Subpopulations Of Lymphoid Cells Research Articles

Monocyte infiltration is an independent positive prognostic biomarker in vulvar squamous cell carcinoma

BackgroundVulvar squamous cell carcinoma (VSCC) arises after an HPV infection or the mutation of p53 or other driver genes and is treated by mutilating surgery and/or (chemo) radiation, with limited success and high morbidity. In-depth information on the immunological make up of VSCC is pivotal to assess whether immunotherapy may form an alternative treatment.MethodsA total of 104 patient samples, comprising healthy vulva (n = 27) and VSCC (n = 77), were analyzed. Multispectral immunofluorescence (15 markers) was used to study both the myeloid and lymphoid immune cell composition, and this was linked to differences in transcriptomics (NanoString nCounter, 1258 genes) and in survival (Kaplan–Meier analyses).ResultsHealthy vulva and VSCC are both well infiltrated but with different subpopulations of lymphoid and myeloid cells. In contrast to the lymphoid cell infiltrate, the density and composition of the myeloid cell infiltrate strongly differed per VSCC molecular subtype. A relative strong infiltration with epithelial monocytes (HLADR−CD11c−CD14+CD68−CD163−CD33−) was prognostic for improved survival, independent of T cell infiltration, disease stage or molecular subtype. A strong infiltration with T cells and/or monocytes was associated with drastic superior survival: 5-year survival > 90% when either one is high, versus 40% when both are low (p < 0.001).ConclusionA hot myeloid and/or lymphoid infiltrate predicts excellent survival in VSCC. Based on the response of similarly high-infiltrated other tumor types, we have started to explore the potential of neoadjuvant checkpoint blockade in VSCC.

A diversity of novel type-2 innate lymphoid cell subpopulations revealed during tumour expansion

Type 2 innate lymphoid cells (ILC2s) perform vital functions in orchestrating humoral immune responses, facilitating tissue remodelling, and ensuring tissue homeostasis. Additionally, in a role that has garnered considerably less attention, ILC2s can also enhance Th1-related cytolytic T lymphocyte immune responses against tumours. Studies have thus far generally failed to address the mystery of how one ILC2 cell-type can participate in a multiplicity of functions. Here we utilized single cell RNA sequencing analysis to create the first comprehensive atlas of naïve and tumour-associated lung ILC2s and discover multiple unique subtypes of ILC2s equipped with developmental gene programs that become skewed during tumour expansion favouring inflammation, antigen processing, immunological memory and Th1-related anti-tumour CTL responses. The discovery of these new subtypes of ILC2s challenges current paradigms of ILC2 biology and provides an explanation for their diversity of function.

Plasticity between type 2 innate lymphoid cell subsets and amphiregulin expression regulates epithelial repair in biliary atresia.

Although a dysregulated type 1 immune response is integral to the pathogenesis of biliary atresia, studies in both humans and mice have uncovered a type 2 response, primarily driven by type 2 innate lymphoid cells. In nonhepatic tissues, natural type 2 innate lymphoid cell (nILC2s) regulate epithelial proliferation and tissue repair, whereas inflammatory ILC2s (iIlC2s) drive tissue inflammation and injury. The aim of this study is to determine the mechanisms used by type 2 innate lymphoid cell (ILC2) subpopulations to regulate biliary epithelial response to an injury. Using Spearman correlation analysis, nILC2 transcripts, but not those of iILC2s, are positively associated with cholangiocyte abundance in biliary atresia patients at the time of diagnosis. nILC2s are identified in the mouse liver through flow cytometry. They undergo expansion and increase amphiregulin production after IL-33 administration. This drives epithelial proliferation dependent on the IL-13/IL-4Rα/STAT6 pathway as determined by decreased nILC2s and reduced epithelial proliferation in knockout strains. The addition of IL-2 promotes inter-lineage plasticity towards a nILC2 phenotype. In experimental biliary atresia induced by rotavirus, this pathway promotes epithelial repair and tissue regeneration. The genetic loss or molecular inhibition of any part of this circuit switches nILC2s to inflammatory type 2 innate lymphoid cell-like, resulting in decreased amphiregulin production, decreased epithelial proliferation, and the full phenotype of experimental biliary atresia. These findings identify a key function of the IL-13/IL-4Rα/STAT6 pathway in ILC2 plasticity and an alternate circuit driven by IL-2 to promote nILC2 stability and amphiregulin expression. This pathway induces epithelial homeostasis and repair in experimental biliary atresia.

