Aastha Khullar, Varun Dhir, Biman Saikia, Ashok Kumar Yadav, Bidyalaxmi Leishangthem, Chandra Bhushan Prasad, Sankar Jayaprakash, Siddharth Jain, G S R S N K Naidu, Shefali K Sharma, Aman Sharma, Sanjay Jain
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Although, higher frequencies of CD4+GMCSF+ T-cells have been reported, there are no data on CD8+GMCSF+ T-cells or polyfunctionality.Our objective was to enumerate frequencies of CD8+GMCSF+ T cells in RA blood and synovial fluid (SF), and assess their polyfunctionality, memory phenotype and cytotoxic ability.</p><p><strong>Methods: </strong>This study included RA patients (blood samples,in some with paired synovial fluid (SF)), healthy controls (HC) (blood) and SpA patients (SF). In some RA patients' blood was sampled twice, before and 16-24 weeks after methotrexate (MTX) treatment. After mononuclear cell isolation from blood and SF, ex-vivo stimulation using PMA/Ionomycin was done, and cells were stained (surface and intracellular after permeabilisation/fixation). Subsequently, frequencies of GMCSF+CD8+ and CD4+ T-cells, polyfunctionality (TNFα, IFNγ, IL-17), phenotype (memory) and perforin/granzyme expression were assessed by flowcytometry.</p><p><strong>Results: </strong>There was no significant difference in frequencies of GMCSF+CD8+ (3.7, 4.1%, p=0.540) or GMCSF+CD4+ T-cells (4.5, 5.2%, p=0.450) inblood of RA and HC. However, there was significant enrichment of both CD8+GMCSF+ (5.8, 3.9%, p=0.0045) and CD4+GMCSF+ (8.5, 4.5%, p=0.0008) T-cells inSF compared to blood in RA patients. Polyfunctional triple cytokine positive TNFα+IFNγ+GMCSF+CD8+T-cells (81, 36%, p=0.049) and CD4+T-cells (48, 32%, p=0.010) was also higher in SF compared to blood in RA. CD8+ T cells showed higher frequency of effector-memory phenotype and granzyme-B expression in RA-SF. On longitudinal follow-up, blood CD4+GMCSF+ T-cells significantly declined (4.6, 2.9%, p=0.0014) post-MTX.</p><p><strong>Conclusions: </strong>We report a novel finding of enrichment of CD8+GMCSF+ in addition to CD4+GMCSF+ T-cells in RA-SF. 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Although, higher frequencies of CD4+GMCSF+ T-cells have been reported, there are no data on CD8+GMCSF+ T-cells or polyfunctionality.Our objective was to enumerate frequencies of CD8+GMCSF+ T cells in RA blood and synovial fluid (SF), and assess their polyfunctionality, memory phenotype and cytotoxic ability.</p><p><strong>Methods: </strong>This study included RA patients (blood samples,in some with paired synovial fluid (SF)), healthy controls (HC) (blood) and SpA patients (SF). In some RA patients' blood was sampled twice, before and 16-24 weeks after methotrexate (MTX) treatment. After mononuclear cell isolation from blood and SF, ex-vivo stimulation using PMA/Ionomycin was done, and cells were stained (surface and intracellular after permeabilisation/fixation). Subsequently, frequencies of GMCSF+CD8+ and CD4+ T-cells, polyfunctionality (TNFα, IFNγ, IL-17), phenotype (memory) and perforin/granzyme expression were assessed by flowcytometry.</p><p><strong>Results: </strong>There was no significant difference in frequencies of GMCSF+CD8+ (3.7, 4.1%, p=0.540) or GMCSF+CD4+ T-cells (4.5, 5.2%, p=0.450) inblood of RA and HC. However, there was significant enrichment of both CD8+GMCSF+ (5.8, 3.9%, p=0.0045) and CD4+GMCSF+ (8.5, 4.5%, p=0.0008) T-cells inSF compared to blood in RA patients. Polyfunctional triple cytokine positive TNFα+IFNγ+GMCSF+CD8+T-cells (81, 36%, p=0.049) and CD4+T-cells (48, 32%, p=0.010) was also higher in SF compared to blood in RA. CD8+ T cells showed higher frequency of effector-memory phenotype and granzyme-B expression in RA-SF. 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引用次数: 0
