Immune Netw.  2014 Apr;14(2):89-99. 10.4110/in.2014.14.2.89.

Kinetics of IFN-gamma and IL-17 Production by CD4 and CD8 T Cells during Acute Graft-versus-Host Disease

• Ju JM
• Lee H
• Oh K
• Lee DS
• Choi EY

• KMID: 2168019
• DOI: http://doi.org/10.4110/in.2014.14.2.89

Abstract

Graft-versus-host disease (GVHD) is a fatal complication that occurs after allogeneic hematopoietic stem cell transplantation. To understand the dynamics of CD4 and CD8 T cell production of IFN-gamma and IL-17 during GVHD progression, we established a GVHD model by transplanting T cell-depleted bone marrow (TCD-BM) and purified T cells from B6 mice into irradiated BALB.B, creating an MHC-matched but minor histocompatibility (H) antigen-mismatched transplantation (B6 --> BALB.B GVHD). Transplantation-induced GVHD was confirmed by the presence of the appropriate compositional changes in the T cell compartments and innate immune cells in the blood and the systemic secretion of inflammatory cytokines. Using this B6 --> BALB.B GVHD model, we showed that the production of IFN-gamma and IL-17 by CD4 T cells preceded that by CD8 T cells in the spleen, mesenteric lymph node, liver, and lung in the BALB.B GVHD host, and Th1 differentiation predated Th17 differentiation in all organs during GVHD progression. Such changes in cytokine production were based on changes in cytokine gene expression by the T cells at different time points during GVHD development. These results demonstrate that both IFN-gamma and IL-17 are produced by CD4 and CD8 T cells but with different kinetics during GVHD progression.

Keyword

GVHD; B6 --> BALB.B GVHD model; Kinetics of IFN-gamma and IL-17 production

MeSH Terms

Animals
Bone Marrow
Cytokines
Gene Expression
Graft vs Host Disease*
Hematopoietic Stem Cell Transplantation
Histocompatibility
Interleukin-17*
Kinetics*
Liver
Lung
Lymph Nodes
Mice
Spleen
T-Lymphocytes*
Cytokines
Interleukin-17

Figure

• 

  Figure 1 Establishment of minor H antigen-disparate graft-versus-host-disease (GVHD) model using T-cell depleted (TCD) bone marrow (BM) and purified T cells. (A) BALB.B (●) or B6 (○) mice were lethally irradiated and infused with 5×106 TCD BM with 6×106 or 3×106 MACS-enriched splenic T cells from B6 mice; n=10 mice/group. Survival rate is shown. (B) BALB.B (■) or B6 (□) mice were lethally irradiated and infused with 5×106 TCD BM with 3×106 MACS-enriched splenic T cells from B6 mice; n=10 mice/group. Sera were collected by centrifugation of the blood from host mice. Host serum cytokine levels of IFN-γ, IL-6, and IL-17 were measured by ELISA on days 4, 7, 14, and 21 post-transplantation. (C) T cells and myeloid cell fractions in the peripheral blood from allogeneic and syngeneic hosts on days 7 and 21. PBLs were pooled and stained with anti-CD4, CD8, Gr-1, and CD11b antibodies and analyzed by flow cytometry. Data representing more than three independent experiments are shown; n=10 mice/group. (D) PBLs from allogeneic and syngeneic hosts on days 7 and 21 were labeled with anti-CD4 and anti-CD8 antibodies, and the CD4/CD8 ratios were compared. Four mice were analyzed in each group. ***p<0.001, **p<0.01. Error bars represent the mean±SD.

