Immunophenotypic Analysis Research Articles

Abstract P1075: Igf-1 Signaling And Nuclear Localization Of Pdgfralpha Tyrosine Kinase Activity Control The Fibrotic Response Of Mesenchymal Stromal Cells To Myocardial Ischemia

Background and Hypothesis: The role of mesenchymal stromal cells expressing the platelet-derived growth factor alpha (PDGFRα + MSCs) has been associated with fibro-adipogenic pathologies. Based on our previous data in regenerating skeletal muscle and the data herein described, we hypothesized that specific and distinct mesenchymal stromal cells exist in the heart, which will retain regenerative capacities by modulating the spatiotemporal distribution of the PDGFRα tyrosine kinase activity and the re-expression of specific regenerative molecules. Methods: Myocardial ischemia/reperfusion (I/R) was induced in mice with standard procedures and in compliance with the regulations of the NIH and the local institutional animal care. Results: Confocal imaging and immunophenotyping analyses showed that cardiac PDGFRα + MSCs exist as two distinct sub-populations expressing markers of specialized and differentiated cells, such as endothelial (60.3%±7.9% expressing CD31) and cardiomyocyte cell-cell anchoring markers (89%± 0.6% of population expressing desmocollin 2/3), while also retaining the expression of mesenchymal markers. Importantly, whereas PDGFRα + MSCs organized in vessel-like structures expressing CD31 during the acute phase after I/R, they transitioned into myofibroblast-like cells in ischemic subacute conditions. Identifying the mechanisms regulating PDGFRα + MSCs dual function remains a critical roadblock. In addressing this gap, we discovered that the transition to myofibroblast-like cells was associated to nuclear shuttling of PDGFRα C-terminal tyrosine kinase domain and ligand-dependent increased nuclear area, suggesting receptor-mediated activation of chromatin remodeling. Furthermore, we discovered that therapeutic re-expression of downregulated paracrine factors, such as the IGF-1Ea splice variant, correlated with long-term organization of PDGFRα + /CD31 + in small capillaries, overcoming the late ischemic transition to fibrotic cells. Conclusions: Taken together, these data suggest that by modulating PDGFRα intracellular shuttling or altering the expression of regenerating genes, may therapeutically direct PDGFRα + MSCs duality to reduce fibrosis, while harnessing their beneficial properties.

Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks

Due to their immunomodulatory properties and in vitro differentiation ability, human mesenchymal stromal cells (hMSCs) have been investigated in more than 1000 clinical trials over the last decade. Multiple studies that have explored the development of gene-modified hMSC-based products are now reaching early stages of clinical trial programmes. From an engineering perspective, the challenge lies in developing manufacturing methods capable of producing sufficient doses of ex vivo gene-modified hMSCs for clinical applications. This work demonstrates, for the first time, a scalable manufacturing process using a microcarrier-bioreactor system for the expansion of gene-modified hMSCs. Upon isolation, umbilical cord tissue mesenchymal stromal cells (UCT-hMSCs) were transduced using a lentiviral vector (LV) with green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) transgenes. The cells were then seeded in 100 mL spinner flasks using Spherecol microcarriers and expanded for seven days. After six days in culture, both non-transduced and transduced cell populations attained comparable maximum cell concentrations (≈1.8 × 105 cell/mL). Analysis of the culture supernatant identified that glucose was fully depleted after day five across the cell populations. Lactate concentrations observed throughout the culture reached a maximum of 7.5 mM on day seven. Immunophenotype analysis revealed that the transduction followed by an expansion step was not responsible for the downregulation of the cell surface receptors used to identify hMSCs. The levels of CD73, CD90, and CD105 expressing cells were above 90% for the non-transduced and transduced cells. In addition, the expression of negative markers (CD11b, CD19, CD34, CD45, and HLA-DR) was also shown to be below 5%, which is aligned with the criteria established for hMSCs by the International Society for Cell and Gene Therapy (ISCT). This work provides a foundation for the scalable manufacturing of gene-modified hMSCs which will overcome a significant translational and commercial bottleneck.Key points• hMSCs were successfully transduced by lentiviral vectors carrying two different transgenes: GFP and VEGF• Transduced hMSCs were successfully expanded on microcarriers using spinner flasks during a period of 7 days• The genetic modification step did not cause any detrimental impact on the hMSC immunophenotype characteristics

Distribution of peripheral blood mononuclear cell subtypes in patients with West syndrome: Impact of synacthen treatment