EXPRESSION PATTERNS OF CD90, CD117, CD123, AND CD147 IN MATURE B CELL NEOPLASMS

Summary. Aim: to analyze the expression patterns of CD90, CD117, CD123, and CD147 on cells of the mature B-cell neoplasms, in particular marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL) and the expression of CD147 on chronic lymphocytic leukemia (CLL) cells. Object and methods: the expression patterns of the surface antigens on the transformed B cells were analyzed in the peripheral blood samples of the MZL, MCL, and CLL patients (34 patients in total) by the flow cytometry using the monoclonal antibodies. Results: at least one of the antigens – CD90, CD117, or CD123 was expressed in 20.0–33.3% of MCL cases and 27.2–54.5% of MZL cases at the level ≥ 1% of the cells in the subpopulation of CD45-positive lymphoid cells. One MCL case demonstrated the high content of CD117-positive cells (31.0%) accompanied by CD90 expression on 3.8% of cells. The high content of the CD147-positive cells was found not only in MZL and MCL but also in CLL. Conclusion: a small fraction of cells of lymphoid neoplasms expresses several antigens of the leukemic stem cells and cells of the myeloid lineage. The ubiquitous pattern of CD147 expression on cells of these neoplasms has been demonstrated. The diagnostic and prognostic significance of the expression of the studied antigens remains unknown necessitating further clinical studies of the clinical features of these diseases taking into account the expression of the atypical markers.

Imbalance of circulating innate lymphoid cell subpopulations in patients with chronic kidney disease

Innate lymphoid cells (ILCs) are a newly identified heterogeneous family of innate immune cells. We conducted this study to investigate the frequency of circulating ILC subsets in various chronic kidney diseases (CKD). In DN, the proportion of total ILCs and certain ILC subgroups increased significantly. Positive correlations between proportion of total ILCs, ILC1s and body mass index, glycated hemoglobin were observed in DN. In LN, a significantly increased proportion of ILC1s was found in parallel with a reduced proportion of ILC2s. The proportions of total ILCs and ILC1s were correlated with WBC count and the level of C3. In all enrolled patients, the proportion of total ILCs and ILC1s was significantly correlated with the levels of ACR and GFR. In the present study, the proportion of circulating ILC subsets increased significantly in various types of CKD and correlated with clinico-pathological features, which suggests a possible role for ILCs in CKD.

Abstract 513: Simultaneous monitoring of immune cell subsets and minimal residual disease in bone marrow aspirate using full-spectrum flow cytometry

Abstract Background: Treatment options for multiple myeloma (MM) patients have expanded and improved outcomes. Autologous stem cell transplantation, immunomodulatory drugs (IMiDs), monoclonal antibodies, and CAR-T cell therapy have direct and indirect immune modulating effects that favor patient responses. However, the majority of patients still relapse due to minimal residual disease (MRD). Current flow cytometry methods that monitor MRD and bone marrow immune cell populations require large cell numbers to achieve assay sensitivity. New approaches are needed to better capture high-resolution data given the limited recovery of bone marrow specimens. Here, we describe the simultaneous monitoring of immune cell subsets in the bone marrow and MRD using a single antibody panel and the Cytek Aurora full-spectrum flow cytometry platform. In a Phase 1/2 study to evaluate an off-the-shelf, allogeneic human placental-derived NK cell product in newly diagnosed MM, this method enables correlative analysis of MRD state with immune profiling. Methods: Bone marrow aspirate (BMA) from 5 patients with newly diagnosed MM following induction therapy were collected for MRD assessment in parallel using next-generation sequencing (NGS) and MRD evaluation by flow cytometry. BMA was lysed of red blood cells and 2x107 cells were stained using TCRγδ, CD16, CD8, CD33, CD117, CD11c, CD3, CD19, CD1c, CD45, CD38, CD14, HLA-DR, CD138, CD141, CD34, CD25, CRTH2, CD56, CD303, CD4, and CD127. Cells were acquired on a Cytek Aurora 4 laser system followed by data analysis using FlowJo software. MRD threshold of 10-5 was used for positivity for both modalities of testing. Results: The 22-parameter panel resolved the majority of immune cell subpopulations present in the bone marrow and peripheral blood including granulocytes, B cells, plasma cells, monocytes, MDSCs, plasmacytoid and classical dendritic cells, NK cells, and T cells (CD4, CD8, NKT, γδT, Treg). Total plasma cells were identified based upon CD38+CD138+ co-staining. Myeloma cells could be identified based on aberrant expression of CD45, CD19, CD56, and CD117. Of the five patients evaluated, one patient was MRD negative by NGS and flow cytometry with no detectable cells. One patient was MRD negative by NGS (0.7x10-5) but positive by flow cytometry (1.53x10-5). The three remaining patients were positive by NGS (6.32x10-4, 7.51x10-4, and 4.78x10-4) and flow cytometry (2.46x10-4, 5.37x10-4, and 9.74x10-4). Conclusion: Utilizing full-spectrum flow cytometry provides streamlined monitoring of MRD while capturing BMA-associated immune cell populations. Compared to validated measures of MRD testing by NGS, our flow cytometry approach provides comparable results while profiling myeloid and lymphoid cell subpopulations, which enables correlative analyses of underlying pathophysiological activities. Citation Format: William van der Touw, Bhavani Stout, James McCauley, Tanel Mahlakõiv, Robert Hariri, Xiaokui Zhang. Simultaneous monitoring of immune cell subsets and minimal residual disease in bone marrow aspirate using full-spectrum flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 513.

