In situ recognition of autoantigen as an essential gatekeeper in autoimmune CD8+ T cell inflammation - PubMed (original) (raw)

In situ recognition of autoantigen as an essential gatekeeper in autoimmune CD8+ T cell inflammation

Jinguo Wang et al. Proc Natl Acad Sci U S A. 2010.

Abstract

A current paradigm states that non-antigen-specific inflammatory cues attract noncognate, bystander T cell specificities to sites of infection and autoimmune inflammation. Here we show that cues emanating from a tissue undergoing spontaneous autoimmune inflammation cannot recruit naive or activated bystander T cell specificities in the absence of local expression of cognate antigen. We monitored the recruitment of CD8(+) T cells specific for the prevalent diabetogenic epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214) in gene-targeted nonobese diabetic (NOD) mice expressing a T cell "invisible" IGRP(206-214) sequence. These mice developed islet inflammation and diabetes with normal incidence and kinetics, but their inflammatory lesions could recruit neither naive (endogenous or exogenous) nor ex vivo-activated IGRP(206-214)-reactive CD8(+) T cells. Conversely, IGRP(206-214)-reactive, but not nonautoreactive CD8(+) T cells rapidly homed to and accumulated in the inflamed islets of wild-type NOD mice. Our results indicate that CD8(+) T cell recruitment to a site of autoimmune inflammation results from an active process that is strictly dependent on local display of cognate pMHC and suggest that CD8(+) T cells contained in extralymphoid autoimmune lesions are largely autoreactive.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

NOD.IGRPK209A/F213A KI/KI mice develop insulitis and diabetes without recruiting IGRP206–214-reactive CD8+ T cells into pancreatic islets. (A) Targeting strategy. The FRT-flanked PGK-neo cassette was removed from targeted ES cells by transient transfection of Flp recombinase-encoding cDNA. (B) Distribution of lymphocyte subsets in thymi and spleens from NOD and NOD.IGRPK209A/F213A KI/KI mice (n = 3 and 5, respectively; 3 independent experiments). DN, double negative; DP, double positive; CD8-SP, CD8 single positive; CD4-SP, CD4 single positive. (C) Frequency of NRP-V7–reactive CD8+ T cells in peripheral blood. Peripheral blood mononuclear cells (PBMCs) from 10-week-old mice (NOD, n = 7; NOD.IGRPK209A/F213A KI/KI, n = 8) were stained with NRP-V7/Kd tetramers and anti-CD8 mAb. Data correspond to 4 independent experiments using one to five mice/experiment. (D) Insulitis scores. Pancreata from nondiabetic 32-week-old mice (NOD, n = 3; NOD.IGRPK209A/F213A KI/KI, n = 5) were examined for islet inflammation. Pancreata were from one cohort of NOD mice and two different cohorts of NOD.IGRPK209A/F213A KI/KI mice. (E and F) CD4+ and CD8+ T cell (E) and NRP-V7/Kd tetramer+ CD8+ T cell content (F) in freshly isolated islets of NOD (n = 6; 3 independent experiments) vs. NOD.IGRPK209A/F213A KI/KI mice (n = 7; 4 independent experiments). (G) Absence of IGRP206–214 -reactive CD8+ T cells in the islet infiltrates of NOD.IGRPK209A/F213A KI/KI (n = 10; 10 independent experiments) vs. NOD mice (n = 3; 3 independent experiments). Islet-associated CD8+ T cells were cultured in IL-2 for 7 days and challenged with peptide-pulsed (10 μM) irradiated NOD splenocytes. The IFNγ content in the supernatants (at 48 h) was measured by ELISA. Data correspond to the means ± SEM. (H) Diabetes incidence in female NOD (n = 56) and NOD.IGRPK209A/F213A KI/KI (n = 27) mice. The average blood glucose levels in newly diagnosed diabetic NOD and NOD.IGRPK209A/F213A KI/KI mice are: 22.3 ± 2.5 vs. 23.7 ± 3.3 mM, respectively. In males, the incidence and average age at onset of disease were also similar in both strains (NOD: n = 15; 40% diabetic at 133 ± 33 days; and NOD.IGRPK209A/F213A KI/KI: n = 20; 50% diabetic at 149 ± 36 days). Data in B_–_F correspond to the means ± SEM. P values in F and G were obtained with Mann-Whitney _U_-test.

Fig. 2.

Fig. 2.

