Fabian Flores-Borja

CD19+CD24hiCD38hi B Cells Exhibit Regulatory Capacity in Healthy Individuals but Are Functionally Impaired in Systemic Lupus Erythematosus Patients

The immunosuppressive function of regulatory B cells has been shown in several murine models of chronic inflammation, including collagen-induced arthritis, inflammatory bowel disease, and experimental autoimmune encephalomyelitis. Despite interest in these cells, their relevance to the maintenance of peripheral tolerance in humans remains elusive. Here, we demonstrate that human CD19(+)CD24(hi)CD38(hi) B cells possessed regulatory capacity. After CD40 stimulation, CD19(+)CD24(hi)CD38(hi) B cells suppressed the differentiation of T helper 1 cells, partially via the provision of interleukin-10 (IL-10), but not transforming growth factor-beta (TGF-beta), and their suppressive capacity was reversed by the addition of CD80 and CD86 mAbs. In addition, CD19(+)CD24(hi)CD38(hi) SLE B cells isolated from the peripheral blood of systemic lupus erythematosus (SLE) patients were refractory to further CD40 stimulation, produced less IL-10, and lacked the suppressive capacity of their healthy counterparts. Altered cellular function within this compartment may impact effector immune responses in SLE and other autoimmune disorders.

CD19+CD24hiCD38hi B Cells Maintain Regulatory T Cells While Limiting TH1 and TH17 Differentiation

Patients with rheumatoid arthritis have fewer regulatory B cells with decreased function, which may be associated with inflammation. Regulatory B Cells Out of Joint In the theater of the adaptive immune system, B cells and T cells are the two leads. Yet, within these cell subsets are many supporting actors, including cells that regulate the function of their more illustrious counterparts. Although regulatory T cells (Tregs) have long stood center stage in the study of autoimmune disease, another subset of regulatory cells—regulatory B cells (Bregs)—remains less well understood. Flores-Borja et al. now compare the function of Bregs in both healthy individuals and patients with rheumatoid arthritis (RA), a chronic inflammatory disease of the joints. The authors found that in healthy individuals, Bregs inhibit the differentiation of naïve T cells into the proinflammatory T helper 1 (TH1) and TH2 subsets and can induce differentiation into Tregs, largely through interleukin-10 secretion. Patients with RA have fewer Bregs, and the Bregs that are present behave differently. Although they maintained the ability to inhibit TH1 cell differentiation, Bregs from RA patients could not inhibit TH17 cell differentiation or promote Treg conversion. These data suggest that in patients with active RA, defects in Bregs may contribute to autoimmunity. The relevance of regulatory B cells in the maintenance of tolerance in healthy individuals or in patients with immune disorders remains understudied. In healthy individuals, CD19+CD24hiCD38hi B cells suppress CD4+CD25− T cell proliferation as well as the release of interferon-γ and tumor necrosis factor–α by these cells; this suppression is partially mediated through the production of interleukin-10 (IL-10). We further elucidate the mechanisms of suppression by CD19+CD24hiCD38hi B cells. Healthy CD19+CD24hiCD38hi B cells inhibited naïve T cell differentiation into T helper 1 (TH1) and TH17 cells and converted CD4+CD25− T cells into regulatory T cells (Tregs), in part through the production of IL-10. In contrast, CD19+CD24hiCD38hi B cells from patients with rheumatoid arthritis (RA) failed to convert CD4+CD25− T cells into functionally suppressive Tregs or to curb TH17 development; however, they maintained the capacity to inhibit TH1 cell differentiation. Moreover, RA patients with active disease have reduced numbers of CD19+CD24hiCD38hi B cells in peripheral blood compared with either patients with inactive disease or healthy individuals. These results suggest that in patients with active RA, CD19+CD24hiCD38hi B cells with regulatory function may fail to prevent the development of autoreactive responses and inflammation, leading to autoimmunity.

Defects in CTLA-4 are associated with abnormal regulatory T cell function in rheumatoid arthritis

The ultimate goal for the treatment of autoimmunity is to restore immunological tolerance. Regulatory T cells (Treg) play a central role in immune tolerance, and Treg functional abnormalities have been identified in different autoimmune diseases, including rheumatoid arthritis (RA). We have previously shown that natural Treg from RA patients are competent at suppressing responder T cell proliferation but not cytokine production. Here, we explore the hypothesis that this Treg defect in RA is linked with abnormalities in the expression and function of CTLA-4. We demonstrate that CTLA-4 expression on Treg from RA patients was significantly reduced compared with healthy Treg and is associated with an increased rate of CTLA-4 internalization. Regulation of T cell receptor signaling by CTLA-4 was impaired in RA Treg and associated with delayed recruitment of CTLA-4 to the immunological synapse. Artificial induction of CTLA-4 expression on RA Treg restored their suppressive capacity. Furthermore, CTLA-4 blockade impaired healthy Treg suppression of T cell IFN-γ production, but not proliferation, thereby recapitulating the unique Treg defect in RA. Our results suggest that defects in CTLA-4 could contribute to abnormal Treg function in RA and may represent a target for therapy for inducing long-lasting remission.