Decreased bone marrow regulatory innate lymphoid cells show a distinctive miRNA profiling in aplastic anemia

ABSTRACT Objectives A new regulatory subpopulation of innate lymphoid cells (ILCs), regulatory innate lymphoid cells (ILCregs), has been identified with both innate lymphoid cells and regulatory cells characteristics. The purpose of this study is to explore ILCregs and its associated miRNAs in patients with aplastic anemia (AA) by evaluating ILCregs frequency, associated miRNA quantification, and their significance. Methods Using 4 color combinations of surface and intracellular antibody staining, the CD45+Lin−CD127+IL-10+ ILCregs from 30 healthy donors and 30 patients newly diagnosed with AA were measured by flow cytometry. Bone marrow cells were studied by next-generation sequence miRNAs quantification. Results Our results showed that the frequency of ILCregs in bone marrow cells from healthy donors (HD) and AA patients were 0.703 ± 0.941 and 0.171 ± 0.233%, respectively. The frequencies of ILCregs in AA patients were significantly lower than that in HD (p <0.05). miRNA detection results showed different expression patterns in the AA patient group comparing with HD. Comparing with HD, there were 52 miRNAs up-regulated and 130 miRNAs down-regulated from AA patients. Analysis of miRNAs from ILCregs associated genes demonstrated different miRNAs expression patterns between HD and AA patient. Conclusion The present study demonstrated the deficiency of ILCregs and differential expression pattern of ILCregs gene-related miRNA in patients with AA. Further studies need to be done to explore the clinical significance of ILCregs and related miRNAs in patients with AA with large samples size and clinical follow-up.

IMMUNOLOGICAL PARAMETERS OF BONE MARROW IN NON-SMALL CELL LUNG CANCER

Introduction Generation of most immunocompetent cells takes place in bone marrow Bone marrow. As well, bone marrow is a peripheral lymphoid organ where antitumor effector cells and memory cells are present. The aim of the work is to estimate peripheral lymphoid cell subpopulations in bone marrow of lung cancer patients. Materials and methods Study has been done in 68 pts with lung cancer: squamous cell cancer (n = 28), adenocarcinoma (n = 38), other forms (n = 2). In all cases standard diagnostic and staging procedures were performed, as well as morphological (myelogram) and immunological study of bone marrow lymphocyte subpopulations. Multicolor Flow cytomtry was used for study of bone marrow lymphocyte populations. We studied T-cells and its subpopulations, B-cells, NK-cells, perforin-positive cells, and CD27-positive cells. Results Squamous cell lung cancer was characterized by higher content of bone marrow mature T-cells (CD3), and CD8 lymphocytes. More typical for adenocarcinoma was mature B-cell reaction (CD20). Effector (perforin-positive) populations of lymphocytes also were related to histological type of cancer: for adenocarcinoma presence of CD4-positive cytotoxic lymphocytes and CD27-expression on effector cells. Perforin-containing lymphoid cells were in opposite correlation to erythrocaryocytes. Conclusion Subpopulational lymphocyte content of bone marrow is related to histological variant of cancer and erythropoiesis in lung cancer patients.

