CD25hi Cells Research Articles

Human CD4+CD25+ T cells expressing a chimeric antigen receptor against aberrant superoxide dismutase 1 trigger antigen-specific immunomodulation

Background aimsAmyotrophic lateral sclerosis (ALS) is a fatal disease associated with motor neuron degeneration, accumulation of aggregated misfolded proteins and neuroinflammation in motor regions of the central nervous system (CNS). Clinical trials using regulatory T cells (Tregs) are ongoing because of Tregs’ immunomodulatory function, ability to traffic to the CNS, high numbers correlating with slower disease in ALS and disease-modifying activity in ALS mouse models. In the current study, a chimeric antigen receptor (CAR) was developed and characterized in human Tregs to enhance their immunomodulatory activity when in contact with an ALS protein aggregate. MethodsA CAR (DG05-28-3z) consisting of a human superoxide dismutase 1 (hSOD1)-binding single-chain variable fragment, CD28 hinge, transmembrane and co-stimulatory domain and CD3ζ signaling domain was created and expressed in human Tregs. Human Tregs were isolated by either magnetic enrichment for CD4+CD25hi cells (Enr-Tregs) or cell sorting for CD4+CD25hiCD127lo cells (FP-Tregs), transduced and expanded for 17 days. ResultsThe CAR bound preferentially to the ALS mutant G93A-hSOD1 protein relative to the wild-type hSOD1 protein. The CAR Tregs produced IL-10 when cultured with aggregated G93A-hSOD1 proteins or spinal cord explants from G93A-hSOD1 transgenic mice. Co-culturing DG05-28-3z CAR Tregs with human monocytes/macrophages inhibited production of tumor necrosis factor alpha and reactive oxygen species. Expanded FP-Tregs resulted in more robust Tregs compared with Enr-Tregs. FP-Tregs produced similar IL-10 and less interferon gamma, had lower Treg-specific demethylated region methylation and expressed higher FoxP3 and CD39. ConclusionsTaken together, this study demonstrates that gene-modified Tregs can be developed to target an aggregated ALS-relevant protein to elicit CAR-mediated Treg effector functions and provides an approach for generating Treg therapies for ALS with the goal of enhanced disease site-specific immunomodulation.

CD51 labels periosteal injury-responsive osteoprogenitors.

The periosteum is a critical source of skeletal stem and progenitor cells (SSPCs) that form callus tissue in response to injury. There is yet to be a consensus on how to identify SSPCs in the adult periosteum. The aim of this study was to understand how potential murine periosteal SSPC populations behave in vivo and in response to injury. We evaluated the in vivo differentiation potential of Sca1-CD51+ and Sca1+CD51+ cells following transplantation. In vitro, the Sca1+CD51+ population appears to be more primitive multipotent cells, but after transplantation, Sca1-CD51+ cells showed superior engraftment, expansion, and differentiation into chondrocytes and osteoblasts. Despite representing a clear population with flow cytometry, we identified very few Sca1+CD51+ cells histologically. Using a periosteal scratch injury model, we successfully mimicked the endochondral-like healing process seen in unstable fractures, including the expansion and osteochondral differentiation of αSMA+ cells following injury. CD51+ cells were present in the cambium layer of resting periosteum and expanded following injury. Sca1+CD51- cells were mainly localized in the outer periosteal layer. We found that injury increased colony-forming unit fibroblast (CFU-F) formation in the periosteum and led to rapid expansion of CD90+ cells. Several other populations, including Sca1-CD51+ and CD34+ cells, were expanded by day 7. Mice with enhanced fracture healing due to elevated Notch signaling mediated by NICD1 overexpression showed significant expansion of CD51+ and CD34hi cells in the early stages of healing, suggesting these populations contribute to more rapid healing. In conclusion, we demonstrate that periosteal injury leads to the expansion of various SSPC populations, but further studies are required to confirm their lineage hierarchy in the adult skeletal system. Our data indicate that CD51+ skeletal progenitor cells are injury-responsive and show good engraftment and differentiation potential upon transplantation.

Peripheral CD4+CD25hiCD127low regulatory T cells are increased in patients with gastrointestinal cancer

BackgroundRegulatory T cells (Tregs) play an important role in regulation of immune response and immunologic tolerance in cancer. Gastrointestinal cancer is still a leading cause of cancer-related death in the world. This study aimed to detect Tregs in patients with gastrointestinal cancer.MethodsIn this study, 45 gastric cancer patients, 50 colorectal cancer patients and 50 healthy controls were enrolled. Flow cytometry was used to detect CD4+CD25hiCD127low Tregs, CD4+CD25hi, and CD4+ cells in peripheral blood. Cytokine interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) in peripheral blood and in the supernatant of Tregs cultures were measured by enzyme linked immunosorbent assay.ResultsCompared with healthy controls, the levels of CD4+CD25hiCD127low Tregs and CD4+CD25hi cells increased significantly in patients with gastrointestinal cancer. Patients with gastrointestinal cancer also showed a significantly increased levels of IL-10 and TGF-β1 in both peripheral blood and CD4+CD25hiCD127low Tregs culture medium.ConclusionThe present study firstly demonstrated that gastrointestinal patients have a compromised immune status where the CD4+CD25hiCD127low Tregs, as well as levels of IL-10 and TGF-β1 are elevated. The data offered new information for understanding the immunological features of gastrointestinal patients, as well as provided new insights into approaches to develop new immunotherapies for patients with gastrointestinal cancer.

