Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

The gut-associated lymphoid tissue (GALT) is the largest mass of lymphoid tissue in the body, and it is a unique line of defense where immune cells are in constant contact with gut commensals and pathogens. B lymphocytes constitute most of the lymphatic tissues of the gastrointestinal (GI) tract and they play a critical role in protecting from infections, promoting tolerance, and shaping microbiota in the gut through the production of immunoglobulins (Ig). Previous studies on GALT B cells in human showed that large B cell clones in the body partition into two broad networks: one includes the blood, bone marrow, spleen, and lung, while the other is confined to tissues within the gastrointestinal tract including the jejunum, ileum, and colon. Moreover, it has been shown that the human colon and ileum contain B cell populations that are not seen in peripheral blood (PB) or other secondary lymphoid organs. These studies highlight the unique nature of B cells in GALT and emphasize the importance of investigating these cells further. In this work, we studied B cells in the human GALT and focused our investigation on two major understudied areas of GALT B cells: 1- the relationships between the different B cell populations present in GALT; 2- the death of GALT B cells. In the first part of this work, we isolated different populations of IgM+ B cells from two human GALT sites, tonsil and appendix, and found a B cell population that is enriched at these sites and absent in other parts of the GI tract. These cells express IgM and lack IgD and CD27 expression. They secrete high levels of IgM spontaneously and are maintained throughout life. These cells show a mature surface phenotype and respond to T-dependent and T-independent stimulation with high levels of IgM secretion and IgA class-switching in vitro. B cell receptor sequence analysis showed that this population shares large clones with IgA and IgG class-switched memory cells, indicating that these cells are major contributors to the memory B cell pool in GALT. In the second part of this thesis research, we investigated a novel death pathway for tonsil B cells. B cells are known to die by apoptosis which is characterized by the nuclear fragmentation and the formation of apoptotic bodies. The morphology of dying tonsil B cells, however, indicated that these cells are dying by another death pathway that involves decondensation of nuclear chromatin, a process termed neutrophil extracellular traps formation or NETosis. A similar cell morphology termed “smudge cell” was observed in the PB of patients with chronic lymphocytic leukemia (CLL) in 1896 and was used since as a diagnostic and prognostic marker in those patients, however, this morphology was never pursued mechanistically. We found that tonsil B cell NETosis requires citrullination of histones (Cit-H3) by peptidyl arginine deaminases (PADs) for the nuclear chromatin decondensation and is enhanced by TNF production. In situ staining for Cit-H3 showed that B cells likely undergo NETosis in the light zone of germinal centers in vivo. We think that this cell death is initiated to limit B cell numbers in the tissue and that the “smudge cells” in CLL patients are B cell dying by NETosis. These data shed light into a novel death pathway for GALT B cells and emphasize the need to investigate the role of B cell NETs further in health and disease.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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