Cell Subsets In Mice Research Articles

Dynamic changes of DC and T cell subsets in mice during Echinococcus multilocularis infection.

The aim of this study was to determine the dynamic changes of dendritic cell (DC) pheno-types and T cell in response to Echinococcus multilocularis (Em) infection in BALB/c mice. Mice comprised the control and Em-infected group. At day 0, 2, 7, 30, 60, 90 and 120 after infection, the size of larval cysts, the phenotype of DC and Th in splenocytes and the expression of CD40, CD86, TLR2 and TLR4, on DCs sulfur were examined. The results show that after 60 days’ infection, larval cysts grow on the surface of liver, and they become larger over time. Compared with the control mice, MHC I and MHC II expressions on DC were significantly increased at day 7 (p < 0.05). At the same time, CD40, CD86, TLR2 and TLR4 increased rapidly, but after that they decreased gradually. At day 120, those markers were lower than in the control group. The ratio of CD4/CD8 was normal during 90 days of infection, while at day 120, a decline in CD4 T cell and increase in CD8 were foundleading to the inversion of the CD4/CD8 ratio. Our findings suggest that within the 120 days of Em infection, the major function of DC is to present antigens. Immune response is provided predominantly by Th1 cells, inducing host immune response against Em. However, after 120 days, DC matured and the function was suppressed. Furthermore, inversion of the CD4/CD8 ratio is beneficial to the growth of Em, thus favoring its immune evasion.

NK Cell Trafficking in Health and Autoimmunity:A Comprehensive Review

Natural killer (NK) cells represent an important subpopulation of lymphocytes that are distributed throughout the body. The development of NK cells primarily occurs in the bone marrow during adult life, involving several putative intermediate stages that finally result in functional maturation. At steady state, NK cell egress from the bone marrow to various peripheral areas is controlled by a network of adhesion molecules, including integrins, selectins, and chemokine receptors and their corresponding ligands. NK cells at different developmental stages express distinct repertoire of adhesion molecules and can therefore be recruited to different sites of the body, including lymphoid and non-lymphoid tissues, and NK cells undergo further differentiation driven by local microenvironmental signals, resulting in unique tissue-specific NK cells. Through their abilities of cytotoxicity and cytokine production, NK cells not only play key roles in the innate immune system, but also participate in shaping adaptive immune responses. On the basis of their heterogeneity in phenotype, function, and tissue distribution, NK cells can be further subdivided into distinct subsets. Under pathological conditions, such as in autoimmune, inflammatory, and infectious diseases, as local microenvironment changes, NK cell subsets would redistribute between tissues and organs and rapidly accumulate at the local pathological sites to exert their effector functions. Here, we describe the development and tissue distribution of NK cell subsets in mice and humans. We focus on the trafficking of NK cell subsets within the bone marrow and emigration into periphery at steady state, and molecular mechanisms involved in their trafficking in autoimmune diseases.

Resveratrol ameliorates high fat diet-induced fatty liver with alteration of Kupffer cell subset in mice. (P1106)

Abstract Background: We gave considerable attention to improve a high-fat diet (HFD)-induced fatty liver by Resveratrol from a standpoint of lifestyle disease prevention. Subject: To determine which protein is strongly regulated by Resveratrol in HFD-induced fatty liver, we investigated the modulation of liver protein and Kupffer cell subset in the HFD mice. Material and Methods: Mice (♂C57BL/6, 7 weeks) were fed HFD, HFD supplemented 0.2% Resveratrol or control diet for 8 weeks. Protein extraction from the liver was analyzed by LCMS-based proteomics. Immunostaining was performed using F4/80, CD68, CD11b and adipophilin (ADFP) Abs, and the Kupffer cell subset was analyzed by flow cytometry. TNF production by Kupffer cells was also measured. Results: Resveratrol decreased liver weights and hepatic triglyceride (TG) levels in the HFD mice without changing food intake, and improved fatty liver. Lipid metabolism was strongly down-regulated by Resveratrol. The most down-regulated protein was the ADFP that plays a role of TG storage in lipid droplets and macrophage. Resveratrol reduced lipid droplets and ADFP in the liver of HFD mice. Concurrently, Resveratrol suppressed increased CD68-CD11b+ Kupffer cell subset with suppressing their TNF production in the HFD mice. Conclusion: Resveratrol altered lipid metabolism, especially ADFP, in HFD mice, and suppressed TNF-producing CD11b+ Kupffer cells, thereby ameliorating fatty liver.

