CD25+FOXP3+CD45RA- regulatory T-cell infiltration as a prognostic biomarker for endometrial carcinoma.

BACKGROUND: Regulatory T (Treg) cells reportedly play crucial roles in tumor angiogenesis as well as antitumor immunity. In order to explore their therapeutic potential, we investigated the precise prognostic impact of Treg markers in endometrial carcinoma. METHODS: We performed multiplexed immunofluorescence and quantitative image analyses of CD25, FOXP3, CTLA4, and CD45RA in tumor specimens from 176 consecutive patients treated at our institution for primary endometrial carcinomas. Bioinformatics analyses were further conducted to corroborate the findings. RESULTS: High CD25+, FOXP3+, and CD25+FOXP3+CD45RA- stromal cell counts correlated with better overall survival (OS) (p = 0.00019, 0.028 and 0.0012) and MSI-high (p = 0.015, 0.016 and 0.047). High CD45RA+ stromal cell count was associated with superficial myometrial invasion (p = 0.0038). Bioinformatics survival analysis by Kaplan-Meier plotter showed that high CD25, FOXP3, CTLA4, and CD45RA mRNA expressions correlated with better OS (p = 0.046, 0.00042, 0.000044, and 0.0022). Univariate and multivariate analyses with various clinicopathologic prognostic factors indicated that high CD25+ or CD25+FOXP3+CD45RA- stromal cell count was significant and independent for favorable OS (p = 0.0053 and 0.0015). We subsequently analyzed the correlations between the multiplexed immunofluorescence results and treatment-free interval (TFI) after primary chemotherapy in recurrent cases, finding no significant associations. Further analysis revealed that high ratio of CD25+ : CD8+ cell count or CD25+FOXP3+CD45RA- : CD8+ cell count correlated with longer TFI (p = 0.021 and 0.021). CONCLUSION: The current observations suggest that the balance between CD25+ or CD25+FOXP3+CD45RA- cells and CD8+ cells, corresponding to promoting or inhibiting effect on tumor angiogenesis, affect tumor chemosensitivity leading to prognostic significance. CD25+FOXP3+CD45RA- effector Treg tumor infiltration may serve as a useful prognostic biomarker and a potential target for immunotherapeutic manipulation of tumor chemosensitivity by novel management for advanced/recurrent endometrial carcinomas.

Analytical and clinical validation of diagnostic tests for the detection of leucospermia in beef bulls.

The objectives of this study were to validate diagnostic tests to detect polymorphonuclear cells (PMNs) in bull semen, and to determine the prevalence of leucospermia in beef bulls with varying semen quality. We hypothesized that all tests have comparable diagnostic value, and that leucospermia is more prevalent in unsatisfactory breeders in association with poor semen quality. For the analytical validation, one ejaculate was obtained from five bulls. Aliquots of 50 × 106 purified sperm were incubated in triplicate with six concentrations of purified bovine PMNs: 1) no PMNs, 2) 0.25 × 106 PMN/ml, 3) 0.5 × 106 PMN/ml, 4) 2.5 × 106 PMN/ml, 5) 5 × 106 PMN/ml, 6) 10 × 106 PMN/ml. The PMNs were quantified using a hemacytometer, cytology, a leucocyte esterase dipstick test (LEDT), a peroxidase test, and CD45 immunolabeling. The number of leucocytes detected with the LEDT differed among treatments (P < 0.0001). The quantitative tests detected differences with the control treatment at a PMN concentration of ≥2.5 × 106 PMN/ml (P < 0.0001). Sperm motion parameters after 4 h of incubation at 38 °C were lower in samples with ≥5 × 106 PMN/ml (P < 0.05). For the clinical validation, semen samples from 305 beef bulls were evaluated. Unsatisfactory breeders (n = 83) had more CD45-positive cells (P = 0.016) and positive LEDT results (P = 0.008) than satisfactory breeders (n = 222). With CD45 immunostaining as the gold standard, the hemacytometer count had the highest clinical sensitivity (64.3 %) but the lowest specificity (73.3 %). A higher specificity was obtained with the peroxidase test (95.1 %) or semen cytology (98.8 %). In conclusion, the presence of ≥5 × 106 PMN/ml was associated with decreased semen quality in beef bulls. The hemacytometer count was the most sensitive bull-side test. But due to the low specificity, positive hemacytometer counts should be confirmed with the identification of peroxidase-positive cells or morphological identification of leucocytes on semen cytology. The CD45 immunostaining is the gold standard for the diagnosis of leucospermia in bulls.