Bone marrow derived long-lived plasma cell phenotypes are heterogeneous and can change in culture

Bone marrow-derived long-lived plasma cells (LLPCs) are thought to be a major source of alloantibody in sensitized transplant patients. However, studies of LLPCs have been hampered not only by the fact that they are rare and difficult to isolate and culture but also due to the lack of consensus regarding a definitive cell-surface phenotype. The goal of the current study was to determine if LLPCs have a specific, stable cell-surface phenotype. PCs were isolated from high-volume (120cc) bone marrow aspirates that were enriched first by negative selection then positive selection using anti-CD38 antibody-coated beads and purified by cell sorting. A typical isolation resulted in >100,000 PCs that were sorted into 4 populations with variable numbers of PCs: CD19+/CD138+/CD38Hi (64.1% of the PCs), CD19-/CD138+/CD38Hi (20.9%), CD19+/CD138-/CD38Hi (10.7%), and CD19-/CD138-/CD38Hi (4.3%). The purity of each subset was 96–99%. Each subset contained PCs secreting IgG and IgA. Measles- and tetanus-specific PCs (i.e. putative IgG secreting, antigen-specific LLPCs). LLPCs were identified in both the CD19+/CD138+/CD38Hi and CD19-/CD138+/CD38Hi subsets and in the smaller CD138- subsets (when pooled). Thus, all CD38Hi subsets contained LLPCs. Cultured PCs maintained viability (>50%) and function and could be retrieved for analyses. During 7 days of culture, cell surface expression changed from baseline in many PCs. For example, approximately 20% of CD19 + CD138+/CD38Hi cells (the largest PC subset) became CD19-. CFSE assays showed no division and only a small percentage of LLPCs were Ki-67 positive suggesting that the cells did not divide in culture and that the antibody detected was not from plasmablasts. We conclude that human bone marrow LLPCs have a heterogeneous expression of CD19 and CD138, which can change during cell culture. The fact that LLPCs were found in all four subsets raises the possibility that a large percentage of PCs in the bone marrow may be LLPCs.

Discovery of surface biomarkers for cell mechanophenotype via an intracellular protein-based enrichment strategy.

Cellular mechanophenotype is often a defining characteristic of conditions like cancer malignancy/metastasis, cardiovascular disease, lung and liver fibrosis, and stem cell differentiation. However, acquiring living cells based on mechanophenotype is challenging for conventional cell sorters due to a lack of biomarkers. In this study, we demonstrate a workflow for surface protein discovery associated with cellular mechanophenotype. We sorted heterogeneous adipose-derived stem/stromal cells (ASCs) into groups with low vs. high lamin A/C, an intracellular protein linked to whole-cell mechanophenotype. Proteomic data of enriched groups identified surface protein candidates as potential biochemical proxies for ASC mechanophenotype. Select surface biomarkers were used for live-cell enrichment, with subsequent single-cell mechanical testing and lineage-specific differentiation. Ultimately, we identified CD44 to have a strong inverse correlation with whole-cell elastic modulus, with CD44lo cells exhibiting moduli three timesgreaterthan that of CD44hi cells. Functionally, these stiff and soft ASCs showed enhanced osteogenic and adipogenic differentiation potential, respectively. The described workflow can be replicated for any phenotype with a known correlated intracellular protein, allowing for the acquisition of live cells for further characterization, diagnostics, or therapeutics.

Early heterogeneity of IL2R□ expression influences the programming of exhausted CD8T cells during chronic infection

