VDJ-recombination of the B-cell Receptor Following Hematopoietic Stem Cell Transplantation

Overview

When a person undergoes a stem cell transplant-an important procedure used to treat serious blood diseases such as leukaemia, lymphoma, or myeloma-the entire immune system is affected. The transplant essentially "resets" the immune system, meaning that the patient loses much of the protection against infections that has been built up over a lifetime. After the transplant, the patient therefore needs to be revaccinated against several diseases, such as tetanus, diphtheria, polio, COVID-19, and pneumococcal disease. In this study, we aim to investigate how B cells-the immune cells that produce antibodies-reconstitute after a stem cell transplant. We are particularly interested in a genetic process called VDJ recombination, through which each B cell develops a unique receptor that enables it to recognize and fight a specific virus or bacterium. This process is what makes our immune system so effective. But what happens to this diversity after the entire immune system has been restarted with new stem cells? Does the body regain the same ability to recognize pathogens? Are there differences between patients who receive their own stem cells (autologous transplantation) and those who receive stem cells from another person (allogeneic transplantation)? To answer these questions, we will follow patients undergoing stem cell transplantation at Umeå University Hospital. We will collect blood samples before and after the transplantation and will assess whether-and how well-patients generate antibodies in response to the vaccines given after transplantation. Using flow cytometry and genetic analyses, we will examine both which B-cell populations are generated and what their genetic architecture looks like. The goal is to understand how the new immune system is rebuilt after transplantation and, ultimately, to help improve vaccination strategies and infection prevention for this vulnerable patient group

Purpose and aims The humoral immune system is critical for mounting protective responses against severe microbial infections. This is a highly specific response, where naïve B cell clones that recognize epitopes on a pathogen are activated, clonally expands and differentiates to memory B cells and antibody producing plasma cells. The specificity of the response is dependent on VDJ recombination that creates naïve B cell clones, each with a distinct B cell receptor that carry unique variable (V), diversity (D) and joining segments. While the process of VDJ recombination is well investigated, it is not well established how/if VDJ recombination is affected in B cells after autologous or allogeneic stem cell transplantation. Such knowledge may improve on strategies to re-vaccinate vulnerable patient groups that require stem cell transplantation to alleviate a number of haematological disorders. Here, we will utilize samples from individuals before and after haematological stem cell transplantation to better define the impact on VDJ recombination and clonal responses to vaccine antigens. Background Stem cell transplantation is a procedure where most/all hematopoietic cells are eradicated from a patient and replaced by new stem cells that are derived from "self" (autologous) or from an MHC matched donor. Upon a successful stem cell transfer, new and healthy hematopoietic cells will emerge. After the procedure immunity to several vaccine preventable diseases are lost or severely impaired putting the patient at risk. Following transplantation patients are vaccinated against several infectious diseases according to an established vaccine schedule. This provides an opportunity to study VDJ recombination in a situation where the immune system is "reset" - compare and contrast the situation to before the procedure as well as differences between autologous and allogenic transplantation. A standardized vaccination schedule based on recommendations by the European Conference on Infections in Leukemia (ECIL) is implemented to HSCT patients. Vaccinations against pneumococcal infections, capsulated haemophilus influenzae, tetanus, diphtheria, polio, pertussis, covid19 and influenzae are recommended to all. Further vaccination may be considered according to the preferences of the patient. B cells are critical for humoral immune responses, as they specifically respond to an antigen and then differentiate into antibody producing plasma cells, and to memory B cells. The importance of this for protection against a primary infection is readily illuminated by susceptibility to severe infectious disease if the humoral immune system is defect or after treatment with B cell depleting drugs, such as anti-CD20 antibodies. In addition, the generation of immunological memory then protects us against re-infection with the same pathogen. Of critical importance for this response is the antigen-specificity of the B-cell receptor (BCR) of naïve B cell clones. The BCR comprise an immunoglobin heavy and light chain with a distinct specificity. The specificity is created by VDJ recombination where one of numerous V, D and J segments are combined into a VDJ region of the heavy chain, and one of numerous V and J segments are combined into a light chain. The combination of these ensures that emerging naïve B cell clones each has a distinct and unique capacity to recognize an epitope. The diversity that is generated ensures that the immune system can respond clonally to almost any foreign pathogen it encounters. By BCR sequencing of B cells, it is therefore possible to determine exactly which V, D or J segments has randomly been recombined to recognize a specific antigen. T cells undergo a similar process of VDJ recombination after maturation in the thymus. After stem cell transplantation, the VJ-segment of the beta chain T cell receptor develop with a relative symmetry but reduced complexity, as compared with the donor. This may explain why B cells may demonstrate reduced T cell-dependent somatic hypermutation after transplantation. However, it is not well understood if a similar set of B cell clones will emerge after autologous stem cell transplantation or that of an allogenic donor. Since stem cell transplant patients undergo vaccination, this will allow us to closely study the process of antigen-specific VDJ recombination pre- and post "reset" of the immune system, and to couple this to antigenspecific responses after vaccination. We hypothesize that this will reveal further information of how/if the nature of the transplant will affect downstream VDJ recombination and lead to an altered capacity or pattern of immune recognition. Moreover, it has been shown that impaired humoral immune responses during ageing may be dependent on extrinsic rather that intrinsic factors that affect Bcl6 and CD39-expression the T cell compartment. The potential availability of samples from allogenic donors with age variation will allow for potential verification of these results in humans. Project description Study structure This is an observational cohort study following patients after stem cell transplantation examining the B-cell development following the standardized and established vaccine schedule given as part of clinical practice. In the current version of this schedule in Västerbotten vaccinations against pneumococci, haemophilus influenzae, COVID-19, tetanus, diphtheria, polio and whopping cough are included. Measles, mumps and rubella vaccination may be given following negative immunity test at 24 months from transplantation. Sampling i. Sampling before immunosuppressive medication is initiated ii. Sampling when admitted for stem cell transplantation iii. Sampling prior to each vaccination time point up until 24 months from transplantation. iv. In allogenic transplantation sampling of related donor if available (sampling once). Blood sample analysis B cell characterization and VDJ sequencing Antigen specific identification of B cells; We will use established methodology to detect antigen specific B cells. In brief, biotin- and fluorochrome labelled SARS-CoV-2 protein or Influenza type A hemagglutinin protein will be utilized to determine vaccine specific CD19+ CD20+ B cells by flow cytometry. We will focus on cells that has undergone thymus-dependent B cell responses (CD27+IgD-) and phenotype these for activation markers such as DAF and CD71. For examination of the VDJ repertoire preand post transplantation, we will mainly rely on bulk sequencing but may subject samples to single cell sequencing to reveal if heavy and light chain pairing is affected by the transplantation. We also aim to dissect transcriptional patterns in B cells to reveal specific patterns or molecules that actively predict efficient establishment of Bmem or plasmablasts after stem cell transplantation. For transcriptional patterns, we will perform 10x Chromium single cell gene expression to provide the single cell transcriptome from up to 10,000 vaccine-specific Bmem cells. By combinatorial analysis of both phenotypic and transcriptomic data, we will be able to identify biomarkers or gene expression patterns that predict robust establishment of "boostable" immune memory after stem cell transplantation.