Both Conventional and Interferon Killer Dendritic Cells Have Antigen-Presenting Capacity during Influenza Virus Infection

Natural killer cells are innate effector cells known for their potential to produce interferon-γ and kill tumour and virus-infected cells. Recently, B220⁺CD11c^intNK1.1⁺ NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220⁻ NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.     

Bone marrow-derived IFN-producing killer dendritic cells account for the tumoricidal activity of unpulsed dendritic cells

The CD11c(int)B220(+)NK1.1(+)CD49(+) subset of cells has recently been described as IFN-producing killer dendritic cells (IKDC), which share phenotypic and functional properties of dendritic cells and NK cells. Herein we show that bone marrow-derived murine dendritic cell preparations contain abundant CD11c(int)B220(+)NK1.1(+)CD49(+) cells, the removal of which results in loss of tumoricidal activity of unpulsed dendritic cells in vivo. Moreover, following s.c. injection, as few as 5 x 10(3) highly pure bone marrow-derived IKDC cells are capable of shrinking small contralateral syngeneic tumors in C57BL/6 mice, but not in immunodeficient mice, implying the obligate involvement of host effector cells in tumor rejection. Our data suggest that bone marrow-derived IKDC represent a population that has powerful tumoricidal activity in vivo.     

17beta-estradiol alters the activity of conventional and IFN-producing killer dendritic cells

Estrogens increase aspects of innate immunity and contribute to sex differences in the prevalence of autoimmune diseases and in response to infection. The goal of the present study was to assess whether exposure to 17beta-estradiol (E2) affects the development and function of bone marrow-derived dendritic cells and to determine whether similar changes are observed in CD11c(+) splenocytes exposed to E2 in vivo. E2 facilitated the differentiation of BM precursor cells into functional CD11c(+)CD11b(+)MHC class II(+) dendritic cells (DCs) with increased expression of the costimulatory molecules CD40 and CD86. Exposure of bone marrow-derived dendritic cells to E2 also enhanced production of IL-12 in response to the TLR ligands, CpG and LPS. In contrast, CD11c(+) cells isolated from the spleens of female C57BL/6 mice that were intact, ovariectomized, or ovariectomized with E2 replacement exhibited no differences in the number or activity of CD11c(+)CD11b(+)MHC class II(+) DCs. The presence of E2 in vivo, however, increased the number of CD11c(+)CD49b(+)NK1.1(low) cells and reduced numbers of CD11c(+)CD49b(+)NK1.1(high) cells, a surface phenotype for IFN-producing killer DCs (IKDCs). Ultrastructural analysis demonstrated that CD11c(+)NK1.1(+) populations were comprised of cells that had the appearance of both DCs and IKDCs. CD11c(+) splenocytes isolated from animals with supplemental E2 produced more IFN-gamma in response to IL-12 and IL-18. These data illustrate that E2 has differential effects on the development and function of DCs and IKDCs and provide evidence that E2 may strengthen innate immunity by enhancing IFN-gamma production by CD11c(+) cells.     

The critical role of IL-15 in the antitumor effects mediated by the combination therapy imatinib and IL-2

The synergistic antitumor effects of the combination therapy imatinib mesylate (IM) and IL-2 depended upon NK1.1- expressing cells and were associated with the accumulation of CD11c(int)B220(+)NK1.1(+) IFN-producing killer dendritic cells (IKDC) into tumor beds. In this study, we show that the antitumor efficacy of the combination therapy was compromised in IL-15 and IFN-type 1R loss-of-function mice. IL-15Ralpha was required for the proliferation of IKDC during IM plus IL-2 therapy. Trans-presentation of IL-15/IL-15Ralpha activated IKDC to express CCR2 and to respond to type 1 IFN by producing CCL2. Moreover, the antitumor effects of the combination therapy correlated with a CCL2-dependent recruitment of IKDC, but not B220(-) NK cells, into tumor beds. Altogether, the IL-15-driven peripheral expansion and the CCL-2-dependent intratumoral chemoattraction of IKDC are two critical parameters dictating the antitumor efficacy of IM plus IL-2 in mice.     