Farnesyltransferase inhibitors differentially affect cytokine secretion from CD4+ T cell subsets in mice (P1004)

Abstract Farnesyltransferase inhibitors (FTIs) have been shown to modulate alloreactive immune responses in mouse models of transplantation (A.E. Gaylo, et al., Transplant Immunology 20 (2009) 163-170) and graft versus host disease (A.-K. Hechinger, et al. Haematoligica 98 (2013) 31-40). FTIs can block cytokine secretion from T cells and this activity may explain the drug’s immunomodulatory mechanism. FTIs could modulate immune responses by blocking T cell cytokine secretion thereby affecting the differentiation of stimulated CD4+ T cells into Th1, Th2, or other effector subtypes. To test this hypothesis, we have examined the ability of FTIs to affect cytokine secreation from various T cell subsets and T cell polarization in mice during an allorejection response. Using qPCR we quantified cytokine gene expression in graft-draining lymph nodes (GDLNs) isolated from mice during the rejection of an MHC class II-mismatched skin allograft (bm12 donors to B6 recipients). Using ELISpot assays, we also measured the number of alloreactive Th1 (IFN-γ producing) and Th2 (IL-4-producing) cells in GDLNs. In vitro T cell polarization of polyclonally-stimulated CD4+ T cells isolated from spleen was measured using IFN-γ, IL-4, IL-17, and FoxP3 intracellular staining. We found that FTI treatment differentially affects cytokine secretion from the CD4+ T cell subsets studied. These effects lead to differences in Th1/Th2 cell polarization that could explain the immunomodulatory activity of FTIs.

CD39 modulates hematopoietic stem cell recruitment and promotes liver regeneration in mice and humans after partial hepatectomy.

To study molecular mechanisms involved in hematopoietic stem cell (HSC) mobilization after liver resection and determine impacts on liver regeneration. Extracellular nucleotide-mediated cell signaling has been shown to boost liver regeneration. Ectonucleotidases of the CD39 family are expressed by bone marrow-derived cells, and purinergic mechanisms might also impact mobilization and functions of HSC after liver injury. Partial hepatectomy was performed in C57BL/6 wild-type, Cd39 ectonucleotidase-null mice and in chimeric mice after transplantation of wild-type or Cd39-null bone marrow. Bone marrow-derived HSCs were purified by fluorescence-activated cell sorting and administered after hepatectomy. Chemotactic studies were performed to examine effects of purinergic receptor agonists and antagonists in vitro. Mobilization of human HSCs and expression of CD39 were examined and linked to the extent of resection and liver tests. Subsets of HSCs expressing Cd39 are preferentially mobilized after partial hepatectomy. Chemotactic responses of HSCs are increased by CD39-dependent adenosine triphosphate hydrolysis and adenosine signaling via A2A receptors in vitro. Mobilized Cd39 HSCs boost liver regeneration, potentially limiting interleukin 1β signaling. In clinical studies, mobilized human HSCs also express CD39 at high levels. Mobilization of HSCs correlates directly with the restoration of liver volume and function after partial hepatectomy. We demonstrate CD39 to be a novel HSC marker that defines a functionally distinct stem cell subset in mice and humans. HSCs are mobilized after liver resection, limit inflammation, and boost regeneration in a CD39-dependent manner. These observations have implications for monitoring and indicate future therapeutic avenues.

