Research

The O’Connor laboratory is part of the Department of Neurology and program in Human Translational Immunology (HTI) at Yale School of Medicine. Our major research interests include human neuroimmunology, autoimmunity and tumor immunology. The aim of our research is to further elucidate the role that the immune system plays in neurological disease. We are specifically interested in defining the mechanisms by which B cells, and the antibodies they produce, affect tissue damage in autoimmunity and participate in tumor biology. To this end we are engaged in determining the specificity of autoantibodies and understanding the mechanisms by which B cells affect tissue damage and organize at sites of such injury. Areas of special interest in our autoimmunity programs include multiple sclerosis, inflammatory myopathy and myasthenia gravis. Our cancer program is currently focused on meningiomas and germ cell tumors.

Multiple Sclerosis

B-cell depletion therapy in MS results in a remarkable reduction in new inflammatory brain lesions and clinical relapses, indicating that B cells contribute to MS pathology. How they do so is not well understood. We have built a program that is focused on furthering the understanding of the role that B cells and antibodies play in the pathology of MS. To this end, my group is investigating the following fundamental questions in MS: What are the antigen targets of MS B cells?Through the application of novel technologies, we demonstrated that autoantibodies to the encephalogenic myelin antigens, MBP and MOG are uncommon in the serum and CSF of adult patients with MS [1-4]. This turned our interest toward the B cells that accumulate at the site of tissue injury, the MS CNS. Here, we are elucidating the antigen specificity of B cells that reside within MS lesions. Our strategy harnesses the adaptive response of the humoral immune system by using the machinery of the B cells present in these tissues to isolate autoantigens [5]. Laser capture micro-dissection is used to isolate single B cells directly from CNS tissue. Single-cell PCR then amplifies the antibody variable regions, sequencing of which provides information concerning expanded clones and evidence of those that have experienced antigen-driven stimulation. Then, large amounts of recombinant, whole IgG from selected clones are produced. The IgG is then used to capture antigens from candidate sources. The precise molecular definition of isolated antigen(s) is then determined through sophisticated mass spectrometric methodologies. To date, we have isolated and identified a number of candidate autoantigens, validation of which is under way. What is the relationship between the antibodies and the B cells present in the CSF and CNS tissue and to those in the periphery?While B cells and antibodies are present in the CSF and CNS tissue of patients with MS, the relationship among them is not understood. We have constructed immunoglobulin transcriptomes and proteomes from CNS tissue and CSF that allowed us to determine that these compartmentalized B cells and antibodies are clonally related [6, 7].

We have now turned our attention toward determining how these cells are related to the peripheral B cell repertoire. We are currently using high-throughput sequencing to build immunoglobulin transcriptomes from the cervical lymph nodes, which will be compared to those derived from CNS lesions and the CSF. How do MS CNS B cells function as antigen-presenting cells?We are developing a model system engineered to examine the role of human MS CNS-derived immunoglobulin in the pathogenic progression of CNS demyelination. Specifically, we will build a mouse model genetically engineered to develop B cells expressing immunoglobulin derived from pervasive human MS CNS clones. This model will allow the study of the contribution that MS CNS-derived immunoglobulin and B cells make to CNS demyelination and tissue injury.


References

1. O'Connor KC, McLaughlin KA, De Jager PL, Chitnis T, Bettelli E, Xu C, Robinson WH, Cherry SV, Bar-Or A, Banwell B, et al: Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein. Nat Med2007, 13:211-217.

2. O'Connor KC, Chitnis T, Griffin DE, Piyasirisilp S, Bar-Or A, Khoury S, Wucherpfennig KW, Hafler DA: Myelin basic protein-reactive autoantibodies in the serum and cerebrospinal fluid of multiple sclerosis patients are characterized by low-affinity interactions. J Neuroimmunol2003, 136:140-148.

3. O'Connor KC, Appel H, Bregoli L, Call ME, Catz I, Chan JA, Moore NH, Warren KG, Wong SJ, Hafler DA, Wucherpfennig KW: Antibodies from inflamed central nervous system tissue recognize myelin oligodendrocyte glycoprotein. J Immunol2005, 175:1974-1982.

4. O'Connor KC, Lopez-Amaya C, Gagne D, Lovato L, Moore-Odom NH, Kennedy J, Krupp L, Tenembaum S, Ness J, Belman A, et al: Anti-myelin antibodies modulate clinical expression of childhood multiple sclerosis. J Neuroimmunol2010, 223:92-99.

5. Owens GP, Bennett JL, Lassmann H, O'Connor KC, Ritchie AM, Shearer A, Lam C, Yu X, Birlea M, DuPree C, et al: Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid. Ann Neurol2009, 65:639-649.

