Research in the McBride lab revolves around three major themes in B cell biology: 1) How B cells in the tumor microenvironment affect patient immunotherapy response; 2) How DNA damage and repair contribute to immunoglobulin/antibody diversity; and 3) Interactions between B cells and oncogenic viruses and how they influence host-pathogen interactions. The lab undertakes a number of biochemical, molecular, and cellular approaches, while also using human tissue samples, mouse models, and a proprietary, McBride Lab-developed, high-throughput, single-cell immunoglobulin (Ig) cloning and antibody characterization pipeline. The pipeline is used for defining the immunoglobulin repertoire (the diverse range of B cell receptors expressed by an individual), and for identifying and selecting specific antigen-binding antibodies for recombinant production. This unique pipeline is key to many of the lab’s projects and collaborations.
In mammals, B cells arise from hematopoietic stem cells in the bone marrow where they differentiate through multiple antigen-independent steps. The resulting naïve, immature B cell migrates to a secondary lymphoid organ, such as the spleen. There, antigen-induced B cell activation is coupled with gene expression changes in the cells of the microenvironment to give rise to the germinal center (GC).
Downstream events in antibody diversification depend on class-switch
DNA recombination (CSR) and somatic hypermutation (SMH). Both CSR and
SMH are influenced by activation-induced cytosine deaminase (AID).
During immune responses, mature B cells diversify immunoglobulin (Ig)
genes through SHM and CSR. SHM alters antibody affinity by introducing
nucleotide changes in the antigen-binding variable region of
antibodies. B cells producing B cell receptors with improved antigen
affinity are positively selected during affinity maturation and before
differentiating into antibody-secreting plasma cells.
The McBride group is breaking new ground in understanding B cell responses in tumor immunology. They are highly focused on comparing the immunoglobulin repertoire from patients who do and do not respond to immune checkpoint blockade (ICB), but they also have many other collaborative projects based on defining the immunoglobulin repertoire in different disease states and experimental conditions.
Tertiary lymphoid structures (TLSs) are lymphoid organs that form in non-lymphoid tissues, often in response to chronic inflammation. In tumors, the presence of TLSs correlates with better overall patient survival and better patient response to immunotherapy. Preliminary evidence from the McBride lab indicates that patients with a positive response to ICB may have B-cells that produce tumor-specific antibodies in the TLS. However, it is unclear how TLSs interact with the tumor microenvironment or how a specific tumor microenvironment impacts B cell function in the TLS/tumor. To better understand these interactions, the McBride Lab is defining the immunoglobulin repertoire of tumor-infiltrating B cells in patients who do and do not respond to immunotherapy in melanoma, renal cell carcinoma, sarcoma, and non-small cell lung cancer.
The unique pipeline developed in the lab for recombinant antibody production has been adapted, as outlined below, for studying human patient samples. In this scheme, human tumor tissue samples are enzymatically digested to release single cells. The cells are sorted and analyzed to identify and isolate B cells recognizing a single antigen through a combination of fluorescence-activated cell sorting (FACS), single cell RNA-seq, and amplification of expressed immunoglobulin genes using RT-PCR. The amplified genes are sequenced and assessed for parameters including mutation profile. The genes are then cloned into expression vectors allowing for recombinant antibody production and testing for a number of applications including immunoblotting, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Overall, this methodology can be used to identify antibodies that recognize tumors, the specific antigens the antibodies recognize, and characterize those antibodies that recognize tumor cell surface antigens. This opens the possibility of identifying and recombinantly producing antibodies with the potential to enhance ICB efficacy in patients who lack a robust response to conventional ICB therapy. (Image created by M. Zelazowska with BioRender.com.)