Epigenetic regulation of NK cell differentiation and effector functions

Upon maturation, natural killer (NK) cells acquire effector functions and regulatory receptors. New insights suggest a considerable functional heterogeneity and dynamic regulation of receptor expression in mature human NK cell subsets based on different developmental axes. Such processes include acquisition of lytic granules as well as regulation of cytokine production in response to exogenous cytokine stimulation or target cell interactions. One axis is regulated by expression of inhibitory receptors for self-MHC class I molecules, whereas other axes are less well defined but likely are driven by different activating receptor engagements or cytokines. Moreover, the recent identification of long-lived NK cell subsets in mice that are able to expand and respond rapidly following a secondary viral challenge suggest previously unappreciated plasticity in the programming of NK cell differentiation. Here, we review advances in our understanding of mature NK cell development and plasticity with regards to regulation of cellular function. Furthermore, we highlight some of the major questions that remain pertaining to the epigenetic changes that underlie the differentiation and functional specialization of NK cells and the regulation of their responses.

How Aging Affects T Lymphocyte-Mediated Immunity

EDITORIAL article Front. Immunol., 24 September 2013Sec. T Cell Biology Volume 4 - 2013 | https://doi.org/10.3389/fimmu.2013.00296

Distinct Drug Responses of CD8+CD4+ and CD8+CD4−leukemic Cell Subsets in Mice with Notch1-Induced T-ALL

AbstractAbstract 1504Leukemic cells are heterogeneous, so defining the differences of leukemic cell sub-populations in response to drug treatment may ultimately help design better strategies to maximize the efficacy of chemotherapeutic agents for patients. We have begun to study this issue with Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL) in mice. The Notch1 signaling pathway plays an essential role in cell growth and differentiation, and activated mutations in the Notch1 gene are frequently observed in human T-ALL. Over-expression of the intracellular Notch1 domain (ICN1) in murine hematopoietic cells is able to induce robust T-ALL in mice. In our current study, we examined T-ALL cells, specifically the CD8 and CD4 double positive (CD8+CD4+) and CD8 single positive (CD8+CD4−) cell sub-populations, for their distinct sensitivities to drug treatment.We first treated T-ALL mice with a cell-cycle nonspecific chemical drug, cyclophosphamide (CTX), or a mTORC1 inhibitor, rapamycin, when the overall T-ALL cell population rose to more than 10% in mouse peripheral blood. We found that CTX treatment effectively reduced the T-ALL cells and led to an increase in the ratio of CD8+CD4+ versus CD8+CD4− T-ALL, indicating that CD8+CD4− T-ALL cells were more sensitive to CTX treatment. In contrast, rapamycin had the opposite effect. When compared with mice treated with rapamycin or CTX alone, the combination of the two drugs led to a significant improvement for T-ALL bearing mice with respect to leukemia initiation, progress and overall survival. We then set out to investigate the mechanism of different sensitivities of CD8+CD4+ and CD8+CD4− T-ALL cells upon CTX or rapamycin treatments. We found that CD8+CD4+ and CD8+CD4−T-ALL cell groups responded similarly to CTX treatment after 12 hours of treatment. However, CD8+CD4−T-ALL cells continued to undergo apoptosis 24 hours after CTX treatment. The expression of the anti-apoptosis gene Bcl-2 in CD8+CD4+ T-ALL cells was significantly higher than that in CD8+CD4− T-ALL cells, while the expression of the pro-apoptosis genes Bax, p53 and Noxa in CD8+CD4+ T-ALL cells was lower than that of CD8+CD4−T-ALL cells after 24 hour CTX treatment. This suggests that CD8+CD4+ and CD8+CD4− T-ALL cells activate apoptosis differently after CTX treatment. Interestingly, rapamycin treatment did not affect apoptosis in the same manner as CTX treatment, but instead, it arrested more T-ALL cells in the G0 phase. More importantly, only CD8+CD4+, but not CD8+CD4−T-ALL cells were sensitive to rapamycin treatment in arresting cell cycle at different time points. The expression levels of CDK2 and CDK4 were significantly lower in CD8+CD4+ T-ALL cells when compared with CD8+CD4− T-ALL cells, while p27 expression in CD8+CD4+ T-ALL cells was higher than that of CD8+CD4− T-ALL cells after rapamycin treatment for 3 or 10 days. These data provide a molecular basis for the distinct apoptotic and cell cycle arrest responses of CD8+CD4+ and CD8+CD4− T-ALL cell subsets to CTX or rapamycin treatment.Taken together, our study documents previously unappreciated, yet distinct properties of CD8+CD4+ and CD8+CD4−Notch1-induced T-ALL cells in the context of specific drug treatment and suggests a therapeutic potential of combining CTX and rapamycin treatment for T-ALL patients. This strategy may also help design better chemotherapeutical regimes for other types of leukemia and cancer. Disclosures:No relevant conflicts of interest to declare.