Abnormal CTLA-4 function in T cells from patients with systemic lupus erythematosus.

CTLA‐4 is a critical gatekeeper of T‐cell activation and immunological tolerance and has been implicated in patients with a variety of autoimmune diseases through genetic association. Since T cells from patients with the autoimmune disease systemic lupus erythematosus (SLE) display a characteristic hyperactive phenotype, we investigated the function of CTLA‐4 in SLE. Our results reveal increased CTLA‐4 expression in FOXP3− responder T cells from patients with SLE compared with other autoimmune rheumatic diseases and healthy controls. However, CTLA‐4 was unable to regulate T‐cell proliferation, lipid microdomain formation and phosphorylation of TCR‐ζ following CD3/CD28 co‐stimulation, in contrast to healthy T cells. Although lupus T cells responded in vitro to CD3/CD28 co‐stimulation, there was no parallel increase in CTLA‐4 expression, which would normally provide a break on T‐cell proliferation. These defects were associated with exclusion of CTLA‐4 from lipid microdomains providing an anatomical basis for its loss of function. Collectively our data identify CTLA‐4 dysfunction as a potential cause for abnormal T‐cell activation in patients with SLE, which could be targeted for therapy.

IL-10 secreting regulatory B cells are potent arbiters of autoimmunity in both mouse and man

Background The existence of B cells with a suppressive function, namely regulatory B cells (Bregs), has been demonstrated in a number of murine models of autoimmunity in the last decade. Adoptive transfer of Bregs has been shown to inhibit the development of several autoimmune diseases via the release of Interleukin-10 (IL-10). Aim To ascertain the role of IL-10 secreting Bregs during the induction and progression of experimental autoimmune disease in mice.Then to use this information to identify an equivalent subset in man and assess the impact of Bregs on inflammatory responses and disease severity. Materials and methods We generated chimeric mice that lack IL-10 specifically in their B cells and used this model to investigate the role of endogenous B cell- derived IL-10 during inflammatory arthritis. Moreover, we have identified and assessed the role of Bregs in healthy individuals as well as patients with rheumatological disorders. Results We have recently investigated the mechanisms utilized by endogenous Breg at the cellular level in an experimental model of arthritis and shown that the IL-10 produced by Bregs is important in the maintenance of regulatory T cells (Tregs) whilst suppressing Th1 and Th17 responses. Indeed chimeric mice specifically lacking IL-10 producing B cells (IL-10 -/- B cell) developed an exacerbated arthritis, displayed reduced absolute numbers of FoxP3+ Tregs, and a marked increase in inflammatory Th1 and Th17 cells compared to chimeric wild type B cell mice. In addition we extended our results from experimental models of arthritis to humans and identified an equivalent population of Breg that produce IL-10, inhibit CD4 + T effector responses and convert effector CD4+T cells into regulatory T cells. Conclusions Our work has demonstrated the importance of Bregs in controlling inflammatory responses and preventing autoimmunity in both experimental arthritis in mice [1,2] and patients with rheumatological disorders [3]. This presentation reports the function of this pivotal B cell subset and describes Bregs in the context of healthy individuals as well as in patients with systemic lupus erythematosus and rheumatoid arthritis.The impact of altered cellular function within the Breg compartment on disease outcome will be also discussed [4,5].

Protein phosphorylation and kinome profiling reveal altered regulation of multiple signaling pathways in B lymphocytes from patients with systemic lupus erythematosus

OBJECTIVE The cause of B lymphocyte hyperactivity and autoantibody production in systemic lupus erythematosus (SLE) remains unclear. Previously, we identified abnormalities in the level and translocation of signaling molecules in B cells in SLE patients. The present study was undertaken to examine the extent of signaling abnormalities that relate to altered B cell responses in SLE. METHODS B lymphocytes from 88 SLE patients and 72 healthy controls were isolated from blood by negative selection. Protein tyrosine phosphorylation and cellular kinase levels were analyzed by Western blotting, flow cytometry, and a kinome array protocol. Changes in protein phosphorylation were determined in ex vivo B cells and following B cell receptor engagement. RESULTS Differences in tyrosine phosphorylation in B cells from patients with SLE, compared with matched controls, were demonstrated. Further, the kinome array analysis identified changes in the activation of key kinases, i.e., the activity of phosphatidylinositol 3-kinase, which regulates survival and differentiation, was up-regulated and the activity of Rac and Rho kinases, which regulate the cytoskeleton and migration, was increased. In contrast, the activity of ATR, which regulates the cell cycle, was down-regulated in SLE patients compared with controls. Differences in signaling pathways were seen in all SLE B lymphocyte subsets that manifested phenotypic features of immature, mature, and memory cells. CONCLUSION This study revealed dysregulation in multiple signaling pathways that control key responses in B cells of SLE patients. Data generated in this study provide a molecular basis for further analysis of the altered B lymphocyte responses in SLE.