Interferon-producing killer dendritic cells provide a link between innate and adaptive immunity

Natural killer (NK) cells and dendritic cells (DCs) are, respectively, central components of innate and adaptive immune responses. We describe here a third DC lineage, termed interferon-producing killer DCs (IKDCs), distinct from conventional DCs and plasmacytoid DCs and with the molecular expression profile of both NK cells and DCs. They produce substantial amounts of type I interferons (IFN) and interleukin (IL)-12 or IFN-gamma, depending on activation stimuli. Upon stimulation with CpG oligodeoxynucleotides, ligands for Toll-like receptor (TLR)-9, IKDCs kill typical NK target cells using NK-activating receptors. Their cytolytic capacity subsequently diminishes, associated with the loss of NKG2D receptor (also known as Klrk1) and its adaptors, Dap10 and Dap12. As cytotoxicity is lost, DC-like antigen-presenting activity is gained, associated with upregulation of surface major histocompatibility complex class II (MHC II) and costimulatory molecules, which formally distinguish them from classical NK cells. In vivo, splenic IKDCs preferentially show NK function and, upon systemic infection, migrate to lymph nodes, where they primarily show antigen-presenting cell activity. By virtue of their capacity to kill target cells, followed by antigen presentation, IKDCs provide a link between innate and adaptive immunity.     

A novel cell subset: Interferon-producing killer dendritic cells

Recent reports introduce a novel cell subset of DCs with antigenic phenotypes shared by both NK cells and B cells, but without surface markers of pDCs and T cells, appearing to be a chimera of NK cells and DCs, namely interferon-producing killer dendritic cells(IKDCs).IKDCs not only secret type I and type II interferons to recognize and kill tumor cells effectively, but also express MHC-II molecules to present antigens.Thus, IKDCs are considered as important immunosurveilance cells for tumors, providing a link between innate and adaptive immunity.     

Trans-presentation of IL-15 dictates IFN-producing killer dendritic cells effector functions

IFN-producing killer dendritic cells (IKDC) were initially described as B220(+)CD11c(+)CD3(-)NK1.1(+) tumor-infiltrating cells that mediated part of the antitumor effects of the combination therapy with imatinib mesylate and IL-2. In this study, we show their functional dependency on IL-15 during homeostasis and inflammatory processes. Trans-presentation of IL-15 by IL-15Ralpha allows dramatic expansion of IKDC in vitro and in vivo, licenses IKDC for TRAIL-dependent killing and endows IKDC with immunizing potential, all three biological attributes not shared by B220(-)NK cells. However, IL-15 down-regulates the capacity of IKDC to induce MHC class I- or II-restricted T cell activation in vitro. Trans-presentation of IL-15 by IL-15Ralpha allows IKDC to respond to TLR3 and TLR4 ligands for the production of CCL2, a chemokine that is critical for IKDC trafficking into tumor beds (as described recently). We conclude that IKDC represent a unique subset of innate effectors functionally distinguishable from conventional NK cells in their ability to promptly respond to IL-15-driven inflammatory processes.     

Potent tumor-specific protection ignited by adoptively transferred CD4+ T cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.     

Immunosurveillance of lung melanoma metastasis in EBI-3-deficient mice mediated by CD8+ T cells

EBV-induced gene 3 (EBI-3) codes for a soluble type I receptor homologous to the p40 subunit of IL-12 that is expressed by APCs following activation. In this study, we assessed the role of EBI-3 in a model of lung melanoma metastasis. Intravenous injection of the B16-F10 cell line resulted in a significant reduction of lung tumor metastasis in EBI-3(-/-) recipient mice compared with wild-type mice. The immunological finding accompanying this effect was the expansion of a newly described cell subset called IFN-gamma producing killer dendritic cells associated with CD8(+) T cell responses in the lung of EBI-3(-/-) mice including IFN-gamma release and TNF-alpha-induced programmed tumor cell death. Depletion of CD8(+) T cells as well as targeting T-bet abrogated the protective effects of EBI-3 deficiency on lung melanoma metastases. Finally, adoptive transfer of EBI-3(-/-) CD8(+) T cells into tumor bearing wild-type mice inhibited lung metastasis in recipient mice. Taken together, these data demonstrate that targeting EBI-3 leads to a T-bet-mediated antitumor CD8(+) T cell responses in the lung.     

