Human Lymphocyte Subsets Research Articles

Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice

Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs which have demonstrated limited effectiveness in OC patients. We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). In the SKOV-3 Luc Hu-mouse model, we assessed the efficacy of PD-1 blockade with pembrolizumab. We observed the presence of human lymphocyte and myeloid cell subsets within the tumors, lymph nodes, blood, and spleens in these models. Notably, these tumors exhibited a high prevalence of tumor-infiltrating macrophages. Furthermore, we identified HDAC class I target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts in the tumors of Hu-mice treated with pembrolizumab. Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer useful information to explore potential mechanisms associated with unresponsive anti-PD-1 treatment in OC.

Abstract 4187: Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice

Abstract Background: Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human cancer and immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs, including immune checkpoint inhibitors (ICIs), which have demonstrated limited effectiveness in OC patients. Methods: We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). We assessed the efficacy of PD-1 blockade with pembrolizumab in the SKOV-3 Luc Hu-mouse model. The immune profiles of the tumors were characterized using colorimetric immunostaining and flow cytometry. Additionally, we analyzed RNA-seq data to investigate the gene expression signature of pembrolizumab refractory tumors. Results: We confirmed tumor development in both OC cell lines within CD34+ Hu-mice. In these models, human lymphocyte and myeloid cell subsets were present in the tumors, draining lymph nodes, blood, and spleens. The SKOV-3 Luc tumor-bearing Hu-mice did not respond to pembrolizumab monotherapy. These tumors exhibited a high presence of tumor-infiltrating macrophages. Tumors in Hu-mice unresponsive to pembrolizumab showed a lower abundance of CD8+ T-cells, memory B cells, plasma cells, and a higher proportion of naïve M0 macrophages and mast cells compared to the PBS control. Furthermore, we identified 43 significantly enriched gene sets in these tumors. The differentially expressed genes (DEGs) were predominantly enriched in HDAC class I, RB1, KLF1/3, TCF21, MYD88, SMARCE1 target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts. Conclusion: Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer potential avenues to explore mechanisms of resistance to PD-1/PD-L1 blockade in OC. Citation Format: Sung Wan Kang, Ji-young Lee, Ok-Ju Kang, Yong-Man Kim, Eun Kyung Choi, Shin-Wha Lee. Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4187.

Comparison of different combinations of antibodies and labeled fluorescein in the detection of lymphocyte subsets by flow cytometry.

Flow cytometry is a classical method for analyzing human peripheral blood lymphocyte subsets. This study aims to explore a new combination of antibody and labeled fluorescein for detecting lymphocyte subsets by comparing the effects of different combinations of antibody and labeled fluorescein in flow cytometry. We conducted a prospective study and enrolled 362 healthy patients undergoing physical examination in the medical examination center of the third hospital of hebei medical university. Venous blood was drawn from volunteers at the same time in the morning and divided into 3 tubes (Tube A, Tube B and Tube C). T lymphocytes were detected by 3-colors method (CD4-FITC/CD8-PE/CD3-PC5) in Tube A, B lymphocytes were detected by 2-colors method (CD19-FITC/CD3-PE) in Tube B, and T lymphocytes and B lymphocytes were detected by 4-colors method (CD4-FITC/CD8-PE/CD3-PC5/CD19-FITC) in Tube C. The repeatability and accuracy of the test scheme for Tube C shall not be inferior to that of Tube A and Tube B. There were no significant difference in the results of CD3 + and CD4+/CD8 + between Tube A and C, as well as in the results of CD3 + and CD19 + between Tube B and C. Pearson correlation analysis showed that the test results of a and C and B and C were highly correlated. The 4-colors method (CD4-FITC/CD8-PE/CD3-PC5/CD19-FITC) can detect T lymphocytes and B lymphocytes at the same time, reduce the use of fluorescence channels and save the detection cost, which is worthy of recommendation.

Reference long-read isoform-aware transcriptomes of 4 human peripheral blood lymphocyte subsets.