BackgroundWest Syndrome (WS) is an epileptic encephalopathy that typically occurs in infants and is characterized by hypsarrhythmia, infantile spasms, and neurodevelopmental impairment. Demonstration of autoantibodies and cytokines in some WS patients and favorable response to immunotherapy have implicated inflammation as a putative trigger of epileptiform activity in WS. Our aim was to provide additional support for altered inflammatory responses in WS through peripheral blood immunophenotype analysis. MethodsEight WS cases treated with synacthen and 11 age- and sex-matched healthy volunteers were included. Peripheral blood mononuclear cells (PBMC) were isolated and immunophenotyping was performed in pre-treatment baseline (8 patients) and 3 months post-treatment (6 patients) samples. The analysis included PBMC expressing NFκB transcription and NLRP3 inflammasome factors. ResultsIn pre-treatment baseline samples, switched memory B cells (CD19+IgD−CD27+) were significantly reduced, whereas plasma cells (CD19+CD38+CD138+) and cytotoxic T cells (CD3+CD8+) were significantly increased. Regulatory T and B cell ratios were not significantly altered. Synacthen treatment only marginally reduced helper T cell ratios and did not significantly change other T, B, NK and NKT cell and monocyte ratios. ConclusionsOur findings lend further support for the involvement of inflammation-related mechanisms in WS. New-onset WS patients are inclined to display increased plasma cells in the peripheral blood. Synacthen treatment does not show a beneficial effect on most effector acquired and innate immunity subsets.

Predicting the clinical efficacy of JAK inhibitor treatment for patients with rheumatoid arthritis based on Fas+ T cell subsets.

Rheumatoid arthritis (RA) is a common autoimmune disease. Janus kinase inhibitors (JAKi) have been approved for the treatment of RA; however, the impact of JAKi on immune cells remains inconclusive. This study investigated the response of immune cells to JAKi treatment to identify biomarkers by which to evaluate and predict clinical outcomes. Blood samples were collected from RA patients before and after JAKi treatment for the analysis of immunophenotypes. Our results revealed that JAKi mainly inhibited Fas+ T cell subsets. The percentage changes of Th Fas+ and Naive Th Fas+ cells were positively correlated with the 28-joint Disease Activity Score with erythrocyte sedimentation rate (DAS28-ESR) values. Following treatment, moderate response (MR) RA patients presented a decrease in the number of Naive Th Fas+ cells (p = 0.0001). Initial percentages of 14 T cell and 20 B cell subsets were correlated with percentage changes in DAS28-ESR. Overall, 16 cell subsets presented significant differences between the non-response (NR) and MR groups. Excluding the multicollinearity of the immune cells, we constructed a JAKi treatment response prediction index (JRPI) using 5 subsets of T/B cells, the results of which were strongly correlated with percentage changes in DAS28-ESR (receiver operating characteristic curve of 1). Note that the NR group was clearly distinguished from the MR group (p = 0.0167). In conclusion, the efficacy of JAKi can be attributed mainly to the suppression of Fas+ T cell subsets. A positive correlation was shown between the therapeutic efficacy of JAKi and the percentage changes in both Th Fas+ cells and Naive Th Fas+ cells. Furthermore, the proposed JRPI could potentially be used as an indicator to predict the efficacy of JAKi prior to treatment in RA patients. These findings may contribute to the development of personalized treatment strategies for RA patients using JAKi.