Blockade of NKG2D signaling prevents the development of murine CD4+ T cell-mediated colitis

It has been recently demonstrated that NKG2D is an activating costimulatory receptor on natural killer (NK) cells, natural killer T (NKT) cells, activated CD8(+) T cells, and gammadelta T cells, which respond to cellular stress, such as inflammation, transformation, and infection. Here we show that intestinal inflammation in colitic SCID mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells is characterized by significant increase of CD4(+)NKG2D(+) T cells and constitutive expression of NKG2D ligands, such as H60, Mult-1, and Rae-1, by lamina propria CD11c(+) dendritic cells. Furthermore, treatment with nondepleting and neutralizing anti-NKG2D MAb after transfer of CD4(+)CD45RB(high) T cells into SCID mice significantly suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of IFN-gamma by lamina propria CD4(+) T cells. These findings demonstrate that NKG2D signaling pathway is critically involved in CD4(+) T cell-mediated disease progression and suggest a new therapeutic target for inflammatory bowel diseases.     

Macrophages, CD4+ or CD8+ cells are each sufficient for protection against Chlamydia pneumoniae infection through their ability to secrete IFN-gamma

By using a T, B, or NK cell-deficient mouse strain (recombinase-activating gene (RAG)-1(-/-)/common cytokine receptor gamma-chain (gamma(C)R)), and T and B cell and IFN-gamma-deficient (RAG-1(-/-)/IFN-gamma(-/-)) mice, we have studied the generation of immunity against infection by Chlamydia pneumoniae. We found that IFN-gamma secreted by innate-cell populations protect against C. pneumoniae infection. However, NK cells were not needed for such IFN-gamma-dependent innate immune protection. Inoculation of wild type, but not IFN-gamma(-/-) bone marrow-derived macrophages protected RAG-1(-/-)/IFN-gamma(-/-) mice against C. pneumoniae infection. In line, pulmonary macrophages from RAG-1(-/-) C. pneumoniae-infected mice expressed IFN-gamma mRNA. Reconstitution of RAG-1(-/-)/gamma(c)R(-/-) or RAG-1(-/-)/IFN-gamma(-/-) mice with CD4(+) or CD8(+) cells by i.v. transfer of FACS sorted wild type spleen cells (SC) increased resistance to C. pneumoniae infection. On the contrary, no protection was observed upon transfer of IFN-gamma(-/-) CD4(+) or IFN-gamma(-/-) CD8(+) SC. T cell-dependent protection against C. pneumoniae was weaker when IFN-gammaR(-/-) CD4(+) or IFN-gammaR(-/-) CD8(+) SC were inoculated into RAG-1(-/-)/IFN-gamma(-/-) mice. Thus both nonlymphoid and T cell-derived IFN-gamma can play a central and complementary role in protection against C. pneumoniae. IFN-gamma secreted by nonlymphoid cells was not required for T cell-mediated protection against C. pneumoniae; however, IFN-gamma regulated T cell protective functions.     

Critical role of NK cells rather than V alpha 14(+)NKT cells in lipopolysaccharide-induced lethal shock in mice

Although macrophages play a central role in the pathogenesis of septic shock, NK1(+) cells have also been implicated. NK1(+) cells comprise two major populations, namely NK cells and V alpha 14(+)NKT cells. To assess the relative contributions of these NK1(+) cells to LPS-induced shock, we compared the susceptibility to LPS-induced shock of beta(2)-microglobulin (beta(2)m)(-/-) mice that are devoid of V alpha 14(+)NKT cells, but not NK cells, with that of wild-type (WT) mice. The results show that beta(2)m(-/-) mice were more susceptible to LPS-induced shock than WT mice. Serum levels of IFN-gamma following LPS challenge were significantly higher in beta(2)m(-/-) mice, and endogenous IFN-gamma neutralization or in vivo depletion of NK1(+) cells rescued beta(2)m(-/-) mice from lethal effects of LPS. Intracellular cytokine staining revealed that NK cells were major IFN-gamma producers. The J alpha 281(-/-) mice that are exclusively devoid of V alpha 14(+)NKT cells were slightly more susceptible to LPS-induced shock than heterozygous littermates. Hence, LPS-induced shock can be induced in the absence of V alpha 14(+)NKT cells and IFN-gamma from NK cells is involved in this mechanism. In WT mice, hierarchic contribution of different cell populations appears likely.     

Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice

The role of mouse liver NK1.1 Ag(+) T (NKT) cells in the antitumor effect of alpha-galactosylceramide (alpha-GalCer) has been unclear. We now show that, whereas alpha-GalCer increased the serum IFN-gamma concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of alpha-GALCER: Injection of Abs to IFN-gamma also inhibited the alpha-GalCer-induced increase in antitumor cytotoxicity of MNCS: alpha-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas alpha-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCS: The alpha-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-gamma. Depletion of CD8(+) T cells did not affect the alpha-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of alpha-GalCer on the survival of B6 mice. Thus, IFN-gamma produced by alpha-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8(+) T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate alpha-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.     

Interferon-gamma contributes to the normalcy of murine pregnancy.

Uterine natural killer (uNK) cells are transient, large, heavily granulated, maternal lymphocytes present on the mesometrial side of the pregnant mouse uterus. These cells contribute to normal implantation site development. Cytokine production, particularly interferon (IFN)-gamma, is a major function of most NK cell subsets. In this study, uNK cells were assessed for IFN-gamma production. Local concentrations of IFN-gamma were measured in the mesometrial regions of murine implantation sites between Days 6 and 16 of gestation. IFN-gamma was detected by ELISA at all days studied in a random-bred (CD1) and an inbred (BALB/c) strain of immune-competent mouse and in two immune-deficient strains, SCID (NK(+), T(-), B(-)) and tgepsilon26 (NK(-), T(-), B(+)). Concentrations of IFN-gamma per implantation site peaked at Day 10 of gestation in NK(+) strains but were low and relatively constant in NK(-) mice. To evaluate the functions of IFN-gamma at murine implantation sites, pregnancy was studied in homozygously mated IFN-gamma(-/-) and IFN-gammaRalpha(-/-) mice and their congenic controls. Primiparous but not multiparous IFN-gamma(-/-) mice experienced significant fetal loss. Primiparous IFN-gammaRalpha(-/-) carried full litters to term. Implantation site pathology was demonstrated in both strains of gene-deleted mice by light microscopy and ultrastructurally. This included elevated numbers of uNK cells that contained fewer and smaller granules and, after Day 10 of gestation, progressive necrosis and loss of decidua. The presence of a fetus able to produce IFN-gamma did not modify the phenotype of pregnant IFN-gamma(-/-) mice. This study indicates that during murine pregnancy, uNK cells are the main source of IFN-gamma on the mesometrial side of the uterus and that IFN-gamma contributes to normal health of the midgestational decidua. Furthermore, evidence is presented that IFN-gamma-producing cells exist in mesometrial regions of implantation sites that are neither NK nor T cells.     

Ectopic CD40 ligand expression on B cells triggers intestinal inflammation

Several studies indicate that CD4(+) T cells, macrophages, and dendritic cells initially mediate intestinal inflammation in murine models of human inflammatory bowel disease. However, the initial role of B cells in the development of intestinal inflammation remains unclear. In this study we present evidence that B cells can trigger intestinal inflammation using transgenic (Tg) mice expressing CD40 ligand (CD40L) ectopically on B cells (CD40L/B Tg). We demonstrated that CD40L/B Tg mice spontaneously developed severe transmural intestinal inflammation in both colon and ileum at 8-15 wk of age. In contrast, CD40L/B TgxCD40(-/-) double-mutant mice did not develop colitis, indicating the direct involvement of CD40-CD40L interaction in the development of intestinal inflammation. The inflammatory infiltrates consisted predominantly of massive aggregated, IgM-positive B cells. These mice were also characterized by the presence of anti-colon autoantibodies and elevated IFN-gamma production. Furthermore, although mice transferred with CD4(+) T cells alone or with both CD4(+) T and B220(+) B cells, but not B220(+) cells alone, from diseased CD40L/B Tg mice, develop colitis, mice transferred with B220(+) B cells from diseased CD40L/B Tg mice and CD4(+) T cells from wild-type mice also develop colitis, indicating that the Tg B cells should be a trigger for this colitis model, whereas T cells are involved as effectors. As it has been demonstrated that CD40L is ectopically expressed on B cells in some autoimmune diseases, the present study suggests the possible contribution of B cells in triggering intestinal inflammation in human inflammatory bowel disease.     