Long-read sequencing technologies such as isoform sequencing can generate highly accurate sequences of full-length mRNA transcript isoforms. Such long-read transcriptomics may be especially useful in investigations of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes are readily available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified 4 lymphocyte populations (CD4+ T, CD8+ T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN > 8) for isoform sequencing and parallel RNA-Seq analyses. Many novel polyadenylated transcript isoforms, supported by both isoform sequencing and RNA-Seq data, were identified within each sample. The datasets met several metrics of high quality and have been deposited to the Gene Expression Omnibus database (GSE202327, GSE202328, GSE202329) as both raw and processed files to serve as long-read reference transcriptomes for future studies of human circulating lymphocytes.

Long-Term Redistribution of Peripheral Lymphocyte Subpopulations after Switching from Calcineurin to mTOR Inhibitors in Kidney Transplant Recipients.

Classical immunosuppression based on steroids, calcineurin inhibitors, and mycophenolate results in several unwanted effects and unsatisfactory long-term outcomes in kidney transplantation (KT). New immunosuppressors search for fewer adverse events and increased graft survival but may have a distinct impact on graft function and immunological biomarkers according to their mechanism of action. This prospective study evaluates the immunological effect of tacrolimus to serine/threonine protein kinase mechanistic target of rapamycin inhibitors (mTORi) conversion in 29 KT recipients compared with 16 controls maintained on tacrolimus. We evaluated renal function, human leukocyte antigen (HLA) antibodies and peripheral blood lymphocyte subsets at inclusion and at 3, 12, and 24 months later. Twenty immunophenotyped healthy subjects served as reference. Renal function remained stable in both groups with no significant change in proteinuria. Two patients in the mTORi group developed HLA donor-specific antibodies and none in the control group (7% vs. 0%, p = 0.53). Both groups showed a progressive increase in regulatory T cells, more prominent in patients converted to mTORi within the first 18 months post-KT (p < 0.001). All patients showed a decrease in naïve B cells (p < 0.001), excepting those converted to mTORi without receiving steroids (p = 0.31). Transitional B cells significantly decreased in mTORi patients (p < 0.001), independently of concomitant steroid treatment. Finally, CD56bright and CD94/NK group 2 member A receptor positive (NKG2A+) Natural Killer (NK) cell subsets increased in mTORi- compared to tacrolimus-treated patients (both p < 0.001). Patients switched to mTORi displayed a significant redistribution of peripheral blood lymphocyte subpopulations proposed to be associated with graft outcomes. The administration of steroids modified some of these changes.

Study on the role and mechanism of β4GalT1 both in vivo and in vitro glioma.

To discuss the role and mechanism of β4GalT1 both in vivo and in vitro glioma, observe whether pathophysiological processes of glioma can be improved after β4GalT1 is knocked down, and study whether β4GalT1 plays a role in malignant biological processes of glioma by regulating the apoptosis and immune processes. Firstly, the distribution difference of β4GalT1 in tumor tissues and normal tissues was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) tumor analysis system to deduce the possible role of β4GalT1 in glioma. Secondly, whether the malignant degree of glioma was related to the expression of β4GalT1 and its immunity using human tumor tissues and blood lymphocyte subsets was analyzed. Thirdly, interfere lentivirus vector with β4GalT1 and knockdown β4GalT1 was analyzed to observe whether the malignant degree of glioma has changed. Fourthly, interfere lentivirus vector with recombinant β4GalT protein and β4GalT1 was analyzed to verify the effect of β4GalT in vitro test. Fifth, interfere lentivirus vector with recombinant β4GalT protein and β4GalT1 was analyzed to verify effect of β4GalT in vivo test. Finally, we discuss whether β4GalT is involved in the biological process of glioma through inflammatory reaction. In the GEPIA tumor analysis system, the expression in tumor was significantly higher than that in normal tissues. The expression of β4GalT1 in glioma tissues was higher than that in normal tissues, and the higher the malignancy of the tumor, the higher the expression of β4GalT1 in the glioma tissues, and the lower the immune level was. The expression of IDH1, MGMT, and ki-67 was reduced, and the survival rate of the mice with glioma was improved after β4GalT1 was knocked down. In vitro tests, the activity of tumor cells and their reproductive ability can be reduced after β4GalT1 was knocked down, the immune level of the body can be improved, and the level of tissue apoptosis can be reduced. After recombinant β4GalT1 was given alone, the result was opposite to that of β4GalT1 knocked down group. In vivo tests, gross tumor volume can be reduced after β4GalT1 was knocked down, the immune level of the body can be improved, and the level of tissue apoptosis can be reduced. After recombinant β4GalT1 was given alone, the result was opposite to that of β4GalT1 knocked down group. After knocking down β4GalT1, the expression of inflammatory factors can be reduced both in vivo and in vitro, and the inflammatory microenvironment of tumors can be improved. After recombinant β4GalT1 was given alone, the result was opposite to that of β4GalT1 knocked down group. The level of β4GalT1 expression in tumor tissues was increased. The malignant degree of glioma is related to the expression of β4GalT1 and its immunity. The level of tumor marker can be decreased, and the survival rate of glioma model mice can be increased after β4GalT1 is knocked down. Apoptosis and immune injury caused by tumor can be improved and gross tumor volume can be deduced after β4GalT1 is knocked down. During the development of glioma, β4GalT1 may play a malignant biological role through inflammatory response.