摘要
研究目的GMCSF+T细胞可能与类风湿性关节炎（RA）的发病机制有关，而多功能性可能是致病性的标志。我们的目的是统计 RA 血液和滑膜液（SF）中 CD8+GMCSF+ T 细胞的频率，并评估它们的多功能性、记忆表型和细胞毒性能力：研究对象包括 RA 患者（血液样本，其中部分样本与滑膜液（SF）配对）、健康对照组（HC）（血液）和 SpA 患者（SF）。对部分 RA 患者的血液采样两次，分别在甲氨蝶呤（MTX）治疗前和治疗后 16-24 周。从血液和 SF 中分离出单核细胞后，使用 PMA/Ionomycin 进行体外刺激，并对细胞进行染色（渗透/固定后的表面和细胞内染色）。随后，用流式细胞仪评估了 GMCSF+CD8+ 和 CD4+ T 细胞的频率、多功能性（TNFα、IFNγ、IL-17）、表型（记忆）和穿孔素/酶的表达：结果：RA和HC血液中GMCSF+CD8+（3.7，4.1%，P=0.540）或GMCSF+CD4+ T细胞（4.5，5.2%，P=0.450）的频率无明显差异。然而，与血液相比，RA 患者血清中的 CD8+GMCSF+ （5.8，3.9%，p=0.0045）和 CD4+GMCSF+ （8.5，4.5%，p=0.0008）T 细胞明显增多。与 RA 患者的血液相比，SF 中多功能三细胞因子阳性 TNFα+IFNγ+GMCSF+CD8+T 细胞（81，36%，p=0.049）和 CD4+T 细胞（48，32%，p=0.010）也更高。在RA-SF中，CD8+T细胞显示出更高的效应记忆表型频率和颗粒酶-B表达。在纵向随访中，MTX后血液中CD4+GMCSF+ T细胞显著下降（4.6，2.9%，P=0.0014）：我们报告了一项新发现：在 RA-SF 中，除了 CD4+GMCSF+ T 细胞外，CD8+GMCSF+ T 细胞也很丰富。这些细胞对 TNFα 和 IFNγ 表现出更高的多功能性和效应记忆表型，表明它们参与了 RA 的发病机制。
Rheumatoid arthritis synovial fluid shows enrichment of T-cells producing GMCSF which are polyfunctional for TNFα and IFNγ.
Objectives: GMCSF+T-cells may be involved in pathogenesis of rheumatoid arthritis (RA), and polyfunctionality may be a marker of pathogenicity. Although, higher frequencies of CD4+GMCSF+ T-cells have been reported, there are no data on CD8+GMCSF+ T-cells or polyfunctionality.Our objective was to enumerate frequencies of CD8+GMCSF+ T cells in RA blood and synovial fluid (SF), and assess their polyfunctionality, memory phenotype and cytotoxic ability.
Methods: This study included RA patients (blood samples,in some with paired synovial fluid (SF)), healthy controls (HC) (blood) and SpA patients (SF). In some RA patients' blood was sampled twice, before and 16-24 weeks after methotrexate (MTX) treatment. After mononuclear cell isolation from blood and SF, ex-vivo stimulation using PMA/Ionomycin was done, and cells were stained (surface and intracellular after permeabilisation/fixation). Subsequently, frequencies of GMCSF+CD8+ and CD4+ T-cells, polyfunctionality (TNFα, IFNγ, IL-17), phenotype (memory) and perforin/granzyme expression were assessed by flowcytometry.
Results: There was no significant difference in frequencies of GMCSF+CD8+ (3.7, 4.1%, p=0.540) or GMCSF+CD4+ T-cells (4.5, 5.2%, p=0.450) inblood of RA and HC. However, there was significant enrichment of both CD8+GMCSF+ (5.8, 3.9%, p=0.0045) and CD4+GMCSF+ (8.5, 4.5%, p=0.0008) T-cells inSF compared to blood in RA patients. Polyfunctional triple cytokine positive TNFα+IFNγ+GMCSF+CD8+T-cells (81, 36%, p=0.049) and CD4+T-cells (48, 32%, p=0.010) was also higher in SF compared to blood in RA. CD8+ T cells showed higher frequency of effector-memory phenotype and granzyme-B expression in RA-SF. On longitudinal follow-up, blood CD4+GMCSF+ T-cells significantly declined (4.6, 2.9%, p=0.0014) post-MTX.
Conclusions: We report a novel finding of enrichment of CD8+GMCSF+ in addition to CD4+GMCSF+ T-cells in RA-SF. These cells showed higher polyfunctionality for TNFα and IFNγ, and effector memory phenotype suggesting their involvement in RA pathogenesis.
期刊介绍：
Clinical and Experimental Rheumatology is a bi-monthly international peer-reviewed journal which has been covering all clinical, experimental and translational aspects of musculoskeletal, arthritic and connective tissue diseases since 1983.
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