B cell-deficient JHD mice were irradiated and reconstituted with 80% JHD BM and 20% 4get BM (B-4get) or with 80% JHD BM and 20% 4getIL-4Rα^-/- BM (B-4getIL-4Rα^-/-). Reconstituted mice were exposed to two rounds of L3 Hp infection and drug treatment and then challenged with 200 L3 Hp. (A) The number of live (PI^neg), EGFP-expressing CD19⁺CD4^neg B cells in the mLNs was determined using flow cytometry. EGFP⁺ B cells present in the MLN of uninfected 4get or 4getIL-4Rα^-/- mice represent ≤0.01% of the total B cell pool or 100-300 B cells/MLN (Harris et al., 2005b). (B) Titers of Hp-specific IgG1 in serum were determined by ELISA. (C) Adult parasites were enumerated by counting. The results shown are representative of 2 independent experiments.

C57BL/6, μMT, μs^-/- or μs^-/-AID^-/- mice were exposed to two rounds of infection with L3 Hp and drug treatment with pyrantel pamoate. Mice were challenged with L3 Hp and the (A) number of CD19⁺PNA^hiFAS⁺ germinal center B cells in the mLNs was determined 13 days later by flow cytometry. (B) Titers of Hp-specific IgM and (C) Hp-specific IgG1 were determined by ELISA. (D) The number of adult parasites present in the intestinal lumen was determined. (E) C57BL/6 and μMT mice were exposed to two rounds of Hp infection and drug treatment. 200 μl of Hp immune serum (IS) or normal mouse serum (NMS) was injected (i.p.) into the memory μMT and B6 recipients 2 days before, the day of, and two days following a challenge infection with Hp. Parasite burdens were determined 19 days later. Data are represented as the mean ± SD (n=10 mice/group). Data are representative of two individual experiments.

B cell deficient μMT mice were lethally irradiated and reconstituted with 4get BM (4get chimeras) or with B cell deficient 4get BM (4getμMT chimeras). (A) The chimeric mice were infected with Hp and the number of EGFP⁺CD62L^lo CD4 cells in the mLNs was determined by flow cytometry at various times. (B) Cells from the mLNs were stimulated with platebound anti-CD3 for 4 hours in the presence of Brefeldin A and the numbers of IL-4⁺EGFP⁺ CD4 T cells were determined using flow cytometry. Data are shown as the mean ± SD of 4-5 mice/group/timepoint. *Statistically significant differences (p≤0.02) between the 4get and 4getμMT groups were found at each of the indicated timepoints. #Statistically significant differences (p≤0.05) between day 0 4getμMT mice and day 8 4getμMT mice are indicated. (C) C57BL/6 and μMT mice were infected with L3 Hp and CD4 cells were purified from pooled mLNs 21 days later. Purified CD4 T cells were restimulated with Hp antigen-pulsed APCs and cytokine levels in the supernatants were determined by cytometric bead array. The results shown are representative of 3 independent experiments.

(A) C57BL/6 and μMT mice or (B) BALB/c and JHD mice were exposed to two rounds of L3 Hp infection and drug treatment with pyrantel pamoate. The memory mice and a group of naive mice (primary) were then challenged with L3 Hp and parasite burdens were determined on day 20. (C) C57BL/6 and μMT mice were infected with L3 Hp and the number of adult parasites in the intestine was determined on days 6, 7, 8, 9, 10 and 21 after infection. The data are shown as the mean ± SD (n=3-4 mice/group/timepoint). (D) C57BL/6 and μMT mice were infected with L3 Hp or were sham-infected. Three weeks later all mice were treated with pyrantel pamoate. Ten days after the last drug treatment, all mice were infected with L3 Hp. The number of adult parasites present in the intestinal lumen of the previously infected mice (memory) and previously naive mice (primary) was determined at the times indicated. The data are shown as the mean ± SD (n=3-4 mice/group/timepoint). The results shown are representative of two or more independent experiments.

