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. 2013 Oct 21;210(11):2403-14.
doi: 10.1084/jem.20130582. Epub 2013 Oct 14.

Pathogenic CD4⁺ T Cells Recognizing an Unstable Peptide of Insulin Are Directly Recruited Into Islets Bypassing Local Lymph Nodes

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Free PMC article

Pathogenic CD4⁺ T Cells Recognizing an Unstable Peptide of Insulin Are Directly Recruited Into Islets Bypassing Local Lymph Nodes

James F Mohan et al. J Exp Med. .
Free PMC article

Abstract

In the nonobese diabetic mouse, a predominant component of the autoreactive CD4(+) T cell repertoire is directed against the B:9-23 segment of the insulin B chain. Previous studies established that the majority of insulin-reactive T cells specifically recognize a weak peptide-MHC binding register within the B:9-23 segment, that to the 12-20 register. These T cells are uniquely stimulated when the B:9-23 peptide, but not the insulin protein, is offered to antigen presenting cells (APCs). Here, we report on a T cell receptor (TCR) transgenic mouse (8F10) that offers important new insights into the biology of these unconventional T cells. Many of the 8F10 CD4(+) T cells escaped negative selection and were highly pathogenic. The T cells were directly recruited into islets of Langerhans, where they established contact with resident intra-islet APCs. Immunogenic insulin had to be presented in order for the T cells to localize and cause disease. These T cells bypassed an initial priming stage in the pancreatic lymph node thought to precede islet T cell entry. 8F10 T cells induced the production of antiinsulin antibodies and islets contained immunoglobulin (IgG) deposited on β cells and along the vessel walls.