Gas6/TAM signaling differentially modulates chronic fungal asthma with the expansion of myeloid regulatory cell subsets in mice (57.7)

Abstract Gas6, a Tyro3, Axl, Mertk (TAM) receptor ligand, is detected in a variety of diseases and has various roles. Herein, we show that Gas6 differentially modulates experimental fungal asthmatic response via the expansion and modulation of myeloid-derived regulatory cells (MDRCs). Aspergillus fumigatus-sensitized mice were challenged with live Aspergillus conidia and received approximately 2 μg (low) or 7 μg (high) of recombinant Gas6 via intranasal installation from days 14 to 28 after conidia challenge. In the low dose Gas6 group, significant airway hyperresponsiveness (AHR), airway remodeling, and whole lung IL-13 were observed compared with the control group. Although high dose Gas6 treatment significantly suppressed AHR and the whole lung levels of inflammatory cytokines compared with control, this treatment exacerbated airway remodeling. MDRCs have both disease enhancing and suppressing cells in asthma. Indeed, low dose Gas6 treatment increased the accumulation of CD11b+F4/80+Ly6C-Ly6G+ MDRC with pro-inflammatory properties into asthmatic lung whereas high dose Gas6 promoted the accumulation of immunosuppressive CD11b+F4/80+Ly6C+Ly6G- MDRC during chronic asthma. Anti-Axl Ab, but not anti-Mer Ab, treatment significantly suppressed not only AHR but also airway remodeling in asthmatic mice compared with IgG control asthmatic groups. Together, these results demonstrate that Gas6-TAM receptor interactions modulate fungal asthma, in part through effects on MDRCs.

Therapeutic effect of tolerogenic dendritic cells in established collagen-induced arthritis is associated with a reduction in Th17 responses.

Tolerogenic dendritic cells (DCs) are antigen-presenting cells with an immunosuppressive function. They are a promising immunotherapeutic tool for the attenuation of pathogenic T cell responses in autoimmune arthritis. The aims of this study were to determine the therapeutic action of tolerogenic DCs in a type II collagen-induced arthritis model and to investigate their effects on Th17 cells and other T cell subsets in mice with established arthritis. Tolerogenic DCs were generated by treating bone marrow-derived DCs with dexamethasone and vitamin D(3) during lipopolysaccharide-induced maturation. Mice with established arthritis received 3 intravenous injections of tolerogenic DCs, mature DCs, or saline. Arthritis severity was monitored for up to 4 weeks after treatment. Fluorescence-labeled tolerogenic DCs were used for in vivo trafficking studies. The in vivo effect of tolerogenic DCs on splenic T cell populations was determined by intracellular cytokine staining and flow cytometry. Tolerogenic DCs displayed a semi-mature phenotype, produced low levels of inflammatory cytokines, and exhibited low T cell stimulatory capacity. Upon intravenous injection into arthritic mice, tolerogenic DCs migrated to the spleen, liver, lung, feet, and draining lymph nodes. Treatment of arthritic mice with type II collagen-pulsed tolerogenic DCs, but not unpulsed tolerogenic DCs or mature DCs, significantly inhibited disease severity and progression. This improvement coincided with a significant decrease in the number of Th17 cells and an increase in the number of interleukin-10-producing CD4+ T cells, whereas tolerogenic DC treatment had no detectable effect on Th1 cells or interleukin-17-producing γ/δ T cells. Treatment with type II collagen-pulsed tolerogenic DCs decreases the proportion of Th17 cells in arthritic mice and simultaneously reduces the severity and progression of arthritis.

