Evidence for increased expression of regulatory cytokine receptors interleukin-12R and interleukin-18R in common variable immunodeficiency

We investigated the expression of T helper (Th)1/Th2 regulatory cytokine receptors on lymphocytes from patients with common variable immunodeficiency (CVID), a disorder associated with raised Th1 cytokine production, comparing the results with those from healthy individuals and atopic asthmatics, the latter generally considered to have a Th2‐driven disease. We proposed that alterations in some of the relevant receptors might be related to the observed imbalances in Th1/Th2 cytokines. Cells from CVID patients showed an increase in the percentages of CD212 [interleukin (IL)‐12Rβ1] cells within the CD4⁺ CD45RA⁺ and CD8⁺ CD45RA⁺ subsets (24% and 41%, respectively), as compared to CD4⁺ CD45RA⁺ and CD8⁺ CD45RA⁺ in healthy subjects (6% and 23%, respectivey). Approximately 21% of the CD4⁺ CD45RA⁺ naïve cells expressed IL‐18Rα, compared with 11% in healthy subjects. In contrast, the cytokine‐receptor expression in asthmatics was similar to that of controls. In spite of the above differences, after 72 h of stimulation with anti‐CD3 and anti‐CD28, cytokine receptor up‐regulation was similar in all three groups, with up to 80% of both CD45RA⁺ and CD45RO⁺ lymphocytes expressing CD212 (IL‐12Rβ1) and IL‐18Rα. Approximately 50% of the ‘naïve’, and 25% of the ‘memory’ subpopulations up‐regulated IL‐12Rβ2. These findings provide further evidence of a polarization towards a Th1 immune response in CVID, the mechanism possibly involving up‐regulation of IL‐12‐mediated pathways.     

Retinoic acid regulates the development of a gut-homing precursor for intestinal dendritic cells

The vitamin A metabolite retinoic acid (RA) regulates intestinal immune responses through immunomodulatory actions on intestinal dendritic cells (DCs) and lymphocytes. Here, we show that RA also controls the generation of gut-tropic migratory DC precursors, referred to as pre-mucosal DCs (pre-μDCs). Pre-μDCs express the gut trafficking receptor α4β7 and home preferentially to the intestines. They develop in the bone marrow (BM), can differentiate into CCR9⁺ plasmacytoid DCs as well as conventional DCs (cDCs), but preferentially give rise to CD103⁺ intestinal cDCs. Generation of pre-μDCs in vivo in the BM or in vitro is regulated by RA and RA receptor α (RARα) signaling. The frequency of pre-μDCs is reduced in vitamin A-deficient animals and in animals treated with RAR inhibitors. The results define a novel vitamin A-dependent, RA-regulated developmental sequence for DCs and identify a targeted precursor for CD103⁺ cDCs in the gut.     

Dendritic Cells from Rheumatoid Arthritis Patient Peripheral Blood Induce Th17 Cell Differentiation via miR‐363/Integrin αv/TGF‐β Axis

Dendritic cells (DCs) are critical regulators of immune responses. This study was to observe the effect of DCs from peripheral blood on the differentiation of Th17 in patients with rheumatoid arthritis (RA). Peripheral blood samples were collected from 30 patients with RA and 20 healthy controls, respectively. Flow cytometry results showed that in contrast to Treg cells, the proportion of Th17 cells in T cells and the Th17/Treg ratio were both increased in patients with RA. The RT‐PCR results showed that Foxp3、ROR γt and miR‐363 expression in PBMC of patients with RA were reduced, but the ITGAV expression was increased, which was negatively related to miR‐363 expression. IL‐17, TGF‐β and IL‐6 levels detected by ELISA were increased in peripheral blood serum of patients with RA. Moreover, we noted that the CD11C⁺αν⁺/CD11C⁺ DCs ratio was obvious increased in patients with RA and has positive correlation to the Th17/Treg ratio. In cocultured system, Th17 cell differentiation was significantly inhibited in the presence of ITGF‐β suggesting that Th17 cell differentiation was controlled by active TGF‐β (aTGF‐β). After DCs transfecting with miR‐363 mimics and cocultured with T cells, Th17 cell number, IL‐17 level and ROR‐γt expression were significantly reduced in the presence of latent TGF‐β (ITGF‐β). In addition, the integrin αv protein expression was both reduced in the presence of aTGF‐β or ITGF‐β. These data demonstrated that DCs induced Th17 cell differentiation through miR‐363/Integrin αv/TGF‐β pathway in patients with RA.     

