Bob1 maintains T follicular helper cells for long-term humoral immunity.

Humoral immunity is vital for host protection, yet aberrant antibody responses can trigger harmful inflammation and immune-related disorders. T follicular helper (Tfh) cells, central to humoral immunity, have garnered significant attention for unraveling immune mechanisms. This study shows the role of B-cell Oct-binding protein 1 (Bob1), a transcriptional coactivator, in Tfh cell regulation. Our investigation, utilizing conditional Bob1-deficient mice, suggests that Bob1 plays a critical role in modulating inducible T-cell costimulator expression and cellular respiration in Tfh cells. This regulation maintains the long-term functionality of Tfh cells, enabling their reactivation from central memory T cells to produce antibodies during recall responses. In a bronchial asthma model induced by house dust mite (HDM) inhalation, Bob1 is observed to enhance HDM-specific antibodies, including IgE, highlighting its pivotal function in Tfh cell regulation. Further exploration of Bob1-dependent mechanisms in Tfh cells holds promise for governing protective immunity and addressing immune-related disorders.

Circulating T follicular helper 2 cells, T follicular regulatory cells and regulatory B cells are effective biomarkers for predicting the response to house dust mite sublingual immunotherapy in patients with allergic respiratory diseases.

The relationships between T follicular helper (Tfh) cells and antigen-specific immunoglobulins (sIgs) in patients with allergic respiratory diseases who are receiving antigen immunotherapy (AIT) have not been fully clarified. Therefore, we started to perform house dust mite sublingual immunotherapy (HDM-SLIT) for 20 patients with atopic asthma comorbid with allergic rhinitis (AA+AR) who were already receiving ordinary treatments including inhaled corticosteroid (ICS). We examined percentages of circulating T follicular helper (cTfh) and regulatory (cTfr) cells and percentages of circulating regulatory T (cTreg) and B (cBreg) cells by FACS and we examined levels of Der-p/f sIgs by ELISA. Based on the symptom score (asthma control questionnaire: ACQ) and medication score ((global initiative for asthma: GINA) treatment step score) in patients with AA, the patients were divided into responders and non-responders. The percentage of cTfh2 cells significantly decreased and the percentage of cTfh1 cells significantly increased within the first year. Der-p/f sIgEs decreased after a transient elevation at 3 months in both groups. Notably, the percentage of cTfh2 cells and the ratio of cTfh2/cBreg cells and Der-p/f sIgEs greatly decreased in responders from 6 months to 12 months. The percentages of cTfr and cTreg cells showed significant negative correlations with the percentage of cTfh2 cells. The percentage of IL-4+ cTfh cells were significantly decreased and the percentage of IFN-γ+ cTfh cells were increased before treatment to 24 months in 6 patients examined (4 responders and 2 non-responders). We performed multi plelogistic regression analysis based on these results, the ratios of cTfh2/cTfr cells and cTfh2/cBreg cells at the start of therapy were statistically effective biomarkers for predicting the response to HDM-SLIT in patients with AA+AR.

Immunohistochemical analysis of arachidonate 5-lipoxygenase expression in B-cell lymphomas: Implication for B cell differentiation and its analogy with lymphomagenesis.

Arachidonate 5-lipoxygenase (ALOX5) is a cardinal enzyme in the synthesis of leukotrienes, which are powerful immune-regulating lipid mediators. We previously reported that ALOX5 is preferentially expressed in B lymphocytes in the mantle zone of human lymphoid tissue. In the context of physiological relevance, the loss of the Alox5 gene in mice significantly impairs the development of follicular B helper T cells and antibody production. However, ALOX5 expression in B-cell lymphomas has not been investigated in detail. In this study, we examined ALOX5 expression in representative B-cell lymphomas and non-neoplastic lymphoid tissues by immunohistochemistry with a commercially available anti-ALOX5 antibody that can be used on formalin-fixed paraffin-embedded specimens. Interestingly, 22/22 cases of mantle cell lymphoma, 7/7 cases of chronic lymphocytic leukemia/small cell lymphoma, and 20/20 cases of follicular lymphoma expressed ALOX5. A small proportion of extranodal marginal zone lymphoma/mucosa-associated lymphoid tissue lymphoma or nodal marginal zone lymphoma cases were positive for ALOX5 (2/13 or 1/3, respectively). In contrast, no cases with diffuse large B-cell lymphoma, regardless of germinal center B cell (GCB) or non-GCB type, expressed ALOX5 (0/25 cases). These findings suggest that ALOX5 may be a novel marker for identifying the cell of origin of B-cell lymphoma. Further investigation is required to clarify the biological significance of ALOX5 expression in lymphoma cells.

