Selective DNAM-1 expression on small peritoneal macrophages contributes to CD4+ T cell costimulation

Mouse peritoneal macrophages consist of two subsets: large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs), defined as CD11bhiF4/80hi and CD11b+F4/80lo cells, respectively. We reveal that SPMs, but not LPMs, have the ability to present antigens to naïve CD4+ T cells. Coculture of SPMs with naïve ovalbumin (OVA) specific CD4+ T cells (OT-II) in the presence of OVA peptide effectively induced CD4+ T cells priming. SPMs, but not LPMs, strongly express DNAM-1, an activating immunoreceptor. Although antigen uptake and processing were comparable between WT and DNAM-1-deficient SPMs, deficiency of DNAM-1 on SPMs or blockade of DNAM-1 and its ligand interaction impaired CD4+ T cells priming by SPMs. Furthermore, T and B cell responses in mediastinal lymph nodes of mice intraperitoneally immunized with trinitrophenyl (TNP)–OVA protein in Alum adjuvant were enhanced by intraperitoneally transferred wild-type, but not DNAM-1-deficient, SPMs. We propose that SPMs are functionally distinct from LPMs, and DNAM-1 plays a costimulatory role in antigen presentation by SPMs.

that SPMs also produce NO and IL-12 after infectious stimulation with Trypanosoma cruzi 9 . However, little else is known about the physiological functions of SPMs and the functional molecules in SPMs remain unclear.
Here, we investigated the functional difference between SPMs and LPMs and revealed that SPMs, but not LPMs, have the ability to present antigens to naïve CD4 + T cells. We further explored that DNAM-1 is highly expressed on SPMs, but not LPMs and play a costimulatory role in antigen presentation to CD4 + T cells by SPMs.

Results
SPMs have the ability to prime antigen specific CD4 + T cells. To investigate the antigen presenting ability of SPMs and LPMs in vitro, peritoneal cavity cells were collected from naïve C57BL/6 mice. CD3 + T cells, CD19 + B cells, CD11c + dendritic cells (DCs), Ly6G + neutrophils, and CD11b lo SSC hi eosinophils were excluded, and then CD11b + macrophages were identified. Among these macrophages, CD11b + F4/80 lo SPMs and CD11b hi F4/80 hi LPMs were distinguished (Fig. 1A) and separately sorted with purity more than 90% (Fig. 1B). CD4 + CD62L hi CD44 dull naïve T cells isolated from OT-II Tg mice were labeled with carboxyfluorescein succinimidyl ester (CFSE) to observe T cells proliferation. After the coculture of T cells with SPMs loaded with 1 µM of pOVA for 5 days, approximately 30% of T cells proliferated (Fig. 1C). The percentages of proliferated T cells were dependent on the concentration of pOVA (Fig. 1D). By contrast, T cells did not proliferate after coculture with LPMs loaded with any of the doses of pOVA examined (Fig. 1C,D). These results indicate that SPMs have ability to prime naïve CD4 + T cells.

DNAM-1 is strongly expressed on SPMs.
To find functional molecules on SPMs, we compared gene expression between the mouse peritoneal macrophage subsets CD115 + B220 − MHCII + F4/80 lo cells and CD115 + B220 − MHCII − F4/80 hi cells by using the microarray data of the Immunological Genome (Immgen) Project 20 , (http://www.immgen.org); the definitions of these subsets share similar features with the definitions of SPM and LPM, respectively 3 . Expression of the Cd226 gene in MHCII + F4/80 lo cells was 58 times more than that in MHCII − F4/80 hi cells (Immgen Population Comparison; MF_II + 480lo_PC vs. MF_II-480hi_PC). In addition, analysis using the Gene Skyline browser of Immgen revealed that Cd226 gene expression was especially high in MHCII + F4/80 lo cells compared with other macrophage subsets, monocytes, and neutrophils (Immgen Gene Skyline; gene symbol = Cd226). Although the definitions of the above mouse peritoneal macrophage subsets share similar features with the definitions of SPM and LPM, there are several differences between them, among which the most important is the exclusion of CD11c + cells in the definition of SPMs. Therefore, we examined DNAM-1 expression on SPMs and LPMs; we found that DNAM-1 protein was strongly expressed on SPMs, but not on LPMs ( Fig. 2A). However, the proportions of SPM in wild-type (WT) and DNAM-1-deficient (Cd226 −/− ) mice were comparable ( Supplementary Fig. 1A). CD11c + peritoneal DCs also express DNAM-1, but at a medium level compared to SPMs (data not shown). Thus, we revealed uniquely high expression of DNAM-1 on SPMs.
Consistent with previous reports 3 , SPMs expressed higher levels of MHCII than did LPMs (Fig. 2B). The expression levels of both MHCII molecules and costimulatory molecules were comparable between WT mice and Cd226 −/− mice (Fig. 2B), indicating that DNAM-1 had no effect on expression levels of either MHCII or costimulatory molecules.