6. Lovato L, Willis SN, Rodig SJ, Caron T, Almendinger SE, Howell OW, Reynolds R, O'Connor* KC, Hafler* DA: Related B cell clones populate the meninges and parenchyma of patients with multiple sclerosis (*co-senior authors). Brain2011, 134:534-541.

7. Obermeier B, Lovato L, Mentele R, Bruck W, Forne I, Imhof A, Lottspeich F, Turk KW, Willis SN, Wekerle H, et al: Related B cell clones that populate the CSF and CNS of patients with multiple sclerosis produce CSF immunoglobulin. J Neuroimmunol2011, 233:245-248.

Myasthenia Gravis

Autoreactive B-cells are thought to play an important role in the immunopathogenesis of MG. Early studies, including one at Yale, indicate that B cell depletion therapy is beneficial to MG patients [8]. It is unclear what specific changes in the immune system are associated with the observed clinical improvement. Accordingly, the rationale for this work is to further understand rituximab’s mechanism of action by measuring its effects on immunity in MG. Molecular and cellular immune system components, which putatively participate in the pathology of MG, will be measured at points prior to, during and after rituximab-mediated B cell depletion. We are developing/adapting immunoassays so that we can measure: Autoantibody titer of MuSK and AChR; Antigen specific B cells and plasma cells; Repertoire and characteristics of antigen specific B cells; Antigen specific T-helper cells; Cytokine profiles of antigen specific T cells. We expect to identify changes in the immune system of MG patients undergoing treatment and these data will be related to measurements of clinical outcome. This work represents a first step toward gaining a more complete understanding of the immune mechanisms underlying treatment of MG with rituximab and will lead to new ways to prevent or treat the disease.

References

8. Nowak RJ, DiCapua DB, Zebardast N, Goldstein JM: Response of patients with refractory myasthenia gravis to rituximab: a retrospective study. Therapeutic Advances in Neurological Disorders 2011, 4:259-266.

The Inflammatory Myopathies

The inflammatory myopathies are a group of autoimmune diseases characterized by progressive skeletal muscle weakness associated with inflammatory cell infiltration within the muscle. The muscle mRNA from a subset of IM patients harbors an abundance of Ig transcripts. Although sparse numbers of CD19 or CD20 B cells are present in IM muscle tissue, we reported that large numbers of CD138 plasma cells are present and that these cells are the source of the Ig transcripts. These findings led us to hypothesize that we would find evidence of an Ag-driven immune response in the tissue of patients with particular IMs. Through investigating the B cell and plasma cell Ig repertoire in muscle biopsies we confirmed that such an antigen-driven response was occurring in this autoimmune tissue [9]. We then set out to identify the antigen driving this response using recombinant IgG derived from plasma cells harbored in the muscle tissue. We are currently evaluating the validity of a novel muscle-associated autoantigen isolated using this strategy.


References

9. Bradshaw EM, Orihuela A, McArdel SL, Salajegheh M, Amato AA, Hafler DA, Greenberg SA, O'Connor KC: A local antigen-driven humoral response is present in the inflammatory myopathies. J Immunol 2007, 178:547-556.

Our Cancer Program

Our cancer program is currently focused on meningiomas and germ cell tumors. These tumors invariably harbor an immune cell infiltrate comprised, in part, of B cells, yet little is known of their role. We are using novel strategies to understand further the characteristics of the B cells commonly found within these tumors. A variety of procedures for the isolation of tumor antigens exist, yet the identification of real and reliable tumor-specific structures is difficult. Thus, our goal is to identify tumor-specific antigen(s) by harnessing the adaptive response of the humoral immune system present in the tumor microenvironment. Such molecular tools represent a sophisticated and reliable strategy both to investigate tumor-associated antibody specificity and to discover novel, tumor-associated antigens that can be used as potential targets in tumor diagnostics or therapeutics. Our work has focused on germ cell tumors, a principal feature of which is that they invariably harbor a prominent immune cell infiltrate. Data collected by our group indicate that germ cell tumors drive tumor-associated B cells toward antibody production that targets specific tumor antigens [10]. To isolate these antigens we have adapted the strategy used in our MS antigen discovery program (described above). The identification of tumor-specific antigens represents a fundamental step toward understanding the biology and the role of the immune system in this tumor family. Moreover, novel tumor antigens may lead to the development of targeted immunotherapy that may be highly efficacious and better tolerated than current treatment strategies.


References

10. Willis SN, Mallozzi SS, Rodig SJ, Cronk KM, McArdel SL, Caron T, Pinkus GS, Lovato L, Shampain KL, Anderson DE, et al: The microenvironment of germ cell tumors harbors a prominent antigen-driven humoral response. J Immunol 2009, 182:3310-3317.