The Suppressive Function of Human CD8+ iTregs is Inhibited by IL‐1β and TNFα

CD8⁺ Tregs display an immunoregulatory activity and may play an essential role in the immunopathology of several diseases. Therefore, their therapeutic potential is exquisite and further studies on their differentiation and function are essential. The aim of this study was to evaluate the role of the innate immune system in CD8⁺ iTreg differentiation and function. Naive human CD8⁺CD25^?CD45RA⁺ T cells were cultured in Treg‐inducing conditions with or without IL‐1β, TNFα or monocyte‐derived dendritic cells (DCs). The differentiation of CD8⁺CD127^?CD25^hiFoxP3^hi‐induced Tregs (CD8⁺ iTregs) is dependent on TGF‐β1 and IL‐2, which had synergistic effect upon their differentiation. CD8⁺ iTregs were also induced in a coculture with allogeneic mature DCs (mDCs). The CD8⁺ iTregs suppressive function was confirmed, which was diminished in the presence of IL‐1β and TNFα. The IL‐1β‐prevented suppressive function was associated with reduced secretion of IL‐10 and IFNγ, whereas the presence of TNFα did not affect their secretion. Furthermore, the presence of TNFα reduced IL‐10 and TGF‐β1 secretion by CD8⁺ iTregs, whereas only IL‐10 secretion was decreased by IL‐1β. Together, these results suggest that IL‐1β and TNFα prevent IL‐2‐ and TGF‐β1‐driven CD8⁺ iTregs suppressive function in human T cells. Such pro‐inflammatory innate immune response possibly mediates its negative tolerogenic effect through reduced IFNγ‐, IL‐10‐ and TGF‐β1‐driven mechanism.     

Healthy Preterm Newborns Show an Increased Frequency of CD4+CD25highCD127lowFOXP3+ Regulatory T Cells with a Naive Phenotype and High Expression of Gut‐Homing Receptors

Treg cells are crucial to prevent immune dysregulation, but little is known about the frequency of these cells in neonates, particularly in very/moderate and late preterm newborns studied as separate groups. The CD4⁺CD25^hiCD127^loFOXP3⁺Treg population was phenotypically characterized to assess maturation markers and gut‐homing integrins by flow cytometry in the cord blood of healthy preterm newborns born at 30–33^6/7 gestation weeks (Group 1), at 34–36^6/7 gestation weeks (Group 2) and term newborns born at 37–41 gestation weeks (Group 3), compared to healthy adults. An inverse correlation of the Treg percentage and gestational age was found, with significantly higher frequencies in Group 1 compared to Groups 2 and 3 and in Group 2 compared to Group 3, and significantly higher Treg frequencies and numbers in the neonates compared to the adults. All of the newborns exhibited increased Treg frequencies with a naive phenotype compared to adults. Cytotoxic T‐lymphocyte‐associated protein 4 CTLA‐4 expression in the naive Treg was decreased in both preterm groups compared with those from term newborns and adults, and in the memory Treg from Group 1 compared with the other groups. The frequencies of Treg expressing α4β7 and α4β1 integrins were higher in both preterm groups, but significantly different only in Group 1, when compared with those from the term newborns and the adults. In conclusion, although a high frequency of Treg is present in newborns, an immature phenotype with a higher expression of CD45RA and α4β7/α4β1 and a lower expression of CTLA‐4 is found, particularly in the very preterm group.     

Amaranthus leucocarpus Lectin Recognizes Human Naive T Cell Subpopulations

Amaranthus leucocarpus lectin (ALL) is specific for GalNAc residue found in the inner core of Galβ1, 3GalNAcα1, O-Ser/Thr disaccharide (T-antigen) or GalNAcα1, O-Ser/Thr (T_n-antigen). Flow cytometric analysis using fluorescein-labeled lectin and monoclonal antibodies against human cell surface markers indicated that 5.7% of mononuclear cells from human healthy donors are recognized by ALL. These cells have the phenotype CD2⁺CD4⁺CD19^? and most of the lymphocytes recognized are also CD27⁺, CD45RA⁺ and CD43⁺. ALL possesses mitogenic activity on lymphocytes after neuraminidase treatment. Our results indicate that the receptors recognized by ALL could be considered surface markers for naive human T lymphocyte subsets.     