DNAM-1-CD155 interaction is involved in costimulation of CD4 + T cells.
Since SPMs are able to prime naïve CD4 + T cells, we examined whether DNAM-1 on SPM is involved in this function. We first compared the CD4 + T cells priming abilities of SPMs isolated from WT mice and Cd226 −/− mice. The frequency of proliferated T cells after coculture with pOVA-loaded Cd226 −/− SPMs was significantly lower than that with WT SPMs (Fig. 3A,B), suggesting that DNAM-1 is involved in T cell priming by SPMs. We next examined whether DNAM-1 is involved in antigen uptake or processing by SPMs; however, both genotypes of SPMs showed comparable levels of phagocytosis and antigen processing ( Supplementary Fig. 1B,C).
Because we previously reported that CD155, a DNAM-1 ligand, on CD4 + T cells plays an important role in costimulation of T cells and promote Th1 differentiation independent of IL-12 21 , we supposed the ligation of DNAM-1 on SPMs with CD155 on T cells induces T cells proliferation. Then, we compared the T cells responses primed by SPMs in the presence of anti-CD155 Ab, anti-DNAM-1 neutralizing Ab or isotype-matched control Ab. The frequency of T cells proliferation was impaired after either coculture of anti-DNAM-1 Ab-pretreated SPMs with CD4 + T cells or SPMs with anti-CD155 Ab-pretreated CD4 + T cells compared to isotype control (Fig. 3C,D). These results indicate that the interaction between DNAM-1 on SPMs and its ligand CD155 on T cells is indispensable for the function of DNAM-1 in SPM-mediated antigen specific T cells priming.
Next, we examined cytokine production from CD4 + T cells after being primed by SPMs from WT mice or Cd226 −/− mice. The frequency of IFNγ + CD4 + T cells after coculture naïve CD4 + T with pOVA-loaded Cd226 −/− SPMs was significantly lower than that with WT SPMs, while frequency of IL-4 + and IL-17 + CD4 + T cells were compatible (Fig. 3E). On the other hand, IL-12 production was compatible between WT and Cd226 −/− SPMs  Supplementary Fig. 1D), indicating that the reduced IFNγ production from Cd226 −/− CD4 + T cells is independent of IL-12 production from SPMs. These results indicate that SPMs activate naïve CD4 + T cells and then promote Th1 differentiation in the dependence on costimulatory signal generated by the interaction of DNAM-1 on SPMs and CD155 on T cells.

DNAM-1 on SPMs contributes to T cells proliferation in the draining lymph nodes after intraperitoneal immunization.
To investigate whether DNAM-1 on SPMs is involved in CD4 + T cell priming in vivo, OT-II Tg mice were subjected to adoptive cell-transfer i.p. with 4 × 10 4 SPMs or LPMs sorted from WT or Cd226 −/− donor mice; at the same time, immunized i.p. with TNP-OVA/Alum. On day 3, CD4 + T cells proliferation in the mediastinal lymph nodes (LNs, draining LNs from peritoneal cavity 22 ) were analyzed by BrdU incorporation (Fig. 4A). T cells proliferation was significantly greater in SPM-transferred mice than in LPM-transferred mice or control mice without cell transfer (Fig. 4B,C). However, this enhanced proliferation of T cells was abolished in mice subjected to cell transfer with Cd266 −/− SPMs (Fig. 4B,C). To confirm the involvement of CD155 on T cells in CD226-mediated T cell proliferation, we used Cd155 −/− OT-II mice. When WT or Cd266 −/− SPMs were transferred into Cd155 −/− OT-II mice, the proliferation of both genotypes of SPM showed comparable ( Supplementary Fig. 2). Together, these results indicate that the interaction between DNAM-1 on SPMs and CD155 on CD4 + T cells contributes to T cells priming in the draining lymph nodes after antigen immunization via the peritoneal cavity.

DNAM-1 on SPMs contributes to the germinal center reaction in mediastinal lymph nodes after immunization.
To investigate whether DNAM-1 on SPMs affects the humoral immune response, we analyzed the GC reaction in the mediastinal lymph nodes after immunization. WT or Cd226 −/− SPMs were transferred i.p. into Cd226 −/− recipient mice. The mediastinal lymph nodes were harvested 7 days after immunization and analyzed for frequency of occurrence of GC B cells (defined as GL7 + PNA + B220 + cells). The GC reaction was greater in SPM-transferred mice than in LPM-transferred mice or control mice without cell transfer (Fig. 5B). Furthermore, the enhanced GC reaction was abolished in mice that had received Cd266 −/− SPMs (Fig. 5A,B). Together with the data in Figs 4 and 5, these results suggest that DNAM-1 expressed on SPMs in the peritoneal cavity is involved in T cell priming and the humoral immune response in the draining lymph nodes after immunization.  Taken together, our results show that SPMs are functionally different from LPMs. They also show that DNAM-1 on SPMs plays an important role in priming T cells in the draining lymph nodes of the peritoneal cavity against antigens present in this cavity.