CD4+CD8+ T follicular helper cells regulate humoral immunity in chronic inflammatory lesions.

T follicular helper (Tfh) cells drive humoral immunity by facilitating B cell responses at the initial and recall phases. Recent studies have indicated the possible involvement of Tfh cells in the process of chronic inflammation. However, the functional role of Tfh cells in persistent immune settings remains unclear. Here, we report that CD4+CD8+ (double-positive, DP; CD3+CD4+CD8+CXCR5hiPD-1hi) Tfh cells, a subset of germinal-center-type Tfh cells, were abundantly present in the fibroinflammatory lesions of patients with immunoglobulin G4-related disease (IgG4-RD). Transcriptome analyses showed that these DP-Tfh cells in the lesions of IgG4-RD preferentially expressed signature genes characteristic of cytotoxic CD8+ T cells, such as Eomes, CRTAM, GPR56, and granzymes, in addition to CD70. Scatter diagram analyses to examine the relationships between tissue-resident lymphocytes and various clinical parameters revealed that the levels of DP-Tfh cells were inversely correlated to the levels of serum IgG4 and local IgG4-expressing (IgG4+) memory B cells (CD19+CD27+IgD-) in patients with IgG4-RD. Cell culture experiments using autologous tonsillar lymphocytes further suggested that DP-Tfh cells possess a poor B-cell helper function and instead regulate memory B cells. Since CD4+ (single positive, SP; CD3+CD4+CD8-CXCR5hiPD-1hi) Tfh cells differentiated into DP-Tfh cells under stimulation with IL-2 and IL-7 as assessed by in vitro experiments, these data imply that SP-Tfh cells are a possible origin of DP-Tfh cells under persistent inflammation. These findings highlight the potential feedback loop mechanism of Tfh cells in immune tolerance under chronic inflammatory conditions. Further studies on DP-Tfh cells may facilitate control of unresolved humoral responses in IgG4-RD pathological inflammation.

Functional Interplay between IL-9 and Peptide YY Contributes to Chronic Skin Inflammation.

Complex interactions between keratinocytes and various cell types, such as inflammatory cells and stromal cells, contribute to the pathogenesis of chronic inflammatory skin lesions. In proinflammatory cytokine‒mediated disease settings, IL-9 plays a pathological role in inflammatory dermatitis. However, IL-9‒related mechanisms remain incompletely understood. In this study, we established tamoxifen-induced keratinocyte-specific IL-9RA-deficient mice (K14CRE/ERTIl9raΔ/Δ mice) to examine the role of IL-9 in multicellular interactions under chronic skin inflammatory conditions. Studies using an imiquimod-induced psoriasis-like model showed that K14CRE/ERTIl9raΔ/Δ mice exhibited a significantly reduced severity of dermatitis and mast cell infiltration compared with control K14WTIl9rafl/fl mice. Transcriptome analyses of psoriasis-like lesions showed that the level of peptide Y-Y (Pyy), a member of the neuropeptide Y family, was markedly downregulated in K14CRE/ERTIl9raΔ/Δ epidermis. Pyy blockade suppressed epidermal thickening and mast cell numbers in imiquimod-treated wild-type mice. Together with in vitro studies indicating that Pyy induced IL-9 production and chemotactic activity in bone marrow‒derived mast cells, these findings suggest that Pyy-mediated interplay between keratinocytes and mast cells contributes to psoriasiform inflammation. Further investigation focusing on the IL-9‒Pyy axis may provide valuable information for the development of new treatment modalities for inflammatory dermatitis.

Cytotoxic Tph-like cells are involved in persistent tissue damage in IgG4-related disease.