Naive 8.3-CD8+ T cells are not recruited into the inflamed pancreatic islets of NOD.IGRPK209A/F213A KI/KI mice. (A) Proliferation of naive 8.3-CD8+ T cells in the PLN. CFSE-labeled naive 8.3-CD8+ T cells were transfused into 10- to 12-week-old NOD (n = 3) or NOD.IGRPK209A/F213A KI/KI (n = 3) recipients (three independent experiments, each using both host types). Dilution of CFSE was measured by flow cytometry 7 days posttransfer. Values correspond to the average percentage of proliferated CD8+ T cells ± SEM. (B) Summary of data described in A. P values were obtained with Mann-Whitney _U_-test. (C) Recruitment and proliferation of naive 8.3-CD8+ T cells from 8.3-NOD.Thy1.1 donor mice in the lymphoid organs and islets of 10- to 12-week-old insulitic NOD and NOD.IGRPK209A/F213A KI/KI hosts 1, 2, and 3 weeks after transfer. CFSE histograms correspond to Thy1.1+CD8+ cells. (D) Mean ± SEM of total numbers of 8.3-CD8+ T cells per million lymphocytes. Data in C and D correspond to three to six experiments/time point and host type (one mouse/time point/host in each experiment). (E) Mean ± SEM of percentages of proliferated cells (for PLNs) or total number of donor lymphocytes (for islet T cell isolates, where all donor-derived T cells were proliferating) (three to six experiments/time point and host type; one mouse/time point/host in each experiment). (F) Absolute numbers of proliferated (for PLNs) or recruited (for islet T cell isolates) 8.3-CD8+ T cells (mean ± SEM) (three to six experiments/time point and host type; one mouse/time point/host in each experiment). P values in D_–_F were obtained by two-way ANOVA.

Fig. 3.

Fig. 3.

Selective recruitment of cognate autoreactive T cells into pancreatic islets of NOD mice. (A) Naive 8.3-CD8+/Thy1.2+ and P14-CD8+/Thy1.2+ T cells were mixed at 1:1 (8 × 106 cells each) and injected i.v. into 10- to 12-week-old NOD.Thy1.1 hosts. Hosts were killed at various time points after transfer and their peripheral lymphoid organs and islet cell suspensions analyzed for presence of NRP-V7/Kd and GP33/Db tetramer+ Thy1.2+ CD8+ T cells. Data correspond to three mice/time point (n = 12 mice) and three independent experiments (one mouse/time point in each experiment) and are shown as mean ± SEM. (B) Data from A presented as absolute numbers of cells (mean ± SEM). P values were obtained with two-way ANOVA.

Fig. 4.

Fig. 4.

The pancreatic islets of NOD.IGRPK209A/F213A KI/KI mice also fail to recruit activated 8.3-CD8+ T cells, even when inflamed. (A) In vitro-activated, CFSE-labeled 8.3-CD8+ T cells were injected i.v. into 3-week-old NOD or NOD.IGRPK209A/F213A KI/KI hosts. Pancreatic sections were stained with anti-insulin antibodies and examined for presence of CFSE+ T cells by confocal microscopy on days 1 and 3 after transfer (three mice/time point; 10–20 islets/mouse; three independent experiments). Representative images are shown. An adjacent tissue section was stained with H&E (Left). (White scale bars, 20 μm.) On NOD hosts’ day 1 samples, CFSE+ T cells were predominantly found in the peri-insular space. Note the near complete depletion of insulin+ cells in the CFSE+ T cell-containing areas of NOD hosts’ day 3 samples. (B) Incidence of diabetes in 3-week-old NOD (n = 7) or NOD.IGRPK209A/F213A KI/KI recipients (n = 6) of 8.3-CTL (two independent experiments, each including three to four mice/strain type). P values were obtained with log rank test. (C) In vitro-activated, CFSE-labeled 8.3-CD8+ Thy1.1+ T cells were injected i.v. into 10- to 12-week-old NOD or NOD.IGRPK209A/F213A KI/KI hosts. Hosts were analyzed for presence of Thy1.1+ CD8+ T cells in different organs. Data correspond to three to four independent experiments/time point and strain (one mouse/time point/strain in each experiment) and are shown as mean ± SEM. (D) Data from C presented as absolute numbers of cells per million lymphocytes (mean ± SEM). P values in C and D were obtained with two-way ANOVA. (E) Analysis of pancreas sections from 10- to 12-week-old mice transfused with CFSE-labeled in vitro-activated 8.3-CD8+ T cells on days 1 and 3 after transfer, for presence of CFSE+ T cells (three independent experiments; one mouse/time point/strain in each experiment). Images of islets are representative of severe insulitis.

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