Exploration of compounds to inhibit the Panton-Valentine leukocidin of Staphylococcus aureus.

The Panton-Valentine leukocidin (PVL) of Staphylococcus aureus is associated with necrotizing infections. After binding to complement 5a receptor (C5aR/CD88) and CD45 it causes cytolysis in polymorphonuclear neutrophils (PMNs) as well as inflammasome activation in monocytes. The objective of this study was to test if (ant)agonists of C5aR and CD45 can attenuate the effect of PVL on PMNs and monocytes. We tested the effect of various concentrations of six C5aR (ant)agonists (avacopan, BM213, DF2593A, JPE-1375, PMX205 and W-54011) and one CD45 antagonist (NQ301) to attenuate the cytotoxic effect of PVL on human PMNs and monocytes in vitro. Shifts in the half-maximal effective concentration (EC50) of PVL to achieve a cytotoxic effect on PMNs and modulation of inflammatory cytokine response from monocytes were determined by flow cytometry and IL-1ß detection. Pre-treatment of PMNs with avacopan, PMX205 and W-54,011 resulted in 3.6- to 4.3-fold shifts in the EC50 for PVL and were able to suppress IL-1ß secretion by human monocytes in the presence of PVL. BM213, DF2593A and NQ301 were unable to change the susceptibility of PMNs towards PVL or reduce inflammasome activation in monocytes. Avacopan, PMX205 and W-54,011 showed protection against PVL-induced cytotoxicity and suppressed IL-1ß secretion by monocytes. Clinical studies are needed to prove whether these substances can be used therapeutically as repurposed drugs.

Microglial heterogeneity in the ischemic stroke mouse brain of both sexes.

BACKGROUND: Ischemic stroke elicits a complex and sustained immune response in the brain. Immunomodulatory treatments have long held promise for improving stroke outcomes, yet none have succeeded in the clinical setting. This lack of success is largely due to our incomplete understanding of how immune cells respond to stroke. The objective of the current study was to dissect the effect of permanent stroke on microglia, the resident immune cells within the brain parenchyma. METHODS: A permanent middle cerebral artery occlusion (pMCAO) model was used to induce ischemic stroke in young male and female mice. Microglia were sorted from fluorescence reporter mice after pMCAO or sham surgery and then subjected to single-cell RNA sequencing analysis. Various methods, including flow cytometry, RNA in situ hybridization, immunohistochemistry, whole-brain imaging, and bone marrow transplantation, were also employed to dissect the microglial response to stroke. Stroke outcomes were evaluated by infarct size and behavioral tests. RESULTS: First, we showed the morphologic and spatial changes in microglia after stroke. We then performed single-cell RNA sequencing analysis on microglia isolated from sham and stroke mice of both sexes. The data indicate no major sexual dimorphism in the microglial response to permanent stroke. Notably, we identified seven potential stroke-associated microglial clusters, including four major clusters characterized by a disease-associated microglia-like signature, a highly proliferative state, a macrophage-like profile, and an interferon (IFN) response signature, respectively. Importantly, we provided evidence that the macrophage-like cluster may represent the long-sought stroke-induced microglia subpopulation with increased CD45 expression. Lastly, given that the IFN-responsive subset constitutes the most prominent microglial population in the stroke brain, we used fludarabine to pharmacologically target STAT1 signaling and found that fludarabine treatment improved long-term stroke outcome. CONCLUSIONS: Our findings shed new light on microglia heterogeneity in stroke pathology and underscore the potential of targeting specific microglial populations for effective stroke therapies.

Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein.

Introduction: The early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins, including the E3/49K protein, which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45, thereby modulating activation of T and NK cells. Methods: Considering that E3/49K represents the most polymorphic viral protein among species D HAdVs, we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus, this feature is conserved regardless of the pathological associations of the respective HAdV types. Results: It appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover, E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45, suggesting E3/49K as a potential tool to investigate CD45 specific functions. Conclusion: We identified B cells as new direct target of E3/49K-mediated immune modulation, representing a novel viral immunosubversive mechanism.

Characterization of non-epithelial cells embedded within the vocal fold epithelial barrier.

The vocal folds vibrate to produce voice, undergoing significant stress due to contact and shearing force. The epithelium operates as the primary protective layer of the tissue against stress and vibratory damage, as well as to provide a barrier against foreign organisms and toxins. Within the vocal fold epithelium, non-epithelial cells were identified that may interrupt the epithelium and compromise the epithelial barrier's protective function. Human vocal fold samples with a variety of pathologies were compared to normal vocal folds. Analysis included the number of cells in the epithelium and epithelial thickness. Vocal fold sections from 10 human tissue samples were assessed via H&E staining and immunofluorescent co-labeling. Three cell populations (vimentin expressing, CD-45 expressing, and cells expressing both) were identified within the epithelium. Statistical analysis revealed that the abnormal samples had a significantly greater number of vimentin-positive cells/area within the epithelium compared to the normal samples. Additionally, normal tissue samples had a significantly greater epithelial depth, suggesting a more robust epithelial barrier compared to tissue with pathology. Knowledge of the function of these cells could lead to a better understanding of how the local immune environment near and within vocal fold epithelium changes in the presence of different pathologies.

Stromal CD4 (+) T Cell Subsets Mediate Antitumor Cytotoxic Immune Responses in Human Colorectal Carcinoma.

BACKGROUND/AIM: The local immune response in colorectal cancer is closely related to prognosis and therapeutic efficacy. In this study, histological analyses were performed to determine the phenotype of tumor-infiltrating lymphocytes (TILs) and their infiltration in the stromal and intratumoral regions, aiming to elucidate their interactions and prognostic effects. PATIENTS AND METHODS: Multiplex fluorescent labeling was performed using surgically resected colorectal cancer specimens to investigate the infiltration of CD45RO (+) TILs, which exhibit cytotoxicity, and subsets of CD4 (+) TILs, identified by their characteristic transcription factor expression. RESULTS: The degree of CD45RO (+) TIL infiltration in the entire observation field or stromal area was not associated with prognosis. However, a high degree of infiltration in the tumor nest (intratumoral area) was significantly associated with a favorable prognosis. CD4 (+) TILs and their subsets were not associated with prognosis. However, stratified analyses revealed that a high degree of infiltration of stromal CD4 (+) TILs and the subsets T helper (Th)1, Th2, Th17, and regulatory T cells is necessary for the association between high intratumoral CD45RO (+) TIL infiltration and favorable prognosis. CONCLUSION: A sufficient degree of infiltration of stromal CD4 (+) TIL subsets is required for intratumoral CD45RO (+) TILs to exert toxicity against cancer cells. This highlights the significance of stromal immune reactions in achieving effective cytotoxic immune responses in the intratumoral area and demonstrates the critical role of the spatial distribution pattern of TILs in exerting their functions.

Expression of CD45 in non-hematopoietic cells: implications in regenerative medicine and disease management.