Projects within this research theme include:
• Defining the immunoglobulin repertoire of tumor-infiltrating B cells in patients undergoing ICB therapy
• Characterizing B cells and the immunoglobulin repertoire in melanoma, head and neck cancer, non-small lung cancer, sarcoma, and ovarian cancer
• Working with immunologists and other collaborators toward the goal of potentiating ICB therapy by evaluating the tumor-recognizing capacity and anti-tumor efficacy of the antibodies identified through the lab's pipeline
B cells and tertiary lymphoid structures promote immunotherapy response. Helmink BA, Reddy SM, Gao J, Zhang S, Basar R, Thakur R, Yizhak K, Sade-Feldman M, Blando J, Han G, Gopalakrishnan V, Xi Y, Zhao H, Amaria RN, Tawbi HA, Cogdill AP, Liu W, LeBleu VS, Kugeratski FG, Patel S, Davies MA, Hwu P, Lee JE, Gershenwald JE, Lucci A, Arora R, Woodman S, Keung EZ, Gaudreau PO, Reuben A, Spencer CN, Burton EM, Haydu LE, Lazar AJ, Zapassodi R, Hudgens CW, Ledesma DA, Ong S, Bailey M, Warren S, Rao D, Krijgsman O, Rozeman EA, Peeper D, Blank CU, Schumacher TN, Butterfield LH, Zelazowska MA, McBride KM, Kalluri R, Allison J, Petitprez F, Fridman WH, Sautès-Fridman C, Hacohen N, Rezvani K, Sharma P, Tetzlaff MT, Wang L, Wargo JA. Nature. 2020 Jan;577(7791):549-555. PMID: 31942075
Mechanisms of Antibody Diversification via DNA Damage and Downstream Repair
The McBride Lab is defining those mechanisms that contribute to antibody diversification with an emphasis on downstream repair mechanisms. When activated, B cells migrate to germinal centers (GC), sites of B cell proliferation, immunoglobulin gene selection, maturation, and death. In the GC, B cells undergo SHM which introduces random DNA mutations into the Ig variable gene. In contrast, CSR rarely takes place in GCs, and is a deletional recombination event in which one heavy chain constant region exon is replaced by another, resulting in isotype switching (e.g. IgG, IgA, IgM) while the variable region is retained, thus also retaining antigen specificity. Both SHM and CSR are initiated and/or catalyzed by activation-induced cytidine deaminase (AID), an enzyme that introduces uracil lesions in single-stranded DNA at the Ig loci. The presence of uracil in DNA is mutagenic, compromises genomic stability, and must be repaired. Uracil lesions are commonly removed by uracil-DNA glycosylases (UNG) via base excision repair resulting in single-nucleotide mutations. Downstream repair of the DNA double-strand breaks created during CSR utilizes non-homologous end-joining mechanisms. Proper repair is needed for generating antibody diversity, maintaining genomic stability, and preventing oncogenic chromosomal translocations (e.g. IgH/MYC).
Projects within this research theme include:
• Identification and analysis of the immunoglobulin repertoire in disease states and other conditions
• Uncovering how mutation-prone versus high-fidelity repair is governed in B cells
• Defining the role of AID phosphorylation in regulating downstream DNA repair and in restricting genomic AID targets
• Characterizing the role of DNA polymerase theta and theta-mediated end-joining in antibody diversification
• Targeting mutagenesis in B cells: Phosphorylation goes beyond AID association. Mu Y, McBride KM. Mol Cell Oncol. 2018 Sep 5;5(5):e1432259. PMID: 30263937
• Phosphorylation promotes activation-induced cytidine deaminase activity at the Myc oncogene. Mu Y, Zelazowska MA, McBride KM. J Exp Med. 2017 Dec 4;214(12):3543-3552. PMID: 29122947
• Mechanism of suppression of chromosomal instability by DNA polymerase POLQ. Yousefzadeh MJ, Wyatt DW, Takata K, Mu Y, Hensley SC, Tomida J, Bylund GO, Doublié S, Johansson E, Ramsden DA, McBride KM, Wood RD. PLoS Genet. 2014 Oct 2;10(10):e1004654. PMID: 25275444
Viral impact on B cell development
B cells, Oncogenic Viruses, and Host-Pathogen Interactions
McBride lab members work at the intersection of virology, oncology, and immunology to discover how members of the gammaherpesvirus (GHV) family subvert B cell function.
Gammaherpesviruses, such as Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KS), establish life-long latent infections that have been linked to the development of lymphomas and other cancers.
Murine herpesvirus 68 (MHV68) infection in mice drives both B
cell proliferation and lymphoma and is as a common model for
studying GHV infection and pathogenesis. It is used by the McBride
lab to identify genes that control GHV pathogenesis and determine
how GHV infection subverts host immune responses.
Upon infection, GHVs lead B lymphocytes to proliferate and diversify their Ig genes while escaping normal immune system controls. After primary infection, both EBV and MHV68 undergo latent expansion in B cells located in germinal centers (GCs) and maintain a pool of virus in isotype class-switched memory B cells. The latent virus will reactive from time-to-time and in some individuals will be triggered to become oncogenic.
