1. Volume 10, Issue 3, Pages 377-386 (March 1999)
A Novel Role for the Major Histocompatibility Complex Class II Transactivator CIITA in the Repression of IL-4 Production 
Tania Gourley, Stacey Roys, Nicholas W Lukacs, Steven L Kunkel, Richard A Flavell, Cheong-Hee Chang 
Immunity 
Volume 10, Issue 3, Pages (March 1999)
DOI: /S (00)

2. Figure 1 Flow Cytometric Analysis of Lymphocytes
Lymphocytes of CIITA−/−xI-E mice express the I-E transgene (A). The transgene expression is sufficient to mediate positive selection of mature CD4 T cells (B), and CD4 lymphocytes express the comparable levels of other cell surface markers (C). The histograms shown in (C) are gated on CD4-positive cells and ten times more cells were collected from CIITA−/− mice. B10.BR mice were used as a control.
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3. Figure 2
Dysregulation of IL-4 Production by CIITA-Deficient CD4 T Cells
(A) Total CD4 T cells from spleen and lymph nodes from the control (B10.BR) and CIITA−/−xI-E mice were stimulated for 4 days with anti-CD3 antibody without exogenous cytokines and restimulated with anti-CD3 for one more day. The supernatant after secondary stimulation was analyzed for IL-4 and IFNγ by ELISA.
(B) Naive CD4 T cells from CIITA−/−xI-E but not control (B10.BR) or Aβ−/−xI-E mice produce IL-4 in the presence of IL-12. The supernatant was assayed for IL-4 or IFNγ by ELISA. Three independent experiments gave comparable results and results of one representative experiment are shown here.
(C) IL-4 gene transcription from IL-12-treated CD4 T cells in the absence of CIITA. Naive CD4 T cells from control (B10.BR)Aβ−/−xI-E, and CIITA−/−xI-E mice were sorted and stimulated for 2 days with anti-CD3 in the presence of cytokines. mRNA was prepared and RT–PCR was performed using primers described in Experimental Procedures.
(D) CIITA-deficient CD4 cells transcribe IL-5 and IL-13 upon stimulation with either IL-4 or IL-12. Cells were treated and assayed as in (C).
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4. Figure 3
The Expression of the CIITA and the MHC Class II Gene in Th1 Cells
(A) Naive CD4 T cells of control mice (C57BL/6) from spleen and lymph nodes were stimulated with anti-CD3 antibody in the presence of either IL-4 or IL-12 for 2 days. The levels of transcripts of the CIITA and the MHC class II (Aαb) gene are inducible in cells stimulated with IL-12 but not IL-4.
(B) IFNγ is necessary to activate CIITA gene transcription. Naive CD4 T cells were stimulated with the same condition as in (A). Anti-IFNγ (H22) was used to block endogenously produced IFNγ in the culture. The PCR products were fractionated on an agarose gel, transferred, and hybridized with radiolabeled CIITA and Aαb genes. Results of one representative experiment of four independent experiments are shown here.
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5. Figure 4
The Effect of CIITA Deficiency on Type 1 Pulmonary Granuloma Size and Cytokine Production
(A) Cross-sectional area of day 4 type 1 bead pulmonary granulomas in mice. There were six mice per group, with a minimun of 20 granulomas measured per animal.
(B) IL-4 and IFNγ production by CD4 T cells from CIITA−/−xI-E and Aβ−/−xI-E mice after challenge with PPD. Two weeks after sensitization with PPD in vivo, 3 × 105 enriched CD4 T cells were stimulated in vitro with PPD (5 μg/ml) or Con A (2.5 μg/ml) in the presence of irradiated B10.BR splenocytes as APC. The supernatant was collected 2 days later and IL-4 and IFNγ production was measured by ELISA.
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6. Figure 5
CIITA Activates the Expression of MHC Class II but Represses IL-4 in D10 Cells
(A) D10 cells were stably transfected with CIITA or DNA encoding the neomycin gene as a control. The transfectants were tested for the expression of T cell receptor (H57) and MHC class II (14.4.4S) by flow cytometry. Three clones showed a similar phenotype, and one of them was used for the experiment.
(B) CIITA downregulates IL-4 production in D10 cells. D10 cells and D10 cells transfected with CIITA were stimulated in the presence of 5 μg/ml of plate-bound anti-CD3 antibody. The supernatant and cells were collected for ELISA and RNA preparation, respectively, at indicated times after stimulation. RT–PCR was performed using different amounts of first strand cDNA product to measure IL-4 transcripts.
(C) CIITA represses transcription of the IL-5 and the IL-13 gene. Cells transfected with the control DNA or CIITA were stimulated with anti-CD3 as in (B). RNA was prepared 2 hr and 24 hr after stimulation, and RT–PCR was performed using primers specific for IL-5 and IL-13.
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7. Figure 6 CIITA Represses IL-4 Gene Transcription in D10 Cells
D10 cells were transfected with the IL-4 promoter–driven luciferase (A) or the MHC class II promoter–driven luciferase (B) with the empty vector DNA, sense CIITA, or antisense CIITA. (C) The 157 bp fragment of the IL-4 promoter is sufficient to be repressed by CIITA. The fragment encompassing 157 bp or 3 kb of the IL-4 promoter was transfected with CIITA or the control DNA. The cells were harvested and analyzed for luciferase activity 2 days after transfection. Expression units were calculated using the luciferase activity of cells transfected with control DNA as 1. The values shown were normalized to β-gal activity (internal control).
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8. Figure 7 The Cascade of CD4 T Cell Differentiation
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