Human ovarian cancer intrinsic mechanisms regulate lymphocyte activation in response to immune checkpoint blockade

Immune checkpoint blocking antibodies are currently being tested in ovarian cancer (OC) patients and have shown some responses in early clinical trials. However, it remains unclear how human OC cancer cells regulate lymphocyte activation in response to therapy. In this study, we have established and optimised an in vitro tumour-immune co-culture system (TICS), which is specifically designed to quantify the activation of multiple primary human lymphocyte subsets and human cancer cell killing in response to PD-1/L1 blockade. Human OC cell lines and treatment naïve patient ascites show differential effects on lymphocyte activation and respond differently to PD-1 blocking antibody nivolumab in TICS. Using paired OC cell lines established prior to and after chemotherapy relapse, our data reveal that the resistant cells express low levels of HLA and respond poorly to nivolumab, relative to the treatment naïve cells. In accordance, knockdown of IFNγ receptor expression compromises response to nivolumab in the treatment naïve OC cell line, while enhanced HLA expression induced by a DNA methyltransferase inhibitor promotes lymphocyte activation in TICS. Altogether, our results suggest a ‘cross resistance’ model, where the acquired chemotherapy resistance in cancer cells may confer resistance to immune checkpoint blockade therapy through down-regulation of antigen presentation machinery. As such, agents that can restore HLA expression may be a suitable combination partner for immunotherapy in chemotherapy-relapsed human ovarian cancer patients.

CD Maps - Dynamic Profiling of CD1 to CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets and their malignant counterparts. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-100 (n=110) on 47 immune cell subsets from blood, thymus and tonsil using an 8-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABC) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as, to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases. CD Maps project is supported with reagents from BD Biosciences, BioLegend and Exbio. TK is supported by Ministry of Health Czech Republic grant 15-26588A and LO1604. KF is supported by Ministry of Health Czech Republic grant NV18-08-00385. Disclosures No relevant conflicts of interest to declare.

CD Maps-Dynamic Profiling of CD1-CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets.

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1–CD100 (n = 110) on 47 immune cell subsets from blood, thymus, and tonsil using an eight-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABCs) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte, and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.

Human cytomegalovirus IL-10 augments NK cell cytotoxicity.

Human cytomegalovirus (HCMV) persistently infects most of the adult population with periods of productive and latent infection differentially orchestrated by multiple HCMV-encoded gene products. One HCMV gene (UL111a) encodes cmvIL-10, a virokine homologous to human IL (hIL)-10. Although the effects of cmvIL-10 on most human lymphocyte subsets have been extensively studied, its impact on NK cell function was unreported prior to this study. We investigated effects of short-term cmvIL-10 exposure on human NK cells and found it substantially enhanced NK cell cytotoxicity through natural cytotoxicity receptors NKp30 and NKp46 as well as through C-type lectin-like receptors NKG2C and NKG2D. Antibody-dependent cell-mediated cytotoxicity triggered through CD16 also increased significantly with short-term cmvIL-10 exposure. These effects of cmvIL-10 on NK cell cytotoxicity were rapid, dose dependent, neutralized by polyclonal anti-cmvIL-10 or monoclonal anti-IL-10 receptor (IL-10R) antibodies and independent of increased perforin synthesis or up-regulation of activating receptors. A low percentage (0.5-5.4%; n=12) of NK cells expressed IL-10R and the impact of cmvIL-10 on NK cells degranulation following CD16 stimulation directly correlated with this percentage (P= 0.0218). Short-term exposure of human NK cells to cmvIL-10 did not introduce phenotypic changes reminiscent of NK adaptation to HCMV infection in vivo. Determining how expression of a viral protein that activates NK cells contributes to their function in vivo will increase understanding of HCMV infection and NK cell biology.

