Increased Frequency of CD4+ Follicular Helper T and CD8+ Follicular T Cells in Human Lymph Node Biopsies during the Earliest Stages of Rheumatoid Arthritis.

Follicular T helper cells (Tfh cells) provide key B-cell help and are essential in germinal center formation and (auto) antibody generation. To gain more insight into their role during the earliest phase of rheumatoid arthritis (RA), we analyzed their frequencies, phenotypes, and cytokine profiles in peripheral blood and lymph node biopsies of healthy controls (HCs), autoantibody-positive individuals at risk for developing RA (RA-risk individuals), and early RA patients. Subsequently, we confirmed their presence in lymph nodes and synovial tissue of RA patients using immunofluorescence microscopy. In the blood, the frequency of Tfh cells did not differ between study groups. In lymphoid and synovial tissues, Tfh cells were localized in B-cell areas, and their frequency correlated with the frequency of CD19+ B cells. Compared to lymphoid tissues of healthy controls, those of RA patients and RA-risk individuals showed more CD19+ B cells, CD4+CXCR5+ follicular helper T cells, and CD8+CXCR5+ follicular T cells. These Tfh cells produced less IL-21 upon ex vivo stimulation. These findings suggest that Tfh cells may present a novel rationale for therapeutic targeting during the preclinical stage of RA to prevent further disease progression.

Human Lymph Node Stromal Cells Have the Machinery to Regulate Peripheral Tolerance during Health and Rheumatoid Arthritis.

BACKGROUND: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. METHOD: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon γ (IFNγ) stimulation (n = 15). RESULTS: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. CONCLUSION: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance.

Impaired lymph node stromal cell function during the earliest phases of rheumatoid arthritis.

BACKGROUND: Systemic autoimmunity can be present years before clinical onset of rheumatoid arthritis (RA). Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) regulate immune responses through their intimate connection with leucocytes. We postulate that malfunctioning of LNSCs creates a microenvironment in which normal immune responses are not properly controlled, possibly leading to autoimmune disease. In this study we established an experimental model for studying the functional capacities of human LNSCs during RA development. METHODS: Twenty-four patients with RA, 23 individuals positive for autoantibodies but without clinical disease (RA risk group) and 14 seronegative healthy control subjects underwent ultrasound-guided inguinal lymph node (LN) biopsy. Human LNSCs were isolated and expanded in vitro for functional analyses. In analogous co-cultures consisting of LNSCs and peripheral blood mononuclear cells, αCD3/αCD28-induced T-cell proliferation was measured using carboxyfluorescein diacetate succinimidyl ester dilution. RESULTS: Fibroblast-like cells expanded from the LN biopsy comprised of fibroblastic reticular cells (gp38+CD31-) and double-negative (gp38-CD31-) cells. Cultured LNSCs stably expressed characteristic adhesion molecules and cytokines. Basal expression of C-X-C motif chemokine ligand 12 (CXCL12) was lower in LNSCs from RA risk individuals than in those from healthy control subjects. Key LN chemokines C-C motif chemokine ligand (CCL19), CCL21 and CXCL13 were induced in LNSCs upon stimulation with tumour necrosis factor-α and lymphotoxin α1ß2, but to a lesser extent in LNSCs from patients with RA. The effect of human LNSCs on T-cell proliferation was ratio-dependent and altered in RA LNSCs. CONCLUSIONS: Overall, we developed an experimental model to facilitate research on the role of LNSCs during the earliest phases of RA. Using this innovative model, we show, for the first time to our knowledge, that the LN stromal environment is changed during the earliest phases of RA, probably contributing to deregulated immune responses early in disease pathogenesis.

Distinctive expression of T cell guiding molecules in human autoimmune lymph node stromal cells upon TLR3 triggering.

Infections are implicated in autoimmunity. Autoantibodies are produced in lymphoid tissue where lymph node stromal cells (LNSCs) regulate lymphocyte function. Infections can alter the interaction between LNSCs and lymphocytes resulting in defective immune responses. In rheumatoid arthritis (RA) autoantibody production precedes clinical disease allowing identification of at risk individuals. We investigated the ability of human LNSCs derived from RA, RA-risk and healthy individuals to sense and respond to pathogens. Human LNSCs cultured directly from freshly collected lymph node biopsies expressed TLR1-9 with exception of TLR7. In all donors TLR3 triggering induced expression of ISGs, IL-6 and adhesion molecules like VCAM-1 and ICAM-1. Strikingly, T cell guiding chemokines CCL19 and IL-8 as well as the antiviral gene MxA were less induced upon TLR3 triggering in autoimmune LNSCs. This observed decrease, found already in LNSCs of RA-risk individuals, may lead to incorrect positioning of lymphocytes and aberrant immune responses during viral infections.

Brief Report: Altered Innate Lymphoid Cell Subsets in Human Lymph Node Biopsy Specimens Obtained During the At-Risk and Earliest Phases of Rheumatoid Arthritis.

OBJECTIVE: Innate lymphoid cells (ILCs) are emerging mediators of immunity, and accumulation of inflammatory ILC populations can occur in inflammatory-mediated conditions. Since early lymph node (LN) activation has been shown in rheumatoid arthritis (RA), we aimed to investigate the frequency and distribution of ILCs in LN biopsy specimens obtained during the earliest phases of RA. METHODS: Twelve patients with early RA, 12 individuals with IgM rheumatoid factor and/or anti-citrullinated protein antibodies without arthritis (RA risk group), and 7 healthy controls underwent ultrasound-guided inguinal LN biopsy. ILC subsets and the expression of vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) by LN endothelial cells and fibroblasts were analyzed by flow cytometry. RESULTS: Although no differences in the frequencies of total ILCs (Lin-CD45+/low CD127+) were found, the distribution of the ILC subpopulations differed among groups. RA patients showed lower numbers of lymphoid tissue-inducer (LTi) cells (c-Kit+NKp44- ILCs) and increased ILC1 (c-Kit-NKp44- ILCs) and ILC3 (c-Kit+NKp44+ ILCs) numbers compared with controls (P < 0.001, P < 0.050, and P < 0.050, respectively). Individuals at risk of RA exhibited an increased frequency of ILC1 compared with controls (P < 0.01). LTi cells paralleled the expression of adhesion molecules on endothelial cells and fibroblasts. CONCLUSION: Our findings indicate that during the at-risk and earliest phases of RA, the ILC distribution in LN changes from a homeostatic profile toward a more inflammatory profile, thereby providing evidence of a role for ILCs in RA pathogenesis.