T cell islet accumulation in type 1 diabetes is a tightly regulated, cell-autonomous event - PubMed (original) (raw)

T cell islet accumulation in type 1 diabetes is a tightly regulated, cell-autonomous event

Greig P Lennon et al. Immunity. 2009.

Abstract

Type 1 diabetes is a T cell-mediated autoimmune disease, characterized by lymphocytic infiltration of the pancreatic islets. It is currently thought that islet antigen specificity is not a requirement for islet entry and that diabetogenic T cells can recruit a heterogeneous bystander T cell population. We tested this assumption directly by generating T cell receptor (TCR) retrogenic mice expressing two different T cell populations. By combining diabetogenic and nondiabetogenic or nonautoantigen-specific T cells, we demonstrate that bystander T cells cannot accumulate in the pancreatic islets. Autoantigen-specific T cells that accumulate in islets, but do not cause diabetes, were also unaffected by the presence of diabetogenic T cells. Additionally, 67% of TCRs cloned from nonobese diabetic (NOD) islet-infiltrating CD4(+) T cells were able to mediate cell-autonomous islet infiltration and/or diabetes when expressed in retrogenic mice. Therefore, islet entry and accumulation appears to be a cell-autonomous and tightly regulated event and is governed by islet antigen specificity.

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Figures

Figure 1

Figure 1. Retroviral-Mediated Stem-Cell Gene Transfer

(A) Schematic representation of the TCRα-2A-TCRβ constructs, including the amino acid sequence of the 2A regions of Thosea asigna virus (T2A) and porcine teschovirus-1 (P2A), GSG linker, and insertion point in the MSCV-IRES-GFP vector. Conserved residues are boxed. The arrow indicates the ‘cleavage’ point between the 2A and 2B peptides. (B) Schematic representation of protocol to produce dual TCR retrogenic mice in NOD/SCID recipients. Retroviral-mediated stem cell gene transfer was performed with MSCV-IRES-GFP and MSCV-IRES-YFP constructs into separate populations of NOD/SCID bone marrow. These were then combined and injected, to produce mice with two different populations of T cells.

Figure 2

Figure 2. HEL-specific “Bystander” CD4+ T cells do not accumulate in the pancreas

Double retrogenic mice, with a diabetogenic T cell labeled with YFP and a HEL-specific “bystander” T cell labeled with GFP. Mice were analyzed 4.5–6.5 weeks post transfer, with the bone marrow, spleen, inguinal lymph nodes, pancreatic lymph nodes and pancreatic islets being extracted, processed, cells counted and subjected to flow cytometric analysis. (A) Representative flow cytometric dot plots from B, gated on live and CD4+ T cells. (B) Mice received bone marrow transduced with either 4.1 or 14H4, or both, at a 50:50 ratio, or a 10:90 ratio. One group received 4.1-YFP bone marrow and bone marrow transduced with empty pMIG vector as a control. Top panel; mean (±SEM) percentage of stem cells for each T cell in the bone marrow, as measured by percentage GFP/YFP expression on Lineage−Sca-1+c-kit+ cells (n=10). Middle and bottom panels, mean (±SEM) numbers of each T cell in the spleen (n=9–10), inguinal lymph nodes (I.L.N, n=7–9), pancreatic lymph nodes (P.L.N., n=6–9) and pancreatic islets (ISLETS, n=10), respectively, as calculated from cell counts and the percentage CD4+FP+ live cells. (C) Representative flow cytometric dot plots from D, showing analysis of T cells from 14H4 HEL-specific mice, injected with 10×106 whole splenocytes from 13 week-old NOD mice, two weeks after bone marrow engraftment, and analyzed 4.5–6.5 weeks post-transplant. Plots are gated on live cells and CD4+ cells. (D) Left panels; percentage of stem cells for each T cell in the bone marrow (n=7–10), as measured by percentage GFP expression on Lineage−Sca-1+c-kit+ cells and total numbers of T cells in the spleen. Right panels show the numbers and ratio to the numbers in the spleen for the pancreatic islets (ISLETS) and pancreatic lymph nodes (P.L.N.), respectively (n=5–9). All figures show mean (±SEM) and numbers were calculated from cell counts and the percentage CD4+GFP+Vβ+ live cells for the retrogenic T cells and the percentage CD4+GFP−TCR+ live cells for the NOD T cells.

Figure 3

Figure 3. An autoantigen-specific CD4+ T cell which does not accumulate in the islet, is increased, but not to a significant extent by the presence of a diabetogenic CD4+ T cell

Retrogenic mice, with diabetogenic 4.1 T cells labeled with YFP and PA17.9G7 GAD-specific T cells labeled with GFP, were analyzed 5.5 weeks post-transplant. (A) Representative flow cytometric dot plots from B, showing 4.1 (YFP) and 9G7 (GFP) T cells in the spleen and pancreatic islets. Plots are gated on live cells and CD4+ cells. (B) Top panels; percentage of stem cells for each T cell in the bone marrow, as measured by percentage GFP/YFP expression on Lineage−Sca-1+c-kit+ cells and total numbers of T cells in the spleen (n=3–5). Next three panels, show numbers and ratio to the numbers in the spleen for the pancreatic islets (ISLETS, n=3–5), inguinal lymph nodes (I.L.N., n=3–5) and pancreatic lymph nodes (P.L.N., n=3–5), respectively. All Figures show mean (±SEM) and numbers were calculated from cell counts and the percentage CD4+FP+ live cells.

