View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Production of IL-12 by DCs. DCs purified from untreated WT (DCB+/+) or µMT (DCB–/–) mice were cultured with medium alone (None) or with fibroblasts transfected (CD40L-3T3) or not (3T3) with CD40L. 48-h and 72-h supernatants were tested for IL-12 p40 and p70 content, respectively. Limits of detection: for p40, 0.3 pg/ml; p70, 0.13 pg/ml. Data shown are the mean ± SD of duplicate cultures. Three independent experiments were performed with similar results, using DCs purified from untreated or FLT3L-treated mice.

View larger version (K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Role of IL-10. (A) RT-PCR analysis of IL-10 expression was performed in spleen cells from untreated wild-type (lanes 1 and 2) and µMT (lanes 3 and 4) mice, using IL-10 and HPRT primers. Anti-CD3–injected mice were used as positive control (lane 5). Left and right sides represent the amplification by PCR of 3 and 1 cDNA, respectively. Similar data were obtained in three independent experiments. (B) RT-PCR analysis of IL-10 expression was performed in unseparated spleen cells (lane 1) and splenic CD19+ cell population (lane 2), which contains 98% B lymphocytes as assessed by FACSTM analysis for IgM expression (C). The T cell hybridoma 3B4.15 was used as negative control (panel B, lane 3). (D) DCs were purified from C57BL/10 (black symbols) or IL-10 knockout (white symbols) mice that were treated for 9 d consecutively with FLT3L. KLH-pulsed DCs were injected into the hind and fore footpads of wild-type recipient animals. Lymph nodes were harvested 6 d later and cultured with KLH (5 µg/ml). The supernatants were collected and cytokine activities were determined by ELISA. Detection limits: for IFN-, 0.3 ng/ml; IL-4, 31.3 pg/ml; IL-5, 31.3 U/ml; IL-10, 1 ng/ml. Each symbol represents production of cytokine of individual animals, and vertical bars are the mean results of five mice. Two independent experiments were performed with similar results. The groups indicated by the double asterisks are significantly different from control (Student's t test, P < 0.01, n = 5).

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Responses of T cells in µMT and C57BL/6 mice to KLH. Wild-type (black symbols) or µMT (white symbols) mice were injected into the footpads with 100 µg KLH emulsified in CFA. Lymph nodes were harvested 5 d later and purified T cells (2 x 105/well) were incubated with graded numbers of irradiated, T-depleted spleen cells from C57BL/6 mice that had been pulsed with KLH (100 µg/ml). Proliferation and cytokine production were measured as indicated in Materials and Methods. Limits of detection: for IL-2, 0.16 U/ml; for IL-4, 31.25 pg/ml; for IFN-, 0.03 ng/ml. The data are representative of four independent experiments using pooled T cells from five C57BL/6 or µMT mice and shown as the mean of duplicate cultures ± SD.

In Vitro Assays.
Unseparated lymph node cells (4 x 10⁵) were cultured in graded doses of KLH. In some experiments, 2 x 10⁵ purified T cells were incubated with graded doses of irradiated, KLH-pulsed spleen cells depleted of T lymphocytes by treatment with anti-Thy1.2 mAb and rabbit complement (BioMérieux s.a.). Lymph node T cells were purified by negative selection: in brief, pooled lymph node cells were incubated with anti–I-A^b (25-917), anti-HSA, anti-FcR, anti-CD45R/B220, and rat anti–mouse IgG2a mAbs at 4°C, followed by goat anti–rat IgG-coated beads (BioMag; PerSeptive Biosystems) and further passed over a magnet. Reanalysis of the selected population confirmed purity of >95%. Proliferation was measured as thymidine incorporation during the last 16 h of a 3-d culture. Culture supernatants were assayed for IL-2 after 24 h of incubation, for IL-4 after 48 h, for IFN- after 72 h, and IL-5 and IL-10 after 96 h. IL-2 and IFN- were measured as previously described 3. IL-4, IL-5, and IL-10 were quantified by two-site ELISA from PharMingen.