RWTC-MBTA: autologous vaccine prevents metastases via antitumor immune responses

BackgroundAutologous tumor cell-based vaccines (ATVs) aim to prevent and treat tumor metastasis by activating patient-specific tumor antigens to induce immune memory. However, their clinical efficacy is limited. Mannan-BAM (MB), a pathogen-associated molecular pattern (PAMP), can coordinate an innate immune response that recognizes and eliminates mannan-BAM-labeled tumor cells. TLR agonists and anti-CD40 antibodies (TA) can enhance the immune response by activating antigen-presenting cells (APCs) to present tumor antigens to the adaptive immune system. In this study, we investigated the efficacy and mechanism of action of rWTC-MBTA, an autologous whole tumor cell vaccine consisting of irradiated tumor cells (rWTC) pulsed with mannan-BAM, TLR agonists, and anti-CD40 antibody (MBTA), in preventing tumor metastasis in multiple animal models.MethodsThe efficacy of the rWTC-MBTA vaccine was evaluated in mice using breast (4T1) and melanoma (B16-F10) tumor models via subcutaneous and intravenous injection of tumor cells to induce metastasis. The vaccine’s effect was also assessed in a postoperative breast tumor model (4T1) and tested in autologous and allogeneic syngeneic breast tumor models (4T1 and EMT6). Mechanistic investigations included immunohistochemistry, immunophenotyping analysis, ELISA, tumor-specific cytotoxicity testing, and T-cell depletion experiments. Biochemistry testing and histopathology of major tissues in vaccinated mice were also evaluated for potential systemic toxicity of the vaccine.ResultsThe rWTC-MBTA vaccine effectively prevented metastasis and inhibited tumor growth in breast tumor and melanoma metastatic animal models. It also prevented tumor metastasis and prolonged survival in the postoperative breast tumor animal model. Cross-vaccination experiments revealed that the rWTC-MBTA vaccine prevented autologous tumor growth, but not allogeneic tumor growth. Mechanistic data demonstrated that the vaccine increased the percentage of antigen-presenting cells, induced effector and central memory cells, and enhanced CD4+ and CD8+ T-cell responses. T-cells obtained from mice that were vaccinated displayed tumor-specific cytotoxicity, as shown by enhanced tumor cell killing in co-culture experiments, accompanied by increased levels of Granzyme B, TNF-α, IFN-γ, and CD107a in T-cells. T-cell depletion experiments showed that the vaccine’s antitumor efficacy depended on T-cells, especially CD4+ T-cells. Biochemistry testing and histopathology of major tissues in vaccinated mice revealed negligible systemic toxicity of the vaccine.ConclusionThe rWTC-MBTA vaccine demonstrated efficacy in multiple animal models through T-cell mediated cytotoxicity and has potential as a therapeutic option for preventing and treating tumor metastasis with minimal systemic toxicity.

Expression profile of HLA-DRB1, RFX5, and CIITA promoters in chronic kidney disease patients from South India

Background The present study elucidated HLA-DRB1 allele frequency, the gene expression profile of HLA-DRB1, CIITA promoters PI, PIV, and RFX5 and their association in chronic kidney disease (CKD). Patients and methods In all, 133 CKD patients and 144 healthy controls were enrolled, and qRT-PCR based expression analysis of HLA-DRB1, CIITA-PI, PIV, and RFX5 promoters was carried out. The typing of HLA-DRB1* alleles was performed by the PCR-SSP method. The immune cell profiling was performed by flow cytometry. Results Out of the 13 HLA-DRB1 alleles genotyped, increased frequencies for DRB1*07 [odds ratio (OR)=2.103] and DRB1*12 (OR=2.50) and decreased frequency for DRB1*10 (OR=0.455) in CKD patients were observed. HLA-DRB1 expression was significantly upregulated in pooled-CKD (Fc: 1.49 ± 0.21; P<0.0001), DRB1*07 (Fc: 3.10 ± 0.70; P<0.057), and DRB1*12 (Fc: 3.62 ± 0.74; P<0.0001) positive CKD patients. Significantly higher levels of expressions were observed for CIITA-PI (Fc: 2.35 ± 0.23; P<0.0005) and PIV (Fc: 1.76 ± 0.23; P<0.0009) in pooled-CKD patients. With HLA-DRB1 alleles, a higher level of expressions of CIITA-PIV was observed in patients with DRB1*12 (Fc: 1.45 ± 0.38; P<0.007). Interestingly, a significantly downregulated expression was observed for CIITA-PIV in patients heterozygous for DRB1*12 (2.15 ± 0.24 vs. 0.16 ± 0.82; P<0.017). An upregulated RFX5 expression was observed for pooled-CKD (Fc: 1.37 ± 0.17; P<0.0001) and DRB1*12 (1.40 ± 0.34; P<0.045) positive patients. Immunophenotyping analysis showed an increased CD3+ and decreased CD19+, CD4+,and CD8+ cell populations in CKD patients compared with controls. Conclusion The study confirmed the increased expression of CIITA-PI, PIV promoters, and RFX5 that in turn led to the upregulation of the DRB1 gene resulting in CKD. Thus, the study concluded the positive association of HLA-DRB1*07 and DRB1*12 alleles, with a differential expression of DRB1 genes as a consequence of upregulation of respective promoters in CKD pathogenesis in South India.

Cellular Immune Responses in Lymphoid Tissues of Broiler Chickens Experimentally Infected with Necrotic Enteritis-Producing Clostridium perfringens Strains.