Figures

Figure 1.
Figure 1.
Analysis of 8F10 mice. (A, left) CD4 and CD8 flow cytometric profiles of NOD and 8F10 thymocytes; (right) percentages of cells found among individual 8F10 mice. (B) Cell surface staining of 8F10 and NOD CD4+ splenocytes with anti-TCR Vβ8.1/8.2 (left) or anti-TCR Vα2 (right). (C) Cell surface staining of 8F10 CD4+ splenocytes with anti-CD44 and -CD62L. (D) Thymic (top left) and splenic (bottom left) profiles, and percentages of T cells among individual 8F10 rag1−/− mice (right). (E) Absolute number of thymocytes and splenocytes from 8F10 and 8F10 rag1−/− mice. (F, left) Foxp3 staining of CD4+ single-positive thymocytes and CD4+ splenocytes of 8F10 and 8F10 rag1−/− mice; (right) percentages of Foxp3+ T cells from individual 8F10 and 8F10 rag1−/− mice. (A–F) Representative flow cytometry plots and cumulative data from two or more independent experiments (error bars, SEM). Statistical analysis: Mann-Whitney U test, (*, P < 0.05; **, P < 0.005).
Figure 2.
Figure 2.
Reactivity of 8F10 CD4+ T cells. (A) Primary proliferation of isolated 8F10 CD4+ T cells in response to B:9-23 peptide and insulin protein presented by CD11c+ DCs. (B) Primary proliferation of isolated 8F10 CD4+ T cells in response to nested register peptides (core peptides) containing a single register of the B:9-23 peptide presented by CD11c+ DCs. (C) Primary proliferation of splenocytes isolated from 8F10 rag1−/− mice incubated with insulin or B:9-23 peptide. (D) Enzyme-linked immunospot (ELISPOT) assay of IL-2 secretion by splenocytes isolated from 8F10 rag1−/− mice pulsed with the B:9-23 peptide, insulin protein, or nested register peptides (core peptides). (A–D) Data representative of two independent experiments (error bars, SEM).
Figure 3.
Figure 3.
Recruitment of 8F10 T cells to islets and islet reactivity. Islet cytology evaluation of 8F10 female mice at 8–10 (A) or 14–19 (B) wk of age. (A and B, left) Number of T cells (CD4+ or Vα8.1/8.2+) per individual islet; bars indicate the median number of T cells per islet. (A and B, right) Percentage of islets positive for CD4+ T cells, Vβ8.1/8.2+ T cells, VCAM-1+ expression on vessels and mouse IgG+ deposition from pooled islets (n = 5 mice per group) and 100 islets screened for each marker. (C) Representative immunofluorescence image of an islet from A showing T cells by Vα8.1/8.2+ staining. Insets show T cell–APC contacts. (D) Representative islet from A showing mouse IgG deposition on the β cells (left). Inset shows IgG+ deposition on β cell membrane. (right) IgG+ deposition found along intra-islet vessels from A. (E) Radiolabeled I-125 insulin response of antiinsulin antibody or 8F10 mouse sera (8–12 wk) in the presence or absence of competing insulin (INS). (F) Unmanipulated controls (NOD and B16A) and bone marrow chimeric mice (8F10/B16A and 8F10/NOD) indicating the number of CD4+ T cells and the percentages of islets containing T cells; bars indicate the median number of T cells per islet. Indicated is diabetes incidence of irradiated B16A and NOD recipients reconstituted with 8F10 rag1−/− bone marrow. (A–F) All data representative of two or more independent experiments (error bars, SEM). Bars, 50 µm. Statistical analysis Mann-Whitney U test (ns, not significant; ****, P < 0.0001).
Figure 4.
Figure 4.
8F10 T cells are specifically recruited to the islets and are highly pathogenic. (A) Insulitis scoring of pancreatic sections from 8F10 mice stained with hematoxylin and eosin (n = 4–7 mice per age group). (B) Hematoxylin and eosin–stained pancreatic section from a representative islet of a 6-wk-old 8F10 mouse showing the periinsulitic lesion. (C) Flow cytometry analysis of islet-infiltrating 8F10 CD4+ T cells and T cells from other anatomical locations (PLN, ILN [inguinal LN], and spleen) in 10-wk-old 8F10 mice. Dispersed islets were pooled from 4 mice. (D) Pancreatic section from a diabetic 5-wk-old 8F10 rag1−/− mouse showing an islet with destructive insulitis. (E) Spontaneous diabetes incidence in 8F10 (n = 60) and 8F10 rag1−/− mice (n = 22). (F) Adoptive transfer of splenocytes from 8F10 rag1−/− mice into NOD rag1−/− recipient mice (106, n = 10; 105, n = 8; 104, n = 4). (A–F) Cumulative data pooled from at least two independent experiments (error bars, SEM).
Figure 5.
Figure 5.
8F10 T cells are not effectively primed in the PLN. CFSE dilution of 8F10 CD4+ T cells in the PLN of recipient NOD mice of the indicated ages at days 3 (A) and 5 (B) after transfer and analyzed by flow cytometry. (C) CFSE dilution of purified transferred CD4+ CD25 8F10 T cells in the PLNs of NOD mice. (D) In vitro CFSE dilution of purified 8F10 CD4+ T cells in the presence of irradiated splenocytes pulsed with B:9-23 peptide. (E) CFSE dilution of 8F10 CD4+ T cells transferred and isolated from the islets of 8-wk-old NOD mice. (F) CFSE dilution of BDC 2.5 CD4+ T cells in the PLN of 8-wk-old NOD mice.(G) Pooled results from multiple experiments depicting percentage of divided 8F10 and BDC 2.5 CD4+ T cells in the ILN, PLN, and islets of NOD recipients. Representative data of at least two independent experiments (A–F) or cumulative data of 2–6 independent experiments (G; error bars, SEM; ns, not significant; **, P < 0.005; ***, P < 0.0005).
Figure 6.
Figure 6.
Islet-infiltrating 8F10 T cells do not require priming in the PLN. (A) Immunofluorescence of a representative islet from 5-wk-old 8F10 nodeless mice. Inserts show T cell–APC contacts. (B) Quantitative analysis showing percentage of infiltrating CD4+ T cells in islets of 5-wk-old 8F10 and 8F10 nodeless mice. (C) Hematoxylin and eosin–stained pancreatic section from a 12-wk-old 8F10 nodeless mouse. (D) Hematoxylin and eosin–stained pancreatic section from a 5-wk-old 8F10 rag1−/− nodeless diabetic mouse. (E) Spontaneous diabetes incidence of 8F10 (n = 10), 8F10 nodeless (8F10 NL, n = 9), 8F10 rag1−/− (8F10 rag1−/−, n = 4), and 8F10 rag1−/− nodeless (8F10 Rag rag1−/− NL, n = 6) mice. Representative (A, C, and D) or cumulative (B and E) data pooled from two to three independent experiments. Bars, 50 µm. Error bars, SEM.

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