Restoring the balance: Harnessing regulatory T cells for therapy in rheumatoid arthritis

Treg play a vital role in the maintenance of tolerance to self antigens, thereby preventing disease through the active suppression of proliferation and pro‐inflammatory cytokine production by autoreactive T cells. Here we discuss strategies aimed at enhancing Treg function in patients with rheumatoid arthritis with the ultimate aim of restoring lasting tolerance but without increasing the risk of infections or cancer.

RORγt+ innate lymphoid cells promote lymph node metastasis of breast cancers

Cancer cells tend to metastasize first to tumor-draining lymph nodes, but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here, we show that in the human breast tumor microenvironment (TME), the presence of increased numbers of RORγt+ group 3 innate lymphoid cells (ILC3) correlates with an increased likelihood of lymph node metastasis. In a preclinical mouse model of breast cancer, CCL21-mediated recruitment of ILC3 to tumors stimulated the production of the CXCL13 by TME stromal cells, which in turn promoted ILC3-stromal interactions and production of the cancer cell motile factor RANKL. Depleting ILC3 or neutralizing CCL21, CXCL13, or RANKL was sufficient to decrease lymph node metastasis. Our findings establish a role for RORγt+ILC3 in promoting lymphatic metastasis by modulating the local chemokine milieu of cancer cells in the TME. Cancer Res; 77(5); 1083-96. ©2017 AACR.

Optical In Vivo Imaging of the Alarmin S100A9 in Tumor Lesions Allows for Estimation of the Individual Malignant Potential by Evaluation of Tumor–Host Cell Interaction

Tumors recruit and reprogram immune cells to support tumor development and spread, the most prominent among them being of monocytic origin such as tumor-associated macrophages (TAM) or myeloid-derived suppressor cells (MDSC). The alarmin S100A8/A9 has been implicated in the induction of TAM and MDSC. We assessed S100A9 as a molecular imaging marker for the activity of tumor-associated immune cells in a syngeneic murine breast cancer model. S100A9 could serve as a surrogate marker for tumor immune crosstalk as a function of malignancy, providing a tool with the potential for both basic research in tumor immunology and clinical stratification of patients. Methods: BALB/c mice were inoculated with murine breast cancer cells of common origin but different metastatic capability. At different times during tumor development, optical imaging was performed using a S100A9-specific probe to visualize activated monocytes. To further explore the impact of tumor-educated monocytes, splenic myeloid cells were isolated from either healthy or tumor-bearing animals and injected into tumor-bearing mice. We analyzed the effect of the cell transfer on immune cell activity and tumor development. Results: We could prove S100A9-driven imaging to sensitively and specifically reflect monocyte activity in primary tumor lesions. The imaging results were corroborated by histology and fluorescence-activated cell sorting analyses. In a prospective experiment, S100A9 imaging proved indicative of the individual tumor growth, with excellent correlation. Moreover, we could show that the monocyte activity as depicted by S100A9 activity in the primary tumor lesion mirrored the tumors metastatic behavior. Treatment with tumor-primed splenic monocytes induced increased tumor growth, accompanied by an augmented infiltration of activated myeloid cells (MDSC and TAM) into the tumor. The consecutive S100A9 expression as depicted by in vivo imaging was significantly increased. Conclusion: S100A9 proved to be a sensitive and specific marker for the activity of tumor-associated immune cells. To our knowledge, S100A9 imaging represents a first in vivo imaging approach for the estimation of recruitment and activity of tumor-associated myeloid immune cells. We demonstrated the potential value of this imaging approach for prediction of local and systemic tumor development.

B-Lymphocyte Signalling Abnormalities and Lupus Immunopathology

Lupus is a complex autoimmune rheumatic disease of unknown aetiology. The disease is associated with diverse features of immunological abnormality in which B-lymphocytes play a central role. However, the cause of atypical B-lymphocyte responses remains unclear. In this article, we provide a synopsis of current knowledge on intracellular signalling abnormalities in B-lymphocytes in lupus and their potential effects on the response of these cells in mouse models and in patients. There are numerous reported defects in the regulation of intracellular signalling proteins and pathways in B-lymphocytes in lupus that, potentially, affect critical biological responses. Most of the evidence for these defects comes from studies of disease models and genetically engineered mice. However, there is also increasing evidence from studying B-lymphocytes from patients and from genome-wide linkage analyses for parallel defects to those observed in mice. These studies provide molecular and genetic explanations for the key immunological abnormalities associated with lupus. Most of the new information appears to relate to defects in intracellular signalling that impact B-lymphocyte tolerance, cytokine production and responses to infections. Some of these abnormalities will be discussed within the context of disease pathogenesis.