Acute cellular rejection in small-bowel transplantation impairs NCR+ innate lymphoid cell subpopulation 3/interleukin 22 axis

Acute cellular rejection (ACR) remains as one of the main causes of graft loss and death in intestinal transplant (ITx) patients. ACR promotes intestinal injury, disruption of the mucosal barrier, bacterial translocation, and organ dysfunction. As epithelial regeneration is critical in reversing these consequences, the functional axis between the innate lymphoid cell subpopulation 3 (ILC3) and interleukin 22 plays an essential role in that process. Natural-cytotoxic-receptor–positive (NCR+) ILC3 cells have been demonstrated to induce intestinal-stem-cell proliferation along with an IL-22–dependent expansion of that population in several intestinal pathologies, though thus far not after ITx. Therefore, we intended to determine the impact of chronic immunosuppression and ACR on ILC3 cells and interleukin-22 (IL-22) production in the lamina propria after that intervention. Materials and methodsWe compared biopsies from healthy volunteers with biopsies from ITx recipients without or with mild-to-moderate ACR, using flow cytometry and the quantitative-PCR. ResultsNCR+ ILC3 cells were found to be unaffected by immunosuppression at different time points posttransplant when patients did not experience ACR, but were diminished upon the occurrence of ACR independently of the post-ITx time. Moreover, IL-22–expression levels were notably reduced in ACR. ConclusionThe NCR+-ILC3/IL-22 axis is impaired during ACR contributing to a delay in or lack of a complete and efficient epithelial regeneration. Thus, our findings reveal that IL-22 analogues could potentially be used as a new complementary therapeutic approach, in conjunction with immunosuppressant drugs, in order to promote mucosal regeneration upon ACR.

Dynamic changes in peripheral type II innate lymphoid cell (ILC2) subpopulation and its clinical significance in children with hay fever during the pollen season

Objective To analyze the dynamic changes in the expression and function of peripheral type Ⅱ innate lymphoid cell (ILC2) subpopulation and the activity of signal transducers and activators of transcription (STAT6) in children with hay fever during pollen season. Methods A total of 10 patients with hay fever, 10 patients with house dust mite (HDM)-sensitized asthma and 12 healthy controls (HC) were enrolled in this study. Changes in peripheral ILC2 and the intracellular expression of Th2-related cytokines were detected by flow cytometry during and outside the pollen season. Peripheral Lin- cell population was isolated from each group and cultured with the presence of IL-25 or IL-33 for 7 d. The concentrations of IL-5 and IL-13 in culture supernatants were measured by ELISA. Expression of phospho-STAT6 at protein level was quantified by Western blot. Results Within the pollen season, the percentage of peripheral ILC2 cells was significantly higher in children with hay fever [(23.09±7.86)%] than in children with HDM-sensitized asthma [(6.84±3.85)%, P<0.05] and healthy children[(1.69±0.87)%, P<0.05]. In the non-pollen season, the peripheral ILC2 cells in children with hay fever presented a decreasing trend [(11.30±2.45)%], but was still higher than that in HDM-sensitized asthmatics [(3.76±1.96)%, P<0.05] and HC [(1.32±0.91)%, P<0.05] at the same time point. Moreover, peripheral IL-13+ ILC2 cells in children with hay fever [(6.94±3.16)% vs(4.17±1.98)%, P<0.05] and in HDM-sensitized asthmatics [(1.89±0.70)% vs(1.44±0.55)%, P<0.05] during the pollen season were significantly higher than those in the non-pollen season. After the in vitro stimulation with IL25 or IL-33, the levels of IL-5 and IL-13 in culture supernatants were both increased in children with hay fever and HDM-sensitized asthmatics, and a synergistic action was observed when IL25 and IL-33 were used in combination. Meanwhile, the protein level of phospho-STAT4 in Lin- cells was significantly up-regulated in the hay fever group after stimulation with IL25 and IL-33. Conclusions During the pollen season, the abnormal number and function of ILC2 subpopulation in children with hay fever might be another cause of the occurrence of clinical symptoms in a short period of time or acute exacerbation. Key words: Hay fever; House dust mite; Artemisia; Signal transducer and activator of transcription 6; Type Ⅱ innate lymphocyte cells

Imbalance of Circulating Innate Lymphoid Cell Subpopulations in Patients With Septic Shock.