OBJECTIVES: The aim of this study was to determine pathological features of T peripheral helper (Tph)-like (PD-1+CXCR5-CD4+ T) cells in IgG4-related disease (IgG4-RD). METHODS: Tph-like cells in the blood and submandibular glands (SMGs) from IgG4-RD patients were analyzed by flow cytometry. Correlations between level of a Tph-like cell subset and clinical parameters of IgG4-RD were investigated. The cytotoxic capacity of Tph-like cells was also examined. Expression profiles of a molecule related to a Tph-like cell subset in IgG4-RD SMGs were assessed by immunohistochemistry. RESULTS: Tph-like cells from IgG4-RD patients highly expressed a fractalkine receptor, CX3CR1. Percentages of circulating CX3CR1+ Tph-like cells were significantly correlated with clinical parameters including IgG4-RD Responder Index, number of involved organs, and serum level of soluble IL-2 receptor. CX3CR1+ Tph-like cells abundantly possessed cytotoxic T lymphocyte-related molecules such as granzyme A, perforin, and G protein-coupled receptor 56. Functional assays revealed their cytotoxic potential against vascular endothelial cells and ductal epithelial cells. Immunohistochemistry showed that fractalkine was markedly expressed in vascular endothelial cells and ductal epithelial cells in IgG4-RD SMGs. CONCLUSION: CX3CR1+ Tph-like cells are thought to contribute to persistent tissue injury in IgG4-RD and are a potential clinical marker and/or therapeutic target for inhibiting progression of IgG4-RD.

Cigarette Smoke Underlies the Pathogenesis of Palmoplantar Pustulosis via an IL-17A-Induced Production of IL-36γ in Tonsillar Epithelial Cells.

Palmoplantar pustulosis (PPP) is characterized by sterile pustules on the palms and soles. A strong association between PPP and tobacco smoking has been reported, and it has been speculated that the IL-17A pathway may play an important role in PPP. Recent studies have suggested that IL-36 plays a pivotal role in the pathogenesis of psoriasis and its subtypes. The relationships among IL-36, smoking, and PPP have not been examined. Here, we investigated the relationships among the smoking index, severity of the clinical condition of PPP, and in vitro dynamics of IL-36 in human tonsillar epithelial cells under the condition of exposure to a cigarette smoke extract. The results demonstrated that the Palmoplantar Pustulosis Area and Severity Index was strongly and positively correlated with the smoking index in female patients. Immunohistochemical examinations showed that IL-36γ was highly expressed in tonsillar epithelial cells from patients with PPP but not in those from patients with recurrent tonsillitis without PPP. The in vitro study revealed that IL-17A synergistically induced a release of IL-36γ under cigarette smoke extract exposure. These results suggest that local production of IL-36γ by epithelial cells induced by cigarette smoke exposure plays an important role in the pathogenesis of PPP.

Activated circulating T follicular helper cells and skewing of T follicular helper 2 cells are down-regulated by treatment including an inhaled corticosteroid in patients with allergic asthma.

BACKGROUND: CXCR5+ T follicular helper (TFH) cells primarily promote B cells to produce an antigen-specific antibody through germinal centers (GCs). TFH cells exist in circulation, and circulating(c) TFH2 cells, a subset of cTFH cells, are able to help naïve B cells produce IgE in healthy individuals. Conversely, IL-10-producing regulatory B (Breg) cells inhibit an accelerated immune response. METHODS: We investigated the roles of cTFH cells and cBreg cells based on a TH2 response in patients with atopic asthma (AA). Thirty-two patients with AA and 35 healthy volunteers (HV) were enrolled. We examined cTFH cells including their subsets, their expression of ICOS and PD-1, and cBreg cells by flow cytometry and their associations with clinical biomarkers. Plasma levels of CXCL13, which is a counterpart of CXCR5, were also measured using ELISA. RESULTS: In patients with AA, cTFH2 cells were increased and cTFH1 cells were decreased compared with those in HV. The expression levels of ICOS on cTFH and their subset cells were elevated and Breg cells were greatly decreased. The plasma levels of CXCL13 in patients with AA were significantly elevated and correlated well with the cTFH2/cBreg ratio. These cells were examined in 10 patients AA before and after inhaled corticosteroid (ICS) treatment. Interestingly, the percentages and numbers of TFH2 and ICOS+ cTFH cells declined after ICS treatment together with improvements in symptoms and clinical biomarkers. CONCLUSIONS: The percentages and numbers of cTFH2 and ICOS+ cTFH cells might be useful as biomarkers of TH2 typed airway inflammation in patients with AA.

Aberrant populations of circulating T follicular helper cells and regulatory B cells underlying idiopathic pulmonary fibrosis.

