Figure 5. PD-1 expression is maintained by PIT and is required for tolerance.

(A) Representative histograms of PD-1 expression by CD4⁺ Tg4 cells sampled as naïve cells, Teff on day of transfer and Teff cells retrieved 4 days after PBS/PIT. (B) PD-1 expression (MFI) gated on CD4⁺ host cells and Tg4 donor cells from spleen 4 days after treatment (four mice per group, from one of four experiments giving consistent results, dotted line represents MFI of isotype control staining). (C) Time course of PD-1 expression on CD4⁺ Tg4 donor cells from PBS/PIT-treated mice (four mice per group). (D) B10.PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4 Teff cells. 4 days later CD4⁺ Tg4 donor cells were FACS-sorted and 2 × 10⁶ were transferred into secondary hosts that were not exposed to PIT (PTX was given on the same day). (E) EAE in secondary hosts (n = 4 for PBS; 21 for PIT, pooled from two experiments). (F) PD-1 expression gated on CD4⁺ Tg4 donor cells from spleens isolated 16 days after secondary transfer (dotted line represents MFI of isotype control staining). (G) EAE in B10.PLxC57BL/6 mice that received PBS/PIT 1 day after transfer of Teff generated from Tg4.PD-1^+/+ or Tg4.PD-1^−/− donors (n = 20–36 mice per group, pooled from three experiments).

DOI: http://dx.doi.org/10.7554/eLife.03416.011

Figure 5—figure supplement 1. Elevated accumulation of Tg4.PD-1^−/− Teff cells following PIT.

B10.PLxC57BL/6 mice received PBS or PIT 1 day after transfer of Tg4.PD-1^+/+ or Tg4.PD-1^−/− Teff cells. (A) Percentage and (B) numbers of CD4⁺ Tg4 cells within the spleen 4 days after PBS or PIT (12 mice per group), data are pooled from three experiments giving consistent results. (C) Percentage of Ki-67+ cells within the Tg4 CD4⁺ population (4 mice per group, from one of two experiments giving consistent results).

DOI: http://dx.doi.org/10.7554/eLife.03416.012

Figure 5—figure supplement 2. PIT does not increase the frequency of Foxp3⁺ Tg4 Teff cells.

B10.PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4.PD-1^+/+ or Tg4.PD-1^−/− Teff cells. (A) Percentage and (B) numbers of Foxp3⁺CD4⁺ Tg4 cells within the spleen 4 days after PBS/PIT. Data are pooled from three experiments giving consistent results (12 mice per group).

DOI: http://dx.doi.org/10.7554/eLife.03416.013

Figure 5—figure supplement 3. PIT limits the frequency of cytokine⁺ Tg4 Teff cells independently of PD-1.

B10.PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4.PD-1^+/+ or Tg4.PD-1^−/− Teff cells. Spleens were taken 4 days later for analysis (A) Percentage and (B) numbers of IFN-γ⁺ and GM-CSF⁺ CD4⁺ Tg4 cells following recall stimulation with 20 μM Ac1-9. Data are pooled from three experiments giving consistent results (12 mice per group).

DOI: http://dx.doi.org/10.7554/eLife.03416.014

Figure 5—figure supplement 4. PIT-exposed Tg4.PD-1^−/− Teff maintain their ability to produce IL-2.

B10.PLxC57BL/6 mice received PBS or PIT 1 day after transfer of Tg4.PD-1^+/+ or Tg4.PD-1^−/− Teff cells. Spleens were taken for analysis 4 days after PIT for retrieval of CD4⁺ Tg4 Teff (FACS-sorting). IL-2 production was measured after 48 hr culture with irradiated B10.PLxC57BL/6 splenic APC and MBP peptide, (dotted lines represent IL-2 levels from unstimulated cultures). Data (4 mice per group) are from one of two experiments giving consistent results).

DOI: http://dx.doi.org/10.7554/eLife.03416.015

Figure 5—figure supplement 5. PD-1 limits CD25 up-regulation following recall stimulation of PIT-exposed Tg4 Teff.

B10.PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4.PD-1^+/+ or Tg4.PD-1^−/− Teff cells. Spleens were taken 4 days later for analysis. CD25 expression within the Tg4 CD4⁺ population (A) directly ex vivo and (B) 12 hr after recall stimulation with 20 μM Ac1-9.

DOI: http://dx.doi.org/10.7554/eLife.03416.016

Figure 5—figure supplement 6. PD-1 limits phosphorylation of STAT5 in Tg4 Teff cells following PIT.

B10.PLxC57BL/6 mice received PBS/PIT 1 day after transfer of Tg4.PD-1^+/+ or Tg4.PD-1^−/− Teff cells. Spleens were taken 4 days later for analysis. Cells were stimulated for 12 hr with 20 μM Ac1-9, to allow upregulation of CD25, before 15-min exposure to IL-2 and detection of pSTAT5. (A) Representative plots of pSTAT5 staining. (B) Percentage of pSTAT5⁺ cells within the Tg4 CD4⁺ population. Data (4 mice per group) are from one of two experiments giving consistent results.

DOI: http://dx.doi.org/10.7554/eLife.03416.017

Figure 5—figure supplement 7. PIT limits effector cytokine production by polyclonal Teff cells.

(A) CD45.1 mice were immunized with pOVA/CFA and Teff cells generated by restimulation of draining lymph node cells with pOVA + cytokines. Teff cells were transferred into C57BL/6 (CD45.2) host mice prior to administration of pOVA or PBS and splenocytes were retrieved on day 4 (B) Gating strategy for identification of CD45.1⁺ donor cells within the spleen. (C) Representative plots of intracellular detection of GM-CSF, IFN-γ, TNF-α and IL-17 within the donor CD4⁺CD45.1⁺ population following recall stimulation with pOVA. (D) Percentage of cytokine⁺ cells within the donor Teff population. Data (6 mice per group) are from one of two experiments giving consistent results.

DOI: http://dx.doi.org/10.7554/eLife.03416.018

Figure 5—figure supplement 8. PIT drives PD-1 expression in polyclonal Teff cells.

Splenocytes from host mice as described in Figure 5—figure supplement 6 and analysed on day 4. (A) Representative plots of PD-1 staining on CD4⁺ donor Teff cells after PIT/PBS. (B) Percentage of PD-1⁺ cells within the donor CD4⁺ population. (C) MFI of PD-1 staining within the PD-1⁺ population. Data (six mice per group) are from one of two experiments giving consistent results.

DOI: http://dx.doi.org/10.7554/eLife.03416.019