Abstract IL-2 signaling at the time of CD8 T cell activation is a critical factor dictating effector and memory CD8 T cell differentiation. However, how IL-2 signaling impacts exhausted CD8 T cell fate outcomes remains poorly defined. Here, we address the role of IL-2 signaling in regulating the differentiation of the stem-like and terminally exhausted CD8 T cells. Prolonged expression of the high affinity IL-2 receptor (IL-2Ra, CD25), and resultant increase in IL-2 signaling drives terminal differentiation of effector cells during acute infection. We hypothesized that curtailing this signal would prevent terminal differentiation and promote the formation of less exhausted stem-cell like TCF-1+CD8T cells in chronic antigenic setting. Our studies show that blocking IL-2 signals at the early phase of chronic infection significantly increased the proportions of TCF-1+CD8T cells. Similarly, knocking down Il2ra augmented the development of TCF-1+CD8T cells. The therapeutic success of PD-1/PD-L1 checkpoint blockade immunotherapy (CBI) relies primarily on stem-cell like cells for vigorous expansion and effector responses. Thus, we next investigated how FACS-purified CD25Hi and CD25Lo cells adoptively transferred into infection-matched chronically infected recipients respond to PD-1 CBI. In contrast to CD25Hi cells, CD25Lo cells underwent vigorous proliferation after PD-1 CBI and produced high amount of effector molecules (GzmB, TNF-α and IFN-γ). Collectively, these data highlight the role of IL-2 signaling in the regulation of CD8 T cell exhaustion, which may be therapeutically exploited to optimize treatments of chronic infections and cancers with immune checkpoint inhibitors. Supported by NIH R21AI154363 NIH R01AI32819

Characterization of immune profile in an aging multiple sclerosis clinic population

BackgroundThere is limited data regarding adaptive immunity in older persons with Multiple Sclerosis (MS). ObjectiveThe aim of the present study was to quantify adaptive immune cells in younger (age less than 50) and older (age greater than 50) with MS in the context of clinical parameters (EDSS, 25-foot walk, SDMT). Subjects were either Untreated (no MS medications in 6 months), taking Injectables (interferons or glatiramer acetate), or Other approved MS treatments. ResultsA total of 72 subjects were enrolled (30 younger and 42 older). Older MS patients that were Untreated or taking Injectables had lower CD8 cell counts. Older MS patients demonstrated increased levels of CD4+CD25hi cells and inflammatory serum cytokines (TNF-α, IL-8). There was suggestion that MS treatments modulated IL-10. Cognition as assessed by SDMT was associated with disease duration and IL-10. ConclusionComponents of adaptive immunity are influenced by aging in MS which may also impact aspects of cognition as measured by SDMT.

Distinct roles of miR-34 family members on suppression of lung squamous cell carcinoma

miR-34, whose mimic was used on phase I clinical trial, has been extensively reported since its dysfunction in various cancers including non-small-cell lung cancer (NSCLC). However, the roles of miR-34 family members in the progression of lung squamous carcinoma (SCC) in patients who have occupational-exposure experience are unclear yet. Here, we comprehensively investigated the expression levels of miR-34 family members in SCC patients and compared the roles of them in SCC in vitro and vivo. The results showed that the average levels of miR-34a and miR-34b/c were decreased in patients. The analysis of miR-34a to miR-34b/c levels in patients graded different stages or metastases or recurrence showed that miR-34b/c was reduced earlier and more significantly than miR-34a. In vitro assays demonstrated that both miR-34a and miR-34b/c inhibits SCC cells proliferation, migration and invasion via Notch1 pathway, while miR-34b/c effects more than miR-34a does. As miR-34a was significantly decreased in cancer recurrence, the further analysis of relationship between miR-34a and stem cell adhesion molecular CD44 showed that miR-34a was significantly correlated with CD44 levels in patients. Knockdown of CD44 significantly blocked miR-34a mediated inhibition of cell migration and invasion. Treating the purified CD44hi cells with miR-34 overexpression lentivirus inhibited the tumor outgrowth. By contrast, anti-miR-34 facilitated tumor development of CD44low cells. Our study showed that miR-34 family members are negative regulator for SCC development, even though the inhibition is mediated by multiple and complicated signal pathways, which provides theoretical basis for SCC treatment and a biomarker candidate for SCC prognosis.

Daratumumab Mitigates B-Cell Receptor Signaling and Enhances Antitumor Activity of Ibrutinib in Chronic Lymphocytic Leukemia (CLL) Cells

