IRF4 ablation in B cells abrogates allogeneic B cell responses and prevents chronic transplant rejection

https://doi.org/10.1016/j.healun.2021.06.008Get rights and content

BACKGOUND

B cells contribute to chronic transplant rejection by producing donor-specific antibodies and promoting T cell response, but how these processes are regulated at the transcriptional level remains unclear. Herein, we investigate the role of transcription factor interferon regulatory factor 4 (IRF4) in controlling B cell response during chronic transplant rejection.

METHODS

We generated the Irf4gfp reporter mice to determine IRF4 expression in B cell lineage. We then used mice with B cell-specific IRF4 deletion to define the role of IRF4 in B cell response after NP-KLH immunization or allogeneic heart transplantation. In particular, graft survival and histology, as well as B and T cell responses, were evaluated after transplantation.

RESULTS

IRF4 is dynamically expressed at different stages of B cell development and is absent in germinal center (GC) B cells. However, IRF4 ablation in the B cell lineage primarily eliminates GC B cells in both naïve and NP-KLH immunized mice. In the transplantation setting, IRF4 functions intrinsically in B cells and governs allogeneic B cell responses at multiple levels, including GC B cell generation, plasma cell differentiation, donor-specific antibody production, and support of T cell response. B cell-specific IRF4 deletion combined with transient CTLA4-Ig treatment abrogates acute and chronic cardiac allograft rejection in naïve recipient mice but not in donor skin-sensitized recipients.

CONCLUSIONS

B cells require IRF4 to mediate chronic transplant rejection. IRF4 ablation in B cells abrogates allogeneic B cell responses and may also inhibit the ability of B cells to prime allogenic T cells. Targeting IRF4 in B cells represents a potential therapeutic strategy for eliminating chronic transplant rejection.

Section snippets

Mice

Balb/c, Irf4fl/fl, and Cd19-Cre mice were purchased from Jackson Laboratory (Bar Harbor, ME). Irf4fl/fl mice were crossed to Cd19-Cre mice to generate Irf4fl/flCd19-Cre+/‒ mice (indicated as Irf4fl/flCd19-Cre mice throughout the text). We designed the Irf4gfp reporter mice and requested Jackson Laboratory Model Generation Services to generate this mouse strain. All animal experiments were approved by the Institutional Animal Care and Use Committee at Houston Methodist Research Institute.

Flow cytometric analysis

Flow

IRF4 is dynamically expressed at different stages of B cell development

To determine the role of IRF4 in allogeneic B cell responses, we first assessed the expression levels of IRF4 in the B cell lineage. To this end, we generated the Irf4gfp reporter mice, in which a P2A.GFP_P2A.DTR_stop cassette was inserted immediately after the last exon of WT B6 mouse Irf4 by using the CRISPR/Cas9 technique (Figure S1).

Irf4gfp reporter mice at 8 weeks of age were used to determine IRF4 expression at different stages of B cell development. Living CD45+ cells from bone marrows

Discussion

We found that IRF4 was most highly expressed in immature B cells and was barely expressed in GC B cells. However, deletion of IRF4 largely spared immature B cells but most significantly eliminated GC B cells. We further found that IRF4 deletion abrogated the expression of GC regulator Bcl6.12 This possibly explains why IRF4 is barely expressed in GC B cells but controls their generation. In consistent with our findings, Willis S et al have indicated that although IRF4 is absent in GC B cells,

Authors’ contributions

GW, WC, and AOG designed the research. GW, DZ, and YW conducted the experiments. GW performed the heart transplantation. GW, DZ, YW, NMG, SGY, XCL, WC, and AOG analyzed the data and interpreted the results. GW, DZ, and WC drafted the manuscript. NMG, SGY, and AOG edited the manuscript.

Disclosure statement

The authors of this manuscript have no conflicts of interest to disclose.

The authors would like to thank the Jackson Laboratory Model Generation Services and the Houston Methodist Flow Cytometry Core Facility for excellent services. The authors appreciate Preston Arnold for helpful scientific discussion.

This study was supported by the US National Institutes of Health grant (#NIH R01AI132492 to WC) and the J.C. Walter Jr. Transplant Center Foundation (to AOG).

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