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Mycobacterial antigen-specific CD4+ Th1 cells have pivotal role in protective immunity against mycobacterial infections including pulmonary tuberculosis. In the course of the infection, Th1 cells differentiate in the lung-draining lymph nodes and migrate into the infected lung. Chemokine receptors on T cells are involved in T cell migration into the intestine and skin. However, role of chemokine receptors in the migration of CD4+ T cells into the lung is not yet established. To address the issue, the role of chemokine receptors in T cell migration into the mycobacteria-infected lung was analyzed using mycobacterial Ag85B peptide 25-specific T cell receptor-transgenic (P25) CD4+ T cells. The P25 T cells in the Mycobacterium bovis BCG-infected lung and lung-draining mediastinal lymph node expressed chemokine receptors CCR5, CCR6, CXCR3 and CXCR5 which bind chemokines expressed by the BCG-infected lung. To further analyze the role of the chemokine receptors in the migration of the BCG-primed P25 T cells into the lung or mediastinal lymph node, the P25 T cells were adoptively transferred into the BCG-infected wild type mice, and their migration into the lung was monitored. Unexpectedly, blocking of chemokine receptor function with pertussis toxin, a G-protein inhibitor, failed to suppress migration of the T cells into the infected lung although the treatment completely blocked migration of the mediastinal lymph node P25 T cells into the recipient lymph node. The results suggest that interaction of chemokine receptors on mycobacterial antigen-specific Th1 cells with chemokines is dispensable in their migration into the mycobacteria-infected lung.

Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, their mechanism of action remains unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages responded to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages responded to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhanced Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerated the elimination of mycobacterial infection, suggesting that TREM2/DAP12 signaling counteracts Mincle/FcRg/CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 to evade the host immunity.

Monocytes and macrophages are important effectors and regulators of inflammation, and both their differentiation and activation are regulated strictly in response to environmental cues. Angiopoietin-like protein 2 (Angptl2) is a multifaceted protein, displaying many physiological and pathological functions in inflammation, angiogenesis, hematopoiesis, and tumor development. Although recent studies implicate Angptl2 in chronic inflammation, the mechanisms of inflammation caused by Angptl2 remain unclear. The purpose of the present study was to elucidate the role of Angptl2 in inflammation by understanding the effects of Angptl2 on monocytes/macrophages. We showed that Angptl2 directly activates resident murine peritoneal monocytes and macrophages and induces a drastic upregulation of the transcription of several inflammatory genes including nitric oxide synthase 2 and prostaglandin-endoperoxide synthase 2, and several proinflammatory cytokine genes such as interleukin (IL)-1β, IL-6, TNFα, and CSF2, along with activation of ERK, JNK, p38, and nuclear factor kappa B signaling pathways. Concordantly, proinflammatory cytokines IL-1β, IL-6, TNFα, and GM-CSF, were rapidly elavated from murine peritoneal monocytes and macrophages. These results demonstrate a novel role for Angptl2 in inflammation via the direct activation of peritoneal monocytes and macrophages.

Degranulation inhibitors in plants are widely used for prevention and treatment of immediate-type allergy. We previously isolated a new ellagic acid glucoside, okicamelliaside (OCS), from Camellia japonica leaves for use as a potent degranulation inhibitor. Crude extracts from leaves also suppressed allergic conjunctivitis in rats. In this study, we evaluated the in vivo effect of OCS using a pure sample and performed in vitro experiments to elucidate the mechanism underlying the extraordinary high potency of OCS and its aglycon. The IC(50) values for degranulation of rat basophilic leukemia cells (RBL-2H3) were 14 nM for OCS and 3 μM for aglycon, indicating that the two compounds were approximately 2 to 3 orders of magnitude more potent than the anti-allergic drugs ketotifen fumarate, DSCG, and tranilast (0.17, 3, and >0.3 mM, respectively). Antigen-induced calcium ion (Ca(2+)) elevation was significantly inhibited by OCS and aglycon at all concentrations tested (p