Effects of microwave ablation combined with intratumoral injection of dendritic cells on T cell subsets of mice bearing myeioma

Objective To study the effects of microwave ablation combined with intratumoral injection of dendritic cells (DC) on T cell subsets of mice with myeloma.Methods BALB/C mouse myeloma model was established subcutaneously.All mice were randomly divided into four groups:control group (B),microwave ablation group(B),microwave ablation+immature DC group(C),microwave ablation combined with mature DC group(D).T cell subsets of peripheral bloed in mice of each group were tested by flow cytomety at different time points(1st,3rd,5th,7th,9th,and 11th day after microwave ablation).Results The proportions of CD3~+,CD4~+ T lymphocytes and ratio of CD4~+/CD8~+ in group A were decreased gradually (P 0.05).The proportions of CD3~+,CD4~+ T lymphoeytes and ratio of CD4~+/CD8~+ in group C were significantly decreased at the 3rd,5th,and 7th day after microwave ablation.then gradually restored at the 9th,and 11th day.The proportions of CD3~+,CD4~+ T lymphocytes and ratio of CD4~+/CD8~+ in group D were significantly increased at the 3rd,5th,and 7th day after microwave ablation.then gradually restored at the 9th,and 11th day,and decreased at the 9th,and 11th day after microwave ablation.Conclusion Microwave ablation combined with intratumoral injection of mature DC can improve the anti-tumor immune response of tumor-bearing mice in approximately one week. Key words: Microwave; Dendritic cells; T cells

CD94 Defines Phenotypically and Functionally Distinct Mouse NK Cell Subsets

Understanding of heterogeneous NK subsets is important for the study of NK cell biology and development, and for the application of NK cell-based therapies in the treatment of disease. Here we demonstrate that the surface expression of CD94 can distinctively divide mouse NK cells into two approximately even CD94(low) and CD94(high) subsets in all tested organs and tissues. The CD94(high) NK subset has significantly greater capacity to proliferate, produce IFN-gamma, and lyse target cells than does the CD94(low) subset. The CD94(high) subset has exclusive expression of NKG2A/C/E, higher expression of CD117 and CD69, and lower expression of Ly49D (activating) and Ly49G2 (inhibitory). In vivo, purified mouse CD94(low) NK cells become CD94(high) NK cells, but not vice versa. Collectively, our data suggest that CD94 is an Ag that can be used to identify functionally distinct NK cell subsets in mice and could also be relevant to late-stage mouse NK cell development.

Imbalance of effector and regulatory T cells in the pathogenesis of experimental eosinophilic esophagitis (80.8)

Abstract RATIONALE: Eosinophilic esophagitis (EE) is an emerging disease and we previously demonstrated that adaptive T cell immunity is critical in the disease pathogenesis. However, the specific subsets of T cells that promote esophageal eosinophilia are not yet understood. Therefore, we focused our studies to examine the role of CD45RB T subsets, as the pathogenic and regulatory activity of T cells depends on the expression levels of CD45RB. METHODS: A flow cytometric analysis was performed to examine the frequency of CD45RBhigh and CD45RBlow T cell subsets in the mouse esophagus. A real time PCR analysis was performed to examine the pathogenic and anti-inflammatory characteristics of both subsets of T cells. In addition, we adoptively transferred these purified cell subsets in mice with experimental EE and examined their role in the disease pathogenesis. RESULTS: An increase of CD4+CD45RBhigh and a decrease of CD4+CD45RBlow cells were observed in mouse esophagus following the induction of experimental EE. A relatively high transcript level of IL-5 and IL-13 in CD4+CD45RBhigh and IL-10 and TGF-β1 in CD4+CD45RBlow spleen purified cells was observed. Further, our initial experimentation revealed that mice adoptively transferred with 4X105 CD4+CD45RBlow T cells/week were protected from the induction of esophageal eosinophilia compared to control mice that did not received adoptively transferred cells. Mice receiving CD4+CD45RBhigh T cell had no significant change in the number of eosinophils in the esophagus compared to control mice following allergen challenge. CONCLUSIONS: CD4+CD45RBlow T cells have a critical role in down-regulating allergen-induced eosinophil recruitment to the mouse esophagus.

