Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD8αα + MHC-II + Cell with Capacity to Terminate Autoimmune Inflammation Is A Novel Antigen-Presenting NK-Like Cell in Rats

doi: 10.4049/jimmunol.1601207

Figure Lengend Snippet: CD8αα+RT1B/D+ cells express both genes associated with professional antigen processing/presenting and NK cytotoxicity. (A) Multi-color flow cytometry analyses show a CD8αα+RT1B/D+ population (purple gate on RT1B-FITC vs CD8α-APC plot, left panel) in rat peripheral blood mononuclear cells (PBMC); right panel shows purified CD8αα+RT1B/D+ population as shown by both SSC vs FSC and CD8α vs RT1B/D plot. (B) Representative flow cytometric plots show each isolated population from glomeruli infiltrating leukocytes as indicated on the top of each plot. MΦ, macrophages. (C) A representative clustering analysis based on DNA microarray shows similar global gene expression patterns among all CD8αα+RT1B/D+ subpopulations from different sources in both LEW and WKY rats; glomeruli-infiltrating macrophages (MΦ) and pan T cells are shown as controls. (D) Summary of expressions of gene clusters associated with NK cells, APCs and T cells. n=4. Inset, RT-PCR detection of Tyrobp and control house keeper gene Gapdh. (E) Summary of expressions of genes associated with Ag processing/presentation for professional APCs; expression levels in (D) and (E) are mean normalized intensities for each gene in DNA microarray. n=4. (F) A representative RT-PCR shows expression of Ag presenting related α chain of rat MHC class II RT1B (Rt1bα) and invariant chain (Clip) in various cell populations as indicated; TcR ε chain (Tcrε) and Gapdh were used as controls.

Article Snippet: Rat DNA microarray assay Total RNA from various cell populations were isolated with a kit (Ambion, Austin, TX) and checked for their quality.

Techniques: Flow Cytometry, Purification, Isolation, Microarray, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD8αα + MHC-II + Cell with Capacity to Terminate Autoimmune Inflammation Is A Novel Antigen-Presenting NK-Like Cell in Rats

doi: 10.4049/jimmunol.1601207

Figure Lengend Snippet: CD8αα+RT1B/D+ cells express NK killing stimulatory receptors Ly49s6. (A) DNA microarray shows expression of various NK receptors in PBMC (P) and glomeruli-infiltrating (G) CD8αα+RT1B/D+ cells, but not in control PBMC T cells and glomeruli-infiltrating macrophages; NK receptor genes are grouped; gene Klrb1a which encodes hallmark of an NK subset NKR-P1B is underlined; note that many Ly49 members were not covered by DNA microarray. (B) RT-PCR detection of mRNAs for representative Ly49 receptors in the indicated cell populations; lower panel shows degenerative PCR products for transmembrane (TM) and intracellular fragment of Ly49 in glomeruli-infiltrating CD8αα+RT1B/D+ cells. (C) Alignment of a.a. sequences of cloned cDNA from degenerative PCR (see lower panel in (B) as connected by lines) demonstrates expression of mainly killing stimulatory receptors in CD8αα+RT1B/D+ cells; numbers of clones for each sequence are indicated at the right with majority of them being Ly49s6; inhibitory motif in intracellular fragment of Ly49 is underlined in red at the bottom; TM, transmembrane. Lower panel shows a.a. sequence of cloned Ly49s6 with transmembrane domain shadowed. (D) Three-color flow cytometry on glomeruli-infiltrating leukocytes after removal of pan T cells shows a RT1B/D+Ly49s6+ population (lower panel) which is also CD8α+ (upper panel for the gated population in lower panel). (E) Immunofluorescence shows expression of both Ly49s6 (red) and RT1B (green) on a CD8αα+RT1B/D+ cell; a nearby T cell is shown as an internal control for staining. bar= 3μm

Article Snippet: Rat DNA microarray assay Total RNA from various cell populations were isolated with a kit (Ambion, Austin, TX) and checked for their quality.

Techniques: Microarray, Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Sequencing, Flow Cytometry, Immunofluorescence, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD8αα + MHC-II + Cell with Capacity to Terminate Autoimmune Inflammation Is A Novel Antigen-Presenting NK-Like Cell in Rats

doi: 10.4049/jimmunol.1601207

Figure Lengend Snippet: Activated autoreactive T cells with elevated level of RT1CE16 are more susceptible to the killing by CD8αα+RT1B/D+Ly49s6+ cells. (A) Summary of classic (RT1A) and non-classic (RT1C/E, M/N and S/T) MHC class I gene cluster expression pattern in PBMC (P) and glomeruli-infiltrating (GIL) T cells; expression level is normalized intensities in DNA microarray. n=4. (B) Flow cytometry shows detection of T cell activation marker CD69 in PBL pan T cells or glomeruli-infiltrating T cells as indicated. (C) Conventional RT-PCR detection of representative non-classic MHC I genes in PBL, splenic (SP), and glomeruli-infiltrating T cells. (D) Quantitative PCR detection of Rt1ce16 expression in glomeruli-infiltrating or PBL T cells. Relative expression levels to house-keeping gene Gapdh are shown. (E) Summary of susceptibility of T cells to CD8αα+RT1B/D+Ly49s6+ cell-mediated killing. Killing efficacy is expressed as % of remaining CFSE-labeled target T cells. T cells alone were used as control. (F) Correlation between RT1CE16 expression level in resting and activated pCol(28–40)-specific T cell lines and their susceptibilities to CD8αα+RT1B/D+Ly49s6+ cell-mediated killing; RT1CE16 expression levels were measured by qPCR and expressed as % of average for activated T cells; linear progression was calculated; each sample was duplicated. (G) Schematic diagram depicts three-step killing of autoreactive T cells by CD8αα+RT1B/D+Ly49s6+ cells (renamed to CD8αα+ AP-NK cell): Step 1, Coupling of CD8αα+ AP-NK cell with effector autoreactive T cell through its Ag presentation. Step 2, Binding of Ly49s/CD8αα on CD8αα+ AP-NK cell to RT1CE16 on the T cell. Step 3, DAP12 mediated release of granule cytotoxic proteins Gzm C and perforin from CD8αα+ AP-NK cell, which induce apoptosis in the target T cell.

Article Snippet: Rat DNA microarray assay Total RNA from various cell populations were isolated with a kit (Ambion, Austin, TX) and checked for their quality.

Techniques: Expressing, Microarray, Flow Cytometry, Activation Assay, Marker, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Labeling, Control, Binding Assay