Interleukin (IL)-17A is a cytokine involved in neutrophilic inflammation but the role of IL-17A in anti-tumor immunity is controversial because both pro- and anti-tumor activities of IL-17A have been reported. We hypothesized that constitutive expression of IL-17A in intestinal environment modifies tumor growth. To address the issue, mice were inoculated into subserosa of cecum (i.c.) with murine EL4 lymphoma expressing a model tumor antigen, and tumor growth was monitored. IL-17A-producing cells were detected both in tumor mass and in normal intestinal tissue of i.c. tumor-bearing wild type mice. Tumor size in the wild-type mice was significantly higher than that in the cecum of IL-17A gene-knockout mice. Furthermore, anti-IL-17A monoclonal antibody treatment of wild-type mice resulted in decreased tumor size in the cecum. Model tumor-antigen-specific interferon-γ production was not modified in draining mesenteric lymph node cells in the absence or after neutralization of IL-17A. All the results suggest that constitutive expression of IL-17A in intestine enhances tumor growth, and anti-IL-17A antibody treatment is a candidate of a new anti-tumor immunotherapy against intestinal tumors.

Methylotrophic yeast (Pichia pastoris) secreted cholera toxin B subunit (CTB) predominantly as a biologically active pentamer (PpCTB) with identical ganglioside binding affinity profiles to that of choleragenoid. Unlike choleragenoid, however, the PpCTB did not induce a footpad edema response in mice. Of the two potential glycosylation sites (NIT4–6 and NKT90–92) for this protein, a N-linked oligosaccharide was identified at Asn4. The oligosaccharide, presumed to extend from the lateral circumference of the CTB pentamer ring structure, was exploited as a site-specific anchoring scaffold for the C-terminal 19-kDa merozoite surface protein-1 (MSP1-19) of the rodent malaria parasite, Plasmodium yoelii. Conjugation of MSP1-19 to PpCTB via its oligosaccharide moiety induced higher protective efficacy against lethal parasite infection than conjugation directly to the PpCTB protein body in both intranasal and subcutaneous immunization regimes. Such increased protection was potentially due to the higher antigen loading capacity of CTB achieved when the antigen was linked to the extended branches of the oligosaccharide. This might have allowed the antigen to reside in more spacious molecular environment with less steric hindrance between the constituent molecules of the fusion complex.

It is generally accepted that cellular immunity plays a critical role in the protection against Mycobacterium tuberculosis, an intracellular pathogen. Recently, however, an increasing number of reports indicate the important contribution of humoral immunity against mycobacterial infection. Since M. tuberculosis establishes its primary lesion in the lung, induction of humoral immunity in the airway tract by mucosal immunization regime could provide protective immunity against tuberculosis. In this study, mycobacterial heparin-binding haemagglutinin adhesin (HBHA) was used as an immunization antigen because HBHA is an essential virulence factor required for the infection of lung epithelial cells and extrapulmonary dissemination of mycobacteria. The effects of intranasal immunization with a yeast-expressed recombinant (r) HBHA co-administered with a mucosal adjuvant cholera toxin (CT) on the induction of humoral and cellular immunity were examined, and its protective efficacy against pulmonary challenge infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) was evaluated. HBHA-specific antibodies were induced in serum and airway tract of immunized mice, which specifically recognized native HBHA expressed on M. bovis BCG. Th1-type immunity against mycobacterial antigens was also enhanced in the lung of immunized mice after pulmonary BCG infection. Furthermore, the immunization suppressed bacterial load in the spleen after pulmonary BCG infection. These results indicate that systemic and local humoral immunity induced by the HBHA-based mucosal vaccine impairs extrapulmonary dissemination, thus providing immune protection against mycobacterial infection.