PCR Analysis of IL-10 Gene Expression.
RNA was extracted from splenocytes from untreated C57BL/6, µMT, or anti-CD3–injected C57BL/6 mice, from B cell-enriched CD19⁺ C57BL/6 spleen cells, and from T cell hybridoma 3B14.15 using the TriPure reagents (Boehringer). The CD19⁺ cells were enriched from spleen cells by incubation with anti-CD19–coupled microbeads and positive selection over a MACS column (Miltenyi Biotec). After preparation of cDNA, PCR was performed essentially as previously described 16. Reactions were incubated in a PerkinElmer/Cetus DNA thermal cycler for 30 cycles for IL-10 gene expression and 24 cycles for housekeeping HPRT gene expression (denaturation: 30 s, 94°C; annealing: 1 min, 57°C; extension: 1 min, 72°C). Primers used were as follows: IL-10 sense primers 5'-TCAAACAAAGGACCAGCTGGACAACATACTGC-3' and antisense 5'-CTGTCTAGGTCCTGGAGTCCAGCAGACTCAA-3' (amplified fragment of 421 bp). HPRT sense primers: 5'-GTTGGTATACAGGCCAGACTTTGTTG-3' and antisense 5'-GAATTTCAACTTGCGCTCATCTTAGGC-3' (amplified fragment of 163 bp).

Production of IL-12 by DCs.
Purified DCs (see above) were cultured in medium alone (RPMI 1640 supplemented with 5% FCS) or in the presence of 3T3 fibroblasts transfected or not with CD40L. The supernatants were assayed for IL-12 p40 after 48 h using a two-site ELISA 3. IL-12 p70 production was monitored on 72 h supernatants by a bioassay based on the ability of IL-12 to induce IFN- production by spleen cells. In brief, culture supernatants were added to 5 x 10⁵ spleen cells and incubated for 72 h. IFN- content from culture supernatants was assayed using two-site ELISA, as described above. A linear standard curve was generated by incubating spleen cells with serial dilutions of recombinant murine IL-12. Specificity of bioassay was confirmed by inhibiting IFN- production with neutralizing anti–IL-12 p40 mAb (C17.8, rat IgG2a).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Responses of T Cells in µMT and B6 Mice to KLH.
We first compared the immune response of mice that are genetically deficient for B lymphocytes (µMT mice) and their control littermates. µMT and wild-type animals were injected in the footpads with KLH emulsified in CFA and the draining lymph nodes were harvested 5 d later. The data in Fig. 1 indicate that KLH-specific T cells were primed in both groups of mice, as assessed by KLH-dependent proliferation in culture. Of note, the analysis of the cytokines released by lymph node cells revealed a differential T helper development in µMT versus control mice. T lymphocytes from wild-type mice secreted IFN- and IL-4 when rechallenged with KLH in vitro, whereas T cells from µMT mice produced higher levels of IFN- but no detectable IL-4 in the same conditions. These observations indicate that B lymphocytes are dispensable for T cell priming but are required for the development of IL-4–secreting cells.

Impaired Ability of DCs from µMT Mice to Promote IL-4 Secretion.
There is increasing evidence that the cell that presents the antigen to T cell may influence the Th1/Th2 balance in vivo. In particular, injection of splenic DCs has been shown to induce the activation of T cells that secrete a large array of cytokines 317. Because DCs are the APCs of the primary immune response, we analyzed the adjuvant function of DCs purified from control and µMT mice. DCs were purified from spleens and pulsed with KLH during overnight culture, as previously described 17. Of note, expression of B7-1, B7-2, and CD40 molecules was similar on DCs from both strains of mice, whereas MHC class II was expressed at slightly higher levels on DCs from µMT animals (data not shown). 3 x 10⁵ DCs were injected into the footpads of wild-type mice, and the lymph nodes were harvested 6 d later. The data in Fig. 2 show that DCs from wild-type and µMT mice differentially regulated the development of Th cells. Injection of DCs from µMT mice induced the differentiation of cells secreting IL-2, IFN-, IL-5, and IL-10, but no detectable IL-4, whereas DCs from control mice prime for IL-4 production in addition to other cytokines.