Effects of Different Stimulators on Proliferation of Peripheral Blood Lymphocyte Subsets

To investigate the effects of different stimultors (PHA, PMA and IL-2) and culture systems (PBMC and whole blood) on the proliferation of human peripheral blood lymphocyte subsets, so as to provide the experimental basis for selecting the appropriate system according to the experimental purposes. A total of 10 ml serum samples were collected from healthy volunteers (n=6). The 300 μl whole blood was directly used to detect lymphocyte subsets by flow cytometry. The 400 μl whole blood were inoculated respectively with 3 different stimulators at 37℃ and 5% CO2 for 60 h; Three different stimulators were also added to the PBMC which were isolated from 2 ml whole blood. Then the proliferation ability of lymphocyte subsets was analyzed by flow cytometry. After the PBMC were stimulated with PHA, CD4-CD8-CD3+ lymphocytes were the most subset; The proportion of CD3+CD4+ T lymphocytes and CD3-CD19+ B lymphocytes decreased after being stimulated by PMA (P<0.01, P<0.05); the lymphocyte subset ratio had no significant change after being stimulated by IL-2. After the whole blood system was stimulated with PHA, the CD4+/CD8+ T lymphoblasts were main subsets, the counts of B lymphocytes and NK cells were reduced; after being stimulated with PMA, the number of CD8+CD3+ T lymphoblast and CD4-CD8-T lymphocytes increased, the B/NK cells were not distinguished with the surface markers; after the whole blood system was stimulated with IL-2, the proportion of NK cells significantly increased (P<0.05). Lymphocyte proliferation stimulated by PMA is the fastest, while the effect of IL-2 on the lymphocyte subset proportion stimulated by IL-2 is the minimal. After being stimulated by PHA the division cycles of lymphocyte are the most.

Expression of glucocorticoid receptor shows negative correlation with human B-cell engraftment in PBMC-transplanted NOGhIL-4-Tg mice.

The humanized mouse system is a promising tool for analyzing human immune responses in vivo. Recently, we developed a new humanized mouse system using the severely immunodeficient NOD/Shi-scid-IL2rγnull (NOG)-hIL-4-Tg mouse, which enabled us to evaluate the human humoral immune response after peripheral blood mononuclear cell (PBMC) transplantation. However, the mechanism by which hIL-4 enhances antigen-specific IgG production in these mice is not clear. In this study, we analyzed the relationship between human lymphocyte subsets and the expression level of the glucocorticoid receptor (GR) to clarify the humoral immune condition in human PBMC-transplanted NOG-hIL-4 mice. The results showed that the human GR mRNA level was significantly lower in NOG-hIL-4-Tg splenocytes than in conventional NOG splenocytes after immunization. Whereas no obvious difference of the proportion of T helper-cell subsets was observed between the NOG and NOG-hIL-4-Tg mouse strains, the B-cell proportion and antigen-specific IgG concentration in plasma showed strong negative correlations with the GR mRNA level. These results suggest that the GR expression level was changed in PBMCs in the humanized NOG-hIL-4-Tg mice, which may support B-cell survival and function in the mouse system.

In Vitro Measles Virus Infection of Human Lymphocyte Subsets Demonstrates High Susceptibility and Permissiveness of both Naive and Memory B Cells.

