Case report
Granulocyte-macrophage colony-stimulating factor (GMCSF) was demonstrated compared with peripheral blood CD4+ Tcells (figure 3A), reflecting the inflammation observed at a clinical and histological level. While the frequency of Treg (CD4+CD127C- D25±FOXP3+) cells was comparable between peripheral blood (6.7%), synovial fluid (7%) and synovial tissue (11%), qualitative differences, where observed, with absence of the more stable naive (CD45RO−CCR7+) Treg cells in the synovial fluid/tissue compared with periphery (figure 4B,C) suggesting a possible defect in their ability to supress T-cell function. At baseline PD-1 expressing CD4+ Tcells were not detectable in the periphery (figure 5B), potentially due to stereochemical blockade of epitope recognition following treatment with pembrolizumab. Interestingly, however, PD-1 expression was detectable (37%) in synovial fluid CD4+ Tcells (figure 5B). Tfh (CXCR5+) cell frequency was comparable between the periphery and synovial fluid, although synovial tissue Tfh cells were predominantly positive for the expression of the chemo- kine receptors CCR6 and CXCR3 indicative of a polyfunctional pathogenic Th1/Th17 mixed phenotype (figure 5C). Frequency of CD19+CD20±Bcell was low in synovial fluid (0.6%) and tissue
(1:400). Following fully informed written consent, serial peripheral blood mononuclear cell (PBMCs) isolates and needle arthroscopy using a 2.7mm (Karl Storz, Germany) were performed under local anaesthetic with full sterile procedure. Synovial tissue biopsies were obtained using a 2mm diameter grasping forceps as previously reported. 6 Routine histology and immunohistochemical analysis (CD3+ cellsand PD-L1 expression) of the original adenocarcinoma biopsy of the lung and the joint synovial tissue, before and after treat- ment, are compared in figure 2. Single cell synovial tissue immune cell analysis was performed using flow cytometry on a 15-colour-BD LSRFortessa with specific antibody panels to identify cytokine expression, T-cell responses and polyfunctionality was further assessed, before and after tofacitinib, by Simplified Presentation Of Incredibly Complex Evaluations (SPICE) algorithm (figure 3). 7 CT-guided biopsy of the right lung nodule showed alveolar infiltrated by a non-small-cell lung carcinoma with solid and acinar architecture (figure 2A). Immunohistochemistry was positive for markers of pulmonary adenocarcinoma (TTF-1 and Napsin) and negative for squamous differentiation markers (p63 and CK5/6). Molecular studies did not detect an epidermal growth factor receptor gene mutation and immunohistochem- istry for anaplastic lymphoma kinase was negative. PD-L1 immunohistochemistry was positive (figure 2B) with a tumour proportion score of 30%. Pretofacitinib synovial biopsy H&E staining demonstrated a hyperplastic synovium with dense lymphoplasmacytic infiltrate (figure 2C) comprising predominantly CD3+ T-lymphocytes (446 lymphocytes/hpf, figure 2D) with 30% immunostaining positive for PD-L1 (figure 2E). Repeat synovial biopsy, 30 days after commencing tofacitinib treatment demonstrated stromal fibrosis with a reduced inflammatory infiltrate (figure 2F) with rare CD3 expression (<1%, figure 2G) and negative staining for PD-L1 (figure 2H). Single cell synovial tissue analysis Gating strategy and flow plots for T cell (figure 4A), T regula- tory (Treg) (figure 4B,C), T follicular helper (Tfh) (figure 5A–C) and B cells in PBMC, synovial fluid mononuclear cell and syno- vial tissue are shown in figure 6A,B. An increase in the frequency of synovial tissue CD4+ T cells producing IL-17A, IL-22 and Figure 2 Histological analysis of synovial tissue before and after treatment with tofacitinib. Needle core biopsy of the left lower lobe lung nodule showed acinar pattern pulmonary adenocarcinoma (A). PD-L1 immunohistochemistry staining showed focal membranous staining in 30% of tumour cells (B). Synovial biopsy H&E staining before tofacitinib treatment demonstrated a hyperplastic synovium with dense lymphoplasmacytic infiltrate (C) comprising predominantly CD3+ T-lymphocytes (446 lymphocytes/hpf, D) with 30% immunostaining positive for PD-L1 (E). Repeat synovial biopsy, following response to tofacitinib treatment demonstrated stromal fibrosis with a reduced inflammatory infiltrate (F) with rare CD3 expression (<1%, G) and negative staining for PD-L1 (H).
Figure 3 Analysis of synovial tissue T cell polyfunctionality using SPICE algorithm. Flow cytometric analysis for the frequency of key proinflammatory cytokine-producing synovial tissue CD4 T cells pretreatment and post-treatment with tofacitinib. Frequencies in red denote increase and frequencies highlighted in blue, decrease in the corresponding proinflammatory cytokine post tofacitinib treatment compared with pretreatment responses (A). Following polyparametric flow cytometric analysis of synovial tissue derived T cells, the supervised data visualisation algorithm SPICE was used (B). The pie segments represent the percentage of cells and the pie arcs the cytokine or marker that is being expressed. Pie segments surrounded by overlapping pie arcs denote CD4 T cell populations expressing multiple cytokines simultaneously. Data presented for synovial tissue CD4 T cells at first arthroscopy—prior to the initiation of tofacitinib treatment and second arthroscopy—post tofacitinib treatment. Frequency of polyfunctional synovial tissue CD4 T cells expressing more than three proinflammatory cytokines simultaneously at time of first and second arthroscopy (C).
Murray K, et al . BMJ Case Rep 2021; 14 :e238851. doi:10.1136/bcr-2020-238851
2
Powered by FlippingBook