5×10⁴ T cell hybridomas were incubated overnight with 5×10⁵ splenocytes, obtained from either WT or DM−/− mice, at the indicated concentration. IL-2 production was measured as described in Material and Methods and is expressed as mean OD. Closed circles represent WT APC, while open circles denote presentation by DM−/− APC.

DM −/− or WT mice were immunized with 20µg/footpad of MalE, HEL or LACK antigens emulsified in CFA. 10 days later 3×10⁵ CD4 T lymphocytes from the draining lymph node were restimulated overnight with 10µM or 1µM of the indicated peptides and 5×10⁵ syngenic splenocytes in IL-2 (thatched bars) or IFNγ (white bars with black dots) ELISPOTS simultaneously. Data is representative of two (HEL) or three (MalE and LACK) individual experiments.

DM −/− (open bars) or WT mice (filled bars) were immunized with 20µg/footpad TS1-MalE (A) or 10 µg/footpad of HEL (B) emulsified in CFA. 10–12 days later 3×10⁵ and 1×10⁵ purified CD4 T lymphocytes from the draining lymph node were restimulated over-night with 10µg/ml of the immunizing antigen or 5µM of the indicated peptides and 5×10⁵ syngenic splenocytes. The number of IL-2 producing cells were quantified from triplicate wells by ELISPOT, and data is expressed A) as the percentage of the response (as described in Figure 1) or B) as the percentage normalized to total IL-2 production upon restimulation with intact Ag (expressed as 100% of the response).

Purified CD4 T lymphocytes (1×10⁵, 5×10⁴, 2.5×10⁴ or 1.25×10⁴ cells/well) from DM−/− or WT mice were stimulated with T cell depleted splenocytes (5×10⁵, 12.5×10⁴, or 3.1×10⁴) from C3H mice in the presence of blocking antibodies against I-A^k (10.2.16), I-E^k (14.4.4S), both antibodies, or control antibody (MK-S4). After overnight incubation the number of IL-2 producing cells were quantified from four replicate wells by ELISPOT. Only the 5×10⁴ T lymphocyte dilution with 5×10⁵ C3H splenocytes is shown, but the pattern of antibody blocking was similar for all cell concentrations. Data is representative of two independent experiments.

WT mice (H-2^k) were immunized with 20µg/footpad of MalE emulsified in CFA. 10 days later 3×10⁵ purified CD4 T lymphocytes from the draining lymph nodes were restimulated over-night with individual peptides (identified from experiments utilizing a MalE matrix) at a final concentration of 3µM with 5×10⁵ splenocytes. IL-2 producing cells were quantified from duplicate wells by ELISPOT. The average IL-2 spots obtained for each indicated peptide from 2–7 independent experiments (depending on the peptide analyzed) are shown. The isotype restrictions of the immunodominant peptides [obtained through restriction mapping using 4×10⁴ I-A^k (RT4.15, black bars) or I-E^k (DCEK, white bar) expressing DAP cells in individual peptide or matrix assays, as well as antibody blocking experiments] are also indicated.

WT H-2^k mice were immunized with 20µg/footpad of the indicated antigens in CFA (or CFA in PBS for PPD responses). 10 days later 4×10⁵ or 1×10⁵ purified CD4 T lymphocytes from the draining lymph node were restimulated over-night with 10µg/ml (βGal, ADH, MalE, HEL, or LACK) or 5µg/ml (PPD) of the immunizing antigen and 5×10⁵ syngenic splenocytes. Blocking antibodies against I-A^k (10.2.16), I-E^k (14-4-4S), both antibodies (10.2.16 and 14-4-4S), or no antibody (antigen alone) were included in the restimulation at 80µg/ml. After overnight incubation the number of IL-2 producing cells were quantified from triplicate wells by ELISPOT. Representative data (from at least two independent experiments) are expressed as spots/1×10⁶ cells after background spots in the absence of antigen are subtracted. * represents no response detected above background.

DM −/− (open bars) or WT mice (filled bars) were immunized with 20µg/footpad of MalE emulsified in CFA. 10 days later 1×10⁵ purified CD4+ T lymphocytes from the draining lymph node were restimulated overnight with four peptide pools at 12.5 uM (A) or individual peptides (B) presented by 5×10⁵ of syngeneic splenocytes. IL-2 producing cells were quantified from triplicate wells by ELISPOT. Data are expressed as the percentage of the total response (individual peptide IL-2 spots/total IL-2 spots) after subtracting responses with control peptide pools as background (A) or as the number of spots per 1×10⁵ CD4 + T cells after subtracting background spots in the absence of peptide (B). Data is representative of at least 4 independent experiments. * Indicates consistent reproducibility for the indicated pools in at least 3 of 4 independent experiments.