Introduction: B-cell receptor (BCR) signaling is a central driver of proliferation in chronic lymphocytic (CLL) cells. BCR engagement leads to recruitment of several intracellular kinases such Lyn, Syk and BTK, that activate ERK, NFkB and induce CLL cell proliferation. While this pathway is effectively targeted with the BTK inhibitor, ibrutinib, various combination treatment strategies that result in greater tumor cell killing are under investigation. On B-cells, the BCR interacts with the CD19 and CD38 cell surface antigens; where the later amplifies intracellular pro-survival signal transmission through the BCR. CD38 is a multifunctional protein, which possess both enzymatic and receptor functions. As such, we hypothesized that concurrent targeting of CD38 and BTK would lead to enhanced mitigation of BCR signaling as well as direct cell death than use of either agent alone.Materials: CD19+ cells were isolated from CLL patients (n=30, collected under an IRB-approved protocol) by magnetic bead sorting. JVM13 (CD38+) and MEC1 (CD38-) cell lines were also used. CD38 expression was measured by flow cytometry with quantification of MFI and surface antibody binding (sAbc). Drug-induced antibody-dependent cellular cytotoxicity (ADCC) in target Calcein AM labeled CLL cells was assessed by co-culturing them with healthy donor PBMCs as effectors (effector: target [E:T] ratio, 50:1) and complement dependent cytotoxicity (CDC) was measured using complement-containing serum from a healthy donor. Antibody dependent cellular phagocytosis (ADCP) was measured by co-culturing calcein AM labeled CLL cells with healthy human macrophages at a 2:1 ratio. Apoptosis by annexin-V/PI dual staining along with mitochondrial outer membrane potential (MOMP) using MitoProbeTMDilC1(5) were assessed by flow cytometric analysis. Immunoblotting was performed using antibodies against Lyn, Syk, BTK, PLCγ2, ERK1/2, AKT, Ikβα, NFAT2 and their phosphorylated isoforms along with GAPDH followed by densitometry analysis.Results: Using a cutoff of 30% (gated CD38 expressing cells), we observed 26.6% (n=8) of CLL pts. to be CD38+ and 73.3% of pts. to be CD38- (n=22). As some pts. deemed CD38- had high CD38 MFI or sAbc, we applied an interquartile range (IQR with 95% confidence interval) for CD38 MFI; re-classifying pts. as CD38lo (56.75 - 199) vs. CD38hi (199 - 355). Using this system, specific-lysis of CLL cells from Dara-mediated ADCC was 23.74±2.55% vs. 14.17±1.27, CDC: 15.93±1.68% vs. 12.29±0.88, ADCP: 8.630.96% vs. 9.50±0.97% and apoptosis: 32.42±3.42 vs. 23.65±1.82 in CD38hi vs. CD38lo pts., respectively. Apoptosis was accompanied by a significant shift in MOMP [21.65±7.90% (CD38hi) vs. 32.52±7.42% (CD38lo), p<0.05]. In CLL cells treated with the combination of Ibr + Dara (ID), ADCC increased to 54.21±4.09% vs. 37.66±2.16; CDC: 34.35±2.47% vs. 28.88±1.51%, ADCP 25.56±2.30% vs. 16.69±1.97%, apoptosis: 61.63±3.14% vs 62.06±3.41% and MOMP: 66.22±3.68% vs 71.13±3.31%: in CD38hi vs. CD38lo pts., respectively. Next we examined the effect of Dara +/- Ibr on BCR signaling in CD38hi and CD38lo CLL cells. In CD38hi cells, single agent Dara significantly decreased phosphorylation Lyn, Syk, BTK and PLCγ2 as well as ERK1/2, AKT, Ikβα and NFAT; the ID combination decreasing these further (except Ikβα and NFAT2) (p<0.01). In CD38lo cells, Dara significantly downregulated phosphorylation of Lyn and PLCγ2, however, insignificant changes were noted in all other proteins. In ID combination treated CD38lo cells, Lyn, Syk, BTK, PLCγ2 and AKT were further decreased.Conclusions: Herein, we provide first evidence on the ability of Dara to modulate BCR signaling and associated proteins in primary CLL cells and that this can be further augmented with Ibr. Dara-induced protein changes were more prominent in CD38hi cells; accompanied by higher % apoptosis vs. that observed in CD38lo cells, yet the ID combination significantly enhanced mitochondrial-mediated apoptosis in both CD38hi and CD38lo cells. This indicates that the combination can potentially overcome lower induction of apoptosis and ADCC in CD38lo cells treated with single agent Dara or Ibr. Studies on how Dara and the ID combination induce direct programmed cell death (independent of modulating BCR signaling) are underway. Overall, our investigations provide rationale for clinical testing of Ibr combined with Dara in CLL. DisclosuresParikh:Pharmacyclics: Research Funding; Pharmacyclics: Honoraria; AstraZeneca: Honoraria. Ding:Merck: Research Funding. Sher:LAM Therapeutics, Inc: Research Funding. Malavasi:Jaansen Pharmaceuticals: Research Funding. Ailawadhi:Novartis: Consultancy, Honoraria; Pharmacyclics: Research Funding; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

