Emily Siniscalco

U.S.A.

Mucosal immunization induces linked local and systemic humoral responses

Emily R. Siniscalco1,2; Hailong Meng3; Gisela Gabernet3; Gaspar. A. Pacheco4,5,6,7; Shahab Saghaei4,5,6,7; Sydney I. Ramirez8,9; L. Benjamin Hills9,10; Farhoud Faraji9,11; Shuting Chen1; Xiangyun Yin1,12; Christine Dien1,3,13,19; Laura R. Hoyt1; Elise G. Liu13; Abel Barrett14; Neima Briggs15; Adam Williams2,16; Vipul Shukla2,17; Shane Crotty8,9, Duane R. Wesemann4,5,6,7; Steven H. Kleinstein1,3,18; Joe Craft1,13; Stephanie C. Eisenbarth1,2,16

1. Department of Immunobiology, Yale School of Medicine; New Haven, CT 06520, USA.
2. Center for Human Immunobiology, Northwestern University Feinberg School of Medicine; Chicago, IL 60611, USA.
3. Department of Pathology, Yale School of Medicine; New Haven, CT 06520, USA.
4. Department of Medicine, Division of Allergy and Clinical Immunology, Division of Genetics, Brigham and Women’s Hospital Boston, MA 02115, USA.
5. Harvard Medical School, Boston, MA 02115, USA.
6. The Broad Institute of MIT and Harvard Cambridge, MA 02142, USA.
7. The Ragon Institute of MGH, MIT, and Harvard Cambridge, MA 02139, USA.
8. Department of Medicine (Infectious Diseases), University of California, San Diego School of Medicine; La Jolla, CA 92093, USA
9. La Jolla Institute for Immunology; La Jolla, CA 92037, USA
10. Department of Pediatrics, Division of Hematology and Oncology, University of California San Diego, La Jolla, CA 92093, USA
11. Department of Otolaryngology-Head and Neck Surgery, University of California San Diego, La Jolla, CA, USA.
12. Department of Ophthalmology and Visual Science, Yale School of Medicine, New Haven, CT, USA
13. Section of Rheumatology, Allergy and Immunology, Yale School of Medicine; New Haven, CT 06520, USA.
14. WovenBit LLC, New Haven, CT 06510, USA
15. Department of Medicine (Infectious Diseases), Yale School of Medicine; New Haven, CT 06520, USA
16. Department of Medicine (Allergy and Immunology); Northwestern University Feinberg School of Medicine; Chicago, IL 60611, USA.
17. Deptartment of Cell and Developmental Biology, Northwestern University Feinberg School of Medicine; Chicago, IL 60611, USA.
18. Program in Computational Biology and Bioinformatics, Yale School of Medicine; New Haven, CT 06520, USA.
19. Yale Center for Systems and Engineering Immunology, Yale School of Medicine, New Haven, CT 06520, USA

Abstract

Background

The intestine is a barrier site where innocuous and harmful stimuli interface with the immune system, resulting in protective and tolerogenic immune responses. IgA is the predominant gut antibody isotype, known to neutralize toxins, bind pathogens and foods, and maintain microbial commensals. Despite IgA’s prevalence, antigen-specific IgA production is poorly-described, particularly with respect to the germinal center (GC) reaction. Such a lack of understanding of gut GCs contrasts the well-defined GC responses in systemic infection or immunization, and indeed, many requirements for canonical GC responses are dispensable in the gut.

Methods

To investigate gut antibody responses, we used a model of oral OVA and cholera toxin immunization, combined with OVA-specific B cell staining to identify antigen-specific responses. We tracked GC participation using the S1PR2CreERT2 Rosa26Lox-Stop-Lox-tdTomato GC fate-mapping mouse model and compared GC contributions to the IgA response over time. We also used single cell BCR and RNA sequencing to define mutational burden and clonal relationships between antigen-specific B cells in GC and nonGC states as well as of multiple antibody isotypes in gut tissues.

Results

We found that antigen-specific B cells enter GCs during the first two weeks of immunization, but do not form GC-derived plasma cells until several weeks later. We also saw an early wave of GC-independent, antigen-specific IgA plasma cells seeding the gut and found that these cells accumulate somatic mutations and undergo clonal selection to the same degree as GC-derived cells. Through clonal analysis of sequenced BCRs in mice and humans, we observed that IgA B cells are frequently related to IgG1 B cells in expanded B cell clones and share mutation patterns. Further molecular analysis of IgG1 and IgA B cells revealed that gut IgG1 B cells class switch to IgA and contribute to the gut IgA response.

Conclusions

This supports a model of IgA generation through both rapid GC-independent IgA plasma cell differentiation and sequential class switching from IgG1 GC B cells. The former pathway describes noncanonical methods of B cell mutation and selection in the gut, while the latter pathway effectively links the specificity of mucosal IgA and systemic IgG1 humoral immunity to gut-derived antigens. Defining such biology is essential for informed design of mucosal vaccines, which need to generate IgA and IgG antibodies for efficient barrier and systemic protection.