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Idd9.2 and Idd9.3 protective alleles function in CD4+ T-cells and nonlymphoid cells to prevent expansion of pathogenic islet-specific CD8+ T-cells.

Hamilton-Williams EE, Wong SB, Martinez X, Rainbow DB, Hunter KM, Wicker LS, Sherman LA - Diabetes (2010)

Bottom Line: Interestingly, the Idd9.1 region, which provides significant protection from disease, did not prevent the expansion of autoreactive CD8(+) T-cells.Idd9 protective alleles are associated with reduced expansion of IGRP-specific CD8(+) T-cells.Protective alleles in the Idd9.2 congenic subregion are required for the maximal reduction of islet-specific CD8(+) T-cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT

Objective: Multiple type 1 diabetes susceptibility genes have now been identified in both humans and mice, yet mechanistic understanding of how they impact disease pathogenesis is still minimal. We have sought to dissect the cellular basis for how the highly protective mouse Idd9 region limits the expansion of autoreactive CD8(+) T-cells, a key cell type in destruction of the islets.

Research design and methods: We assess the endogenous CD8(+) T-cell repertoire for reactivity to the islet antigen glucose-6-phosphatase-related protein (IGRP). Through the use of adoptively transferred T-cells, bone marrow chimeras, and reconstituted severe combined immunodeficient mice, we identify the protective cell types involved.

Results: IGRP-specific CD8(+) T-cells are present at low frequency in the insulitic lesions of Idd9 mice and could not be recalled in the periphery by viral expansion. We show that Idd9 genes act extrinsically to the CD8(+) T-cell to prevent the massive expansion of pathogenic effectors near the time of disease onset that occurs in NOD mice. The subregions Idd9.2 and Idd9.3 mediated this effect. Interestingly, the Idd9.1 region, which provides significant protection from disease, did not prevent the expansion of autoreactive CD8(+) T-cells. Expression of Idd9 genes was required by both CD4(+) T-cells and a nonlymphoid cell to induce optimal tolerance.

Conclusions: Idd9 protective alleles are associated with reduced expansion of IGRP-specific CD8(+) T-cells. Intrinsic expression of protective Idd9 alleles in CD4(+) T-cells and nonlymphoid cells is required to achieve an optimal level of tolerance. Protective alleles in the Idd9.2 congenic subregion are required for the maximal reduction of islet-specific CD8(+) T-cells.

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Intrinsic expression of Idd9 genes within the CD8+ T-cell does not mediate CD8+ tolerance by Idd9 genes. Purified NOD or Idd9 Thy1.1+ CD8+ T-cells (4 × 106) from 3-week-old donor mice were cotransferred with partially (90%) CD8+ T-cell–depleted Thy1.2+ NOD spleen and LN cells (30 × 106) into NOD-SCID mice. After 10 weeks, mice were infected with Vac-IGRP and IGRP-tetramer+ CD8+ T-cells measured in the spleen 7 days later. A: Frequency of IGRP-specific cells among all CD8+ T-cells; pooled data from two experiments are shown and indicated by circles and triangles; horizontal lines depict mean values. B: Thy1.1 and IGRP-specific tetramer staining of CD8+ T-cells; one representative animal shown.
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Figure 5: Intrinsic expression of Idd9 genes within the CD8+ T-cell does not mediate CD8+ tolerance by Idd9 genes. Purified NOD or Idd9 Thy1.1+ CD8+ T-cells (4 × 106) from 3-week-old donor mice were cotransferred with partially (90%) CD8+ T-cell–depleted Thy1.2+ NOD spleen and LN cells (30 × 106) into NOD-SCID mice. After 10 weeks, mice were infected with Vac-IGRP and IGRP-tetramer+ CD8+ T-cells measured in the spleen 7 days later. A: Frequency of IGRP-specific cells among all CD8+ T-cells; pooled data from two experiments are shown and indicated by circles and triangles; horizontal lines depict mean values. B: Thy1.1 and IGRP-specific tetramer staining of CD8+ T-cells; one representative animal shown.