Mice treated with a contraceptive dose of beta-estradiol (E2) demonstrated changes in their macrophage (Mphi) number and functions. While E2 increased and decreased the Mphi number in PBMC and PEC respectively, it enhanced the in vitro phagocytosis of FITC-labeled beads by both cells. E2 treatment also enhanced the phagocytic function of Mphi as assessed by the in vivo carbon clearance assay. In contrast, the in vitro intracellular killing function of adherent cells in peritoneal exudate cells (PEC) against Listeria monocytogenes decreased after E2 treatment. In line with the decrease in the intracellular killing function, the E2-treated mice showed an impaired protection against L. monocytogenes infection. To clarify the mechanism of the E2-mediated suppression of the protective response against L. monocytogenes infection, we next analyzed the cytokine expression by PEC in E2-treated L. monocytogenes-infected mice. On day 5 of the infection, the expression of IL-12, TNF-alpha and IL-10 by adherent PEC from the E2-treated mice was lower than that from the control-infected mice. The decrease in the cytokine expression by adherent PEC of E2-treated mice coincided with the decrease of IFN-gamma expression, and the increase in the IL-4, IL-10 and TGF-beta expressions by non-adherent PEC. These results revealed two aspects of the effects of E2 on Mphi. Even though E2 was found to enhance Mphi phagocytosis, the anti-bacterial function was suppressed. This suppression may be mediated by the inhibition of both IL-12 and TNF-alpha which play important roles in the protective response against intracellular bacteria.

In the present report, we demonstrated that extrathymic gamma(delta) T cells proliferated in the mixed culture of lymph node cells of BALB/c nu/nu mice and irradiated syngeneic spleen cells (syngeneic MLR). Reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that V(delta)5 and V(delta)6 were dominantly expressed by the gamma(delta) T cells from the syngeneic MLR. Furthermore, the syngeneic MLR may expand V(delta)5+ T cells with BALB invariant delta (BID) sequence which is specifically detected in BALB background mice because V(delta)5 RT-PCR products from syngeneic MLR were digested with an AluI restriction enzyme which cut the D(delta)2-J(delta)1 junction of the BID sequence but not those of most non-BID V(delta)5 sequences. Interestingly, anti-mycobacterial and mammalian heat shock protein (Hsp) 60 mAb ML30 suppressed the generation of V(delta)5 with an AluI site in syngeneic MLR. These results suggest that extrathymically derived V(delta)5+ and V(delta)6+ gamma(delta) T cells recognize BALB/c self antigens and that the Hsp60 or an Hsp60-related molecule may be involved in the anti-self response of V(delta)5+ BID+ gamma(delta) T cells.

Using two- and three-color flow cytometry, we investigated the surface makers of regional lymph node lymphocytes (RLNL) in 54 patients with primary lung cancer in order to determine whether or not RLNL are in an activation state when compared with the corresponding peripheral blood lymphocytes (PBL) for the purpose of clarifying the characteristics of RLNL. RLNL showed a cell composition that was different from the corresponding PBL with a high proportion of CD3 + cells, CD4 + cells and CD20 + (B) cells as well as a low proportion of CD8 + cells and CD16 + (NK) cells. RLNL also contained a significantly higher proportion of CD45RO + T cells and a lower proportion of CD45RA + T cells in comparison to the corresponding PBL. Furthermore, we explored the activation-related molecules such as the interleukin-2 receptor (IL-2R) α chain, the IL-2R β chain, HLA-DR and leukocyte function-associated antigen 1 (LFA-1) on CD4 + and CD8 + cells. The data showed that the expression of CD45RO, the IL-2R α chain, HLA-DR on CD4 + cells, and those of CD45RO and HLA-DR on CD8 + cells were significantly higher in RLNL than in PBL. On the other hand, PBL showed a higher expression of the IL-2R β chain and LFA-1 only on CD8 + cells, which are thought to include CD8 + NK cells. When these activation-related molecules were analyzed on CD45RO + T cells, which are thought to be memory T cells, then the expression of the IL-2R α chain, HLA-DR on CD4 + CD45RO + cells and HLA-DR on CD8 + CD45RO + cells were significantly higher in RLNL than in PBL. Lastly, we analyzed the surface molecules according to such clinical factors as metastasis to the lymph nodes and the stage of lung cancer. Interestingly, RLNL with lymph node metastasis showed an increase in the percentage of CD20 + cells and a decrease in the percentage of CD4 + cells in comparison to those with benign lung disease, while these differences were not observed in PBL. The expression of CD45RO and LFA-1 on CD4+ and LFA-1 on CD8 + cells on RLNL in stage I + II is significantly higher in comparison with benign lung disease. Furthermore, RLNL in stage III + IV revealed the reduced expression of CD45RO, HLA-DR and LFA-1 on both CD4 + and CD8 + cells compared with those in stage I+II. These results thus demonstrated that RLNL were in a more activated state, particularly in stages I + II, than PBL. Whether these activation states in RLNL are directed against autologous tumor cells or not can hopefully be elucidated after the completion of further ongoing studies.