The ��E(CD103)��7 integrin interacts with oral and skin keratinocytes in an E-cadherin-independent manner

The integrin αE(CD103)β7 (αEβ7) is expressed by intraepithelial lymphocytes, dendritic cells and regulatory T cells. It plays an important role in the mucosal immune system by retaining lymphocytes within the epithelium and is involved in graft rejection, immunity against tumours and the generation of gut‐homing effector cells. In gut and breast, the ligand for αEβ7 is E‐cadherin but in human oral mucosa and skin, there is evidence that lymphocytes use an alternative, unknown, ligand. In the present study, the I domain of the human αE subunit, which contains the E‐cadherin‐binding site, was locked in a highly active, ‘open’ and an inactive, ‘closed’ conformation by the introduction of disulphide bonds and these domains were expressed as IgG Fc fusion proteins. αE fusion proteins recognize E‐cadherin, the only known ligand for αEβ7. This interaction was inhibited by an antibody that blocks the αE‐binding site on E‐cadherin and by the omission of Mn²⁺, which is essential for integrin function in vitro. The locked ‘open’ conformation of αE adhered to human oral and skin keratinocytes, including the E‐cadherin‐negative H376 cell line, and this was not inhibited by blocking antibody against the αEβ7‐binding site on E‐cadherin, providing further evidence for the existence of an alternative ligand for αEβ7 in skin and oral mucosa. The interaction with E‐cadherin and the alternative ligand was Mn²⁺ dependent and mediated by the metal ion‐dependent coordination site (MIDAS) of the locked ‘open’αE I domain, independently of the β7 subunit.     

Rheumatoid arthritis fibroblast‐like synoviocytes co‐cultured with PBMC increased peripheral CD4+CXCR5+ICOS+ T cell numbers

‘Circulating’ T follicular helper cells (Tfh), characterized by their surface phenotypes CD4⁺chemokine receptor 5 (CXCR5)⁺ inducible co‐stimulatory molecule (ICOS)⁺, have been identified as the CD4⁺ T cell subset specialized in supporting the activation, expansion and differentiation of B cells. Fibroblast‐like synoviocytes (FLS) are critical in promoting inflammation and cartilage destruction in rheumatoid arthritis (RA), and the interaction between FLS and T cells is considered to facilitate FLS activation and T cell recruitment. However, it remains unknown whether RA‐FLS co‐cultured with activated peripheral blood mononuclear cells (PBMC) has immunoregulatory effects on peripheral Tfh. In the present study, we co‐cultured RA‐FLS with or without anti‐CD3/CD28‐stimulated PBMC. The results showed that RA‐FLS co‐cultured with stimulated PBMC could increase the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells of RA PBMC possibly via the production of interleukin (IL)‐6, a critical cytokine involved in the differentiation of Tfh cells. We also observed increased reactive oxygen species (ROS) levels in the co‐culture system of RA‐FLS and PBMC. The percentage of CD4⁺CXCR5⁺ICOS⁺ T cells was decreased when ROS production was inhibited by N‐acetyl‐L‐cysteine (NAC), a specific inhibitor which can decrease ROS production. In addition, we showed that the higher levels of tumour necrosis factor (TNF)‐α and IL‐1β in the co‐culture system and the blocking of TNF receptor 2 (TNF‐R2) and IL‐1β receptor (IL‐1βR) both decreased the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells. Our study reveals a novel mechanistic insight into how the interaction of RA‐FLS and PBMC participates in the RA pathogenesis, and also provides support for the biologicals application for RA.     

Retinoic acid receptors exhibit cell‐autonomous functions in cranial neural crest cells

Previous work has emphasized the crucial role of retinoic acid (RA) in the ontogenesis of the vast majority of mesenchymal structures derived from the neural crest cells (NCC), which migrate through, or populate, the frontonasal process and branchial arches. Using somatic mutagenesis in the mouse, we have selectively ablated two or three retinoic acid receptors (i.e., RARα/RARβ, RARα/RARγ and RARα/RARβ/RARγ) in NCC. By rigorously analyzing these mutant mice, we found that survival and migration of NCC is normal until gestational day 10.5, suggesting that RAR‐dependent signaling is not intrinsically required for the early steps of NCC development. However, ablation of Rara and Rarg genes in NCC yields an agenesis of the median portion of the face, demonstrating that RARα and RARγ act cell‐autonomously in postmigratory NCC to control the development of structures derived from the frontonasal process. In contrast, ablation of the three Rar genes in NCC leads to less severe defects of the branchial arches derived structures compared with Rar compound null mutants. Therefore, RARs exert a function in the NCC as well as in a separated cell population. This work demonstrates that RARs use distinct mechanisms to pattern cranial NCC. Developmental Dynamics 238:2701–2711, 2009. © 2009 Wiley‐Liss, Inc.     