DCs from µMT Mice Produce Higher Levels of Bioactive IL-12.
Because IL-12 appears to be the dominant cytokine favoring the differentiation of Th1 cells over Th2 cells, we measured the production of IL-12 by DCs upon in vitro stimulation. The data in Fig. 3 show that DCs from µMT mice produced higher levels of IL-12 homodimer (p40) and heterodimer (p70) than did DCs from wild-type mice. This observation suggests that the lack of IL-4 priming upon immunization with DCs from B cell–deprived mice may result from increased production of IL-12 by transferred DCs. By contrast, DCs from wild-type mice release lower amounts of IL-12 and have the capacity to promote IFN- and IL-4 production upon adoptive transfer. We next compared the number of CD8⁺ and CD8^– DCs in wild-type and µMT mice, since only the subset of DCs expressing CD8 has been shown to produce IL-12 1819. The data in Table show that the proportion of DC subsets is similar in both strains of mice.

Role of the Microenvironment.
To test whether the microenvironment in which priming of T cells occurs may influence the character of the immune response, KLH-pulsed DCs from wild-type animals were transferred into control and B cell–deprived recipient mice. As shown in Fig. 5, immunization of µMT mice resulted in sensitization of cells producing high levels of IFN- and low levels of IL-4, whereas priming of wild-type mice induced lower IFN- and higher IL-4 production. Evidence that T cells from µMT mice have the capacity to produce IL-4 is provided by the observation that injection of Th2-prone, IL-10–treated DCs into µMT mice results in the development of IL-4–secreting cells (Table ).

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 5 B cells from recipient animals are required for IL-4 priming. KLH-pulsed DCs from untreated C57BL/10 mice were injected into the hind and fore footpads of wild-type (black symbols) or µMT (white symbols) recipients. Lymph nodes were harvested 6 d later and purified T cells were cultured with various numbers of -irradiated, T cell–depleted spleen cells from C57BL/10 mice, which have been pulsed with KLH. Proliferation and lymphokine production were measured as indicated in Materials and Methods. Detection limits: for IL-2, 0.16 U/ml; IFN-, 0.3 ng/ml; IL-4, 16 pg/ml. The results represent the mean of duplicate cultures (± SD). Similar data were obtained in three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although we do not have direct evidence, several features suggest that IL-10 may be involved in this immunoregulatory process: splenocytes from µMT mice express reduced levels of IL-10 mRNA than wild-type animals; DCs from IL-10 knockout mice display properties similar to DCs from µMT mice, i.e., they have lost the capacity to induce the development of IL-4–secreting cells; treatment of DCs from µMT mice with IL-10 restored the generation of IL-4–producing cells in vivo; and numerous reports have shown that IL-10 inhibits the production of IL-12 heterodimer by DCs in vitro and in vivo 310. Collectively, these observations suggest that the level of IL-12 released by DCs is regulated by B lymphocytes, presumably via the production of IL-10. Our data show that B lymphocytes may produce IL-10 constitutively. B cell–depleted spleen cells also express mRNA for IL-10 (data not shown), suggesting that other cell populations may indirectly control the level of IL-12.

There is evidence that the level of IL-12 released by transferred DCs determines the Th1/Th2 balance in vivo. DCs from wild-type animals produce intermediate levels of IL-12 and induce the development of T cells producing IL-2, IFN-, IL-4, IL-5, and IL-10 (this paper and reference 17). DCs, treated with IL-10 in culture, secrete decreased amounts of IL-12 and direct the differentiation of Th2-type cells 3, similar to the response induced by DCs from IL-12–deficient mice 3. Conversely, DCs from IL-10– or B cell–deficient mice release increased levels of IL-12 and do not promote IL-4 secretion (this paper). Coadministration of IL-12 with the wild-type DCs results in sensitization of Th1-type cells only 17. Therefore, the development of IL-4–secreting T lymphocytes appears as a default when T cells are sensitized in the presence of low levels of IL-12. Whether IL-12 directly or indirectly inhibits the development of Th2-type cells is still a matter of speculation.