Daratumumab Efficiently Targets NK/T Cell Lymphoma with High CD38 Expression

CD38 expression can be detected on a majority of NK/T cell lymphoma (NKTCL). Based on the clinical data of 94 patients with ENKTL, high CD38 surface expression was an adverse prognostic factor for progression free survival, and these patients had significantly inferior outcomes (Wang, 2015). This corroborates with our own studies showing that relapsed and refractory NKTCL patients almost always exhibit high CD38 expression, thereby proposing that it may be worthwhile to target CD38 as a novel therapeutic option.Daratumumab is a human anti-CD38 monoclonal antibody which has shown significant anti-cancer activity in multiple myeloma. In this study, we investigated the potential cytotoxic effects and mechanisms of actions of Daratumumab in a panel of 10 NKTCL cell lines. Firstly, we identified that cell lines can be stratified into CD38Hi, CD38Mid and CD38Lo as quantified by mRNA expression and CD38 protein surface expression which was qualitatively analysed by FACS and IHC. Daratumumab is able to induce antibody dependent cellular cytotoxicity (ADCC) in a CD38-dependent manner and in a dose-dependent fashion. We also observe a greater induction of antibody dependent cellular phagocytosis (ADCP) activation in correlation with CD38 expression. Furthermore, complement-dependent cytotoxicity (CDC) was significantly triggered by Daratumumab in CD38Hi and CD38Mid cells. However, we found evidence of an additional level of regulation provided by the expression of complement inhibitory proteins(CIPs) CD46, CD55 and CD59 on NKTCL cell lines which can limit and inhibit the induction of CDC regardless of CD38 surface expression levels.All-trans retinoic acid (ATRA) is a vitamin A derivative that binds to retinoic acid receptor alpha leading to the upregulation of CD38 expression. Treatment with ATRA increases CD38 mRNA and surface protein expression in all CD38Hi-Lo NKTCL cell lines. Interestingly CD38Lo cell lines showed the greatest fold increase of CD38 mRNA and surface protein expression. Notably, addition of ATRA to SNK-10, a CD38LoCD55Hi cell line, prior to Daratumumab treatment was able to improve the induction of ADCC but not CDC. This suggests a critical role for CIPS in mediating the induction of CDC in NKTCL.Subsequent investigation for the efficacy of Daratumumab in vivo is currently being performed.We also discovered a synergistic cytotoxic effect in NKTCL CD38Hi cell lines mediated by L-Asparaginase (a component of the current chemotherapeutic regimen in NKTCL) treatment in combination with Daratumumab. Altogether,these results propose a rationale therapeutic option for single or combined Daratumumab treatment in the clinic especially for relapsed and refractory patients. DisclosuresJin:Janssen China R&D: Employment. Yu:Janssen China R&D: Employment. Chen:Janssen China R&D: Employment. Yang:Janssen China R&D: Employment. Chng:Janssen China R&D: Research Funding.

Memory CD4 T Cells Induce Graft Versus Host Disease.

In a complete MHC mismatched allogeneic model of mouse bone marrow transplantation (BMT), naive CD4+ T cells from the C57BL/6 donor spleen combined with T cell depleted bone marrow cells induce graft versus host disease (GVHD) in lethally irradiated BALB/c hosts, leading to death within 10–20 days. This is characterized by acute injury to the large bowel with severe diarrhea. It has been reported that naive CD4+ CD62L hi CD44 lo T cells induce severe GVHD, but that effector memory CD4+ CD62Llo CD44hi T cells obtained from untreated normal donors do not induce GVHD in this model. We hypothesized that the poor GVHD-inducing capacity of effector memory cells from untreated donors may reflect their lack of previous exposure to host alloantigens. We tested this hypothesis by comparing the ability of effector memory T cells obtained from untreated donors and donors immunized to host alloantigens to induce GVHD. Donors were immunized by injecting 50 x106 host spleen cells i.p. and after one week with 10x 106 cells. We sorted naive (CD62Lhi CD44 lo) and effector memory (CD62LloCD44 hi) CD4+ T cell subsets from C57BL/6 donor mice four weeks after immunization, and compared their GVHD-inducing capacity to the same T cells subsets sorted from unimmunized C57BL/6 donors. We found that CD62Llo CD44hi cells from unimmunized donors failed to induce GVHD in 85% of the hosts over 100 days while CD62LloCD44hi cells from immunized donors caused progressive weight loss and death in 100% of hosts (p <0.001). Whereas naive CD4+ Tcells from unimmunized donors responded vigorously to host stimulator cells in the mixed leukocyte reaction (MLR), and accumulated rapidly in the lymph nodes and spleen of irradiated hosts, effector memory CD4+ T cells had markedly reduced activities in these assays. We are currently comparing the activity of memory CD4+ T cells from immunized and unimmunized donors in these assays. In conclusion, memory CD4+ T cells from donors immunized to host alloantigens are able to induce lethal GVHD, but memory cells from unimmunized donors were not. We are testing whether the increased potency of memory T cells from immunized donors is due to their increased reactivity to alloantigens and increased ability to accumulate in the host target tissues of GVHD.