Discussion
Previous reports demonstrated that peritoneal macrophages engulf pathogens, induce neutrophils recruitment into the peritoneal cavity and thus play important roles in the control of infections [23][24][25] ; the macrophages analyzed in these reports are likely LPMs on the basis of their surface markers. In contrast, we revealed that SPMs, a newly defined subset of peritoneal macrophages, but not LPMs, play an important role in adaptive immunity against i.p. injected antigen. Note that SPMs are distinct from peritoneal DCs; the former have much less expression of CD11c and different levels of expression of CD11b and F4/80 3 . Some reports have defined two populations of mouse peritoneal macrophages, MHCII lo F4/80 hi and MHCII hi F4/80 lo 6,26,27 . MHCII lo F4/80 hi cells in these reports likely correspond to LPMs, considering the reported gating strategy used in the flow cytometry analysis. In contrast, MHCII hi F4/80 lo cells in these reports appear to be mixtures of SPMs and DCs; DCs have a CD11b mid MHCII hi F4/80 − phenotype (1), which is somewhat similar to the CD11b + MHCII hi F4/80 lo phenotype of SPMs, but DCs can be distinguished from SPMs by their stronger expression of CD11c, which was not assessed in these reports. Therefore, we suspect that the antigen presenting ability of MHCII hi F4/80 lo cells, which has been reported previously 27 , reflects the function of DCs. Our finding demonstrates direct evidence for the antigen presenting ability of SPMs.
We showed that SPMs were involved in T cell priming and GC B cell development in vivo. Because CD4 + helper T cells are essential for GC maintenance 28 , our data indicate that SPMs are involved in CD4 + T cell proliferation in response to antigen immunization, which in turn enhances the humoral immune response. Given that peripheral tissue immunization with antigen in adjuvant induces antigen uptake by local DCs and priming of T cells in the draining lymph nodes 29 , it is speculated that SPMs take up antigen in the peritoneal cavity, migrate into the draining lymph nodes, and present antigen to naïve CD4 + T cells 3 . In support of this idea, we showed in this report that SPMs are able to uptake and process antigens in vivo, consistent with previous reports 3,7,9 . Moreover, Ghosn et al. reported that half of SPMs express the lymphocyte homing receptor CD62L; they also suggested that SPMs migrate into lymph nodes 3 . Although our data suggest that SPMs migrate into the mediastinal lymph nodes and present antigen to T cells, further investigations are required to demonstrate the migration of SPMs into draining lymph nodes.
We showed here that DNAM-1 is expressed on SPMs at an exclusively high level among peritoneal macrophages. A recent report by Kim et al. has demonstrated that DNAM-1 served as a distinct surface marker of MHCII + resident peritoneal macrophages compared to F4/80 + counterpart 30 . Because MHCII + resident peritoneal macrophages and SPMs share MHCII molecule as a surface marker, both populations in the peritoneal cavity may overlap each other. More interestingly, we revealed that DNAM-1 enhances the antigen-specific CD4 + T cell priming by SPMs. Whereas the function of DNAM-1 as an activating receptor on T cells and NK cells had been well studied [10][11][12][13][16][17][18][19] , its function in macrophages had not yet been reported. Our report provides new understanding that DNAM-1 is one of the molecules characterizing particular functions of heterogeneous macrophage subsets.
DNAM-1-deficient SPMs showed markedly lower antigen presentation ability than WT SPMs. In addition, T cells proliferation was impaired when the T cells were pretreated with anti-CD155 neutralizing Ab in vitro and mice deficient in CD155 transferred with SPM were analyzed. Consistent with our recent findings that CD155 mediated a costimulatory signal in naïve CD4 + T cells and induce Th1 differentiation 21 , these results indicate that interaction between DNAM-1 and its ligand, CD155, is indispensable for the function of DNAM-1 in the antigen presentation of SPMs. Whereas DNAM-1 acts as a signaling molecule on T cells and NK cells 10,31 , it is unclear whether DNAM-1 on SPMs mediates an activating signal in SPMs and induces changes in the function of SPMs. Even though we do not exclude the possibility of intracellular signaling of DNAM-1 on SPMs, our data suggest that DNAM-1 on SPMs interacts with CD155 expressed on CD4 + T cells, upregulates T cells proliferation and promotes Th1 differentiation via CD155-mediated costimulatory signals.
We propose that SPMs are functionally distinct from LPMs and that the DNAM-1-CD155 axis plays an important role in costimulation of antigen-specific CD4 + T cells. In this context, the exclusively high expression of DNAM-1 on SPMs characterizes the function of SPMs among heterogeneous subsets of peritoneal macrophages.