Host cellular responses against Clostridium perfringens (CP), the causative agent of necrotic enteritis (NE) in chickens, are poorly understood. In the present study, we first tested the NE-producing ability of seven netB+ CP strains (CP5, CP18, CP26, CP64, CP67, CP68, and NCNE-1), using an experimental infection model of broiler chickens. Evaluation of intestinal gross lesions showed that all the strains, except CP5, were able to produce NE, while CP26 and CP64 strains produced relatively more severe lesions when compared with other groups. Next, cellular responses in the cecal tonsil (CT), bursa of Fabricius, and spleen were evaluated in chickens infected with strains representing variation in the level of virulence, namely, avirulent CP5, virulent CP18, and a relatively more virulent CP26 strain. Immunophenotyping analysis showed that CT or splenic macrophage frequencies were significantly higher in CP18- and CP26-infected chickens compared with uninfected controls, while the frequencies of γδ T-cells and B-cells in the CT of CP26-infected chickens were significantly higher than those in the uninfected, CP5- or CP18-infected groups. The T-cell analysis showed that chickens infected with CP18 and CP26 had a significantly higher number of splenic CD4+ and CD8+ T-cells expressing CD44 and CD28 activation molecules, while CP26-infected chickens also had significantly increased CT frequency of these activated CD4+ and CD8+ T-cells when compared with uninfected or CP5-infected groups. Collectively, our findings suggested that cellular responses, including activation of T-cells, are selectively induced against virulent CP strains and that the NE-producing characteristics of this pathogen may influence the outcome of immunity to NE.

Oligosymptomatic long-term carriers of SARS-CoV-2 display impaired innate resistance but increased high-affinity anti-spike antibodies

The vast spectrum of clinical features of COVID-19 keeps challenging scientists and clinicians. Low resistance to infection might result in long-term viral persistence, but the underlying mechanisms remain unclear. Here, we studied the immuneresponse of immunocompetent COVID-19 patients with prolonged SARS-CoV-2 infection by immunophenotyping, cytokine and serological analysis. Despite viral loads and symptoms comparable to regular mildly symptomatic patients, long-term carriers displayed weaker systemic IFN-I responses and fewer circulating pDCs and NK cells at disease onset. Type 1 cytokines remained low, while type-3 cytokines were in turn enhanced. Of interest, we observed no defects in antigen-specific cytotoxic Tcell responses, and circulating antibodies displayed higher affinity against different variants of SARS-CoV-2 Spike protein in these patients. The identification of distinct immune responses in long-term carriers adds up to our understanding of essential host protective mechanisms to ensure tissue damage control despite prolonged viral infection.

Development of Stable CHO-K1 Cell Lines Overexpressing Full-Length Human CD20 Antigen.

CD20 is a differentiation-related antigen exclusively on the membrane of B lymphocytes. CD20 amplification is observed in numerous immune-related disorders, making it an ideal target for immunotherapy of hematological malignancies and autoimmune diseases. MAb-based therapies targeting CD20 have a principal role in the treatment regimens. Fc gamma receptors mediate CD20 internalization in hematopoietic cells; therefore, this study aimed to establish non-hematopoietic stable cell lines overexpressing full-length human CD20 antigen as an in vitro model for CD20-related studies. CD20 gene was cloned into the transfer vector. The lentivirus system was transfected to packaging HEK 293T cells, and the supernatants were harvested. CHO-K1 cells were transduced using recombinant viruses, and a stable cell pool was developed by the antibiotic selection. CD20 expression was confirmed at the mRNA and protein levels. Simultaneous expression of GFP protein facilitated the detection of CD20-expressing cells. Immunophenotyping analysis of stable clones demonstrated expression of CD20 antigen. In addition, the mean fluorescence intensity was significantly higher in the CD20-CHO-K1 clones than the wild-type CHO-K1 cells. This study is the first report on using second-generation lentiviral vectors for the establishment of a non-hematopoietic cell-based system, which stably expresses full-length human CD20 antigen. Results of stable CHO cell lines with different levels of CD20 antigen are well suited to be used for CD20-based investigations, including binding and functional assays.