BACKGROUND: T follicular helper (Tfh) cells have been identified as a new category of helper T cells, which express CXCR5 on their surface and induce the production of antigen-specific antibodies. Many investigations have found morbid proliferation and/or activation of Tfh cells in systemic autoimmune and allergic diseases. It is also known that Tfh cells are regulated by regulatory B (Breg) cells in the deteriorating such diseases. Recently, CXCL13, a ligand of CXCR5, has been reported to increase in the peripheral blood and lungs of patients with idiopathic pulmonary fibrosis (IPF). This study aimed to investigate the involvement of Tfh cells and Breg cells in IPF. METHODS: Peripheral blood samples were obtained from 18 patients with IPF. We isolated heparinized peripheral blood mononuclear cells and investigated the proportions of Breg cells, Tfh cells, PD-1+ICOS+ Tfh cells (activated form of Tfh cells), and the Tfh-cell subsets by flow cytometry. These cell profiles were compared with those of 21 healthy controls. Furthermore, we investigated the correlations between profiles of lymphocytes and lung physiology. RESULTS: The median proportions of Tfh cells per total CD4+ T cells and of PD-1+ICOS+ proportion of Tfh cells per total Tfh cells was significantly more in the IPF patients (20.4 and 5.2%, respectively) compared with healthy controls (15.4 and 2.1%, respectively; p = 0.042 and p = 0.004, respectively). The proportion of Tfh2 cells per total Tfh cells was significantly higher and the proportion of Tfh17 was smaller in the IPF patients than healthy controls. The percentage of Breg cells to total B cells was significantly decreased in the IPF patients (median, 8.5%) compared with that in the controls (median, 19.7%; p < 0.001). The proportion of Breg cells was positively correlated with the annual relative change in diffusing capacity of the lungs for carbon monoxide in the IPF patients (r = 0.583, p = 0.018). CONCLUSION: Proliferation and activation of Tfh cells and a decrease in Breg cells were observed in the peripheral blood of patients with IPF. The profile of the Tfh-cell subset also changed. Specific humoral immunity aberration would likely underlie complicated pathophysiology of IPF.

Bob1 enhances RORγt-mediated IL-17A expression in Th17 cells through interaction with RORγt.

POU domain class 2-associating factor 1 (also called Bob1), which is mainly expressed in B cells, regulates B cell homeostasis and controls humoral immune responses. Although Bob1 is known to function reliably in T cell subsets including follicular helper T cells, Th1 cells and Th2 cells, it is unknown whether Bob1 functions in other T cell subsets. In this study, we found that Bob1 knock out (KO) mice are resistant to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide and that Bob1 KO T cells are defective in Th17 differentiation. Importantly, Bob1 interacts with retinoid acid receptor-related orphan receptor (ROR) gamma t (RORγt), a signature transcription factor for Th17 cells, through the ligand-binding domain of RORγt, thereby enhancing IL-17A transcription activity. IL-17A induction by Bob1 requires the ability for its formation of a DNA-Oct1-Bobl ternary complex. Thus, our findings demonstrate that Bob1 enhances IL-17A expression in vivo and in vitro by interacting with RORγt in Th17 cells, suggesting that Bob1 plays a pivotal role in Th17-mediated autoimmune disease.

IL-10+ T follicular regulatory cells are associated with the pathogenesis of IgG4-related disease.

IgG4-related disease (IgG4-RD) is a chronic fibroinflammatory disease characterized by elevation of serum IgG4 level as well as infiltration of IgG4+ plasma cells in various affected organs. The etiology of IgG4-RD is still not fully understood. Since IgG4-RD is more prevalent in the elderly, aging in itself is considered to be an important risk factor of IgG4-RD. However, the relationship between the pathogenesis of IgG4-RD and immunosenescence remains unknown. To clarify age-related features underlying IgG4-RD, we focused on T follicular regulatory (Tfr) cells, which share forkhead box P3 with regulatory T cells, since the percentage of Tfr cells is known to depend on age. Studies of blood specimens from patients with IgG4-RD and from healthy volunteers demonstrated a marked elevation of circulating Tfr (cTfr) cells in patients with IgG4-RD. Moreover, the percentage of cTfr cells was significantly correlated with various clinical parameters including the level of serum IgG4 and the number of involved organs in IgG4-RD patients. The percentages of tonsillar and blood Tfr cells were increased with aging in healthy volunteers, whereas the suppressive effect of cTfr cells on B cell function in elderly subjects was impaired in comparison with that in young subjects due to a defect in the production of a regulatory cytokine, IL-10. Given that the number of IL-10-producing cTfr cells in IgG4-RD patients was markedly increased compared with that in healthy elderly subjects, these findings suggest that an abnormal aging process of Tfr cells may be related to the pathogenesis of IgG4-RD.

Retroperitoneal Fibrosis Diagnosed as IgG4-related Disease after 35 Years.