A unique population of extrathymically derived alpha beta TCR+CD4-CD8- T cells with regulatory functions dominates the mouse female genital tract

A better understanding of the regulatory role of genital tract T cells is much needed. In this study, we have analyzed the phenotype, distribution, and function of T lymphocytes in the female genital tract of naive, pregnant, or Chlamydia trachomatis-infected C57BL/6 mice. Unexpectedly, we found that the dominant lymphocyte population (70-90%) in the genital tract was that of CD3(+)alphabetaTCR(int)CD4(-)CD8(-) T cells. Moreover, these cells were CD90(low) but negative for the classical T cell markers CD2 and CD5. The CD3(+)B220(low) cells were NK1.1 negative and found in nude mice as well as in mice deficient for MHC class II, beta(2)-microglobulin, and CD1, indicating extrathymic origin. They dominated the KJ126(+)Vbeta8.2(+) population in the genital tract of DO11.10 OVA TCR-transgenic mice, further supporting the idea that the CD3(+)B220(low) cells are truly T cells. The function of these T cells appeared not to be associated with immune protection, because only CD4(+) and CD8(+) T cells increased in the genital tract following chlamydial infection. Notwithstanding this, the infected, as well as the uninfected and the pregnant, uterus was dominated by a high level of the CD3(+)CD4(-)CD8(-)B220(low) cells. Following in vitro Ag or polyclonal stimulation of the CD3(+)CD4(-)CD8(-)B220(low) cells, poor proliferative responses were observed. However, these cells strongly impaired splenic T cell proliferation in a cell density-dependent manner. A large fraction of the cells expressed CD25 and produced IFN-gamma upon anti-CD3 plus anti-CD28 stimulation, arguing for a strong regulatory role of this novel T cell population in the mouse female genital tract.     

Protective effect of NK1.1(+) T cells as well as NK cells against intraperitoneal tumors in mice

Peritoneal resident cells of mice normally contain small populations of NK cells and NK1.1(+) alphabetaT cells. These populations increased after either 3LL or EL4 tumor inoculations into the peritoneal cavity. In vivo depletion of NK cell alone by anti-asialo GM1 (ASGM1) Ab significantly decreased survival time of tumor-injected mice, while depletion of both NK cells and NK1.1(+) T cells by anti-NK 1.1 Ab greatly shortened mouse survival time. NK1. 1(+) T cells in peritoneal cavity consist of a larger proportion of double-negative T cells and smaller populations of CD4(+) T cells and Vbeta8(+) T cells compared with liver NK1.1(+) T cells and normally lack Vbeta2(+) T cells. Tumor inoculation induced rapid IL-12 and IFN-gamma mRNA in tumor-infiltrating mononuclear cells (TIM). Although anti-NK1 Ab pretreatment in vivo abrogated IFN-gamma mRNA expression and IFN-gamma production of TIM, NK cell depletion alone by anti-ASGM1 Ab pretreatment retained IFN-gamma mRNA expression and partly inhibited IFN-gamma production of TIM. Peritoneal NK cells as well as NK1.1(+) T cells but not NK1.1(-) T cells of 3LL cell- or EL4 cell-injected mice showed cytotoxicities against the same tumor cells. Further, either anti-IL-12 Ab or anti-IFN-gamma Ab ip injection significantly shortened EL4 cell-inoculated mouse survival time. Our findings suggest that peritoneal macrophages activated by tumors produce IL-12 which activates NK cells and NK1.1(+) T cells to produce IFN-gamma and both NK cells and NK1.1(+) T cells are important in suppressing the growth of the intraperitoneal tumors.     