A method for expansion and retroviral transduction of mouse regulatory T cells

Adoptive cell therapy with genetically modified regulatory T cells (Tregs) is under clinical investigation for the treatment of transplant rejection and various autoimmune conditions. A limitation of modelling this approach in mice is the lack of optimized protocols for expanding and transducing mouse Tregs. Here we describe a protocol for purifying, expanding and retrovirally transducing mouse Tregs with a vector encoding a chimeric antigen receptor as a model transgene. We found that isolation of Tregs from C57Bl/6J Foxp3EGFP mice solely based on eGFP expression resulted in sufficiently pure cells; co-sorting of CD25hi cells was not essential. Although expansion with rapamycin reduced Treg expansion, it promoted maximal in vitro suppressive activity. Retroviral transduction of Tregs following 2 days of stimulation with anti-CD3/CD28 beads achieved a transduction efficiency of ~40% and did not impair their suppressive capacity. When injected into a conventional T cell (Tconv)-transfer-induced colitis model, transduced Tregs inhibited colitis progression at ratios as low as 1 Treg to 100 Tconvs, and maintained Foxp3 and transgene expression throughout an 8-week period. This method facilitates the study of transduced Tregs in animal models and will enable the study of genetically engineered Treg therapy for a variety of inflammatory diseases.

CXCL12γ induces human prostate and mammary gland development.

Epithelial stem cells (ESCs) demonstrate a capacity to maintain normal tissues homeostasis and ESCs with a deregulated behavior can contribute to cancer development. The ability to reprogram normal tissue epithelial cells into prostate or mammary stem-like cells holds great promise to help understand cell of origin and lineage plasticity in prostate and breast cancers in addition to understanding normal gland development. We previously showed that an intracellular chemokine, CXCL12γ induced cancer stem cells and neuroendocrine characteristics in both prostate and breast adenocarcinoma cell lines. However, its role in normal prostate or mammary epithelial cell fate and development remains unknown. Therefore, we sought to elucidate the functional role of CXCL12γ in the regulation of ESCs and tissue development. Prostate epithelial cells (PNT2) or mammary epithelial cells (MCF10A) with overexpressed CXCL12γ was characterized by quantitative real-time polymerase chain reaction, Western blots, and immunofluorescence for lineage marker expression, and fluorescence activated cell sorting analyses and sphere formation assays to examine stem cell surface phenotype and function. Xenotransplantation animal models were used to evaluate gland or acini formation in vivo. Overexpression of CXCL12γ promotes the reprogramming of cells with a differentiated luminal phenotype to a nonluminal phenotype in both prostate (PNT2) and mammary (MCF10A) epithelial cells. The CXCL12γ-mediated nonluminal type cells results in an increase of epithelial stem-like phenotype including the subpopulation of EPCAMLo /CD49fHi /CD24Lo /CD44Hi cells capable of sphere formation. Critically, overexpression of CXCL12γ promotes the generation of robust gland-like structures from both prostate and mammary epithelial cells in in vivo xenograft animal models. CXCL12γ supports the reprogramming of epithelial cells into nonluminal cell-derived stem cells, which facilitates gland development.

P0336INHIBITION C-C CHEMOKINE RECEPTOR TYPE 2 (CCR2) ON ACTIVATED KIDNEY PARIETAL EPITHELIAL CELLS REVEALS A NEW THERAPEUTIC APPROACH FOR CHRONIC KIDNEY DISEASES

Abstract Background and Aims We have recently demonstrated that small molecule inhibitors of CCR2 rapidly reduced proteinuria and preserved renal structure in animal models of diabetic nephropathy (DN) and focal segmental glomerular sclerosis (FSGS). Similar beneficial effects of CCR2 inhibition were also observed in DN patients in a large diabetic nephropathy Phase 2 clinical trial. Despite its rapid and robust effects in these kidney diseases, the mechanism by which CCR2 inhibition affords such renal protection remains unclear. CCR2 is well-known to be expressed on cells of the immune system, where it mediates inflammation and immunological functions by controlling the trafficking of monocytes and T cells. However, immune cell expression of CCR2 may not adequately explain the rapid pharmacologic benefits of CCR2 inhibitors in DN and FSGS because these diseases do not exhibit appreciable kidney inflammation. To better understand the protective mechanism(s) of CCR2 inhibition in renal diseases, we have used a variety of in vivo nephropathy models to determine if CCR2 is expressed on kidney parenchymal cells. Our results suggest that a subset of CD45 negative, renal parietal epithelial cells express CCR2. Method To characterize CCR2 expression in the kidney we used CCR2 RFP heterozygous mice (CCR2RFP/+) in which one copy of CCR2 was replaced by red fluorescent protein (RFP), whose expression is under the control of the endogenous CCR2 promoter. This approach provided a highly sensitive method for CCR2 detection by both flow cytometry and immunofluorescence, and compensated for the lack of validated, commercially available anti-CCR2 antibodies. Enriched glomerular cells were obtained by a non-enzymatic disruption of kidneys, followed by centrifugation at low speed (80gs), and sieving through 300µm and 150µm meshes. The Adriamycin (ADR)-nephropathy model with CCR2 RFP heterozygous mice was used to determine CCR2 expression on renal cells. 5/6 nephrectomy models of FSGS, as well as the db/db diabetic nephropathy model were used to access the change of activated parietal epithelial cells and proteinuria, both in the presence and absence of a CCR2 inhibitor in the setting of kidney injury. Results We observed dramatic increases in the number of activated parietal epithelial cells (PECs, identified as CD133+CD24+CD44hi cells by flow cytometry) in all three renal disease models. In the ADR nephropathy model, we found that in 30-40% of activated PEC cells within an enriched population of glomerular cells expressed CCR2/RFP. CD44hi RFP+ increased much more dramatically than CD44hi RFP- cells in response to ADR, and this was highly correlated with proteinuria, suggesting that CCR2 plays an important role in PEC cell activation and kidney pathology in ADR induced kidney injury. Furthermore, we found that blocking CCR2 with the CCR2-specific antagonist CCX872 significantly reduced both proteinuria and the number of activated PECs in the 5/6 nephropathy and db/db diabetic nephropathy models. Conclusion This is the first report that CCR2 is present on a subset of activated renal PECs. This population is markedly upregulated during kidney injury in a variety of murine models of nephropathy. Changes in the abundance CCR2+ activated PECs in the glomerular enriched preparations in response to CCR2 inhibition suggests that CCR2 antagonism may represent a novel approach for the treatment of chronic kidney disease.