CD4 +CD45RB Low T Cells Are Critical In Regulating Esophageal Eosinophilia In Mice

RATIONALE: Eosinophilic esophagitis (EE) is an emerging disease and we previously demonstrated that adaptive T cell immunity is critical in the disease pathogenesis. However, the specific subsets of T cells that promote esophageal eosinophilia are not yet understood. Therefore, we focused our studies to examine the role of CD45RB T subsets, as the pathogenic and regulatory activity of T cells depends on the expression levels of CD45RB. METHODS: A flow cytometric analysis was performed to examine the frequency of CD45RBhigh and CD45RBlow T cell subsets in the mouse esophagus. A real time PCR analysis was performed to examine the pathogenic and anti-inflammatory characteristics of both subsets of T cells. In addition, we adoptively transferred these purified cell subsets in mice with experimental EE and examined their role in the disease pathogenesis. RESULTS: An increase of CD4+CD45RBhigh and a decrease of CD4+CD45RBlow cells were observed in mouse esophagus following the induction of experimental EE. A relatively high transcript level of IL-5 and IL-13 in CD4+CD45RBhigh and IL-10 and TGF-β1 in CD4+CD45RBlow spleen purified cells was observed. Further, our initial experimentation revealed that mice adoptively transferred with 4X105 CD4+CD45RBlow T cells/week were protected from the induction of esophageal eosinophilia compared to control mice that did not received adoptively transferred cells. Mice receiving CD4+CD45RBhigh T cell had no significant change in the number of eosinophils in the esophagus compared to control mice following allergen challenge. CONCLUSIONS: CD4+CD45RBlow T cells have a critical role in down-regulating allergen-induced eosinophil recruitment to the mouse esophagus.

Unidirectional development of CD8+ central memory T cells into protective Listeria‐specific effector memory T cells

Three distinct subsets of antigen-experienced CD8(+) T cells have been identified so far: short-living effector T cells (T(EC)) and two long-living subsets, described as central (T(CM)) and effector memory (T(EM)) T cells. The lineage relationships of these subpopulations as well as their involvement in protection have not yet been conclusively determined. We recently described a novel marker combination (CD127 and CD62L) to identify all three major CD8(+) T cell subsets in mice infected with Listeria monocytogenes (L.m.). Extensive lineage relationship analyses on highly purified subpopulations after in vitro and in vivo stimulation demonstrated that T(CM) can develop into T(EM) or T(EC), whereas T(EM) can only progress to T(EC) cells. Short-living T(EC) never regained a T(EM) or T(CM) phenotype. These data strongly suggest a hierarchical and unidirectional order of developmental stages. In vivo priming protocols that preferentially induced one of the different CD8(+) T cell subsets demonstrated that predominance of T(EM) (CD40 stimulation) correlated best with effective protection against L.m., whereas generation of neither T(CM) (by immunization with heat-killed L.m.) nor T(EC) (by systemic co-administration of CpG during primary infection) conferred substantial long-term protective immunity. These findings have important implications for the design of more effective T cell-based vaccines.

Mechanisms by Which NK T Cells Become the Predominant T Cell Subset in Mice after Irradiation.