Figure 4

Figure 4. Expression of an antigen under the control of a rat-insulin promoter (RIP) allows an antigen-specific CD4+ T cell which does not normally accumulate in the islet, to accumulate

NOD.Cg-Prkdc_scid_ Tg(Ins2-GAD2)2Lt/LtJ transgenic mice and NOD/SCID littermate controls were used as recipients, to generate single retrogenic mice, with either PA21.14H4 “bystander” T cells, 10.23 I-A2-specific T cells, PA17.9G7 GAD-specific T cells or PA19.9G11 GAD-specific T cells. Mice were analyzed at 4.5–6.5 weeks post-transplant. (A) Representative flow cytometric dot plots from B, showing T cells in the spleen and pancreatic islets. Plots are gated on live cells. (B) Top four panels; mean (±SEM) numbers of each T cell in the spleen, inguinal lymph nodes (I.L.N.) pancreatic lymph nodes (P.L.N.), and pancreatic islets (ISLETS), respectively, as calculated from cell counts and the percentage CD4+GFP+ live cells (all n=1–5). Third panel, mean (±SEM) ratio of CD4+ T cells in the islets, relative to the numbers in the spleen. Bottom panel: Percentage insulitis, at 6.5 weeks post-transplant (n=3–6).

Figure 5

Figure 5. An autoantigen-specific CD4+ T cell which accumulates in the islet poorly, is increased, but not to a significant extent by the presence of a diabetogenic CD4+ T cell

Retrogenic mice, with diabetogenic 4.1 T cells labeled with YFP and Phogrin18 phogrin-specific T cells labeled with GFP, were analyzed 4.5–6.5 weeks post-transplant. (A) Representative flow cytometric dot plots from B, showing 4.1 (YFP) and Phogrin18 (GFP) T cells in the spleen and pancreatic islets. Plots are gated on live cells and CD4+ cells. (B) Top panels; percentage of stem cells for each T cell in the bone marrow, as measured by percentage GFP/YFP expression on Lineage−Sca-1+c-kit+ cells and total numbers of T cells in the spleen (n=12–15). Next three panels, show numbers and ratio to the numbers in the spleen for the pancreatic islets (ISLETS, n=12–15), inguinal lymph nodes (I.L.N., n=10–15) and pancreatic lymph nodes (P.L.N., n=10–14), respectively. All Figures show mean (±SEM) and numbers were calculated from cell counts and the percentage CD4+FP+ live cells. * p=0.016, t=2.526, 27 d.f., t-test.

Figure 6

Figure 6. An autoantigen-specific CD4+ T cell which accumulates in the islet but does not cause destruction, does not accumulate in higher numbers in the presence of a diabetogenic CD4+ T cell or T cells from whole NOD splenocytes

Retrogenic mice, with diabetogenic 4.1 T cells labeled with YFP and 12-4.4V1 insulin-specific T cells labeled with GFP, were analyzed 4.5–6.5 weeks post-transplant. (A) Representative flow cytometric dot plots from B, showing 4.1 (YFP) and 12-4.4V1 (GFP) T cells in the spleen and pancreatic islets. Plots are gated on live cells and CD4+ cells. (B) Top panels; percentage of stem cells for each T cell in the bone marrow (n=7–8), as measured by percentage GFP/YFP expression on Lineage−Sca-1+c-kit+ cells and total numbers of T cells in the spleen. Next three panels, show numbers and ratio to the numbers in the spleen for the pancreatic islets (ISLETS), inguinal lymph nodes (I.L.N.) and pancreatic lymph nodes (P.L.N.), respectively (n=6–9). All Figures show mean (±SEM) and numbers were calculated from cell counts and the percentage CD4+FP+ live cells. (C) Diabetes incidence. (D) Representative flow cytometric dot plots from E, showing analysis of T cells from 12-4.4V1 Insulin-specific mice, injected with 10×106 whole splenocytes from 13 week-old NOD mice, two weeks after bone marrow engraftment, and analyzed 4.5–6.5 weeks post-transplant. Plots are gated on live cells and CD4+ cells. (E) Left panels; percentage of stem cells for each T cell in the bone marrow (n=7–10), as measured by percentage GFP expression on Lineage−Sca-1+c-kit+ cells and total numbers of T cells in the spleen. Right panels show the numbers and ratio to the numbers in the spleen for the pancreatic islets (ISLETS) and pancreatic lymph nodes (P.L.N.), respectively (n=5–9). All figures show mean (±SEM) and numbers were calculated from cell counts and the percentage CD4+GFP+Vβ+ live cells for the retrogenic T cells and the percentage CD4+GFP−TCR+ live cells for the NOD T cells.

Figure 7

Figure 7. TCRs isolated from NOD islets-infiltrating CD4+ T cells can mediate T cell accumulation in the islets of retrogenic mice

Vα2+Vβ6+ and Vα2+Vβ10+ CD4+ T cells were single-cell sorted from the spleen and islets of a 13 week-old female NOD mouse. The TCR chains were then amplified using a nested RT-PCR, sequenced and cloned into a 2A-linked retroviral vector. Retrogenic mice generated using these vectors, were analyzed 8-10 weeks after engraftment (n=2–11). Shown are numbers of CD4+GFP+ T cells in the spleen (top panel) and pancreatic islets (bottom panel). Plots are gated on live cells. Islet derived clones are indicated by the black bars and the “I” in their name, splenic clones by open bars and the “S” in their name. NY4.1 is included as a control and is indicated by the gray bar. The * indicates that mice in this group were diabetic when analyzed at 8–10 weeks after engraftment. GL25.10I1 T cells induced rapid diabetes, and these mice were analyzed at 5–6 weeks post engraftment.

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