Interleukin-4-producing CD4+ NK1.1+ TCRα/βintermediate liver lymphocytes are down-regulated by Listeria monocytogenes

Experimental infection of mice with the intracellular bacterium, Listeria monocytogenes, provides a paragon model for immune defence dominated by T helper type 1 (Th1) responses. Potent production of interleukin (IL)-12 by infected macrophages is considered the determining factor in Th1 cell development. In contrast, it is assumed that IL-4 producers remain virtually unstimulated in listeriosis. In the liver, the major target organ of listeriosis, an unusual T lymphocyte population exists with the intriguing phenotype CD4⁺NK1.1⁺ TCRα/β^intermediate (TCRα/β^int). Here we show that IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes are down-regulated early in listeriosis. We assume that curtailment of IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes promotes unconstrained development of Th1 cells which are central to protection against intracellular bacteria.     

Evidence for autoantibody-induced CD4 depletion mediated by apoptotic and non-apoptotic mechanisms in HIV-positive long-term surviving haemophilia patients

It is believed that autoimmune phenomena and apoptosis contribute to CD4 depletion. We investigated 11 long‐term (>20 years) HIV‐infected haemophilia patients and 10 healthy controls. Using four‐colour‐fluorescence flow cytometry, we studied the proportions of CD3⁺CD4⁺ and CD3⁺CD4^– blood lymphocytes that were CD95⁺, CD95L⁺, immune complex⁺ (IC⁺, consisting of IgM, IgG, C3d and/or gp120), and were viable or non‐viable (propidium iodide⁺ = PI⁺). In addition, we studied viability of CD4⁺IgG⁺ patient lymphocytes using the apoptosis marker annexin and the permeability indicator 7‐amino actinomycin D (7‐AAD). HIV⁺ patients had a higher proportion of CD3⁺CD4⁺IgG⁺PI⁺ lymphocytes than healthy controls (median: 3·7%versus 0·3%; P = 0·00001). These non‐viable IgG‐coated lymphocytes might have been killed in vivo by ADCC or complement lysis; 9·1% of the circulating CD3⁺CD4⁺ blood lymphocytes were IgG⁺PI^– (controls: 2·5%; P = 0·001). These viable IgG‐coated lymphocytes might be targets for phagocytosis or anti‐CD95 autoantibody‐mediated apoptosis. Because HIV⁺ patients and healthy controls had similar proportions of PI⁺ or PI^– CD3⁺CD4⁺ lymphocytes that carried CD95L on the surface, and because CD3⁺CD4⁺CD95L⁺ cells that were IgG⁺, C3d⁺ and/or gp120^– were increased in HIV⁺ patients, the role of CD95L‐induced apoptosis in long‐term HIV‐infected haemophilia patients remains unclear. The findings that HIV⁺ patients had higher proportions of CD3⁺CD4⁺CD95⁺ (PI⁺: 6·5%versus 1·4%; P = 0·00002; PI^–: 55·8%versus 44·4%; P = 0·04) blood lymphocytes and that the proportion of CD4⁺IgG⁺Annexin⁺7‐AAD^– blood lymphocytes was associated inversely with peripheral CD4 counts (r = ?0·636; P < 0·05) suggest that attachment of IgG to CD4⁺ blood lymphocytes (anti‐CD95?) induces in some lymphocytes apoptosis with subsequent depletion of these IgG‐coated apoptotic CD4⁺ lymphocytes from the circulation. We found supporting evidence for the contention that autoantibody‐induced apoptotic and non‐apoptotic mechanisms contribute to CD4 depletion in long‐term HIV‐infected haemophilia patients.     