Mentions: The fact that NOD-8.3 CD8+ T-cells were tolerized in Idd9 hosts suggested that tolerance was caused by expression of Idd9 genes in the host. It was also possible that intrinsic expression of Idd9 genes by CD8+ T-cells contributed to tolerance. To test this possibility, CD8+ T-cells from 3-week-old Idd9-Thy1.1+ mice (4 × 106) were purified and adoptively transferred into NOD-SCID hosts together with CD8+-depleted (90% reduction) NOD-Thy1.2+ splenocytes (30 × 106), from 3-week-old mice. Splenocytes from 3-week-old NOD and Idd9 mice contained only background frequencies of IGRP-specific CD8+ T-cells as assessed by tetramer staining. A second group of mice was prepared similarly but with CD8+ T-cells from NOD-Thy1.1+ donors. After 10 weeks, the mice were infected with Vac-IGRP and the presence of IGRP-tetramer binding CD8+ T-cells determined (Fig. 5A and B). In both groups of mice, approximately half of the CD8+ T-cells were Thy1.1+ at the conclusion of the experiment (supplementary Fig. 3A). We also confirmed that the majority of the CD4+ T-cell population was of NOD-Thy1.2+ origin (supplementary Fig. 3B). Thus, Idd9-derived cells contributed significantly only to the CD8+ T-cell population and not to other cell types. When Idd9-Thy1.1+ CD8+ T-cells were mixed with NOD-Thy1.2+ spleen and lymph node cells, a high frequency of IGRP-specific CD8+ T-cells expanded (Fig. 5A). In both groups, the IGRP-specific cells were derived from both the Thy1.1+ and Thy1.2+ populations, as shown in Fig. 5B. Although individual mice differed in whether the IGRP+ cells were predominantly derived from the Thy1.1+ or Thy1.2+ population, the overall frequency of IGRP-specific cells was comparable between the groups (Fig. 5A). Thus, the peripheral repertoire of 3-week-old Idd9 mice contains autoreactive IGRP-specific CD8+ T-cells that are able to expand and survive when transferred into mice that express susceptible NOD alleles on all other cell types. These results directly demonstrate that intrinsic expression of Idd9 genes within the CD8+ T-cells does not prevent their expansion in a NOD host.


Idd9.2 and Idd9.3 protective alleles function in CD4+ T-cells and nonlymphoid cells to prevent expansion of pathogenic islet-specific CD8+ T-cells.

Hamilton-Williams EE, Wong SB, Martinez X, Rainbow DB, Hunter KM, Wicker LS, Sherman LA - Diabetes (2010)