MDB-25. LARGE CELL/ANAPLASTIC MEDULLOBLASTOMA REPRESENTS MORE THAN ONE DISEASE ENTITY

Abstract Medulloblastoma (MB) with predominant large cell or anaplastic cytological features have been identified to be associated to an aggressive clinical course and poor survival. Because cases can show mixed cytologies, these histologically defined MB types were merged in the recent WHO classification of tumors of the CNS to large cell/anaplastic (LC/A) MB. In this study, we collected FFPE samples of 137 LC/A MB of all age groups (0-39 years) diagnosed at the DGNN Brain Tumor Reference Center in Bonn, Germany, and performed a comprehensive histological, immunophenotypical, genetic and epigenetic analysis by next-generation panel sequencing (NGS), 450k/850k methylation bead-array hybridization and molecular inversion probe (MIP) technology. 73% of cases showed predominant anaplastic histology, 27% predominant large cell histology. Methylation-based subtyping and sequencing showed that the cohort of LC/A MB represented different biological entities: 6% WNT-MB, 7% SHH-MB-TP53-wildtype, 10% SHH-MB-TP53-mutant, and 77% non-WNT/non-SHH MB (with predominant epigenetic subtype II). High-resolution copy number profiling by MIP demonstrated MYC amplification in 40% of non-WNT/non-SHH MB. MYC amplification was highly associated to infant disease and predominant large cell histology. On the contrary, predominant anaplastic histology was highly associated to non-infant disease, MYCN amplification and SHH activation. By NGS we detected recurrent mutations in 37 MB-related genes; the frequency of mutations of individual MB-related genes differed significantly between the histologically and genetically defined MB types. Our data clearly demonstrate that large cell MB and anaplastic MB represent different biological entities. Therefore, and also in respect to the future development of targeted treatment approaches to these highly malignant MB types, they should be precisely diagnosed as separate MB entities.

CD81 and Its Relationship to Treatment Response in Patients With Acute Myeloid Leukemia at a Hospital in Hanoi, Vietnam.

Acute myeloid leukemia (AML) has the proliferation of poorly differentiated immature myeloid cells. New studies on immune markers also consider them as one of the factors that affect the prognosis or the patient's ability to respond to drugs. Our study was designed to determine the rate of remission and mortality, and the ability to respond to drugs in newly diagnosed AML patients withpositive CD81. A total of 50 patients diagnosed with AML (excluding acute promyelocytic leukemia) underwent immunophenotyping analysis using flow cytometry. Following the initial diagnosis, the patients received induction therapy, followed by three cycles of consolidation therapy. The patients were then followed up for a period of six months. The treatment efficacy was assessed at two timepoints: on day 28 after the first chemotherapy course and on day 28 after the fourth chemotherapy course. Out of the 50 newly diagnosed AML patients, 40 (80%) were found to be CD81 positive. This CD81-positive group had a high mortality rate after the first course of chemotherapy (17.5%)and after the fourth course of chemotherapy(52.5%), while no patients died in the CD81-negative group. The CD81-positive group had a worse drug response rate with 22.5% and 18.2% in CD81 positive group versus 30% and 40% in the CD81-negative group achieving complete remission after the first course and fourth course, respectively. The CD81 immunological marker was found to be highly prevalent among AML patients in Vietnam. Overexpression of CD81 in patients with AML is associated with an unfavorable prognosis, characterized by higher mortality rates and poorer treatment response.

Plasma Cell Leukemia-Clinicopathological Profile from a Tertiary Care Center in Western India.

Poornima ManimaranIntroduction Plasma cell leukemia (PCL) is very uncommon and aggressive neoplasm constituting 2 to 4% of all plasma cell dyscrasias. By definition, clonal plasma cells should make up 20% of peripheral blood or have an absolute plasma cell count of 2 × 10 9 cells/cu.mm. PCL can be primary or secondary. In this study, the clinicohematological features of PCL, and correlation of immunophenotypic profile and conventional therapies with overall survival was analyzed. Materials and Methods This retrospective study involved PCL patients who were diagnosed across a 12-year period, from 2010 to 2021, at a tertiary care center in western India. Clinical, biochemical, peripheral smear, bone marrow aspirate, immunophenotyping, and molecular analysis were performed. Results Total 39 PCL patients were included in the study among which 36 were primary PCL patients. Splenomegaly (10/27), hepatomegaly (6/26), and lymphadenopathy (5/23) were noted. At presentation, all patients had anemia (<11g/dL), thrombocytopenia (33/39), hypercalcemia (>11mg/dl) 10/33 (30.3%) and lytic lesions was noted in 18/26 (69.2%). Immunophenotype of these patients showed CD 38 positivity, CD 138 positivity, CD56 positivity, and CD 117 negativity were 100, 62, 41.6, and 89%, respectively. Overall survival of our patients was 4.1 months and overall survival of patients treated with VTD (bortezomib, thalidomide, dexamethasone) and VCD (bortezomib, cyclophosphamide, dexamethasone) regimen was 3.4 and 4.1 months, respectively, which was not statically significant ( p -value 0.816). CD117 and CD56 markers were also not having any prognostic significance ( p -value 1.000 and 0.873, respectively). Conclusion Because of rarity of the disease, prospective studies are very limited and hence management and outcome of the disease are difficult to analyze. The current treatment protocols have no survival advantage and hence newer therapeutic approach is mandatory to attain better outcome.