CD45 plays a crucial role in the regulation of hematopoiesis. However, a comprehensive understanding of its role in non-hematopoietic cells is lacking. Several tissue precursors express CD45, indicating its crucial role in tissue regeneration. These precursors would fall prey to the recent therapies involving CD45 as a target. CD45+ double-positive tumor cells contribute to cancer progression, but whether CD45 is involved in the process needs to be investigated. Recently, we showed that aging induces CD45 expression in mesenchymal stromal cells and affects their differentiation potential. In this review, we, for the first time, unravel the important implications of the expression of CD45 in non-hematopoietic cells and provide novel insights into its potential therapeutic target in regenerative medicine and disease management.

[Box: see text].

Safety and Efficacy of High-Dose Memory CD45RO+ Donor Lymphocyte Infusion in Pediatric Recipients after Hematopoietic Stem Cell Transplantation.

Memory T selected cells (CD45RA-/RO+) as donor lymphocyte infusion are less capable of producing alloreactivity and graft versus host disease (GvHD) compared with naïve T cells. The objective of this study was to evaluate the safety and efficacy of high-dose memory (CD45RA-/RO+) donor lymphocyte infusion (mDLI) after allogeneic hematopoietic cell transplantation (HCT). Indications for mDLI were "as needed" and "as prophylactic regimen." Sixty-one children diagnosed with malignant (82%) and non-malignant diseases (18%) received 241 mDLIs. Patients received a median of three infusions (range 1‒13) of mDLI with a median infused dose of 1.35 × 107/kg CD45RO+ containing 8.96 × 106/kg CD3+CD45RO+ and 3.81 × 103/kg CD3+CD45RA+. De novo GvHD developed in 7 patients following 4% of the mDLI infusions. Among patients with GvHD before mDLI, this condition worsened following 6 infusions (11%) in the 3 patients with grade II-IV acute GvHD. A decrease in cytomegalovirus viral load followed 65% of mDLI infusions. Two-year overall survival (OS) for the total cohort was 64% (95% CI 57%‒72%). For patients receiving prophylactic mDLI, the two-year non-relapse mortality was 10% (95% CI 9%‒11%). In summary, high-dose mDLI is feasible and safe, with a relatively low risk of severe GvHD even in patients with active GvHD. Importantly, mDLI was associated with positive effects, including enhanced control of CMV viremia.

Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing.

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.

Dual targeting negative enrichment strategy for highly sensitive and purity detection of CTCs.

Circulating tumor cells (CTCs) have significant clinical value in early tumor detection, dynamic monitoring and immunotherapy. CTC detection stands out as a leading non-invasive approach for tumor diagnostics and therapeutics. However, the high heterogeneity of CTCs and the occurrence of epithelial-mesenchymal transition (EMT) during metastasis pose challenges to methods relying on EpCAM-positive enrichment. To address these limitations, a method based on negative enrichment of CTCs using specific leukocyte targets has been developed. In this study, aiming to overcome the low purity associated with immunomagnetic beads targeting solely the leukocyte common antigen CD45, we introduced CD66b-modified immunomagnetic beads. CD66b, a specific target for neutrophils with abundant residues, was chosen as a complementary approach. The process involved initial collection of nucleated cells from whole blood samples using density gradient centrifugation. Subsequently, magnetically labeled leukocytes were removed by magnetic field, enabling the capture of CTCs with higher sensitivity and purity while retaining their activity. Finally, we selected 20 clinical blood samples from patients with various cancers to validate the effectiveness of this strategy, providing a new generalized tool for the clinical detection of CTCs.

The VISTA/VSIG3/PSGL-1 axis: crosstalk between immune effector cells and cancer cells in invasive ductal breast carcinoma.