Role of CD44 in tumor-initiating cells of salivary gland pleomorphic adenoma: More than a surface biomarker.

CD44, a cell-surface glycoprotein, functions as a receptor for hyaluronic acid. Our research group has previously shown that CD44 is a biomarker for the CD44hi cells (tumor-initiating cells; TICs) in murine salivary gland tumors. However, little is known concerning the biological roles of CD44 in the tumorigenesis of pleomorphic adenoma. The present study is aimed to investigate the effects of CD44 on the proliferation, invasive capability, and apoptosis of TICs in vitro, as well as the tumorigenicity of TICs in vivo. The results demonstrated that knockdown of CD44 attenuated the malignant phenotype of TICs. Furthermore, in vivo xenograft studies indicated that CD44 knockdown inhibited tumorigenesis of pleomorphic adenoma. In addition, neither the CD44low cells nor the CD44-modified CD44low cells developed neo-tumors, which indicated that overexpression of CD44 did not enable the CD44low cells to be transformed into TICs. Taken together, these data demonstrate that CD44 not only acts as a biomarker, but also functions as a key player in the tumor-initiating capacity of TICs. These results shed light on the pathogenesis of salivary gland tumors and provide a potential therapeutic target for treating pleomorphic adenoma.

PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1+CD38hi cells and anti-PD-1 resistance.

Understanding resistance to antibody to programmed cell death protein 1 (PD-1; anti-PD-1) is crucial for the development of reversal strategies. In anti-PD-1-resistant models, simultaneous anti-PD-1 and vaccine therapy reversed resistance, while PD-1 blockade before antigen priming abolished therapeutic outcomes. This was due to induction of dysfunctional PD-1+CD38hi CD8+ cells by PD-1 blockade in suboptimally primed CD8 cell conditions induced by tumors. This results in erroneous T cell receptor signaling and unresponsiveness to antigenic restimulation. On the other hand, PD-1 blockade of optimally primed CD8 cells prevented the induction of dysfunctional CD8 cells, reversing resistance. Depleting PD-1+CD38hi CD8+ cells enhanced therapeutic outcomes. Furthermore, non-responding patients showed more PD-1+CD38+CD8+ cells in tumor and blood than responders. In conclusion, the status of CD8+ T cell priming is a major contributor to anti-PD-1 therapeutic resistance. PD-1 blockade in unprimed or suboptimally primed CD8 cells induces resistance through the induction of PD-1+CD38hi CD8+ cells that is reversed by optimal priming. PD-1+CD38hi CD8+ cells serve as a predictive and therapeutic biomarker for anti-PD-1 treatment. Sequencing of anti-PD-1 and vaccine is crucial for successful therapy.

The skewed frequency of B-cell subpopulation CD19+ CD24 hi CD38 hi cells in peripheral blood mononuclear cells is correlated with the elevated serum sCD40L in patients with active systemic lupus erythematosus.

CD19+ CD24hi CD38hi cells play an essential role in maintaining immune homeostasis. CD40 signaling is involved in regulating the induction and function of CD19+ CD24hi CD38hi cells. Changes in B-cell subpopulations and CD19+ CD24hi CD38hi cells have been observed in systemic lupus erythematosus (SLE) patients. Whether changes in the B-cell subpopulation are related to the aberrant CD40 signaling in SLE patients remains unclear. In this study, we examined changes in the levels of CD19+ CD24hi CD38hi cells and CD19+ CD24hi CD38low cells in peripheral blood mononuclear cells and the serum level of soluble CD40 ligand (sCD40L) in 30 patients with SLE. Through routine biochemical assays and flow cytometry assay, we found that (1) the CD19+ CD24hi CD38hi cell subset was upregulated in SLE patients compared to that in healthy controls (HCs) (P < 0.05); (2) the CD19+ CD24hi CD38low cell subset was downregulated in SLE patients compared with that in HCs; and (3) CD38 expression was positively correlated with SLE manifestations and the serum sCD40L level (P < 0.05). In conclusion, the relative level of Bregs is significantly higher in SLE patients than in HCs and is positively correlated with disease activity and sCD40L level.