Background: Septic shock, a major cause of death in critical care, is the clinical translation of a cytokine storm in response to infection. It can be complicated by sepsis-induced immunosuppression, exemplified by blood lymphopenia, an excess of circulating Treg lymphocytes, and decreased HLA-DR expression on circulating monocytes. Such immunosuppression is associated with secondary infections, and higher mortality. The effect of these biological modifications on circulating innate lymphoid cells (ILCs) has been little studied.Methods: We prospectively enrolled patients with septic shock (Sepsis-3 definition) in the intensive care unit (ICU) of Timone CHU Hospital. ICU controls (trauma, cardiac arrest, neurological dysfunction) were recruited at the same time (NCT03297203). We performed immunophenotyping of adaptive lymphocytes (CD3+ T cells, CD19+ B cells, CD4+CD25+FoxP3+ Treg lymphocytes), ILCs (CD3−CD56+ NK cells and helper ILCs – ILC1, ILC2, and ILC3), and monocytes by flow cytometry on fresh blood samples collected between 24 and 72 h after admission.Results: We investigated adaptive and innate circulating lymphoid cells in the peripheral blood of 18 patients in septic shock, 15 ICU controls, and 30 healthy subjects. As expected, the peripheral blood lymphocytes of all ICU patients showed lymphopenia, which was not specific to sepsis, whereas those of the healthy volunteers did not. Circulating CD3+ T cells and CD3−CD56+ NK cells were mainly concerned. There was a tendency toward fewer Treg lymphocytes and lower HLA-DR expression on monocytes in ICU patients with sepsis. Although the ILC1 count was higher in septic patients than healthy subjects, ILC2, and ILC3 counts were lower in both ICU groups. However, ILC3s within the total ILCs were overrepresented in patients with septic shock. The depression of immune responses has been correlated with the occurrence of secondary infections. We did not find any differences in ILC distribution according to this criterion.Conclusion: All ICU patients exhibit lymphopenia, regardless of the nature (septic or sterile) of the initial medical condition. Specific distribution of circulating ILCs, with an excess of ILC1, and a lack of ILC3, may characterize septic shock during the first 3 days of the disease.

C-Kit-positive ILC2s exhibit an ILC3-like signature that may contribute to IL-17-mediated pathologies.

Here we identify a group 2 innate lymphoid cell (ILC2) subpopulation that can convert into interleukin-17 (IL-17)-producing NKp44- ILC3-like cells. c-Kit and CCR6 define this ILC2 subpopulation that exhibits ILC3 features, including RORγt, enabling the conversion into IL-17-producing cells in response to IL-1β and IL-23. We also report a role for transforming growth factor-β in promoting the conversion of c-Kit- ILC2s into RORγt-expressing cells by inducing the upregulation of IL23R, CCR6 and KIT messenger RNA in these cells. This switch was dependent on RORγt and the downregulation of GATA-3. IL-4 was able to reverse this event, supporting a role for this cytokine in maintaining ILC2 identity. Notably, this plasticity has physiological relevance because a subset of RORγt+ ILC2s express the skin-homing receptor CCR10, and the frequencies of IL-17-producing ILC3s are increased at the expense of ILC2s within the lesional skin of patients with psoriasis.

698 – Distinctive Subpopulation of Innate Lymphoid Cells Effluxed from Intestinal Mucosa by Different Intestinal Inflammations

698 – Distinctive Subpopulation of Innate Lymphoid Cells Effluxed from Intestinal Mucosa by Different Intestinal Inflammations

Implementation of Mass Cytometry for Immunoprofiling of Patients with Solid Tumors.

Monitoring immune responses to solid cancers may be a better prognostic tool than conventional staging criteria, and it can also serve as an important criterion for the selection of individualized therapy. Multiparametric phenotyping by mass cytometry extended possibilities for immunoprofiling. However, careful optimization of each step of such method is necessary for obtaining reliable results. Also, with respect to procedure length and costs, sample preparation, staining, and storage should be optimized. Here, we designed a panel of 31 antibodies which allows for identification of several subpopulations of lymphoid and myeloid cells in a solid tumor and peripheral blood simultaneously. For sample preparation, disaggregation of tumor tissue with two different collagenases combined with DNase I was compared, and removal of dead or tumor cells by magnetic separation was evaluated. Two possible procedures of barcoding for single-tube staining of several samples were examined. While the palladium-based barcoding affected the stability of several antigens, the staining with two differently labeled CD45 antibodies was suitable for cells isolated from a patient's blood and tumor. The storage of samples in the intercalation solution for up to two weeks did not influence results of the analysis, which allowed the measurement of samples collected within this interval on the same day. This procedure optimized on samples from patients with head and neck squamous cell carcinoma enabled identification of various immune cells including rare subpopulations.