A checkpoint protein called the V-domain Ig suppressor of T cell activation (VISTA) is important for controlling immune responses. Immune cells that interact with VISTA have molecules, or receptors, known as VISTA receptors. Immune system activity can be modified by the interaction between VISTA and its receptors. Since targeting VISTA or its receptors may be beneficial in certain conditions, VISTA has been studied in relation to immunotherapy for cancer and autoimmune illnesses. The purpose of this study was to examine the expression levels and interactions between VISTA and its receptors, VSIG3 and PSGL-1, in breast cancer tissues. IHC analysis revealed higher levels of proteins within the VISTA/VSIG3/PSGL-1 axis in cancer tissues than in the reference samples (mastopathies). VISTA was found in breast cancer cells and intratumoral immune cells, with membranous and cytoplasmic staining patterns. VISTA was also linked with pathological grade and VSIG3 and PSGL-1 levels. Furthermore, we discovered that the knockdown of one axis member boosted the expression of the other partners. This highlights the significance of VISTA/VSIG3/PSGL-1 in tumor stroma and microenvironment remodeling. Our findings indicate the importance of the VISTA/VSIG3/PSGL-1 axis in the molecular biology of cancer cells and the immune microenvironment.

Lymphocyte Phosphatase-Associated Phosphoprotein (LPAP) as a CD45 Protein Stability Regulator.

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a binding partner of the phosphatase CD45, but its function remains poorly understood. Its close interaction with CD45 suggests that LPAP may potentially regulate CD45, but direct biochemical evidence for this has not yet been obtained. We found that in the Jurkat lymphoid cells the levels of LPAP and CD45 proteins are interrelated and well correlated with each other. Knockout of LPAP leads to the decrease in the surface expression of CD45, while its overexpression, on the contrary, caused its increase. No such correlation was found in the non-lymphoid K562 cells. We hypothesize that LPAP regulates expression level of CD45 and thus can affect lymphocyte activation.

Memory T-cell enriched haploidentical transplantation with NK cell addback results in promising long-term outcomes: a phase II trial.

BACKGROUND: Relapse remains a challenge after transplantation in pediatric patients with hematological malignancies. Myeloablative regimens used for disease control are associated with acute and long-term adverse effects. We used a CD45RA-depleted haploidentical graft for adoptive transfer of memory T cells combined with NK-cell addback and hypothesized that maximizing the graft-versus-leukemia (GVL) effect might allow for reduction in intensity of conditioning regimen. METHODS: In this phase II clinical trial (NCT01807611), 72 patients with hematological malignancies (complete remission (CR)1: 25, ≥ CR2: 28, refractory disease: 19) received haploidentical CD34 + enriched and CD45RA-depleted hematopoietic progenitor cell grafts followed by NK-cell infusion. Conditioning included fludarabine, thiotepa, melphalan, cyclophosphamide, total lymphoid irradiation, and graft-versus-host disease (GVHD) prophylaxis consisted of a short-course sirolimus or mycophenolate mofetil without serotherapy. RESULTS: The 3-year overall survival (OS) and event-free-survival (EFS) for patients in CR1 were 92% (95% CI:72-98) and 88% (95% CI: 67-96); ≥ CR2 were 81% (95% CI: 61-92) and 68% (95% CI: 47-82) and refractory disease were 32% (95% CI: 11-54) and 20% (95% CI: 6-40). The 3-year EFS for all patients in morphological CR was 77% (95% CI: 64-87) with no difference amongst recipients with or without minimal residual disease (P = 0.2992). Immune reconstitution was rapid, with mean CD3 and CD4 T-cell counts of 410/µL and 140/µL at day + 30. Cumulative incidence of acute GVHD and chronic GVHD was 36% and 26% but most patients with acute GVHD recovered rapidly with therapy. Lower rates of grade III-IV acute GVHD were observed with NK-cell alloreactive donors (P = 0.004), and higher rates of moderate/severe chronic GVHD occurred with maternal donors (P = 0.035). CONCLUSION: The combination of a CD45RA-depleted graft and NK-cell addback led to robust immune reconstitution maximizing the GVL effect and allowed for use of a submyeloablative, TBI-free conditioning regimen that was associated with excellent EFS resulting in promising long-term outcomes in this high-risk population. The trial is registered at ClinicalTrials.gov (NCT01807611).

Tumor-associated CD8+T cell tolerance induced by erythroid progenitor cells.

