e r a c q u i r e d f r o m t h e N I H 419 T e t r a m e r F a c i l i t y . 2 m i l l i o n c e l l s p e r w e l l w e r e i n c u b a t e d f o r 1 h o u r a t R T w i t h p 7 9 420 e a c t i o n p e r f o r m e d w i t h 5 0 0 n g p e r r e a c t i o n i n d u p l i c a t e w e l l s . c D N A q u a n t i f i e d 441 w i t h a s p e c t r o p h o t o m e t e r . q P C R w a s p e r f o r m e d u s i n g i Q T M S Y B R ® G r e e n s u p e r m i x ( B i o v i r a l c y t o p a t h i c e f f e c t s ( C P E ) . D a t a w e r e p l o t t e d a s s i g m o i d a l d o s e c u r v e s a n d i n t e r p o l a t i o n w a s 456 u s e d t o d e t e r m i n e t h e c e l l d e n s i t y p e r s a m p l e a t w h i c h 6 3 . 2 % o f w e l l s w e r e p o s i t i t e s t s , s i g n i f i c a n c e ( p -v a l u e ) , s a m p l e s i z e ( n , n u m b e r o f m i c e p e r g r o u p ) a n d n u m b e r o f 478 e x p e r i m e n t a l r e p l i c a t e s a r e s t a t e d i n t h e f i g u r e l e g e n d s . S t a t i s t i c a l a n a l y s e s i n c l u d e d : t w o -w a F i g u r e 1 A B C s e x p a n d i n t h e b l o o d a n d s p l e e n t h r o u g h o u t γHV68 i n f e c t i o n 673 B l o o d a n d s p l e e n c o l l e c t e d f r o m C 5 7 B L / 6 ( J ) m i c e ( 6 -8 -w e e k -o l d a t i n f e c t i o n ) m o c k -i n f e c t e F i g u r e 2 C y t o k i n e p r o d u c t i o n b y A B C s i n t h e s p l e e n d u r i n g γHV68 a n d A C R T A -γHV68 i n f e c t i o n 688 ( A -C ) C 5 7 B L / 6 ( J ) m i c e ( 6 -8 -w e e k -o l d a t i n f e c t i o n ) m o c k -i n f e c t e d w i t h m e d i a ( n a ï F i g u r e 3 A n a l y s i s o f A B C i n f e c t i o n s t a t u s w i t h f l u o r e s c e n t γHV68 s t r a i n 710 F e m a l e C 5 7 B L / 6 ( J ) m i c e ( 6 t o 8 -w e e k -o l d a t i n f e c t i o n ) i n f e c t e d F i g u r e 4 A B C s a r e n o t r e q u i r e d f o r c o n t r o l l i n g l y t i c i n f e c t i o n a n d a c h i e v i n g l a t e n c F i g u r e 5 M i c e w i t h o u t A B C s d i s p l a y a d y s r e g u l a t e d a n t i -γHV68 a n t i b o d y r e s p o n s e , t h o u g h n o 749 a l t e r a t i o n s t o t h e c e l l p o p u l a t i o n s i n f e c t e d w i t A B-cell subset uniquely 496 responsive to innate stimuli accumulates in aged mice Toll-like receptor 7 (TLR7)-driven 499 accumulation of a novel CD11c+ B-cell population is important for the development of 500 autoimmunity Age-Associated B Cells Age-associated B cells in viral infection unique type of B-cell activation essential for effective viral 507 clearance Cell Production of IFNγ in Response to 509 TLR7/IL-12 Stimulates Optimal B Cell Responses to Viruses T-bet+ B cells are induced by human viral infections 512 and dominate the HIV gp140 response The Transcription Factor T-bet Resolves 515 Memory B Cell Subsets with Distinct Tissue Distributions and Antibody Specificities in 516 Mice and Humans. 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