Journal: The Journal of Experimental Medicine

Article Title: CD8 T Cells Are Required for the Formation of Ectopic Germinal Centers in Rheumatoid Synovitis

doi: 10.1084/jem.20011565

Figure Lengend Snippet: Follicular TCR β-chain sequences preferentially derive from CD8 T cells. Synovial tissue was selected from five patients with RA who had multiple ectopic GCs in the synovial membrane. (A) The distribution of CD4 T cells (left), CD20 B cells (middle), and IgD + cells (right) is shown in a representative example. The mantle zone is indicated by an asterisk (right). Follicles were microdissected and TCR β-chain sequences were obtained ( and ). In parallel, synovial tissue and peripheral CD4 and CD8 cells were purified by FACS ® . cDNA was tested for the presence of these TCR β-chain sequences by PCR-ELISA using N-D-N–specific oligonucleotides as probes. (B) In four of the five patients, the majority of the TCR β-chain sequences were detected in the CD8, and not the CD4, population. (C) To define the localization of the CD8 cells in synovial follicles, tissue sections were stained with anti-CD8 (brown) and anti-CD23 (red, expressed on follicular dendritic cells) mAb. CD8 T cells were found in the perifollicular zone, sometimes within the mantle zone, and only occasionally in the GC. Original magnifications: (left) × 100 and (right) × 400.

Article Snippet: Total human IgG in the serum of human synovium-SCID mouse chimeras was measured by solid-phase capture sandwich ELISA (AlerChek).

Techniques: Membrane, Purification, Enzyme-linked Immunosorbent Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: CD8 T Cells Are Required for the Formation of Ectopic Germinal Centers in Rheumatoid Synovitis

doi: 10.1084/jem.20011565

Figure Lengend Snippet: Functional profiles of synovial CD8 + T cell subsets. TCR sequences that were derived from synovial follicles were found in purified synovial CD8 + CD40L + T cells, suggesting that follicular CD8 + T cells may have a distinct functional profile . Synovial tissue CD8 + T cells were separated into CD40L + and CD40L − populations by FACS ® and analyzed for the transcription of perforin, granzyme A, and IFN-γ. (A) Results for two patients are shown. Expression of perforin and granzyme A was restricted to CD8 + CD40L − T cells. (B) Immunohistochemistry confirmed that the majority of CD8 T cells (blue, yellow arrows) in the synovial lymphoid follicles lacked perforin expression. Perforin-positive cells (brown, black arrows) were absent in most follicles (left) and only occasionally seen in others (right). These perforin-positive cells did not coexpress CD8 and were presumably NK or NK T cells. Original magnification: × 200. (C) IFN-γ–specific transcripts were produced at significantly higher levels in CD40L + than CD40L − T cells. IFN-γ was semiquantified by PCR-ELISA and is shown as mean copy numbers ± SD of triplicate measurements.

Article Snippet: Total human IgG in the serum of human synovium-SCID mouse chimeras was measured by solid-phase capture sandwich ELISA (AlerChek).

Techniques: Functional Assay, Derivative Assay, Purification, Expressing, Immunohistochemistry, Produced, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: CD8 T Cells Are Required for the Formation of Ectopic Germinal Centers in Rheumatoid Synovitis

doi: 10.1084/jem.20011565

Figure Lengend Snippet: Depletion of synovial CD8 T cells suppresses IFN-γ and TNF-α production. Synovial tissues from patients with RA were engrafted into NOD-SCID mice. Chimeras were treated with anti-CD8 mAb; synovial tissue grafts were explanted after 7 d and analyzed for cytokine transcription. Anti-CD8 mAb treatment effectively depleted CD8 cells from the synovial tissue. (A) Transcripts for the CD8 β-chain were amplified by PCR in tissue extracts prepared from the grafts of sham or anti-CD8 treated chimeras. (B) After depletion of synovial CD8 T cells, in situ transcription of IFN-γ and TNF-α was significantly diminished. Results from six experiments with anti-CD8 mAb– and sham-treated mice are shown. Transcript numbers are adjusted relative to 2 × 10 6 β-actin transcripts. Data are given as the mean ± SD of triplicate measurements by PCR-ELISA. (C) Immunohistochemical analysis of tissues retrieved from anti–CD8–treated mice (right) demonstrated that the tissues were depleted of IFN-γ + cells (brown) in contrast to sham-treated mice (left). Ab-mediated depletion of CD8 T cells resulted in the disintegration of synovial follicles and the formation of cell clusters composed of dysmorphic lymphocytes.

Article Snippet: Total human IgG in the serum of human synovium-SCID mouse chimeras was measured by solid-phase capture sandwich ELISA (AlerChek).

Techniques: Amplification, In Situ, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining

Journal: The Journal of Experimental Medicine

Article Title: CD8 T Cells Are Required for the Formation of Ectopic Germinal Centers in Rheumatoid Synovitis

doi: 10.1084/jem.20011565

Figure Lengend Snippet: Ig production in synovial GCs is CD8 T cell dependent. Human synovium-SCID mouse chimeras were either sham-treated or treated with anti-CD8 mAb or isotype-matched control IgG as described in . Serum and synovial tissues were collected from the mice 7 d after the treatment.(A) cDNA from the tissues were amplified with Igκ- and IgG-specific primer sets in multiplex PCR. Anti-CD8 mAb treatment but not treatment with control IgG clearly diminished the transcription of Ig H and L chains. (B) Human IgG was quantified in serum from the treated and sham-treated mice by ELISA. Tissue depleted of CD8 T cells secreted minimal amounts of human IgG. Results are shown as mean ± SD of triplicate measurements. Similar results were seen in experiments with tissues from two additional patients.

Article Snippet: Total human IgG in the serum of human synovium-SCID mouse chimeras was measured by solid-phase capture sandwich ELISA (AlerChek).

Techniques: Control, Amplification, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: CD8 T Cells Are Required for the Formation of Ectopic Germinal Centers in Rheumatoid Synovitis

doi: 10.1084/jem.20011565

Figure Lengend Snippet: Depletion of CD8 T cells disrupts synovial GCs. GCs depend on the production of LT-α1β2 and the presence of FDCs. LT-β transcripts were semiquantified by PCR-ELISA. (A) Treatment with anti-CD8 mAb significantly reduced the relative number of LT-β transcripts in the tissues. Results from one of three experiments are shown as mean ± SD of triplicate measurements. CD8 T cell depletion abrogated CD21L transcription, confirming that FDC networks were no longer present. (B) Synovial tissues retrieved from control and anti-CD8–treated chimeras were assayed for the presence of CD21L transcript, which is exclusively produced by FDCs.

Article Snippet: Total human IgG in the serum of human synovium-SCID mouse chimeras was measured by solid-phase capture sandwich ELISA (AlerChek).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Produced