Introduction: CD8+T cell tolerance plays an important role in tumor escape. Recent studies have shown that CD45+ erythroid progenitor cells (CD45+EPCs) generated through splenic extramedullary erythropoiesis suppress tumor immunity. However, the mechanism underlying how CD45+EPCs mediate CD8+T cell tolerance remains incompletely understood and requires further research. Methods: In this study, the antigen-processing abilities of CD45+EPCs was verified through both in vitro and in vivo experiments. We have used the method of co-culture in vitro and adoptive transfer experiments in vivo to explore the effects of CD45+EPCs on CD8+T cell tolerance. RNA-sequencing analysis and blocking experiments were used to evaluate the role of ROS in the CD45+EPC mediated tolerance of CD8+T cells. Finally, we incorporated uric acid into the adoptive transfer experiments to rescue the CD45+EPC mediated tumor-promoting effect. Results and discussion: We found that CD45+EPCs take up soluble proteins, present antigenic epitopes on their surface, and induce antigen-specific CD8+T cell anergy. In addition, we found that CD45+EPC directly nitrates tyrosine within the TCR/CD8 complex via the production of reactive oxygen species and peroxynitrite, preventing CD8+ T cells from responding to their specific peptide antigens. Furthermore, uric acid treatment effectively abolished the immunosuppressive effects of CD45+EPCs during CD8+T cell adoptive transfer, thereby enhancing the anti-tumor efficacy. These results demonstrated that CD8+T cell tolerance in tumor-bearing mice is induced by CD45+EPCs. The results of this study have direct implications for tumor immunotherapy.

High sodium, rather than high blood pressure, induces immune cell activation and renal infiltration in ovariectomized adult Wistar rats.

We used an animal model of salt-sensitive hypertension (SSH) in which ovariectomized (oVx) rats developed hypertension with high salt (HS) intake. Hypertension is accompanied by changes in the percentage of CD4+ T lymphocytes, immune CD45+ cell infiltration into renal tissue, and changes in Na+, K+- ATPase (NKA) expression in both renal tissue and peripheral blood mononuclear cells (PBMCs). To determine whether the observed changes resulted from HS intake, high blood pressure, or both, hydralazine (HDZ) was used to lower blood pressure. The oVx HS rats received two HDZ schedules either to prevent or to treat hypertension. NKA was overexpressed in the kidneys of all oVx groups and in PBMCs of oVx HS rats. This pattern was not altered with HDZ treatment. Changes in CD4+ T lymphocytes and renal infiltration of CD45+ cells were not reversed either. High salt, but not high blood pressure, induces immune cell activation and renal infiltration. Overexpressed NKA is the primary event, and HS is the perturbation to the system in this model of SSH, which resembles the postmenopausal state.

Single-tube Ptprc SNP genotyping of JAXBoy (CD45.1) and C57BL/6J (CD45.2) mice by endpoint PCR and gel electrophoresis.

Allelic variation at the Ptprc gene, which encodes the pan-leukocyte marker CD45/Ly5, is commonly exploited to track hematopoietic reconstitution by flow cytometry in mixed bone marrow chimera transplant experiments. Historically, this was accomplished using bone marrow from C57BL/6 (Ptprcb/CD45.2/Ly5.2) and congenic B6.SJL-PtprcaPepcb/Boy (Ptprca/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprca allele in C57BL/6J mice. This new isogenic strain, termed JAXBoy, differs from wild-type C57BL/6J mice by two nucleotides, compared to the biologically significant 37 megabase (Mb) SJL interval retained in B6.SJL-PtprcaPepcb/Boy/J mice. Currently, Ptprc/CD45 variants are identified by flow cytometry or allele-specific real-time PCR, both of which require specialized workflows and equipment compared to standard genotyping of endpoint PCR products by gel electrophoresis. Here, we employed allele-specific oligonucleotides in conjunction with differential incorporation of a long non-specific oligo 5'-tail to allow for simultaneous identification of the Ptprca and Ptprcb alleles using endpoint PCR and gel electrophoresis. This method allows for integration of Ptprc genotyping into standard genotyping workflows, which use a single set of thermocycling and gel electrophoresis conditions. Importantly, the strategy of primer placement and tail addition described here can be adapted to discriminate similar single- or multi-nucleotide polymorphisms at other genomic loci.