Epstein–Barr virus‐specific CD8+ T lymphocytes from diffuse large B cell lymphoma patients are functionally impaired

Epstein–Barr virus (EBV) is a persistent virus with oncogenic capacity that has been implicated in the development of aggressive B cell lymphomas, primarily in immunosuppressed individuals, although it can be present in immunocompetent individuals. Changes in the function and clonal diversity of T lymphocytes might be implied by viral persistence and lymphoma development. The aim of the present study was to evaluate the frequency, phenotype, function and clonotypical distribution of EBV‐specific T cells after peripheral blood stimulation with a virus lysate in newly diagnosed patients with diffuse large B cell lymphoma (DLBCL) aged more than 50 years without prior histories of clinical immunosuppression compared with healthy controls. Our results showed impaired EBV‐specific immune responses among DLBCL patients that were associated primarily with decreased numbers of central and effector memory CD8⁺ T lymphocytes. In contrast to healthy controls, only a minority of the patients showed CD4⁺/tumour necrosis factor (TNF)‐α⁺ T cells expressing T cell receptor (TCR)‐Vβ17 and CD8⁺/TNF‐α⁺ T cells with TCR‐Vβ5·2, Vβ9 and Vβ18 in response to EBV. Notably, the production of TNF‐α was undetectable among TCR‐Vβ5·3⁺, Vβ11⁺, Vβ12⁺, Vβ16⁺ and Vβ23⁺ CD8⁺ T cells. In addition, we observed decreased numbers of CD4⁺/TNF‐α⁺ and CD8⁺/TNF‐α⁺, CD8⁺/interleukin (IL)‐2⁺ and CD8⁺/TNF‐α⁺/IL‐2⁺ T lymphocytes in the absence of T cells capable of producing TNF‐α, IL‐2 and IFN‐γ after EBV stimulation simultaneously. Moreover, DLBCL patients displayed higher IL‐10 levels both under baseline conditions and after EBV stimulation. These findings were also observed in patients with positive EBV viral loads. Prospective studies including a large number of patients are needed to confirm these findings.     

Retinoic acid receptor signaling levels and antigen dose regulate gut homing receptor expression on CD8+ T cells

Recent studies have highlighted a central role for intestinal dendritic cells (DCs) and vitamin A metabolite retinoic acid (RA) in the generation of α4β7⁺ CCR9⁺“gut tropic” effector T cells. Here, using RA-responsive element reporter mice, we demonstrate that both splenic and mesenteric lymph node (MLN) DCs enhanced retinoic acid receptor (RAR) signaling in CD8⁺ T cells; however, only a subset of MLN DCs, expressing the integrin α-chain CD103, induced an early RAR signal that is required for efficient CCR9 induction. MLN-primed CD8⁺ T cells also received enhanced RAR-dependent signals compared with splenic-primed CD8⁺ T cells in vivo. Further DC-mediated induction of gut homing receptors was inhibited at a high antigen dose without influencing RAR signaling events, and resulted in less efficient CD8⁺ T-cell entry into the small intestinal mucosa. These results highlight a complex interplay between antigen dose and DC subset-induced RAR signaling events in the generation of tissue tropic effector T-cell subsets.     

Cross-presentation of cell-associated antigens by CD8alpha+ dendritic cells is attributable to their ability to internalize dead cells

In the mouse, cross‐presentation is an exclusive property of the CD8α⁺ subset of dendritic cells (DC) but the basis for this selectivity remains unclear. Here we report that splenic CD8α⁺ DC are much superior to other DC subsets in internalizing dying cells in vitro. In contrast, CD8α⁺, CD8α^– CD4⁺ and CD8α^– CD4^– DC subsets phagocytose bacteria or latex beads to a similar extent. Although CD8α⁺ DC are better than CD4⁺ DC at presenting ovalbumin (OVA)‐loaded splenocytes to naïve OT‐I T lymphocytes, CD4⁺ DC are better at presenting OVA‐expressing Escherichia coli to the same T cells. In both cases, presentation is abrogated by lactacystin. These results show that both splenic CD8α⁺ and CD8α^– DC can present exogenous antigens on major histocompatibility complex (MHC) class I via a proteasome‐dependent pathway and suggest that the specialized cross‐presenting function of CD8α⁺ DC is a result of their ability to endocytose dying cells rather than a unique pathway for handling endosomal contents.     

The human immunomodulatory CD25+ B cell population belongs to the memory B cell pool