In 1982, we reported a case of retroperitoneal fibrosis (RPF) exhibiting various clinical manifestations. Our current understanding of immunoglobulin G4 (IgG4)-related disease led us to consider it as a possible diagnosis because all of the patient's clinical features could be explained by this disease entity. To confirm our hypothesis, were investigated the histopathological findings of resected specimens that had been stored for 35 years postoperatively. Typical pathological findings together with predominant IgG4+ plasma cell infiltration confirmed a potential diagnosis of IgG4-related RPF. Furthermore, we observed positive immunohistochemical staining for several molecules associated with T regulatory and T follicular helper cells.

New insights into IgG4-related disease: emerging new CD4+ T-cell subsets.

PURPOSE OF REVIEW: New insights into IgG4-related disease (IgG4-RD) have recently been obtained. A better understanding of the mechanisms underlying this disease is important for identification of therapeutic targets, which will lead to the development of specific strategies for treatment. RECENT FINDINGS: Infiltration of activated T follicular helper (Tfh) cells is observed in affected tissues of IgG4-RD. Such Tfh cells have a greater capacity than tonsillar Tfh cells to help B cells produce IgG4. Circulating PD-1CXCR5 peripheral T helper (Tph)-like cells are also increased in patients with IgG4-RD. Because Tph-like cells express high levels of chemokine receptors and granzyme A, they have the capacity to infiltrate affected tissues and exert a cytotoxic function. Tph-like cells can also produce CXCL13, and CXCR5 Tfh cells and B cells are therefore preferentially recruited to form ectopic lymphoid structures in the sites. Tph cells may have a role to ignite inflammation and maintain persistent fibroinflammation in collaboration with Tfh cells in lesions of IgG4-RD. SUMMARY: Recent advances in understanding the pathogenesis of IgG4-RD are remarkable. In this review, we summarize and discuss the possible pathologic role of CD4 T-cell subsets in IgG4-RD.

Interleukin 5-producing ST2+ memory Th2 cells in IgG4-related dacryoadenitis and sialadenitis.

Objectives: Immunoglobulin (Ig) G4-related disease (IgG4-RD) is often complicated by allergic disorders. This study was conducted to investigate the mechanism of type 2 helper T-inflammation (Th2-inflammation) in IgG4-related dacryoadenitis and sialadenitis (IgG4-DS). Methods: We separated and analyzed the proportion of growth stimulation expressed gene 2 (ST2)+ memory Th2 cells among the peripheral blood mononuclear cells by flow cytometry in cases with IgG4-DS and healthy individuals. Finally, we identified the role of ST2+ memory Th2 cells in the involved tissues. Results: The proportion of circulating ST2+ memory Th2 cells was much higher in the patients with IgG4-DS than in the healthy controls. Abundant infiltration of ST2+ memory Th2 cells was detected in the involved salivary glands and lymph nodes, and these cells produced interleukin-5. Conclusion: We demonstrated that there is an increase of interleukin-5 producing ST2+ memory Th2 cells in the involved tissues in IgG4-DS. This subset of cells is considered to be an important player in inducing the inflammatory Th2 environment characteristic of IgG4-DS.

Mucosal Immune Response in Nasal-Associated Lymphoid Tissue upon Intranasal Administration by Adjuvants.

The nasal administration of vaccines directed against diseases caused by upper respiratory tract infections of pathogens, such as the influenza virus, mimics the natural infection of pathogens and induces immunoglobulin A (IgA) production in the nasal cavity to effectively protect viral entry. Therefore, the development of a nasally administered vaccine is a research objective. Because the antigenicity of influenza split vaccines is low, nasal inoculation with the vaccine alone does not induce strong IgA production in the nasal cavity. However, the addition of adjuvants activates the innate immune response, enhancing antigen-specific IgA production and the T-cell response. Although the development of suitable adjuvants for nasal vaccinations is in progress, the mechanism by which adjuvants promote the immune response is still unclear. In this review, we discuss the mucosal immune response, especially in the nasal-associated lymphoid tissue, induced in response to the intranasal inoculation of an influenza vaccine and adjuvants in animal models.

Stage classification of IgG4-related dacryoadenitis and sialadenitis by the serum cytokine environment.