ABSTRACTMeasles is characterized by a transient immune suppression, leading to an increased risk of opportunistic infections. Measles virus (MV) infection of immune cells is mediated by the cellular receptor CD150, expressed by subsets of lymphocytes, dendritic cells, macrophages, and thymocytes. Previous studies showed that human and nonhuman primate memory T cells express higher levels of CD150 than naive cells and are more susceptible to MV infection. However, limited information is available about the CD150 expression and relative susceptibility to MV infection of B-cell subsets. In this study, we assessed the susceptibility and permissiveness of naive and memory T- and B-cell subsets from human peripheral blood or tonsils to in vitro MV infection. Our study demonstrates that naive and memory B cells express CD150, but at lower frequencies than memory T cells. Nevertheless, both naive and memory B cells proved to be highly permissive to MV infection. Furthermore, we assessed the susceptibility and permissiveness of various functionally distinct T and B cells, such as helper T (TH) cell subsets and IgG- and IgA-positive memory B cells, in peripheral blood and tonsils. We demonstrated that TH1TH17 cells and plasma and germinal center B cells were the subsets most susceptible and permissive to MV infection. Our study suggests that both naive and memory B cells, along with several other antigen-experienced lymphocytes, are important target cells of MV infection. Depletion of these cells potentially contributes to the pathogenesis of measles immune suppression.IMPORTANCE Measles is associated with immune suppression and is often complicated by bacterial pneumonia, otitis media, or gastroenteritis. Measles virus infects antigen-presenting cells and T and B cells, and depletion of these cells may contribute to lymphopenia and immune suppression. Measles has been associated with follicular exhaustion in lymphoid tissues in humans and nonhuman primates, emphasizing the importance of MV infection of B cells in vivo. However, information on the relative susceptibility of B-cell subsets is scarce. Here, we compared the susceptibility and permissiveness to in vitro MV infection of human naive and memory T- and B-cell subsets isolated from peripheral blood or tonsils. Our results demonstrate that both naive and memory B cells are more permissive to MV infection than T cells. The highest infection levels were detected in plasma cells and germinal center B cells, suggesting that infection and depletion of these populations contribute to reduced host resistance.

Suppression of Lymphocyte Functions by Plasma Exosomes Correlates with Disease Activity in Patients with Head and Neck Cancer.

Purpose: Head and neck cancers (HNCs) often induce profound immunosuppression, which contributes to disease progression and interferes with immune-based therapies. Body fluids of patients with HNC are enriched in exosomes potentially engaged in negative regulation of antitumor immune responses. The presence and content of exosomes derived from plasma of patients with HNC are evaluated for the ability to induce immune dysfunction and influence disease activity.Experimental Design: Exosomes were isolated by size-exclusion chromatography from plasma of 38 patients with HNC and 14 healthy donors. Morphology, size, numbers, and protein and molecular contents of the recovered exosomes were determined. Coculture assays were performed to measure exosome-mediated effects on functions of normal human lymphocyte subsets and natural killer (NK) cells. The results were correlated with disease stage and activity.Results: The presence, quantity, and molecular content of isolated, plasma-derived exosomes discriminated patients with HNC with active disease (AD) from those with no evident disease (NED) after oncologic therapies. Exosomes of patients with AD were significantly more effective than exosomes of patients with NED in inducing apoptosis of CD8+ T cells, suppression of CD4+ T-cell proliferation, and upregulation of regulatory T-cell (Treg) suppressor functions (all at P < 0.05). Exosomes of patients with AD also downregulated NKG2D expression levels in NK cells.Conclusions: Exosomes in plasma of patients with HNC carry immunosuppressive molecules and interfere with functions of immune cells. Exosome-induced immune suppression correlates with disease activity in HNC, suggesting that plasma exosomes could be useful as biomarkers of HNC progression. Clin Cancer Res; 23(16); 4843-54. ©2017 AACR.