We have shown that human CD20⁺25⁺ B cells display immunomodulatory properties. The aim of this study was to investigate if CD25⁺ B cells are found within the CD27 memory B cell population, and to analyse pattern of their cytokine production. B cells isolated from healthy subjects, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients were analysed regarding the frequency of CD25⁺ B cells within certain B cell subsets. Purified CD25⁺ B cells from healthy subject were used in vitro to evaluate their production of immunomodulatory cytokines. In healthy subjects the majority (60%) of memory B cells (CD20⁺27⁺) also co‐expressed CD25 while only 10–20% of the naïve B cells (CD20⁺27⁻) and plasmablasts (CD20–27⁺) expressed CD25. In RA and SLE patients, we found that 51% and 48%, respectively, co‐expressed CD25 in the memory population, whereas only 11% and 9% co‐expressed CD25 in the naïve B cell population. Phenotypic analysis of the CD20⁺25⁺27⁺ and CD20⁺25⁺27⁻ cells using CD10, CD24, CD38, CD45, CD71, CD80, CD86, CD95, CD138, BAFF‐R, TACI, IgA, IgD, IgG and IgM showed that CD20⁺25⁺27⁺ B cells preferentially represent highly activated, Ig class switched memory B cells. Cytokine profile analysis showed that CD25⁺ B cells secreted significantly higher levels of IL‐10 versus CD25⁻ B cells. In contrast, TGF‐β1 secretion was similar between the CD25⁺ and CD25⁻ sub‐populations. In conclusion, CD20⁺25⁺ B cells constitute a unique subpopulation preferentially occurring among CD20⁺27⁺ memory B cells. We suggest that CD25 can be used as a marker for a memory B cell subset.     

Co-expression of the CD45RA and CD45RO antigens on T lymphocytes in chronic arthritis

The site of T lymphocyte activation in chronic arthritis is unknown. Peripheral blood (PB) lymphocytes from chronic arthritis patients are in a ‘naïve’ or non-activated state, as defined by expression of the CD45RA antigen and lack of HLA class II expression. In contrast, most synovial fluid (SF) T lymphocytes express a ‘memory’ or activated phenotype, as defined by the CD45RO antigen and high HLA class II expression. Following stimulation, naive cells lose CD45RA and gain CD45RO expression to become memory cells with a transitional stage of dual CD45RA, CD45RO antigen expression. To localize where this change in phenotype occurs we used dual colour immunofluorescence labelling to compare the percentage of dual CD45RA, CD45ROpositive T lymphocytes in PB and SF from chronic arthritic patients and from normal PB, assuming this population would be increased at the primary site of T lymphocyte activation. Expression of the intermediate and late activation marker. HLA-DR, was also analysed using dual colour immunofluorescence labelling. The percentage of dual positive T lymphocytes was similar between arthritic PB, SF. and normal PB, as was the density of both CD45RA and CD45RO antigens. Thus, CD45 isoform expression did not indicate where T lymphocytes were activated. However, we identified a previously unreported population of CD45RA⁺ CD45RO⁺ HLA-DR^- T lymphocytes in arthritic and normal PB. In SF, this population was absent, but a substantial number of dual CD45RA, CD45RO-positive HLA-DR⁺ T lymphocytes were identified. This population would not be predicted by the current model of T lymphocyte activation. Division of T lymphocytes into functional groups on the basis of CD45 isoform expression is likely to be more complicated than previously thought. Based on our findings we propose an alternative model of T lymphocyte differentiation.     

Combined use of etanercept and MTX restores CD4+/CD8+ ratio and Tregs in spleen and thymus in collagen-induced arthritis

Objective

To further explore the mechanism of etanercept (ENT, rhTNFR:Fc) and methotrexate (MTX) in the combined treatment of rheumatoid arthritis (RA), we investigated whether thymic and splenic T-cell subsets and their related cytokines imbalance could be restored by ETN/MTX treatment.

Methods

The effect of ETN/MTX on collagen-induced arthritis (CIA) was evaluated by arthritis scores, joint and spleen histopathology, as well as indices of thymus and spleen. T lymphocytes proliferation was determined by [³H]-TdR incorporation. Levels of TNF-α, LT-α, IL-1β, RANKL, IL-10, IL-17, IFN-γ and IL-6 were detected by enzyme linked immunosorbent assay. The subsets of T lymphocytes including CD4⁺, CD8⁺, CD3⁺CD4⁺, CD4⁺CD25⁺, CD4⁺CD62L⁺ and CD4⁺CD25⁺Foxp3⁺ cells were quantified using flow cytometry.

Results

Combined administration of ETN/MTX significantly inhibited the proliferation of T lymphocytes, decreased serum IL-6, TNF-α, IL-1β, RANKL and macrophage supernatant IL-17, LT-α, increased serum IFN-γ and macrophage supernatant IL-10. Moreover, the combined administration could restore CD4⁺/CD8⁺ ratio and Treg cells of CIA thymus and spleen.

Conclusion

Taken together, our findings suggest that ENT/MTX may modify the abnormal T lymphocytes balance from central to peripheral lymphoid organs, which may partially, explained the mechanism of the combined administration.