Characterization of lymphocyte subsets in the bronchiolar lymph nodes of BALB/c mice infected with cilia-associated respiratory bacillus

Cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative, extracellular bacterium that causes chronic respiratory tract disease in rodents. Infected mice develop microscopic lesions characterized by a primary lymphocytic response followed by macrophage and neutrophilic infiltration. To characterize the lymphocytic subsets that respond to CAR bacillus infection, BALB/c mice were inoculated with 10(5) CAR bacillus bacteria. At seven weeks after inoculation, mice were euthanized and the tracheobronchiolar and hilar lymph nodes were collected and stained for cell surface markers to T cells (CD3, CD4, and CD8), B cells (B220, CD5), natural killer (NK) cells (pan-NK) and intracellular interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). Flow cytometric analysis of lymph nodes from CAR bacillus-infected mice revealed 11% increase in frequency of B cells (R220+), 12% increase in the frequency of double-negative (CD4-CD8-CD3+) T cells, and slight increase in the B-1 subset of B cells (B220+CD5+). There was no change in the frequency of NK cells. The CAR bacillus-infected mice had an overall decrease in the frequency of T cells. Intracellular cytokine staining revealed distinct populations of T cells producing IL-10 and IFN-gamma, and IL-10 production from B cells; NK cells were not a substantial source of IFN-gamma. To our knowledge, this is the first characterization of lymphocytic responses and suggestion that B cells and double-negative T cells may be principally responsible for the lesions associated with CAR bacillus infection.     

Induction of tumor regression by administration of B7-Ig fusion proteins: mediation by type 2 CD8+ T cells and dependence on IL-4 production

CD28 signals contribute to either type 1 or type 2 T cell differentiation. Here, we show that administration of B7.2-Ig fusion proteins to tumor-bearing mice induces tumor regression by promoting the differentiation of antitumor type 2 CD8(+) effector T cells along with IL-4 production. B7.2-Ig-mediated regression was not induced in IL-4(-/-) and STAT6(-/-) mice. However, it was elicited in IFN-gamma(-/-) and STAT4(-/-) mice. By contrast, IL-12-induced tumor regression occurred in IL-4(-/-) and STAT6(-/-) mice, but not in IFN-gamma(-/-) and STAT4(-/-) mice. Moreover, B7.2-Ig treatment was effective in a tumor model not responsive to IL-12. B7.2-Ig administration elicited elevated levels of IL-4 production. Tumor regression was predominantly mediated by CD8(+) T cells, although the induction of these effector cells required CD4(+) T cells. Tumor regression induced by CD8(+) T cells alone was inhibited by neutralizing the IL-4 produced during B7.2-Ig treatment. Thus, these results indicate that stimulation in vivo of CD28 with B7.2-Ig in tumor-bearing mice results in enhanced induction of antitumor type 2 CD8(+) T cells (Tc2) leading to Tc2-mediated tumor regression.     

TLR2-dependent induction of IL-10 and Foxp3+ CD25+ CD4+ regulatory T cells prevents effective anti-tumor immunity induced by Pam2 lipopeptides in vivo

16 S-[2,3-bis(palmitoyl)propyl]cysteine (Pam2) lipopeptides act as toll-like receptor (TLR)2/6 ligands and activate natural killer (NK) cells and dendritic cells (DCs) to produce inflammatory cytokines and cytotoxic NK activity in vitro. However, in this study, we found that systemic injection of Pam2 lipopeptides was not effective for the suppression of NK-sensitive B16 melanomas in vivo. When we investigated the immune suppressive mechanisms, systemic injection of Pam2 lipopeptides induced IL-10 in a TLR2-dependent manner. The Pam2 lipopeptides increased the frequencies of Foxp3(+)CD4(+) regulatory T (T reg) cells in a TLR2- and IL-10- dependent manner. The T reg cells from Pam2-lipopeptide injected mice maintained suppressor activity. Pam2 lipopeptides, plus the depletion of T reg with an anti-CD25 monoclonal antibody, improved tumor growth compared with Pam2 lipopeptides alone. In conclusion, our data suggested that systemic treatment of Pam2 lipopeptides promoted IL-10 production and T reg function, which suppressed the effective induction of anti-tumor immunity in vivo. It is necessary to develop an adjuvant that does not promote IL-10 and T reg function in vivo for the future establishment of an anti-cancer vaccine.