(A) Irradiated μMT mice were reconstituted with 100% μMT BM (μMT chimeras), with 75% μMT BM and 25% C57BL/6 BM (B-WT chimeras) or with 75% μMT BM and 25% IL-4^-/- BM (B-IL-4^-/- chimeras). The reconstituted mice were exposed to 2 rounds of L3 Hp infection and drug treatment and were then challenged with L3 Hp. Parasite burden was determined on day 21. (Panel B, D, F) Irradiated μMT mice were reconstituted with 75% μMT BM and 25% IL-2^-/- BM (B-IL-2^-/- chimeras), with 100% IL-2^-/- BM (IL-2^-/- chimeras), with 100% μMT BM (μMT chimeras) or with 75% μMT BM and 25% C57BL/6 BM (B-WT chimeras). (Panel C, E, G) Irradiated μMT mice were reconstituted with 75% μMT BM and 25% TNFα^-/- BM (B-TNFα^-/- chimeras), with 100% TNFα^-/- BM (TNFα^-/- chimeras), with 100% μMT BM (μMT chimeras) or with 75% μMT BM and 25% C57BL/6 BM (B-WT chimeras). In separate experiments, the cytokine chimeras along with control B-WT and μMT chimeras were infected and drug-treated twice and then challenged with L3 Hp. Parasite burden in the lumen was determined on (B) day 12, or (C) day 10 post-infection. (D-E) Cells from mLNs of cytokine chimeras and controls were stimulated in the presence of Brefeldin A for 5 hours on anti-CD3 coated plates and the number of IL-4-producing CD4 T cells was determined by flow cytometry. (F-G) Serum titers of Hp-specific IgG1 were determined by ELISA. All data is shown as the mean ± SD of ≥ 5 mice/group. The results shown are representative of 2 or more independent experiments.

(A) Irradiated μMT mice were reconstituted with 75% 4getμMT BM and 25% μMT BM (μMT chimeras), with 75% 4getμMT BM and 25% C57BL/6 BM (B-WT chimeras) or with 75% 4getμMT BM and 25% μs^-/-AID^-/- BM (B-μs^-/-AID^-/- chimeras). The chimeric mice were infected with Hp, drug treated and then challenged with 200 L3 Hp. EGFP⁺CD62L^lo CD4 T cells in the mLNs were enumerated by flow cytometry 6 days after challenge. (B-D) Irradiated μMT mice were reconstituted with 75% μMT BM and 25% μMT BM (μMT chimeras), with 75% μMT BM and 25% C57BL/6 BM (B-WT chimeras) or with 75% μMT BM and 25% MHCII^-/- BM (B-MHCII^-/- chimeras). The reconstituted mice were exposed to two rounds of Hp infection and drug treatment and were then challenged with 200 L3 Hp. (B) Parasite burdens were determined for individual animals on day 21 and (C) serum titers of Hp-specific IgG1 were determined by ELISA. (D) CD4 T cells were purified from pooled mLNs and stimulated with platebound anti-CD3 overnight. IL-4 levels in the T cell supernatants were determined by ELISA. (E-F) B6 mice and MD4μMT mice were exposed to two rounds of Hp infection and drug treatment and were then challenged with L3 Hp. (E) Parasite burdens were determined for individual animals. (F) Cells from the mLNs of each group were pooled and the CD4 T cells were purified and stimulated on anti-CD3 coated plates. Cytokine levels in the culture supernatants were determined by cytokine bead array at 24 hr post-restimulation. The data are shown as the mean ± SD of 4-5 mice/group. The results shown are representative of 2 or more independent experiments.

B cell deficient μMT mice were irradiated and reconstituted with BM from either 4get mice (4get chimeras) or B cell deficient 4get mice (4getμMT chimeras). Chimeric mice were infected with Hp and drug treated on day 21. On day 60 a cohort of mice was analyzed (memory timepoint) and the remainder of the mice were challenged with L3 Hp and mLN cells were analyzed for expression of CD4, EGFP and CD62L by flow cytometry. (A-B) Plots gated on live CD4⁺ cells and are representative of mice at the (A) memory timepoint and (B) 5 days post-challenge. The numbers in each quadrant indicate the percentage of CD4⁺ cells that express EGFP or CD62L. (C) The number of EGFP⁺CD62L^lo CD4 T cells present in the mLNs at the memory timepoint and various times post-challenge was determined. (D-E) Cells from mLNs of (D) memory and (E) day 5 post-challenged mice were restimulated with platebound anti-CD3 in the presence of Brefeldin A for 4 hours and the expression of IL-4 and EGFP on the CD4 T cells was determined by flow cytometry. The percent of EGFP⁺CD4⁺ cells co-expressing IL-4 is indicated in the plot (mean ±SD of 5 mice/group). (F) The number of EGFP⁺IL-4⁺ CD4 T cells present in the mLNs at the memory timepoint and various times post-challenge was determined. Data in panels C and F are shown as the mean ± SD of 4-5 mice/group. *Statistically significant differences (p≤0.02) between the 4get and 4getμMT groups were found at each of the indicated timepoints. #Statistically significant differences (p≤0.03) between day 0 4getμMT mice and day 3 4getμMT mice are indicated. (G) C57BL/6 and μMT mice were infected with Hp, drug treated and then challenged with 200 L3 Hp. Ten days later, CD4 cells were purified from pooled mLNs and restimulated with Hp antigen-pulsed APCs overnight. Cytokines in the supernatants were quantified by cytometric bead array. The results shown are representative of 3 independent experiments.