Comprehensive Analysis of Activation and Proliferation Kinetics and Effector Functions of Human Lymphocyte Subsets in Xenogeneic GvHD Model

To better understand the biology of graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT) and develop novel treatment strategies, accurate and clinically relevant experimental animal models are indispensable. The majority of our understanding of this potentially lethal complication is based on mouse models of bone marrow transplantation in major histocompatibility complex antigens (MHC) and/or minor histocompatibility antigen mismatched settings. These mouse models of GvHD provide us with an immeasurable wealth of information; however, many findings obtained from mouse models are not necessarily correlated with clinical GVHD in humans. In addition, these mouse models are not suitable for predicting the effectiveness of recent human-specific therapies such as monoclonal anti-human antibodies and adaptive cell immunotherapies. To overcome these limitations, xenogeneic GvHD models using severe immunodeficient mice are currently being used in worldwide and allow us to investigate in vivo human immune reactions.The major aim of our study is to clarify the precise mechanism of immune response in vivo and to determine the principle components responsible for xenogeneic GvHD. We first observed that immunodeficient NOG mice receiving either no or sublethal irradiation consistently showed gradual body weight loss and eventual severe GvHD following injection of human unmanipulated peripheral blood mononuclear cells (PBMCs). Histopathology performed at the late phase of GvHD showed extensive infiltration of human CD45+ mononuclear cells, most of which were T-cells, and tissue destruction in lungs, bone marrow, liver, and spleen, with a smaller number detected in gut and skin unlike in human GvHD. We identified the infiltration of human T-cells exclusively in lungs and spleen before the onset of GvHD. Flow cytometric analysis showed a marked reduce of naïve (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) T-cells and an increase of effector memory (CD45RA-CCR7-) T-cells within CD4+ subset in lungs, suggesting that xenogeneic response by human cells in lungs occur preceding systemic inflammation. Of note, the distribution of human cells was similar between intraperitoneal and intravenous injection. These results indicated that the acute lung injury is not associated with cell trapping within mouse pulmonary microvasculature by intravenous delivery.We next subdivided human lymphocyte subsets by magnetic cell sorting before adoptive transfer to characterize human cells responsive for xenogeneic GvHD development. CD4+ T-cells mediated more rapid and severe GvHD than did an equal number of CD8+ T-cells, whereas neither natural killer cells nor γδT cells caused any symptoms of GvHD. All antigen presenting cells (APCs)-depleted PBMCs mediated GvHD as well, suggesting that human T-cells are activated independently of their own APCs in this model.CFSE cell division assay showed more rapid proliferation of CD4+ rather than CD8+ T-cells. It is a noteworthy that the proliferation of and the expression of early activation marker CD69 on CD8+ T-cells was accelerated in the presence of CD4+ T-cells, indicating that CD4+ T-cells are required for the rapid and more efficacious response of CD8+ T-cells. We also found a strong increase of human IL-2, IFN-γ, and TNF-α in serum of mice injected with whole T-cells. In contrast, we observed very low levels of these inflammatory cytokines in mice injected with isolated CD8+ T-cells alone.Taken together, these results suggest that the early activation of human naïve CD4 T-cells by recognition of foreign antigen on mouse APCs following cytokine production play a critical role in triggering systemic inflammation in xenogeneic GvHD model. This in vivo response was confirmed in vitro by demonstrating that human CD4 T-cells rapidly proliferated and expressed CD69 with significantly increase of IL-2 and IFN-γ in response to mouse dendritic cells. These findings are helpful for adequate evaluation of immune response and creation of optimal experimental conditions in xenogeneic GvHD model. DisclosuresKanda:Otsuka Pharmaceutical: Honoraria, Research Funding.

C-type lectin-like NKC-encoded immunoreceptors NKp80 and NKp65 are selectively expressed by human innate lymphocyte subsets, uniquely signal via hemITAMs and facilitate tissue-specific immunosurveillance via their genetically linked ligands AICL and KACL

