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PIK3IP1 Promotes Extrafollicular Class Switching in T-Dependent Immune Responses.

ABSTRACT: PI3K plays multiple roles throughout the life of a B cell. As such, its signaling is tightly regulated. The importance of this is illustrated by the fact that both loss- and gain-of-function mutations in PI3K can cause immunodeficiency in humans. PIK3IP1, also known as TrIP, is a transmembrane protein that has been shown to inhibit PI3K in T cells. Results from the ImmGen Consortium indicate that PIK3IP1 expression fluctuates throughout B cell development in a manner inversely correlated with PI3K activity; however, its role in B cells is poorly understood. In this study, we define the consequences of B cell-specific deletion of PIK3IP1. B cell development, basal Ig levels, and T-independent responses were unaffected by loss of PIK3IP1. However, there was a significant delay in the production of IgG during T-dependent responses, and secondary responses were impaired. This is likely due to a role for PIK3IP1 in the extrafollicular response because germinal center formation and affinity maturation were normal, and PIK3IP1 is not appreciably expressed in germinal center B cells. Consistent with a role early in the response, PIK3IP1 was downregulated at late time points after B cell activation, in a manner dependent on PI3K. Increased activation of the PI3K pathway was observed in PIK3IP1-deficient B cells in response to engagement of both the BCR and CD40 or strong cross-linking of CD40 alone. Taken together, these observations suggest that PIK3IP1 promotes extrafollicular responses by limiting PI3K signaling during initial interactions between B and T cells.

SUBMITTER: Ottens K

PROVIDER: S-EPMC7541772 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200kb downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH. Activation Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB. However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high throughput genome wide translocation sequencing (HTGTS) into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor. We performed high-throughput genome-wide translocation sequencing (HTGTS) with different B cell genotypes that induces either I-SceI or AID-initiated DSBs as bait to study their joining pattern to AID-initiated S region breaks Please note that the 'ΔSγ1_2xI/ΔSµ_2xI-3' raw data files were analyzed twice with different reference assemblies to address specific points, and associated with both 'ΔSγ1_2xI_ΔSµ_2xI-3'.txt and Sγ1_2xI_ΔSµ_2xI-3'_trans-Sm.txt processed data files. As for the reference genome mm9_129_IgHC, it is a custom built generated based on mm9 (Bl6 line based mouse genome) and the partial genome sequence of another mouse line 129sv. It is not currently available in any public database however can be easily obtained either from the authors or self-built using information provided in the associated manuscript.

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PIK3IP1 Promotes Extrafollicular Class Switching in T-Dependent Immune Responses.

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