Phase 1 dose escalation study of SL-172154 (SIRPα-Fc-CD40L) in platinum-resistant ovarian cancer.

5544 Background: SIRPα-Fc-CD40L is a hexameric, bi-functional fusion protein consisting of SIRPα linked to CD40L via an inert Fc linker. This molecule competitively inhibits CD47 to enhance tumor cell phagocytosis and activates CD40 to increase antigen processing and cross-presentation by antigen presenting cells (APCs) to CD8 T cells, thus bridging innate and adaptive immunity. Methods: This first-in-human, Phase 1 dose escalation study evaluated SL-172154 in patients (pts) with advanced platinum resistant ovarian, fallopian tube and primary peritoneal cancers (PROC). SL-172154 was administered intravenously across 5 dose levels (0.1, 0.3, 1.0, 3.0, 10mg/kg). Dose escalation followed a modified toxicity probability interval 2 design. Objectives included evaluation of safety, dose-limiting toxicity (DLT), recommended phase 2 dose, pharmacokinetic and pharmacodynamic (PD) parameters, and antitumor activity per RECIST 1.1. Results: As of 3 Jan 2023, 27 pts (median age 66 years [range 33-85]; median of 4 prior systemic therapies [range 2-9]) with ovarian (70%), fallopian tube (15%) or primary peritoneal (15%) cancer were dosed. Treatment-emergent AEs (&gt;15%) regardless of grade (G) included infusion related reaction (IRR) (67%), fatigue (44%), nausea (33%), back pain (26%), constipation, diarrhea (both 22%), decreased appetite and pruritus (both 19%). There was one DLT of G3 ALT increased at 10mg/kg requiring dose interruption for resolution. A maximum tolerated dose was not reached. G3/4 treatment-related AEs (&gt;1pt) were AST increased (G3) and lymphopenia (G4), each in 2 pts (7%); all were fully resolved with no dose modifications. There were no fatal AEs, no AEs that led to drug discontinuation and no events of cytokine release syndrome. The frequency of IRR events increased with increasing dose, and slowing the rate of infusion was utilized for mitigation. SL-172154 Cmax and AUC increased with dose with greater than proportional exposure noted at 3 and 10mg/kg potentially due to target saturation. These findings were supported by dose-dependent target engagement of CD47 and CD40 on CD4 T cells and B cells respectively, approaching 100% by 3 mg/kg. Rapid dose-dependent egress of CD40+ B cells was maximal at ≥3mg/kg. SL-172154 induced dose-dependent responses in IL-12, CXCL-8, CXCL-10, IL-10, CCL2, CCL20, and CCL22, which also approached a plateau at ≥3mg/kg. Analysis of peripheral and tumor immunophenotypes and anti-drug antibodies in response to SL-172154 is ongoing and will be presented. Best response was stable disease in 6/27 response evaluable (22%) pts. Conclusions: SL-172154 is well tolerated in heavily pretreated PROC pts. IRRs were readily manageable. Maximal CD47 and CD40 target engagement and CD40-dependent PD effects were observed with ≥3mg/kg SL-172154. 3mg/kg is a safe, tolerable, and pharmacologically active dose for evaluation in combination studies in pts with PROC. Clinical trial information: NCT04406623 .

Immunophenotypic analysis of human adipose-derived stem cells through multi-color flow cytometry.

Immunophenotypic analysis of human adipose-derived stem cells through multi-color flow cytometry.

Oncolytic DNX-2401 virotherapy plus pembrolizumab in recurrent glioblastoma: a phase 1/2 trial

Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2–20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1–69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7–13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05–0.87). A total of 56.2% (95% CI 41.1–70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406).

