The role of B cells in the development of CD4 effector T cells during a polarized Th2 immune response

Abstract

Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-gamma, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4-/- recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4-/- B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-gamma-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.

FIGURE 1

B cell blockade inhibits Nb-induced Th2 response without triggering Th1 response. A–E, WT and B cell-deficient mice were immunized intracutaneously in the ear with 300 third-stage Nb larvae (L3). Seven days later, cells from CLNs were collected. The total number of IL-4-secreting cells per 10⁶ CLN cells (A) and the number of IL-4-secreting CD4 T cells per 10⁶ sorted CD4⁺ T cells (B) were assessed by ELISPOT. IL-4 mRNA (C), IL-13 mRNA (D) and IFN-γ mRNA (E) from sorted CD4 T cells in the CLN at day 7 after Nb inoculation were assessed by quantitative real-time RT-PCR. F–H, WT and B cell-deficient mice were intracutaneously immunized with Nb in the ears. Four days later, mice were again challenged, and 7 days after primary immunization, cells from CLNs were collected and CD4 T cells were positively sorted by magnetic CD4-beads for quantitative real-time RT-PCR analysis of IL-4 (F), IL-13 (G) and IFN-γ (H) mRNA. Results represent the mean and SE of each treatment group (four to five animals per treatment group), and similar results were obtained in two experiments. **, p < 0.001. Sorted cells were obtained from pooled samples from each treatment group.

FIGURE 2

B cells are required for Ag-specific Th2 cell development in vivo. WT and B cell-deficient mice were adoptively transferred with naive DO11.10 T cells on day 0. Two days later, these mice were intracutaneously immunized with Nb plus OVA peptide and 7 days after immunization, cells from CLNs were collected. A, The total number of DO11.10 T cells (CD4⁺ KJ1-26⁺) per CLN is shown. B, CLN cells were restimulated with OVA peptide in vitro and then stained for cytoplasmic IL-4. C and D, KJ1-26⁺ cells were positively selected by magnetic bead cell sorting and IL-4 and IFN-γ mRNA from KJ1-26⁺ cells were determined by quantitative real-time RT-PCR. The experiment was repeated three times with similar results.

FIGURE 3

B cell blockade partially inhibits expansion but not activation of transferred Ag-specific T cells following Nb plus OVA inoculation. Following DO11.10 T cell transfer, WT and B cell-deficient recipient mice were immunized with Nb plus OVA as previously described. At day 7 postimmunization, CLN cells were collected from recipient mice, and cell suspensions were stained for CD4-CyChrome, KJ1-26-FITC, and CD44-PE, CD69-PE, or CD62L-PE. The gated CD4⁺ KJ1.26⁺ population (A) was analyzed for CD44 (B), CD69 (C), and CD62L (D). E, naive DO11.10 T cells were labeled with CFSE and transferred into B cell-deficient and WT BALB/c mice. Two days later, the mice were immunized with Nb plus OVA. At day 7 postimmunization, the CLN cells were collected. Cell cycle progression of donor DO11.10 T cells was determined according to the dilution of CFSE fluorescence. The experiment was repeated three times with similar results.

FIGURE 4

IL-4 message expressed by OVA-specific Th2 cells is first elevated in T:B zone on day 2 postimmunization. Naive DO11.10 T cells were adoptively transferred into IL-4^−/− mice. Two days later, recipient mice were intracutaneously immunized in the ears with Nb plus OVA (Nb+OVA). At days 2, 3, and 4 postimmunization, CLNs were collected. A, Tissue sections from CLNs were stained for KJ1-26-PE and B220-Alexa Fluor 647 at each time point and whole lymph node images obtained as described in Materials and Methods. B, Tissue samples from T zone, B zone, and T:B zone were collected by LCM and assessed for IL-4 mRNA by quantitative real-time RT-PCR.

FIGURE 5

IL-4 protein produced by OVA-specific DO11.10 Th2 cells is first detectable in T:B zone by day 3 postimmunization. Naive DO11.10 T cells were transferred to IL-4^−/− recipients, which were intracutaneously immunized with Nb plus OVA 2 days later. At days 3, 4, 5, and 7 postinoculation, CLNs were frozen, sectioned, and stained with PE-conjugated KJ1-26 Ab (DO11.10 T cells, red) and Alexa Fluor 647-conjugated anti-mouse B220 (B cells, blue). IL-4 protein (green) was detected by using two Alexa Fluor 488-conjugated anti-IL-4 mAbs, which recognize distinct epitopes of IL-4, as described in Materials and Methods. The B zone, T:B zone, and T zone microenvironments were then digitally imaged at a magnification of ×1000. Representative samples from untreated (A–C), day 3 (D–F), day 4 (G–I), day 5 (J–L), and day 7 (M–O) are shown. Alexa Fluor 488-conjugated rat IgG1 was used as an isotype control for IL-4 staining, with day 4 shown (P–R). To facilitate comparison, individual panels (for each given day) were normalized for brightness and contrast. S, Enlarged image of day 3 T:B zone (E).

FIGURE 6

Reconstitution of B cell-deficient mice with untreated WT B cells restores Ag-specific T cell expansion and IL-4 production after Nb plus OVA inoculation. CFSE-labeled naive DO11.10 T cells were adoptively transferred to WT and B cell-deficient mice. Some B cell-deficient mice also received WT naive B cells. Two days later, recipient mice were inoculated with Nb plus OVA. A, At day 7 postimmunization, CLN cell suspensions were surface stained with anti-CD4-PerCp and KJ1-26-PE, and stained intracellularly with anti-IL-4-allophycocyanin. B, Cell division of transferred DO11.10 T cells was analyzed by flow cytometric analysis of CFSE fluorescence. The experiment was repeated twice with similar results.

FIGURE 7

B7, but not IL-4, expression by B cells is important in mediating Th2 cell development in vivo. A and B, Naive DO11.10 T cells were adoptively transferred to WT and B cell-deficient mice. Some B cell-deficient mice also received IL-4^−/− B cells (A) or B7-1/B7-2-deficient B cells (B), which were compared with B cell-deficient mice receiving WT B cells. C, Naive DO11.10 T cells were transferred to WT and B7-1/B7-2-deficient mice. Some B7-1/B7-2-deficient mice also received WT naive B cells. In all experiments, at day 2 after cell transfer mice (five mice per treatment group) were inoculated with Nb and OVA and at day 7 postimmunization, CLN cell suspensions were surfaced stained with anti-CD4-PerCp and KJ1-26-PE, and stained intracellularly with anti-IL-4-allophycocyanin. These experiments were repeated twice with similar results.