OBJECTIVES: Patients with immunoglobulin-G4 related disease (IgG4-RD) diagnosed according to the comprehensive diagnostic criteria (CDC) show varied therapeutic responses and prognoses. We assumed that there are clinical stages in IgG4-RD and have verified it using serum cytokine levels in the groups classified by lesion distribution. METHODS: Definite IgG4-related dacryoadenitis and sialadenitis (IgG4-DS) cases were divided according to the CDC for IgG4-RD into 11 cases with focal type and 30 cases with systemic type. The levels of serum interleukin (IL)-4, IL-5, IL-6, IL-10, IL-13, IL-15, IL-21, interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1, and monocyte chemotactic protein (MCP)-1 were measured in healthy controls, allergic patients, probable IgG4-RD cases, and focal and systemic type cases. The cytokine environment was analyzed in each group. The 52 definite IgG4-RD cases were next classified into four groups with cluster analysis in terms of therapeutic responses and prognosis. The relationships between each cytokine level and therapeutic responses were also analyzed. RESULTS: Both serum IL-5 and IFN-α concentrations were very low in healthy controls, but they increased in the allergic cases, probable cases, and focal and systemic type cases. The level of serum IL-5 was significantly higher in definite cases than in healthy controls. The serum IL-5 level was also significantly increased in the groups with a poor prognosis than in the good prognosis group. CONCLUSION: These results suggest that there are clinical stages in IgG4-RD, and serum IL-5 play roles in the pathogenesis of IgG4-RD.

Predicting therapeutic response in IgG4-related disease based on cluster analysis.

To bring the clinical practice of immunoglobulin (Ig)G4-related disease (IgG4-RD) close to personalized medicine, we classified the patient groups and clarified the therapeutic responses of each group. A total of 147 patients enrolled in our registry were classified into four groups by cluster analysis with the software. The therapeutic responses and prognosis of each group were examined. The cluster analysis classified the subjects into four groups: Cluster 1, patients who presented with prominent hypergammaglobulinemia, elevated levels of serum IgG4, and hypocomplementemia; Cluster 2, patients who presented with eosinophilia, elevated concentrations of serum IgG, IgG4, and IgE, and in whom CRP tended to be positive; Cluster 3, patients with younger onset and serum levels of IgG, IgG4, and IgE and peripheral eosinophil counts lower than the other clusters; and Cluster 4, patients with elder onset and low peripheral eosinophil counts. The amounts of glucocorticoid for maintenance treatment were from 5 to 7 mg/d in all groups, but the amounts were significantly greater in Cluster 1 (patients with hypergammaglobulinemia, elevated levels of serum IgG4, and hypocomplementemia) than in Cluster 4 (elder onset patients, relatively low concentrations of peripheral eosinophils). With regard to the use of immunosuppressants and the relapse rate, there were high frequencies in Cluster 1 and Cluster 3 (younger onset patients who presented with mild elevations of serum IgG and IgG4). On the other hand, Cluster 4 showed a low rate of relapse and often could discontinue steroids. The present results suggest that personalized medicine could be provided in IgG4-RD by classifying patients based on their clinical features.

Circulating PD-1+CXCR5-CD4+ T cells underlying the immunological mechanisms of IgG4-related disease.

OBJECTIVE: The aim was to study the pathological role of lymphocytes with a peripheral T helper-cell-like phenotype (PD-1+CXCR5-CD4+) in IgG4-related disease (IgG4-RD). METHODS: PD-1+CXCR5-CD4+ T cells in the blood of patients with IgG4-RD (n = 53), patients with SS (n = 16) and healthy volunteers (n = 34) as controls were analysed by flow cytometry. Correlations between results obtained by flow cytometry and clinical parameters relevant to IgG4-RD were also analysed. RESULTS: The percentage and absolute number of PD-1+CXCR5- cells within total CD4+ T cells in IgG4-RD patients were significantly increased compared with those in healthy volunteers. Further analysis showed that there were marked positive correlations of the percentage of PD-1+CXCR5-CD4+ T cells with the serum level of IgG4 and the number of organs involved. Interestingly, granzyme A (GZMA)+ cells were enriched in PD-1+CXCR5-CD4+ T cells, and the percentage and absolute number of GZMA+PD-1+CXCR5-CD4+ T cells were significantly elevated in IgG4-RD patients. Although no obvious change was observed in the percentage of total CD4+ T cells, the percentage and absolute number of PD-1+CXCR5-CD4+ T cells decreased in accordance with a reduction of serum IgG4 level after treatment with glucocorticoids. CONCLUSION: In IgG4-RD, circulating CD4+ T-cell populations were composed of PD-1+CXCR5- cells, and the ratios of these cells were correlated with clinical manifestations of IgG4-RD. Further analysis of GZMA+PD-1+CXCR5-CD4+ T cells might lead to a deeper understanding of the pathogenesis of ectopic lymphoid follicles and the persistent inflammation in IgG4-RD.