Abstract NKp80 is an activating C-type lectin-like immunoreceptor (CTLR) on human NK cells. Upon ligation by its cognate ligand ‘activation-induced C-type lectin’ (AICL) up-regulated on PAMP-stimulated myeloid cells and interleukin-experienced NK cells, NKp80 stimulates NK cytotoxicity and cytokine secretion. Unlike other NK receptors, NKp80 uses an atypical cytoplasmic hemITAM for cellular activation. In contrast to “classical” hemITAMs of related myeloid CTLR, we showed that the NKp80 hemITAM does not recruit Syk upon phosphorylation. However, we now provide evidence for specific recruitment of other signaling molecules by NKp80. Alike its close relative NKp80, NKp65 is a homodimeric CTLR with a cytoplasmic hemITAM and encoded in the Natural Killer Gene Complex (NKC). NKp65 binds with high affinity to its cognate genetically linked ligand KACL almost exclusively expressed by human keratinocytes. We previously showed that NKp65/KACL interaction activates cytotoxicity by NK-92 cells. However, while NKp80 is expressed by virtually all human NK cells, physiological expression of NKp65 remained elusive. Generation of NKp65-specific mAb enabled us to trace NKp65 expression to a minor subset of human innate lymphocytes. Transcriptomics revealed strict correlation between NKp65 expression ILC hallmark genes. Hence, NKp65 expression defines that ILC subset and enables a tissue-specific cross-talk with human keratinocytes. Moreover, we show that NKp80 and NKp65 are mutually exclusive expressed by NK cells and that ILC subset, qualifying NKp80 and NKp65 as markers of these 2 distinct innate lymphocyte lineages and raising questions for their respective subset-associated function.

Abstract 3158: Ex vivo expansion and activation of human lymphocytes with a selective activator of effector cells

Abstract RDB 1450 is an engineered fusion protein of circularly permuted IL-2 and IL-2Rα that is selective for the intermediate-affinity IL-2 receptor. The intermediate-affinity receptor is comprised of the IL-2 receptor beta chain (CD122) and common gamma chain (CD132) expressed on lymphocytes with effector function, whereas the high-affinity receptor, which additionally includes the alpha chain (CD25), is expressed primarily on immunosuppressive CD4+CD25+ regulatory T cells (Tregs). Ex vivo proliferation of subsets of human lymphocytes in response to RDB 1450 or rhIL-2 were evaluated. RDB 1450 supports the selective activation and expansion of effector lymphocytes based on higher observed memory CD8 T/Treg and NK/Treg ratios. Both RDB 1450 and IL-2 drive expansion of NK cells, however, a greater proportion of CD25+ NK cells are observed in response to RDB 1450. Furthermore, unlike IL-2, RDB 1450 does not effectively expand highly suppressive ICOS+ Tregs. The immunological profile resulting from the ex vivo treatment of human PBMCs with RDB 1450 supports its use as a novel immunotherapy for cancer. Citation Format: Jared E. Lopes, Jan L. Fisher, Heather C. Losey, Marc S. Ernstoff, Juan C. Alvarez. Ex vivo expansion and activation of human lymphocytes with a selective activator of effector cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3158. doi:10.1158/1538-7445.AM2015-3158

Glucocorticoid-induced S-Adenosylmethionine Enhances the Interferon Signaling Pathway by Restoring STAT1 Protein Methylation in Hepatitis B Virus-infected Cells

Patients with chronic hepatitis B usually exhibit a low response to treatment with interferon α (IFN-α). An alternative approach to increase the response rate of IFN-α might be to immunologically stimulate the host with glucocorticoids (GCs) before treatment with IFN-α, but the underlying mechanism remains unclear. We hypothesized that the GCs enhance IFN signaling by inducing S-adenosylmethionine (AdoMet) when hepatitis B virus (HBV) replication was effectively suppressed by IFN-α. Here, we investigated the effect of GCs and IFN-α on AdoMet production and methionine adenosyltransferase 1A (MAT1A) expression in vitro. Furthermore, we determined whether post-transcriptional regulation is involved in HBV-repressed MAT1A expression and AdoMet production induced by dexamethasone (Dex). We found that AdoMet homeostasis was disrupted by Dex and that Dex directly regulated MAT1A expression by enhancing the binding of the glucocorticoid receptor (GR) to the glucocorticoid-response element (GRE) of the MAT1A promoter. HBV reduced AdoMet production by increasing methylation at GRE sites within the MAT1A promoter. The X protein of hepatitis B virus led to hypermethylation in the MAT1A promoter by recruiting DNA methyltransferase 1, and it inhibited GR binding to the GRE in the MAT1A promoter. Dex could increase an antiviral effect by inducing AdoMet production via a positive feedback loop when HBV is effectively suppressed by IFN-α, and the mechanism that involves Dex-induced AdoMet could increase STAT1 methylation rather than STAT1 phosphorylation. These findings provide a possible mechanism by which GC-induced AdoMet enhances the antiviral activity of IFN-α by restoring STAT1 methylation in HBV-infected cells.