Invasion of chronic lymphocytic leukemia (cll/sll) bloodstream tumor cells by borrelia spirochetes

A sixty-six-year-old woman with a strong family history of breast cancer noted gradual onset of axillary lymph node enlargement in 2019. Axillary lymph node biopsies disclosed Lymph node involvement by Chronic Lymphocytic Leukemia (CLL/SLL). Immunophenotype analysis of nodal tissue and of bloodstream leukemic cells in immunohistochemistry and flow cytometry and next generation molecular diagnostics confirmed CLL/SLL. Replicate absolute peripheral blood lymphocyte counts in excess of 5.0x10e3/microliter confirmed the numerical threshold for diagnosis of Chronic Lymphocytic Leukemia in the bloodstream. FISH studies disclosed an immunoglobulin “mutation negative” CLL/SLL genotype without a light chain restriction pattern. No translocations or germline mutations were present. Borrelia spirochetes in the bloodstream were detected with simultaneous high spirochetemia of both borrelia miyamotoi and borrelia burgdorferi. FISH hybridization of leukemic lymphocytes disclosed extensive borrelia invasion of the cytoplasm and of the nucleus compartments of CLL/SLL tumor cells. Retrospective studies of the patient’s archival stored blood smears obtained three years prior in 2016 at age 63 disclosed no evidence of CLL/SLL but confirmed asymptomatic bloodstream borrelia miyamotoi and borrelia burgdorferi borrelia infection. Lyme serology had been reported as negative in 2016. Additionally, retrospective FISH DNA hybridizations focused on preleukemic bloodstream Lymphocytes disclosed rare borrelia spirochetal adherence to benign blood lymphocytes in the year 2016 in the preleukemic blood smears. This case report is the very first to describe precursor chronic asymptomatic Lyme borreliosis and Miyamotoi borrelia bloodstream infections three years prior to the diagnosis of Rai Stage 1 Chronic Lymphocytic leukemia.

Value of negatively correlated miR-205-5p/HMGB3 and miR-96-5p/FOXO1 on the diagnosis of breast cancer and benign breast diseases

BackgroundMicroRNA (miRNA) and mRNA levels in matching specimens were used to identify miRNA–mRNA interactions. We aimed to integrate transcriptome, immunophenotype, methylation, mutation, and survival data analyses to examine the profiles of miRNAs and target mRNAs and their associations with breast cancer (BC) diagnosis. MethodsBased on the Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA), differentially expressed miRNAs and targeted mRNAs were screened from experimentally verified miRNA-target interaction databases using Pearson's correlation analysis. We used real-time quantitative reverse transcription polymerase chain reaction to verify BC and benign disease samples, and logistic regression analysis was used to establish a diagnostic model based on miRNAs and target mRNAs. Receiver operating characteristic curve analysis was performed to test the ability to recognize the miRNA-mRNA pairs. Next, we investigated the complex interactions between miRNA-mRNA regulatory pairs and phenotypic hallmarks. ResultsWe identified 27 and 359 dysregulated miRNAs and mRNAs, respectively, based on the GEO and TCGA databases. Using Pearson's correlation analysis, 10 negative miRNA-mRNA regulatory pairs were identified after screening both databases, and the related miRNA and target mRNA levels were assessed in 40 BC tissues and 40 benign breast disease tissues. Two key regulatory pairs (miR-205-5p/High mobility group box 3 (HMGB3) and miR-96-5p/Forkhead Box O1 (FOXO1)) were selected to establish the diagnostic model. They also had utility in survival and clinical analyses. ConclusionsA diagnostic model including two miRNAs and their respective target mRNAs was established to distinguish between BC and benign breast diseases. These markers play essential roles in BC pathogenesis.

Deep Phenotyping of Neurologic Postacute Sequelae of SARS-CoV-2 Infection.