Lack of Adipocytes Alters Hematopoiesis in Lipodystrophic Mice.

Adult hematopoiesis takes place in the perivascular zone of the bone cavity, where endothelial cells, mesenchymal stromal/stem cells and their derivatives such as osteoblasts are key components of bone marrow (BM) niches. Defining the contribution of BM adipocytes to the hematopoietic stem cell niche remains controversial. While an excess of medullar adiposity is generally considered deleterious for hematopoiesis, an active role for adipocytes in shaping the niche has also been proposed. We thus investigated the consequences of total adipocyte deletion, including in the BM niche, on adult hematopoiesis using mice carrying a constitutive deletion of the gene coding for the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ). We show that PpargΔ/Δ lipodystrophic mice exhibit severe extramedullary hematopoiesis (EMH), which we found to be non-cell autonomous, as it is reproduced when wild-type donor BM cells are transferred into PpargΔ/Δ recipients. This phenotype is not due to a specific alteration linked to Pparg deletion, such as chronic inflammation, since it is also found in AZIPtg/+ mice, another lipodystrophic mouse model with normal PPARγ expression, that display only very moderate levels of inflammation. In both models, the lack of adipocytes alters subpopulations of both myeloid and lymphoid cells. The CXCL12/CXCR4 axis in the BM is also dysregulated in an adipocyte deprived environment supporting the hypothesis that adipocytes are required for normal hematopoietic stem cell mobilization or retention. Altogether, these data suggest an important role for adipocytes, and possibly for the molecular interactions they provide within the BM, in maintaining the appropriate microenvironment for hematopoietic homeostasis.

IL-7-dependent maintenance of ILC3s is required for normal entry of lymphocytes into lymph nodes.

IL-7 is essential for the development and homeostasis of T and B lymphocytes and is critical for neonatal lymph node organogenesis because Il7-/- mice lack normal lymph nodes. Whether IL-7 is a continued requirement for normal lymph node structure and function is unknown. To address this, we ablated IL-7 function in normal adult hosts. Either inducible Il7 gene deletion or IL-7R blockade in adults resulted in a rapid loss of lymph node cellularity and a corresponding defect in lymphocyte entry into lymph nodes. Although stromal and dendritic cell components of lymph nodes were present in normal numbers and representation, innate lymphoid cell (ILC) subpopulations were substantially decreased after IL-7 ablation. Testing lymphocyte homing in bone marrow chimeras reconstituted with Rorc-/- bone marrow confirmed that ILC3s in lymph nodes are required for normal lymphocyte homing. Collectively, our data suggest that maintenance of intact lymph nodes relies on IL-7-dependent maintenance of ILC3 cells.

Experimental asthma persists in IL-33 receptor knockout mice because of the emergence of thymic stromal lymphopoietin–driven IL-9+ and IL-13+ type 2 innate lymphoid cell subpopulations

IL-33 plays an important role in the development of experimental asthma. We sought to study the role of the IL-33 receptor suppressor of tumorigenicity 2 (ST2) in the persistence of asthma in a mouse model. We studied allergen-induced experimental asthma in ST2 knockout (KO) and wild-type control mice. We measured airway hyperresponsiveness by using flexiVent; inflammatory indices by using ELISA, histology, and real-time PCR; and type 2 innate lymphoid cells (ILC2s) in lung single-cell preparations by using flow cytometry. Airway hyperresponsiveness was increased in allergen-treated ST2 KO mice and comparable with that in allergen-treated wild-type control mice. Peribronchial and perivascular inflammation and mucus production were largely similar in both groups. Persistence of experimental asthma in ST2 KO mice was associated with an increase in levels of thymic stromal lymphopoietin (TSLP), IL-9, and IL-13, but not IL-5, in bronchoalveolar lavage fluid. Expectedly, ST2 deletion caused a reduction in IL-13+ CD4 T cells, forkhead box P3-positive regulatory T cells, and IL-5+ ILC2s. Unexpectedly, ST2 deletion led to an overall increase in innate lymphoid cells (CD45+lin-CD25+ cells) and IL-13+ ILC2s, emergence of a TSLP receptor-positive IL-9+ ILC2 population, and an increase in intraepithelial mast cell numbers in the lung. An anti-TSLP antibody abrogated airway hyperresponsiveness, inflammation, and mucus production in allergen-treated ST2 KO mice. It also caused a reduction in innate lymphoid cell, ILC2, and IL-9+ and IL-13+ ILC2 numbers in the lung. Genetic deletion of the IL-33 receptor paradoxically increases TSLP production, which stimulates the emergence of IL-9+ and IL-13+ ILC2s and mast cells and leads to development of chronic experimental asthma. An anti-TSLP antibody abrogates all pathologic features of asthma in this model.