Novel Mechanisms Of Immune Modulation By Alpha-1-Antitrypsin

S U N D A Y 484 CD4+ and CD8+ T Cells Of Allergic Humans Express Increased Phosphorylated p38 MAP Kinase (p38MAPK), Substance P Suppresses T Cell Expression Of p38MAPK and Memory IgE Responses Bryan McCarthy, BS, Jonathan I. Silverberg, MD, PhD, MPH, Seto M. Chice, MS, Ankuri Desai, BS, Dr. Tamar A. Smith-Norowitz, PhD, Mark Stewart, MD, PhD, Dr. Rauno Joks, MD, Helen G. Durkin, PhD; Center for Allergy and Asthma Research at SUNY Downstate, Brooklyn, NY, Center for Allergy and Asthma Research, State University of New York Downstate Medical Center, Brooklyn, NY. RATIONALE: Magnetic/electrical stimulation of IgE+ human/rat left TPO cortex increases serum/blood substance P (2 hr) and CD4+/CD8+ T cell numbers (4 hr), while suppressing IgE responses (4 days). Thymus supplied T cells to blood because thymectomy or cutting of spinal cord at T2 (thymic innervation level) abrogated their appearance. Substance P suppresses specific murine IgE responses in vivo/vitro, and a relationship between p38MAPK and IL-4 has been reported. The effect of substance P on p38MAPK expression by human lymphocyte subsets and memory IgE responses has not been studied. METHODS: Distributions of p38MAPK+ lymphocytes (CD4+,CD8+,CD19+,CD16+) in PBMC obtained from ragweed sensitized IgE+ humans were determined6 15-30 min incubation with PMA6 substance P (flow cytometry). In addition, PBMC were cultured for 0-12 days 6 ragweed antigen 6 substance P and IgE levels in supernatants determined (ELISA). RESULTS: Before PMA stimulation, virtually no p38MAPK+ T cells (CD4+, CD8+), B or NK cells were detected in PBMC (<5%). After PMA, virtually all T cells, and CD4+ T cells which had been purified from PBMC, were p38MAPK+ (90-98%), with no change in p38MAPK+ B or NK cells.When PBMCor purified CD4+ T cells were incubatedwith PMA + substance P, p38MAPK+ CD4+ and CD8+ T cell numbers were suppressed (50-75%) When PBMC were cultured with ragweed antigen, peak IgE responses were induced on day 8; inclusion of substance P in culture prevented induction of these responses on all days. CONCLUSIONS: Taken together, our results indicate that brain can suppress IgE responses by releasing substance P.

Dynamicly Observing Influence of Ambient PM&lt;sub&gt;2.5&lt;/sub&gt; on Human Lymphocyte Subsets

AbstractObjective: To dynamicly observe the impact of changes of ambient PM2.5 on lymphocyte subsets of healthy young people. Method: During the 2008 Olympic Games in Beijing, we dynamic detected changes of lymphocyte subsets from125 healthy young people living in the center area of Beijing, and detected changes of lymphocytes’ immunophenotype by flow cytometry (FCM). Air samplers installed in the same area monitored continuously changes of ambient PM2.5. Results: The PM2.5 in August was obviously decreased comparing with that in June and in October. Repeated measures ANOVA suggested that the changes of PM2.5 were statistically significant (P&lt;0.05). With the improvement of ambient PM2.5, under the total number of lymphocytes, T cell number, the total number of Naïve T cells not change significantly (P&gt;0.05), helper T cell (TH) increased, suppressor T cell (TS) decreased, the ratio of TH/TS increased, regulatory T cell (RT) increased, NK cells decreased. The Pearson correlation analysis showed that the correlation between PM2.5 and TH, TH/TS, RT was negatively statistically significant (P&lt;0.05), whereas no statistically significant correlation with TS, NK cell. Conclusion: The changes of ambient PM2.5 in the shortterm effected obviously on healthy human lymphocyte subsets. With the improvement of PM2.5, on behalf of immune surveillance function of TH cell increased, TH/TS and RT cell increased, whereas representative excessive immune TS cell, NK cell reduced.