A unique human cord blood CD8+CD45RA+CD27+CD161+ T-cell subset identified by flow cytometric data analysis using Seurat.

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

T-cells and CD45-cells discovery in the central nervous system of healthy and nodavirus-infected teleost fish Dicentrarchus labrax.

To achieve insights in antiviral immune defense of the central nervous system (CNS), we investigated T cells and CD45 cells in the marine fish model Dicentrarchus labrax infected with the CNS-tropic virus betanodavirus. By employing markers for pan-T cells (mAb DLT15) and CD45-cells (mAb DLT22) in immunofluorescence (IIF) of leukocytes from brain, we obtained 3,7 ± 2.3 % of T cells and 7.3 ± 3.2 % of CD45+ cells. Both IIF and immunoelectron microscopy confirmed a leukocyte/glial morphology for the immunoreactive cells. Quantitative immunohistochemistry (qIHC) of brain/eye sections showed 1.9 ± 0.8 % of T+ cells and 2 ± 0.9 % of CD45+ cells in the brain, and 3.6 ± 1.9 % and 4.1 ± 2.2 % in the eye, respectively. After in vivo RGNNV infection the number of T cells/CD45+ leukocytes in the brain increased to 8.3 ± 2.1 % and 11.6 ± 4.4 % (by IIF), and 26.1 ± 3.4 % and 45.6 ± 5.9 % (by qIHC), respectively. In the eye we counted after infection 8.5 ± 4.4 % of T cells and 10.2 ± 5.8 % of CD45 cells. Gene transcription analysis of brain mRNA revealed a strong increase of gene transcripts coding for: antiviral proteins Mx and ISG-12; T-cell related CD3ε/δ, TcRß, CD4, CD8α, CD45; and for immuno-modulatory cytokines TNFα, IL-2, IL-10. A RAG-1 gene product was also present and upregulated, suggesting somatic recombination in the fish brain. Similar transcription data were obtained in the eye, albeit with differences. Our findings provide first evidence for a recruitment and involvement of T cells and CD45+ leukocytes in the fish eye-brain axis during antiviral responses and suggest similarities in the CNS immune defense across evolutionary distant vertebrates.

TCRαß-depleted hematopoietic stem cell transplant and third-party CD45RA+ depleted adoptive cell therapy for treatment of post-transplant parvovirus B19 aplastic crisis.

This is a case report of a 6-year-old girl with relapsed B cell acute lymphoblastic leukemia in which adoptive cell therapy was applied successfully to treat refractory human parvovirus (HPV) B19 infection. Allogenic chimeric antigen receptor (CAR) T-cell therapy (bispecific CD19/CD22) was bridged to hematopoietic stem cell transplantation (HSCT) using a haploidentical paternal donor. However, HPV B19 DNAemia progressed and transfusion-related graft versus host disease occurred. After finding a third-party related donor with a better HLA match, haploidentical HPV B19-seropositive CD45RA+ depleted cells (16.5 × 106/kg) were administered and paternal TCRαß+ depleted stem cell were retransplanted. The HPV B19 DNAemia became negative within 1 week and the reticulocyte, neutrophil, hemoglobin, and platelet counts gradually normalized. The patient remained stable during the 1-year outpatient follow-up period. Thus, our case report highlights that persistent B19 infection can lead to pancytopenia, aplastic crisis, and graft rejection and TCRαß+ depleted haplo-HSCT is an effective means of hematopoiesis recovery. CD45RO memory T-cell therapy is the key to treating and preventing the development of refractory severe HPV B19 infection.