Interleukin-17 receptor A (IL-17RA) is a key regulator of type 2 inflammatory responses involved in maintaining immune system homeostasis. (IRC7P.431)

Abstract IL-17 cytokine family signaling is a key mediator of skin inflammatory disorders including atopic dermatitis and psoriasis. It has previously been shown that deficiency of IL-17 producing cells and especially TCRγδ T cells attenuates the skin inflammatory phenotype observed in murine models of psoriasis. Mice lacking IL-17 receptor A expression, through which IL-17A and other IL-17 family cytokines signal, develop severe and progressive skin inflammation. This spontaneous skin inflammation was characterized by increased epidermal thickness with cellular infiltration, which was accompanied with lymphadenopathy, and type 2 responses, including eosinophilia and increased serum levels of IgE. On a cellular level a significant increase in the numbers of innate lymphoid cell subpopulations was observed. Additionally IL-17RA-/- mice have increased γδ T cells with an activated memory-like phenotype. To further elucidate the role of IL-17 family cytokines in atopic dermatitis (AD) we crossed IL-17RA-/- mice with mice, carrying a mutation previously associated with human AD, that results in a spontaneous atopic dermatitis-like disease. The absence of IL-17RA signaling resulted in a severely accelerated AD like inflammation. This highlights that IL-17RA signaling is required for maintaining homeostasis including the regulation of Th2 responses.

Dual effects of indoleamine 2,3-dioxygenase inhibitors on the therapeutic effects of cyclophosphamide and cycloplatam on Ehrlich ascites tumor in mice.

Ethyl pyruvate, an inhibitor of indoleamine 2,3-dioxygenase, slightly suppressed the growth of transplantable Ehrlich tumor in mice and significantly potentiated the therapeutic effect of cyclophosphamide. Another inhibitor amidoxime produced a similar effect. However, both ethyl pyruvate and amidoxime significantly reduced the effect of cycloplatam therapy. The observed changes can be stipulated by different effects of cyclophosphamide and cycloplatam on the subpopulations of lymphoid cells taking part in the formation of antitumor immunity and resistance to tumors.

Investigation of Tissue-Specific Expression and Functions of MLF1-IP during Development and in the Immune System

Myeloid leukemia factor 1-interacting protein (MLF1-IP) has been found to exert functions in mitosis, although studies have been conducted only in cell lines up to now. To understand its roles during ontogeny and immunity, we analyzed its mRNA expression pattern by in situ hybridization and generated MLF1-IP gene knockout (KO) mice. MLF1-IP was expressed at elevated levels in most rudimentary tissues during the mid-gestation stage, between embryonic day 9.5 (e9.5) and e15.5. It declined afterwards in these tissues, but was very high in the testes and ovaries in adulthood. At post-natal day 10 (p10), the retina and cerebellum still expressed moderate MLF1-IP levels, although these tissues do not contain fast-proliferating cells at this stage. MLF1-IP expression in lymphoid organs, such as the thymus, lymph nodes, spleen and bone marrow, was high between e15.5 and p10, and decreased in adulthood. MLF1-IP KO embryos failed to develop beyond e6.5. On the other hand, MLF1-IP+/− mice were alive and fertile, with no obvious anomalies. Lymphoid organ size, weight, cellularity and cell sub-populations in MLF1-IP+/− mice were in the normal range. The functions of MLF1-IP+/− T cells and naïve CD4 cells, in terms of TCR-stimulated proliferation and Th1, Th17 and Treg cell differentiation in vitro, were comparable to those of wild type T cells. Our study demonstrates that MLF1-IP performs unique functions during mouse embryonic development, particularly around e6.5, when there was degeneration of epiblasts. However, the cells could proliferate dozens of rounds without MLF1-IP. MLF1-IP expression at about 50% of its normal level is sufficient to sustain mice life and the development of their immune system without apparent abnormalities. Our results also raise an intriguing question that MLF1-IP might have additional functions unrelated to cell proliferation.