Intrinsic expression of Idd9 genes within the CD8+ T-cell does not mediate CD8+ tolerance by Idd9 genes. Purified NOD or Idd9 Thy1.1+ CD8+ T-cells (4 × 106) from 3-week-old donor mice were cotransferred with partially (90%) CD8+ T-cell–depleted Thy1.2+ NOD spleen and LN cells (30 × 106) into NOD-SCID mice. After 10 weeks, mice were infected with Vac-IGRP and IGRP-tetramer+ CD8+ T-cells measured in the spleen 7 days later. A: Frequency of IGRP-specific cells among all CD8+ T-cells; pooled data from two experiments are shown and indicated by circles and triangles; horizontal lines depict mean values. B: Thy1.1 and IGRP-specific tetramer staining of CD8+ T-cells; one representative animal shown.
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Figure 5: Intrinsic expression of Idd9 genes within the CD8+ T-cell does not mediate CD8+ tolerance by Idd9 genes. Purified NOD or Idd9 Thy1.1+ CD8+ T-cells (4 × 106) from 3-week-old donor mice were cotransferred with partially (90%) CD8+ T-cell–depleted Thy1.2+ NOD spleen and LN cells (30 × 106) into NOD-SCID mice. After 10 weeks, mice were infected with Vac-IGRP and IGRP-tetramer+ CD8+ T-cells measured in the spleen 7 days later. A: Frequency of IGRP-specific cells among all CD8+ T-cells; pooled data from two experiments are shown and indicated by circles and triangles; horizontal lines depict mean values. B: Thy1.1 and IGRP-specific tetramer staining of CD8+ T-cells; one representative animal shown.
Mentions: The fact that NOD-8.3 CD8+ T-cells were tolerized in Idd9 hosts suggested that tolerance was caused by expression of Idd9 genes in the host. It was also possible that intrinsic expression of Idd9 genes by CD8+ T-cells contributed to tolerance. To test this possibility, CD8+ T-cells from 3-week-old Idd9-Thy1.1+ mice (4 × 106) were purified and adoptively transferred into NOD-SCID hosts together with CD8+-depleted (90% reduction) NOD-Thy1.2+ splenocytes (30 × 106), from 3-week-old mice. Splenocytes from 3-week-old NOD and Idd9 mice contained only background frequencies of IGRP-specific CD8+ T-cells as assessed by tetramer staining. A second group of mice was prepared similarly but with CD8+ T-cells from NOD-Thy1.1+ donors. After 10 weeks, the mice were infected with Vac-IGRP and the presence of IGRP-tetramer binding CD8+ T-cells determined (Fig. 5A and B). In both groups of mice, approximately half of the CD8+ T-cells were Thy1.1+ at the conclusion of the experiment (supplementary Fig. 3A). We also confirmed that the majority of the CD4+ T-cell population was of NOD-Thy1.2+ origin (supplementary Fig. 3B). Thus, Idd9-derived cells contributed significantly only to the CD8+ T-cell population and not to other cell types. When Idd9-Thy1.1+ CD8+ T-cells were mixed with NOD-Thy1.2+ spleen and lymph node cells, a high frequency of IGRP-specific CD8+ T-cells expanded (Fig. 5A). In both groups, the IGRP-specific cells were derived from both the Thy1.1+ and Thy1.2+ populations, as shown in Fig. 5B. Although individual mice differed in whether the IGRP+ cells were predominantly derived from the Thy1.1+ or Thy1.2+ population, the overall frequency of IGRP-specific cells was comparable between the groups (Fig. 5A). Thus, the peripheral repertoire of 3-week-old Idd9 mice contains autoreactive IGRP-specific CD8+ T-cells that are able to expand and survive when transferred into mice that express susceptible NOD alleles on all other cell types. These results directly demonstrate that intrinsic expression of Idd9 genes within the CD8+ T-cells does not prevent their expansion in a NOD host.

Bottom Line: Interestingly, the Idd9.1 region, which provides significant protection from disease, did not prevent the expansion of autoreactive CD8(+) T-cells.Idd9 protective alleles are associated with reduced expansion of IGRP-specific CD8(+) T-cells.Protective alleles in the Idd9.2 congenic subregion are required for the maximal reduction of islet-specific CD8(+) T-cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT

Objective: Multiple type 1 diabetes susceptibility genes have now been identified in both humans and mice, yet mechanistic understanding of how they impact disease pathogenesis is still minimal. We have sought to dissect the cellular basis for how the highly protective mouse Idd9 region limits the expansion of autoreactive CD8(+) T-cells, a key cell type in destruction of the islets.

Research design and methods: We assess the endogenous CD8(+) T-cell repertoire for reactivity to the islet antigen glucose-6-phosphatase-related protein (IGRP). Through the use of adoptively transferred T-cells, bone marrow chimeras, and reconstituted severe combined immunodeficient mice, we identify the protective cell types involved.

Results: IGRP-specific CD8(+) T-cells are present at low frequency in the insulitic lesions of Idd9 mice and could not be recalled in the periphery by viral expansion. We show that Idd9 genes act extrinsically to the CD8(+) T-cell to prevent the massive expansion of pathogenic effectors near the time of disease onset that occurs in NOD mice. The subregions Idd9.2 and Idd9.3 mediated this effect. Interestingly, the Idd9.1 region, which provides significant protection from disease, did not prevent the expansion of autoreactive CD8(+) T-cells. Expression of Idd9 genes was required by both CD4(+) T-cells and a nonlymphoid cell to induce optimal tolerance.

Conclusions: Idd9 protective alleles are associated with reduced expansion of IGRP-specific CD8(+) T-cells. Intrinsic expression of protective Idd9 alleles in CD4(+) T-cells and nonlymphoid cells is required to achieve an optimal level of tolerance. Protective alleles in the Idd9.2 congenic subregion are required for the maximal reduction of islet-specific CD8(+) T-cells.

Show MeSH
Related in: MedlinePlus