Background & Aims: Intraepithelial leukocytes (IELs) residing in the intestinal epithelium are thought to recognize bacterial antigens and play an important role in providing protection against the invasion of microorganisms through the intestinal wall. This report studies the effect of various bacterial antigens on the proliferation of cultured IELs. Methods: Bacterial antigens, mycobacterial heat shock protein (hsp) 60 or 70, staphylococcal enteroxin A (SEA), or purified protein derivative (PPD) from Mycobacterium tuberculosis were added to mixed cultures of IELs and irradiated syngeneic or allogeneic spleen cells, and the proliferative response was evaluated. Monoclonal antibodies to CD4, CD8, and major histocompatibility complex class I or class II molecules were added to assess the role of these proteins in the proliferative response. Results: The proliferation of IELs isolated from mature rats in mixed culture was significantly enhanced by the addition of PPD, hsp 60, hsp 70, or SEA. The proliferation of mixed cultures of IELs isolated from young rats was enhanced only by the addition of SEA. In mixed cultures of mature rat IELs and syngeneic spleen cells containing PPD, proliferating populations were of the CD4 − CD8 + and CD4 + CD8 + phenotypes. Addition of anti-CD4 monoclonal antibody suppressed the proliferative response to PPD to control levels. Conclusions: IELs isolated from mature rats contain mycobacterial antigen-specific T cells, and these cells may be selected by long-term exposure to common intestinal bacterial antigens such as hsp. These cells may contain populations that recognize bacterial antigens in a CD4-dependent manner.

The Th1 type CD4 + T cell clone (MH2), which is capable of recognizing purified protein derivative from Mycobacterium tuberculosis (PPD), was examined for its anti-metastatic activity against melanoma. In using an in vitro proliferative assay, MH2 was able to recognize PPD-derived antigen in a major histocompatibility complex class 11-restricted manner. MH2 showed neither any natural killer (NK) activity nor cytolytic activity against syngeneic B16 melanoma. This clone produced interferon-γ, tumor necrosis factor and interleukin-2, but not interleukin-4, when co-cultured with PPD and irradiated syngeneic C57BL/6 spleen cells, suggesting that this clone could thus be assigned to the Th1 subset. An intraperitoneal (i.p.) co-injection of 2 x 10 6 MH2 and 50μg PPD increased the NK activity of the peritoneal exudate cells (PEC) and the percentage of NK1.1 + cells in the PEC. These activated NK cells showed a low but significant cytolytic activity against B16 melanoma. The augmented NK activity induced by the co-injection of MH2 and PPD was maintained by the weekly additional i.p. injections of PPD alone. Using a murine metastatic model, an i.p. co-injection of MH2 and PPD-induced antimetastatic activity against B16 melanoma. This anti-metastatic activity was then abrogated by the in vivo administration of anti-asialo GMt serum. In addition, the NK activity in both peripheral blood and metastatic lungs was significantly augmented in the mice which were co-injected with MH2 and PPD. Taken together, these findings indicate that the in vivo activation of Tht type CD4+ T cells augmented the NK activity in vivo and thus could potentially be an efficient immunotherapeutic weapon against metastasis of melanoma. These results also imply that adoptive immunotherapy could induce antimetastatic activity through cytokine production but not through any direct cytolytic activity.