SARS-CoV-2 infection has been associated with a syndrome of long-term neurologic sequelae that is poorly characterized. We aimed to describe and characterize in-depth features of neurologic postacute sequelae of SARS-CoV-2 infection (neuro-PASC). Between October 2020 and April 2021, 12 participants were seen at the NIH Clinical Center under an observational study to characterize ongoing neurologic abnormalities after SARS-CoV-2 infection. Autonomic function and CSF immunophenotypic analysis were compared with healthy volunteers (HVs) without prior SARS-CoV-2 infection tested using the same methodology. Participants were mostly female (83%), with a mean age of 45 ± 11 years. The median time of evaluation was 9 months after COVID-19 (range 3-12 months), and most (11/12, 92%) had a history of only a mild infection. The most common neuro-PASC symptoms were cognitive difficulties and fatigue, and there was evidence for mild cognitive impairment in half of the patients (MoCA score <26). The majority (83%) had a very disabling disease, with Karnofsky Performance Status ≤80. Smell testing demonstrated different degrees of microsmia in 8 participants (66%). Brain MRI scans were normal, except 1 patient with bilateral olfactory bulb hypoplasia that was likely congenital. CSF analysis showed evidence of unique intrathecal oligoclonal bands in 3 cases (25%). Immunophenotyping of CSF compared with HVs showed that patients with neuro-PASC had lower frequencies of effector memory phenotype both for CD4+ T cells (p < 0.0001) and for CD8+ T cells (p = 0.002), an increased frequency of antibody-secreting B cells (p = 0.009), and increased frequency of cells expressing immune checkpoint molecules. On autonomic testing, there was evidence for decreased baroreflex-cardiovagal gain (p = 0.009) and an increased peripheral resistance during tilt-table testing (p < 0.0001) compared with HVs, without excessive plasma catecholamine responses. CSF immune dysregulation and neurocirculatory abnormalities after SARS-CoV-2 infection in the setting of disabling neuro-PASC call for further evaluation to confirm these changes and explore immunomodulatory treatments in the context of clinical trials.

Preliminary analysis of double-negative T, double-positive T, and natural killer T-like cells in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the expansion of CD5+ malignant B lymphocytes. Recent discoveries have shown that double-negative T (DNT) cells, double-positive T (DPT) cells, and natural killer T (NKT)-cells may be involved in tumor surveillance. A detailed immunophenotypic analysis of the peripheral blood T-cell compartment of 50 patients with B-CLL (classified in three prognostic groups) and 38 healthy donors (as controls) matched for age was performed. The samples were analyzed by flow cytometry using a stain-lyse-no wash technique and a comprehensive six-color antibody panels. Our data confirmed a reduction in percentage values and an increase in absolute values of T lymphocytes in patients with B-CLL, as already reported. In particular, DNT, DPT, and NKT-like percentages were significantly lower than in the controls, except for NKT-like in the low-risk prognostic group. Moreover, a significant rise in the absolute counts of DNT cells in each prognostic group and in the low-risk prognostic group of NKT-like cells was found. A significant correlation of the absolute values of NKT-like cells in the intermediate-risk prognostic group versus B cells was observed. Furthermore, we analyzed whether the increase in T cells was related to the subpopulations of interest. Only DNT cells were positively correlated with the increase in CD3+ T lymphocytes, regardless of the stage of the disease, supporting the hypothesis that this T-cell subset plays a key role in the immune T response in B-CLL. These early results supported that DNT, DPT, and NKT-like subsets may be related to disease progression and should encourage further studies aimed at identifying the potential immune surveillance role of these minority T subpopulations.

Keratinocyte FABP5-VCP complex mediates recruitment of neutrophils in psoriasis

Abstract Psoriasis is a common chronic inflammatory skin disease. One of the hallmarks of psoriasis is the abundance of neutrophil infiltration in the skin lesions. However, the detailed molecular mechanisms of neutrophil chemotaxis and activation remains unclear. Here we demonstrated an upregulation of epidermal fatty acid binding protein (E-FABP, also known as FABP5) in skin tissue from both human psoriasis and imiquimod (IMQ)-induced psoriatic mouse model. Genetic deletion of FABP5 in global knockout mice and keratinocyte conditional (Krt6a-Cre) knockout mice, but not in myeloid cell conditional (Lysm-Cre) knockout mice, attenuated the skin erythema, thickness, and desquamation. Immunophenotypic analysis showed that FABP5 deficiency reduced the recruitment of Ly6G+ neutrophils in the dermis and ear of IMQ-treated mice. Mechanistically, activated keratinocytes in IMQ-treated mice produced chemokines and cytokines (e.g., CXCL8, IL-36γ) that triggered neutrophil chemotaxis and activation in a FABP5-dependent manner. Proteomic and IP analyses further identified that FABP5 interacted with a valosin-containing protein (VCP), a key player in NFκB signaling pathway in human keratinocytes. Silencing of FABP5 inhibited VCP-mediated IκBα phosphorylation and NFκB signaling activation. Collectively, these data suggest that FABP5 in keratinocytes interacts with VCP to activate NFκb signaling, thus playing an essential role in the neutrophil infiltration in psoriatic skin lesions. R01AI137324, #LINK#R01CA180986#ENDLINK#