Previously, we found that the percentage of NK T cells among all T cells in the spleen of mice treated with fractionated irradiation to the lymphoid tissues (Total lymphoid irradiation; TLI) with a total dose of 4,080 cGy increased markedly due to greater reduction in the absolute number of non-NK T cells as compared to NK T cells. The underlying mechanisms of the change in the T cell subsets after irradiation remained to be established. In the current study, C57BL/6 mice were given escalating single doses of 240, 1,000, 2,000 and 3,000 cGy total body irradiation (TBI). Splenocytes were harvested at 4 or 24 hours after irradiation, and the percentage and absolute number of NK T and non-NK T cells was determined. At the same time, the intracellular level of the anti-apoptotic protein, Bcl-2 was assayed by flow cytometry. In some studies, the turnover rate of NK T cells and non-NK T cells was examined by injection of BrdU and intracellular staining. At 4 hours after all doses of irradiation, neither the NK T nor non-NK T cell subset had a significant change in percentage or absolute number as compared to untreated controls. However, at 24 hours the percentage of NK T cells among all T cells had progressively increased with increased doses of TBI from 3% in the untreated controls to 65% in mice given 3,000 cGy. Whereas the absolute number of non-NK T cells decreased at least 1000 fold, the absolute number of NK T cells decreased approximately 50 fold after 3,000 cGy. The BrdU incorporation of NK T cells from irradiated mice was markedly reduced as compared to untreated mice, and was similar to that of non NK T cells in these irradiated mice. 8–12% of NK T cells and non NK T cells in untreated mice expressed a high level Bcl-2. As the dose of TBI increased progressively, the percentage of Bcl-2hi cells increased progressively to 89% amongst NK T cells and 70% amongst non-NK T cells. At each irradiation dose, the percentage of Bcl-2hi cells amongst NK T cells was higher than amongst non-NK T cells. There were 40×103 Bcl-2hi NK T cell and 10×103 Bcl-2hi non-NK T cells surviving per spleen at 24 hours after 3000 cGy TBI. The absolute number of Bcl-2hi NK T cells decreased by about two fold while the absolute number of Bcl-2hi non-NK T cells decreased by about 100 fold. These results indicate that the increased percentage of NK T cells amongst all T cells after irradiation is due to greater radioresistance rather than to more rapid replenishment of NK T cells as compared to non-NK T cells. We are investigating whether Bcl-2 plays a critical role in the extraordinary radioresistance of the NK T cells.

Association of B-1 B Cells with Follicular Dendritic Cells in Spleen

Although CD5(+) B-1 B cells have been recognized as an infrequent B cell subset in mice for many years, attempts to identify their histologic location in normal mouse spleen have proven difficult due to both their paucity and low level expression of CD5. In this study we have studied V(H)11/D(H)/J(H) gene-targeted mice, V(H)11t, that develop elevated numbers of CD5(+) V(H)11/V(k)9 B cells with an anti-phosphatidylcholine (anti-PtC) autoreactive specificity, allowing B-1 B cell detection by anti-PtC Id-specific Abs in spleen section staining. Using this approach we found that anti-PtC B-1 cells first appear within the white pulp in neonates, expand in association with follicular dendritic cells (FDC), and localize more centrally than other (non-B-1) IgD(high) follicular B cells in adults. Among neonatal B cells, CD5(+) B-1 cells in both normal and V(H)11t mouse spleen and peritoneal cavity express the highest levels of CXCR5, which is important for FDC development. Injection of purified spleen or peritoneal B-1 cells into RAG knockout mice resulted in B-1 cell follicle formation in spleen, inducing FDC development and plasma cell generation. These results indicate that B-1 B cells are the first B cells to express fully mature levels of CXCR5, thereby promoting the development of FDC.

