Gunilla Karlsson Hedestam Group Projects
B cell repertoire analysis following immunization
B cell responses are the basis by which most effective anti-viral vaccines provide protection against infection and disease. Promising candidates are still lacking for many human pathogens and in other cases vaccines exist but do not provide sufficiently broad or long-lasting responses http://www.ncbi.nlm.nih.gov/pubmed/18197170. A robust understanding of B cell biology and the mechanisms underlying B cell activation and persistence is needed to design improved immunization approaches. The circulating B cell repertoire is comprised of naïve B cells with potential to respond, expand and differentiate into memory B cells or antibody-producing plasma cells after antigen exposure.
Examination of antigen-specific memory B cell repertoires comprehensively surveys the B cell clones engaged by a specific antigen following infection or immunization. In this project, we study responses elicited by recombinant HIV-1 envelope glycoproteins (Env), an important neutralizing antibody target. Currently, there is limited information about the genetic and functional properties of antibody specificities elicited by immunization that can be found archived in peripheral memory B cells or in long-lived bone marrow-resident plasma cells. Whether the peripheral memory B cell repertoire is qualitatively different from the bone marrow plasma cell repertoire against a given antigen is insufficiently understood. In this project, we address these questions using single cell and deep sequencing approaches.
Isolation and analysis of vaccine-elicited monoclonal antibodies
HIV-1 is highly resistant to antibody neutralization and understanding why is a necessary first step toward the design of effective vaccine candidates. Characterization of vaccine-induced monoclonal antibodies provides higher resolution information about the response, which together with analysis of unfractionated polyclonal plasma samples delineate the response elicited by a given vaccine candidate. Until recently, approaches to isolate monoclonal antibodies (MAbs) were relatively cumbersome but with improved methods to sort antigen-specific B cell populations by flow cytometry, coupled with growing genetic databases of antibody heavy and light chain germline gene segments and the availability of vectors for efficient expression of antibodies in mammalian cells, improved opportunities to investigate antigen-specific antibody repertoires and to isolate MAbs have become available. In this project, we examine HIV-1 Env-specific memory B cells and use single cell sorting protocol to allow sequencing and cloning of matching V(D)J transcripts for genetic, functional and structural analyses of elicited MAbs. By using this approach, we have characterized a panel of MAbs directed against the CD4 binding site (CD4bs) of HIV-1 and compared their binding specificities to those of broadly neutralizing infection-induced antibodies including VRC01 http://www.ncbi.nlm.nih.gov/pubmed/22786681 and http://www.ncbi.nlm.nih.gov/pubmed/24550318. These results suggest that differences in how antibodies access the highly conserved CD4bs play a major role for their capacities to bind and neutralize the functional Env spike on infectious HIV virions. Furthermore, for vaccine-induced responses to be effective, B cells with appropriate sub-specificities need to be archived in the peripheral memory B cell pool and in the long-lived plasma cell compartment in the bone marrow. The efficiency by which B cells targeting conserved epitopes on HIV-1 Env are recruited into these compartments following immunization is not known. The availability of monoclonal antibodies will allow Ab lineage tracing using the larger data set generated from deep sequencing providing new and highly detailed information about the development of antigen-specific antibodies.
Mechanistic studies of mice with defects in humoral immune responses
Antibody-producing B cells are divided into B-1 and B-2 subsets. B-1 cells are produced first during ontogeny, while conventional B-2 cells are produced in later developmental steps. B-2 cells are further subdivided into follicular B cells and marginal zone B cells (MZB). B-1 cells are important for providing effective immune responses against a number of pathogens and are also major IgM and IL-10 producing cells, thus playing a role in homeostasis and immune regulation. Whilst the developmental pathways of B-2 cells have been described in detail, less is known about B-1 cell development. In this project, we are analyzing mouse strains identified from an ENU screen for mice that are unable to respond to T-dependent (TD) and/or T-independent (TI) antigens. Our current work focuses on a strain known as bumble, which displays normal numbers of follicular B cells but severely reduced numbers of B-1 cells and reduced numbers of MZB cells. http://www.ncbi.nlm.nih.gov/pubmed/22761313. The bumble mice carry a point mutation in IkBNS, a relatively recently identified regulator of the NFkB pathway. We are currently investigating where the developmental block in bumble B-1 cells occurs http://www.ncbi.nlm.nih.gov/pubmed/25228759. As part of this project, we are also using other mouse strains with mutations in components of the canonical or the non-canonical NFkB pathway to define the roles of these signaling pathways for B cell development, function and homeostatis. The ENU screen for mice displaying defective Ab responses to TD and TI antigens is performed in collaboration with the Beutler laboratory at UT Southwestern, see http://mutagenetix.utsouthwestern.edu, using new methods for automated mapping of point-mutations allowing real-time resolution of genetic causes of the immune dysfunctions identified in the screen.
Genetic characterization of B cell receptor repertoires in cancer
With the development of high-throughput sequencing approaches of immunoglobulins and analysis tools based on the IgDiscover platform, we are now in a position to define immunoglobulin repertoires of B cell populations in cancer. The unique sequence identity of the B cell receptor (BCR) provides a powerful identity tag to characterize cellular origins, clonal expansions and evolution of B cell responses in the context of tumorigenesis. Building upon our ongoing collaborations with the laboratories of Mikael Karlsson (MTC) and Robert Månsson (HERM), we are applying high-throughput immunoglobulin repertoire sequencing to human leukemia samples as well as to tumor-infiltrating B cells in mouse models. The aims are to investigate if a) human B cell chronic lymphocytic leukemia (CLL) is a progenitor-derived disease comprised of several malignant B cell clones and b) whether a limited number of B cell specificities are recruited to the tumor microenvironment to modulate tumor growth in models of solid tumors, including melanoma and breast cancer, and whether the encoded antibodies have a protective role. The IgDiscover pipeline is optimized for both Ig heavy (H) and light (Igk/l) chain analysis and includes stringent methods for error correction using unique molecular identifiers (UMIs) build into the primer sequences. The output provides data on Ig VDJ segment usage, clonal relationship and somatic hypermutation (SHM), offering a state-of-the-art approach to Ig repertoire analysis in tumorigenesis.