Increased Numbers of CD4+CD25+ and CD8+CD25+ T-Cells in Peripheral Blood of Patients with Rheumatoid Arthritis with Parvovirus B19 Infection.

To investigate T-cell subpopulations in peripheral blood of human parvovirus B19 DNA-positive (B19+) and -negative (B19-) patients with rheumatoid arthritis (RA) and healthy persons. Blood samples were collected from 115 patients with RA and 47 healthy volunteers; 27 patients with RA and nine controls were B19+ Cluster of differentiation (CD) 4, 8, 25 and 45RA were analyzed on blood cells. CD25 expression on CD4+CD45RA+, CD4+CD45RA-, CD8+CD45RA+, CD8+CD45RA- subsets were analyzed by flow cytometry. The percentage of CD25low and CD25hi cells was increased on CD4+CD45RA+, CD4+CD45RA- T-cells and the percentage of CD25+ cells was increased on CD8+CD45RA+, CD8+CD45RA- T-cells of B19+ patients with RA in comparison with B19- patients and controls. Raised levels of CD4 and CD8 regulatory T-cells in B19+ RA patients could cause down-regulation of antiviral clearance mechanisms and lead to activation of persistent human parvovirus B19 infection in patients with RA.

Human B-1 and B-2 B Cells Develop from Lin-CD34+CD38lo Stem Cells.

The B-1 B cell population is an important bridge between innate and adaptive immunity primarily because B-1 cells produce natural Ab. Murine B-1 and B-2 cells arise from distinct progenitors; however, in humans, in part because it has been difficult to discriminate between them phenotypically, efforts to pinpoint the developmental origins of human B-1 and B-2 cells have lagged. To characterize progenitors of human B-1 and B-2 cells, we separated cord blood and bone marrow Lin-CD34+ hematopoietic stem cells into Lin-CD34+CD38lo and Lin-CD34+CD38hi populations. We found that transplanted Lin-CD34+CD38lo cells, but not Lin-CD34+CD38hi cells, generated a CD19+ B cell population after transfer into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1wjl/SxJ neonates. The emergent CD19+ B cell population was found in spleen, bone marrow, and peritoneal cavity of humanized mice and included distinct populations displaying the B-1 or the B-2 cell phenotype. Engrafted splenic B-1 cells exhibited a mature phenotype, as evidenced by low-to-intermediate expression levels of CD24 and CD38. The engrafted B-1 cell population expressed a VH-DH-JH composition similar to cord blood B-1 cells, including frequent use of VH4-34 (8 versus 10%, respectively). Among patients with hematologic malignancies who underwent hematopoietic stem cell transplantation, B-1 cells were found in the circulation as early as 8 wk posttransplantation. Altogether, our data demonstrate that human B-1 and B-2 cells develop from a Lin-CD34+CD38lo stem cell population, and engrafted B-1 cells in humanized mice exhibit an Ig-usage pattern comparable to B-1 cells in cord blood.

Elevated level of circulating CD4+Helios+FoxP3+ cells in primary Sjogren’s syndrome patients

Objective: This study was designed to investigate the expression of regulatory T cells in primary Sjögren’s Syndrome (pSS) and to evaluate the clinical role of CD4 + Helios+ FoxP3+ cells in pSS patients.Methods: CD4 + FoxP3+ T cells in the peripheral blood of 39 pSS patients and 30 healthy controls were measured by flow cytometry and CD25 and Helios expression were also analyzed. The repression ability of CD4 + CD25hi cells was tested in vitro. Clinical information of pSS patients was retrospectively collected and their correlations with circulating Treg cells were analyzed. Cytokine levels in plasma were measured by ELISA and correlations with Helios+ FoxP3+ cells were also detected.Results: Circulating FoxP3+ and Helios+ FoxP3+ cells were elevated in pSS patients compared with controls. The suppression function of CD4 + CD25hi cells is not different between two groups. There are inverse correlations between Helios+ FoxP3+ percentage and ESR, IgG, IgM and ESSDAI. Anti-SSB− patients possess higher level of Helios+ FoxP3+ cells than anti-SSB+ patients. IL-6, IFNγ and IFNα levels were increased in pSS plasma and there were positive correlations between the levels of IFNγ/IFNα and percentage of Helios+ FoxP3+ cells.Conclusion: Circulating Helios + FoxP3+ cells were elevated in pSS patients and may contribute to suppressing autoimmunity in pSS patients.