Inhibitors of TLR-9 Act on Multiple Cell Subsets in Mouse and Man In Vitro and Prevent Death In Vivo from Systemic Inflammation

In parallel with the discovery of the immunostimulatory activities of CpG-containing oligodeoxynucleotides, several groups have reported specific DNA sequences that could inhibit activation by CpG-containing oligodeoxynucleotides in mouse models. We show that these inhibitory sequences, termed IRS, inhibit TLR-9-mediated activation in human as well as mouse cells. This inhibitory activity includes proliferation and IL-6 production by B cells, and IFN-alpha and IL-12 production by plasmacytoid dendritic cells. Our studies of multiple cell types in both mice and humans show the optimal IRS to contain a GGGG motif within the sequence, and the activity to require a phosphorothioate backbone. Although the GGGG motif readily itself leads to formation of a tetrameric oligodeoxynucleotide structure, inhibitory activity resides exclusively in the single-stranded form. When coinjected with a CpG oligodeoxynucleotide in vivo, IRS were shown to inhibit inflammation through a reduction in serum cytokine responses. IRS do not need to be injected at the same site to inhibit, demonstrating that rapid, systemic inhibition of TLR-9 can be readily achieved. IRS can also inhibit a complex pathological response to ISS, as shown by protection from death after massive systemic inflammation induced by a CpG-containing oligodeoxynucleotides.

Secretoglobins SCGB3A1 and SCGB3A2 define secretory cell subsets in mouse and human airways.

Clara cell secretory protein (CCSP) is expressed abundantly within the conducting airway epithelium and is thought to have immunoregulatory functions. Differences in the localization of CCSP between mouse and human airways led us to hypothesize that functional homologues of CCSP may compensate for the lack of CCSP expression in proximal airway locations. We previously identified an expressed sequence tag (W82219) whose expression is induced within Clara cells of CCSP knockout mice. Expressed sequence tag W82219 is distantly related to CCSP and represents a member of a new subfamily of secretoglobins (MmSCGB3A2). Another member of the mouse SCGB3 family (MmSCGB3A1) as well as human orthologues (HsSCGB3A1 and HsSCGB3A2) that possess structural homology to CCSP were identified, suggesting they may share common functional properties. SCGB3A1 messenger RNA localizes to a subset of SCGB3A2-expressing cells within bronchi of both mouse and neonatal human lungs. CCSP, SCGB3A1, and SCGB3A2 were decreased in airways of neonates with bronchopulmonary dysplasia and in mice after airway injury. We conclude that secretory cells of the conducting airway epithelium express distinct members of the secretoglobin family in a partially overlapping fashion. Altered expression of secretoglobins in airway disease may contribute to immunoregulatory perturbations commonly seen in chronic airway disease.

Effects of administration of progenipoietin 1, Flt-3 ligand, granulocyte colony-stimulating factor, and pegylated granulocyte-macrophage colony-stimulating factor on dendritic cell subsets in mice

AbstractWe studied the effects of administration of several cytokines, including progenipoietin-1 (ProGP-1), Flt-3 ligand (FL), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor in a pegylated form (pGM-CSF), on dendritic cell (DC) populations in mouse spleen. ProGP-1 produced the most striking increase in overall DC numbers, apparently more than its constituent FL and G-CSF components. However, the expansion in DC numbers was strongly subpopulation selective, with ProGP-1 and FL producing selective expansion of CD8+ DCs, whereas pGM-CSF produced selective expansion of CD8− DCs. Surprising differences were observed between the effects of murine and human recombinant FL preparations on murine DCs. Many of the biologic functions of the DC subpopulations expanded by cytokines remained intact, including the capacity of the ProGP-1– and FL-expanded CD8+ DCs to produce the T-helper-1–biasing cytokine interleukin 12 (IL-12). However, the expanded DCs from all but G-CSF–treated mice were deficient in the ability to make interferon γ, and the CD8+ DCs produced with pGM-CSF treatment had an abrogated capacity to form bioactive IL-12. Such selective expansion of DC populations and alterations in their cytokine-secretion capacity have implications for clinical use of the studied cytokines in immune modulation.