A) DM −/− or WT mice were immunized with 5nmoles/mouse of the indicated peptides (shown at the right of the figure) restricted to either I-A^d (MalE_69-81, MalE_269–285) and I-E^d (MalE_89–104 and HEL_107–118) emulsified in CFA. 10 days later purified CD4 T lymphocytes from the draining lymph node were restimulated overnight with the indicated concentration of each peptide presented by 5×10⁵ splenocytes from either WT or DM-deficient mice. The numbers of IL-2 producing cells were quantified from triplicate wells by ELISPOT and the normalized spot number from each concentration were averaged. Results from individual mice are shown. B) DM −/− or WT mice were immunized with a mixture of I-A^d (HA1_26–138, MalE_69–81, OVA_273–288, OVA_323–339) and I-E^d (MalE_89–104 and HEL_107–118) restricted peptides emulsified in CFA. 10 days later 0.5×10⁵, 1×10⁵, 2×10⁵, and 4×10⁵ purified CD4 T lymphocytes from the draining lymph node were restimulated over-night with 5µM of each peptide presented by 5×10⁵ syngenic splenocytes. The numbers of IL-2 producing cells were quantified from triplicate wells by ELISPOT and the normalized spot number from each concentration were averaged. Data are expressed as spots/1×10⁶ cells after background spots in the absence of antigen are subtracted, and is representative of at least seven independent experiments.

A) Amino acid sequence of the I-A^d-restricted MalE_69–83 peptide, illustrating the introduced amino acid substitutions (Q72T, L75A, underlined), known class II pocket interactions (bold), and kinetic stability of the two peptides with class II at endosomal pH5.3. B) WT mice were immunized with 20 µg/footpad of WT MalE (black bars) or Q72T, L75A MalE (thatched bars) emulsified in CFA. 10 days later purified CD4 T lymphocytes from the draining lymph node were restimulated over-night with 10µM of the MalE peptides [(69–81), (103–118), (269–285) or (89–104)] or 20µg/ml of the immunizing antigen presented by 5×10⁵ syngeneic splenocytes. IL-2 producing cells were quantified from triplicate wells by ELISPOT. Data are expressed as the percentage of the total antigen response (individual peptide IL-2 Spots/total IL-2 spots to antigen). Data is representative of two independent experiments. The same results were obtained when CD4 T lymphocytes were restimulated with the MalE Q72T, L75A peptide.

A) DM −/− or WT mice were immunized with 20µg/footpad of β-Galactosidase, Alcohol Dehydrogenase or LACK antigens emulsified in CFA or PBS:CFA alone (PPD). 10 days later 3×10⁵ or 1.5×10⁵ purified CD4 T lymphocytes from the draining lymph node were restimulated overnight with 10µg/ml (pGal, ADH, or LACK) or 5µg/ml (PPD) of the immunizing antigen and 5×10⁵ syngenic splenocytes. Blocking antibodies against I-A^d (MK-D6), I-E^d (14-4-4S), both antibodies (MKD6 and 14-4-4S), or no antibody (antigen alone) were included in the restimulation at 80µg/ml. After overnight incubation the number of IL-2 producing cells were quantified from triplicate wells by ELISPOT. Representative data are expressed as spots/1×10⁶ cells after background spots in the absence of antigen are subtracted. The percentages of isotype-restriction (calculated as in Figure 2) are also included. * represents no response detected above background. B) DM−/− mice were immunized with 5nmoles/mouse of LACK (33–52) or LACK (257–276) emulsified in CFA. 10 days later 4×10⁵ purified CD4 T lymphocytes from the draining lymph node were restimulated over-night with various doses of the immunizing peptide [uM] and I-A^d (RT2.3B2) or I-E^d (RT10.3C5) DAP cells. The number of IL-2 producing cells were quantified from triplicate wells by ELISPOT after background spots obtained using class II−/− DAP cells were subtracted. Data is representative of two independent experiments.

A) 5×10⁴ T hybridoma cells were incubated with 5µM of the indicated MalE or HEL peptide (positive control for I-E^d presentation) and an equal number of I-A^d (RT2.3B2) or I-E^d (RT10.3C5) expressing DAP cells. IL-2 production was measured as described in the Material and Methods and is expressed as mean OD. The origin of each hybridoma is indicated below each peptide in parentheses. Data is representative of three independent experiments. B) DM −/− or WT mice were immunized with 20µg/footpad of MalE emulsified CFA. 10 days later 3×10⁵ purified CD4+ T lymphocytes from the draining lymph node were restimulated overnight with 20µg/ml MalE antigen. Blocking antibodies against I-A^d (MKD6), I-E^d (14-4-4S), both antibodies (MKD6 and 14-4-4S), or control antibody (10-2-16) were included in the restimulation at 80µg/ml. The percentages of isotype-restriction are also included; calculated from the number of spots in the presence of either MKD6 (% of I-E^d-restriction) or 14.4.4S (% of I-A^d- restriction)/total response to antigen alone (10.2.16 control antibody). Data is representative of four independent experiments. C and D) FACs analysis of splenocytes isolated from WT or DM−/− animals. Cells were incubated with directly conjugated C) anti-I-A^d/FITC, I-E/PE, D) 25.9.17/biotin followed by SA/PerCP-Cy5.5(1:1000, BD Pharmingen), or culture supernatants followed by rat anti-mouse IgG2a/2b for thirty minutes. Cells were washed 2–3 times between incubations and before analysis on FACs Aria using Cell Quest software.