Our observations indicate that DCs from B cell–deprived mice have lost the ability to prime for IL-4 production but have retained the capacity to induce the development of cells producing diminished but still significant amounts of IL-5 and IL-10. The lack of association of expression among IL-4, IL-5, and IL-10 has been recently reported by Kelso et al. 22, who analyzed the single-cell expression patterns in type 1– or type 2–polarized responses.

Our data also show that DCs from wild-type mice injected in a µMT host induce the development of T cells secreting IL-2 and IFN-, but not IL-4. Whether B lymphocytes limit IL-12 synthesis by transferred DC homing in the T cell area of lymph nodes remains to be determined. Additional studies, including injection of labeled DCs in the footpad and staining of lymph node sections for IL-12 p70, would help clarify this point. Interestingly, Wykes et al. 23 have shown that DCs can retain unprocessed antigen and directly interact with B lymphocytes to initiate antibody synthesis. Thus, B cells recognizing the antigen expressed by DCs form short-lived clusters and could give regulatory signals to the transferred DCs. Alternatively, direct B to T cell signaling may promote IL-4 synthesis in this experimental model. It is noteworthy that T cells from µMT mice have the capacity to produce IL-4, as T cells from µMT mice produced high amounts of IL-4 in response to Schistosoma mansoni eggs 8 or Onchocerca volvulus 24. The capacity of T cells from µMT mice to secrete IL-4 is further illustrated by the observation that injection of DCs that have been treated with IL-10 to enhance their capacity to promote Th2 development 3 induces the production of significant levels of IL-4 in µMT recipient mice (Table ).

We and others have reported that subclasses of DCs directed the development of distinct T helper cells in vivo 1819. Thus, CD8⁺ and CD8^– DCs, purified from spleens, have the potential to differentially regulate the Th1/Th2 balance: CD8^– DCs induced the activation of cells secreting high levels of IL-4, IL-5, and IL-10, and low levels of IL-2 and IFN-, whereas CD8⁺ DCs sensitize cells producing IL-2 and IFN-, but little IL-4, IL-5, or IL-10. We therefore compared the numbers of DCs of either subset in µMT and wild-type mice. Little difference was found between these strains, suggesting that B lymphocytes did not alter the distribution of DC subsets (Table ). Of note, the number of DCs was consistently reduced (by approximately twofold) in B cell–deprived mice as compared with wild-type animals, an observation that may result from a lack of survival or maturation signals and/or chemoattractants factors such as B cell–derived chemokines macrophage inflammatory protein (MIP)-1 and MIP-1β 25.Our results are consistent with the observations of Stockinger et al. 10 that the B cell is the crucial APC for the development of Th2 responses. Stockinger et al. also recently have identified a feedback loop triggered by IL-12 that promotes Th2 differentiation 26. Thus, delivery of IL-12 by DCs during B cell activation induces the secretion of IL-6 and IL-10 by activated B cells. IL-6 and IL-10 confer the capacity to induce IL-4 expression in T cells to these B lymphocytes. These data were interpreted to indicate that DCs, through IL-12 secretion, enhance the ability of B cells to influence T cell differentiation toward Th2. Our data further extend these observations by showing that DCs themselves, after interaction with B cells, show higher IL-4–inducing activity.

Interestingly, our results may shed new light on the enigmatic observation that µMT mice showed enhanced CTL activity to tumor cells in comparison to their control littermates 27. Qin et al. reported that the presence of B cells in the priming phase resulted in disabled help for CTL-mediated tumor immunity, and interpreted these data by a competition between B cells and other APCs for antigen. Our data suggest that the absence of B cells may enhance the generation of tumor immunity by promoting a polarized Th1 response.

In conclusion, our observations suggest that at the steady state level, IL-10 released by B lymphocytes may control the level of IL-12 released by DCs, which upon encounter with an antigen would induce an unpolarized immune response. In addition, there is evidence that presentation of antigen by B lymphocytes may preferentially direct the development of Th2-type cells 2829. Therefore, B cells may directly (upon interaction with T cells) or indirectly (by controlling IL-12 production via DCs) favor the development of Th2-type cells that provide helper activity for antibody synthesis, thereby promoting their own effector function.