In vitro 5-day cultures of naive spleen cells with viable Listeria monocytogenes (VLM), but not heat-killed L. monocytogenes, induced CD4+ T cells that produced IFN-gamma upon secondary antigen stimulation. The VLM-induced Listeria-specific T cells produced IFN-gamma but lacked expression of IL-2 and IL-4. To study the role of IFN-gamma in the induction of the IFN-gamma-producing T cells, we added anti-IFN-gamma mAb to the primary culture and analyzed IFN-gamma production upon secondary antigen stimulation. Addition of anti-IFN-gamma mAb to the culture suppressed generation of IFN-gamma-producing CD4+ T cells, suggesting that IFN-gamma is important in the induction of IFN-gamma-producing CD4+ T cells. Furthermore, our results showed that depletion of NK cells from spleen cells by anti-asialo GM1 antibody plus complement before culture enhanced induction of IFN-gamma-producing CD4+ T cells. Although NK cells are known to produce IFN-gamma, the results indicate that NK cell-derived IFN-gamma may not be important in induction of the Listeria-specific IFN-gamma-producing CD4+ T cells in the culture system. In addition, we demonstrated that IFN-gamma expression was high in CD4+ T cells from cultures of spleen cells with VLM at the primary culture level. These results suggest that IFN-gamma derived from T cells may enhance production of IFN-gamma by CD4+ T cells, while NK cells rather suppress the induction of IFN-gamma producing CD4+ T cells.

We have reported that Listeria monocytogenes-specific IFN-gamma-producing CD4+ T cells are induced by in vitro 5-day culture of naive spleen cells with viable L. monocytogenes (VLM), but not induced by culture with heat-killed L. monocytogenes (HKLM). In the present study, the role of B cells in the regulation of induction of IFN-gamma-producing CD4+ T cells in the in vitro system was investigated. We found that L. monocytogenes-specific IFN-gamma-producing CD4+ T cells were not generated when B cells were depleted from spleen cells before culture with VLM although Ag-specific proliferative response was retained. IFN-gamma production by CD4+ T cells was restored by addition of B cells cultured with VLM to the culture of B cell-depleted spleen cells and VLM. In contrast, B cells cultured with HKLM could not restore the induction of IFN-gamma production when added in culture of B cell-depleted spleen cells and VLM. Analysis of cytokine gene expression by reverse transcription-polymerase chain reaction method revealed that expression of interleukin-10 (IL-10) was higher but TNF-alpha was lower in B cells cultured with HKLM when compared with that in B cells cultured with VLM. Furthermore, addition of neutralizing anti-IL-10 mAb into culture of naive spleen cells with HKLM resulted in appearance of IFN-gamma-producing cells. These results suggest that B cells have positive and negative roles in the induction of IFN-gamma-producing CD4+ T cells. The inhibition of induction of IFN-gamma-producing CD4+ T cells may depend on B cell-derived IL-10.

Fetal thymus grafting into athymic nude mice has been used as an experimental model of T cell development. To understand the early events of T cell development, we have examined the sequence of appearance of T cell subsets in lymph nodes (LN) of BALB/c nu/nu mice after grafting with syngeneic fetal thymus. T cells expressing T cell receptor (TCR) alpha/beta or gamma/delta increased in LN from 1 week after grafting, although no host-derived CD3+ T cells were detected in the grafted thymus and no donor thymus-derived T cells were detected in the LN. The early appearing T cells of both TCR alpha/beta and TCR gamma/delta showed a CD4-CD8- phenotype. V region usage analysis of the early appearing TCR alpha/beta T cells revealed that they contained cells bearing V beta 3 or V beta 11, which are potentially reactive to self-superantigen Mls-2a or Dvb11, respectively, and are deleted in the course of T cell development in the thymus of euthymic BALB/c mice. The early appearing T cells showed neither mixed lymphocyte reaction nor cytotoxic T cell activity against allogeneic cells. In contrast, lymphokine-activated killer cells from early appearing T cells, which contained high percentages of TCR gamma/delta T cells, exhibited higher cytotoxic activity against P815 mastocytoma than those from euthymic mice or untreated nude mice. All these results suggest that the early appearing T cells are developed extrathymically. We propose that the thymus may induce extrathymic T cell development without direct cell-to-cell interaction. It seems likely that the extrathymically developed T cells, especially TCR gamma/delta T cells, induced by the thymus have some role in the defense mechanism in the absence of conventional thymus-derived T cells.