• 

  Figure 2 The local dynamics of cellular composition during mild acute GVHD. Leukocytes infiltrating the spleen, mesenteric lymph node (mLN), liver, and lung of the GVHD hosts received TCD-BM cells. A total of 3×106 splenic T cells from B6 mice were prepared on days 7 and 21 post-transplantation and T cell fractions were analyzed using flow cytometry after staining with anti-CD4 and CD8 antibodies. (A) Representative flow cytometry data for CD4+ and CD8+ T cells obtained from allogeneic and syngeneic hosts on days 7 and 21 are shown. (B) Absolute numbers of CD4 and CD8 T cells in the secondary lymphoid organs (spleen and mLN) and target organs (liver and lung) from allogeneic and syngeneic hosts on days 7 and 21 are plotted. Data representing more than three independent experiments are shown. (C) T cells from the spleen, mLN, liver, and lung target organs from allogeneic or syngeneic hosts on day 7 and day 21 were labeled with anti-CD4 and anti-CD8 antibodies, and the CD4/CD8 ratios were compared. Four mice were analyzed in each group. ***p<0.001, **p<0.01, *p<0.05. Error bars represent the mean±SD.

• 

  Figure 3 Dynamics of IFN-γ production by CD4 and CD8 T cells infiltrating the organs of GVHD hosts. Lymphocytes were prepared from secondary lymphoid organs (spleen and mLN) and target organs (liver and lung) of BALB.B and B6 host mice that received TCD BM and 3×106 splenic T cells from B6 mice on days 7 and 21 post-transplantation. The lymphocytes were analyzed for cytokine production by flow cytometry after intracytoplasmic staining with anti-IFN-γ in combination with anti-CD4 and anti-CD8 antibodies. (A) IFN-γ and CD4 profiles in CD4 T cells (top) and percentages and absolute numbers of the IFN-γ-producing CD4 T cells (bottom) from the spleen, mLN, liver, and lung target organs on day 7 and 21 post-GVHD progression are plotted. (B) IFN-γ and CD8 profiles in CD8 T cells (top) and percentages and absolute numbers of the IFN-γ-producing CD8 T cells (bottom) from the spleen, mLN, liver, and lung target organs on days 7 and 21 post-GVHD progression are plotted. (C) T cells from spleen, mLN, liver, and lung target organs from allogeneic and syngeneic hosts on days 7 and 21 were labeled with anti-CD4, CD8, and IFN-γ antibodies, and the CD4/CD8 ratios of the IFN-γ+ cells were compared. Four mice were analyzed in each group. The data represent more than three independent experiments. ***p<0.001, **p<0.01, *p<0.05. Error bars represent the mean±SD.

• 

  Figure 4 Dynamics of IL-17 production by CD4 and CD8 T cells infiltrating the organs of GVHD hosts. Lymphocytes were prepared as Fig. 3. The lymphocytes were analyzed for cytokine production by flow cytometry after intracytoplasmic staining with anti-IL-17 in combination with anti-CD4 and anti-CD8 antibodies. (A) IL-17 and CD4 profiles of CD4 T cells (top) and percentages and absolute numbers of IL-17-producing CD4 T cells (bottom) from the spleen, mLN, liver, and lung target organs on days 7 and 21 post-GVHD progression are plotted. (B) IL-17 and CD8 profiles of CD8 T cells (top) and percentages and absolute numbers of the IL-17-producing CD8 T cells (bottom) from the spleen, mLN, liver, and lung target organs on days 7 and 21 post-GVHD progression are plotted. (C) T cells from the spleen, mLN, liver, and lung target organs of allogeneic and syngeneic hosts on days 7 and 21 were labeled with anti-CD4, CD8 and IL-17 antibodies, and the CD4/CD8 ratios of IL-17+ cells were compared. Four mice were analyzed in each group. The data represent more than three independent experiments. ***p<0.001, **p<0.01, *p<0.05. Error bars represent the mean±SD.

• 

  Figure 5 Dynamics of cytokine production in CD4 and CD8 T cells. Levels of IFN-γ (A) and IL-17 (B) mRNA transcripts were determined using quantitative real-time RT-PCR with MACS-enriched splenic CD4 and CD8 T cells from each host on days 7 and 21. Fold changes in IFN-γ and IL-17 mRNA expression levels compared to β-actin as the internal control were calculated using the 2-ΔΔCt method. The data represent more than three independent experiments. **p<0.01, *p<0.05. Data performed in triplicate are presented as the mean±SD; n=6 mice/group.

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