The lab is defining the role of uracil DNA glycosylases (UNGs) in GHV pathogenesis and B cell development as well as the roles of host (endogenous) and viral UNG (vUNG) in establishing viral latency/chronic infection. They have discovered that the vUNG of MHV68 alters somatic hypermutation. By using single-cell and GC cell population analysis, they showed that GHV-infected germinal center B cells express a distinct and abnormal immunoglobulin repertoire. Their evidence indicates that the virus alters the immunoglobulin repertoire of the host by subverting host B cell selection and immunoglobulin evolution.
Projects within this research theme include:
• Characterization of the B cell subtype inhabited by
gammaherpesviruses in vivo
• Analyzing mechanisms of gammaherpesvirus subversion of host immunoglobulin repertoire
• Identification and characterization of viral genes involved in host immune subversion
• Defining the differences in how gammaherpesvirus vUNG and host UNG aid DNA repair by biochemically characterizing vUNG
• Dangerous Liaisons: Gammaherpesvirus Subversion of the Immunoglobulin Repertoire. Zelazowska MA, McBride K, Krug LT. Viruses. 2020 Jul 23;12(8):788. PMID: 32717815
• Gammaherpesvirus-infected Germinal Center Cells Express a Distinct Immunoglobulin Repertoire. Zelazowska MA, Dong Q, Plummer JB, Zhong Y, Liu B, Krug LT, McBride KM. Life Sci Alliance. 2020 Feb 6;3(3):e201900526. PMID: 32029571
• Combinatorial Loss of the Enzymatic Activities of Viral Uracil-DNA Glycosylase and Viral dUTPase Impairs Murine Gammaherpesvirus Pathogenesis and Leads to Increased Recombination-Based Deletion in the Viral Genome. Dong Q, Smith KR, Oldenburg DG, Shapiro M, Schutt WR, Malik L, Plummer JB, Mu Y, MacCarthy T, White DW, McBride KM, Krug LT. mBio. 2018 Oct 30;9(5):e01831-18. PMID: 30377280
• Absence of the uracil DNA glycosylase of murine
gammaherpesvirus 68 impairs replication and delays the establishment
of latency in vivo. Minkah N, Macaluso M, Oldenburg DG,
Paden CR, White DW, McBride KM, Krug LT.
J Virol. 2015 Mar;89(6):3366-79. PMID:
The Mark Foundation For Cancer Research: Harnessing tertiary lymphoid structures for improved immunotherapeutic strategies in cancer patients (02/01/2022-1/31/2025)
Cancer Prevention Research Institute of Texas (CPRIT) High Impact High Risk Award, Project number RP200574: Targeting B cells to enhance responses to immune checkpoint blockade (08/31/2020-08/30/2022)
Cancer Prevention Research Institute of Texas (CPRIT) Core Facility Support Award, Project number RP190507: Recombinant Antibody Production Core (RAPC) 08/31/2019-08/30/2024
Leukemia & Lymphoma Society Specialized Center of Research Program Award, Project number SCOR-12206-17: Role of WWOX Loss of Function in High-Risk Multiple Myeloma 10/01/2017-09/30/2022
NIH/NIAID Research Project Grant Program Award, Project number R01AI121403: Modeling ICF syndrome in mice: Role of Zbtb24 in DNA methylation and antibody production (09/26/2016-08/31/2022)
NIH/NIAID Research Project Grant Program Award, Project number R01AI125397: Uracil DNA Glycosylases in Gammaherpesvirus Pathogenesis and B Cell Development (06/01/2016-05/31/2022)
Recombinant Antibody Production Core (RAPC)
Antibodies are a central tool in biomedical science: they allow researchers to identify, purify, and track molecular targets; however, it is not always possible to obtain a supply of replenishable, consistently reliable, and molecularly characterized antibodies. Our process uses single-cell cloning technology to purify cells expressing antigen-specific antibodies via fluorescence-activated cell sorting directly from immunized mice. Immunoglobulin genes are amplified from single cells, cloned into expression vectors, and expressed in vitro to produce antibodies.
Pilot project funding supported establishing and optimizing the protocols used to clone immunoglobulin genes and produce recombinant antibodies. During the pilot phase, the Recombinant Monoclonal Antibody Production Service produced several antibodies and refined their methods for isolating specific immunoglobulin genes for future antibody production. The pilot antibody production service is transforming into a multi-service CPRIT-funded core, under grant RP190507.
Advantages of recombinant monoclonal antibody production technology over traditional hybridoma production include:
- Potential for limitless supply with consistent quality
- Antibody reconfiguration or fusion to epitope tags, fluorescent protein or HRP
- Better suited to producing modification-specific antibodies, as screening can potentially occur prior to cloning and production
Please see our RAPC website for more information.