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.

Selective haematological cancer eradication with preserved haematopoiesis.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells1. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies2. However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs2-5. Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies.

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.

Aging and asthma - changes in CD45RA, CD29 and CD95 T cells subsets

Background Aging is associated with thymus involution leading to a reduction in naive T cells and to an accumulation of effector-memory cells. Apoptosis is a key mechanism to clear the immune system from activated and harmful cells. In asthma the stimulation of T cells by environmental antigens can decrease naive cells and sustain activated cells. The aim of this work was to evaluate the imbalance between CD45RA and CD29 cells during the aging process and their changes in elderly asthma and to evaluate how elderly and chronic diseases like asthma can affect susceptibility to apoptosis. Methods Elderly and young adult healthy volunteers and elderly asthmatic patients were submitted to skin prick tests, immunoglobulin determination and flow cytometry analyses of CD3, CD4, CD8, CD45RA, CD29, and CD95. Results Serum IgE was increased in allergic patients (p=0.0001). Asthmatics presented an increase in CD4 cells (p<0.05). CD45RA was significantly decreased in elderly individuals (p<0.05) and this decrease was higher in asthmatics (p<0.05). CD29 was increased in elderly healthy individuals compared to the control young group (p=0.0001). A negative correlation between CD29 and CD45RA (p<0.05) was observed. CD95 lymphocytes increased in elderly (p=0.0001) and a positive correlation between age and CD95 (p<0.05) was found. Asthmatic patients showed significant decreases in CD95 (p=0. 0001). Conclusions Naive cells are key cells in the defence against infections and their decrease in the elderly and in asthma is a bad prognosis factor. The reduction of apoptosis markers can promote the persistence of activated cells involved in chronic conditions(AU)

Two-signal blockade with anti-CD45RB and anti-CD154 monoclonal antibodies inhibits graft rejection via CD4-dependent mechanisms in allogeneic skin transplantation

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low)and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low)T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two-signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.

Producción de interferón gamma por linfocitos pas-positivos infiltrados en corazones de pacientes chagasicos en los sitos de lesiones / Interferon gamma production by pas-positive T Lymphocytes of chagastic patients infiltrated into human hearts in the sites of lesions

En sangre de chagásicos en trabajos previos se halló un número alto de linfocitos T productores de substancia P.A.S.- positiva, más numerosos en chagásicos con electrocardiograma anormal. Luego los hallamos infiltrados en el corazón chagásico. En este trabajo consideramos nuestra hipótesis de que esa substancia linfocitaria sería Interferongamma. Material y Métodos. Muestras de corazones de 8 chagásicos fallecidos por muerte cardiaca. Se utilizaron anticuerpos monoclonales anti-IFN gamma humano; para linfocitos T activados (CD45 ROJ, y la reacción del P.A.S, respectivamente. Las células positivas se contaron en 50 campos a 400x y el estado de miocardio se comparó con datos clínicos. También. en frotis de sangre de pacientes chagásicos con ECG anormal, investigamos inmunomarcación con anti interferon gamma y la P.A.S.-positividad, respectivamente. Resultados El mayor número de células infiltrantes intracardiacas (65-75%) resultaron positivos para IFN gamma, y similares valores para P.A.S.-positividad y para CD45 RO. En sangre hallamos 41 % ±7 de linfocitos P.A.S.-positivos: y similar de positivos para IFN-gamma. Conclusiones: Los datos muestran que los linfocitos P.A.S.-positivos de los chagásicos producen IFN-gamma tanto en sangre. Como infiltrados en tejidos cardíacos. e indican una fuerte respuesta T-helper l. También explican la gran afluencia de macrófagos en dichos tejidos cardíacos. No se vieron parásitos T. cruzi ni formas intracelulares. Esos hechos refuerzan los datos que indican existencia de autoinmunidad en Chagas. Abrimos una pregunta: ¿a que antígeno/s responden en el corazón chagásico los linfocitos T P.A.S.-positivos productores de IFN-gamma?(AU)

In the blood of chagasic patients, a high number of T-lymphocytes producers of a P.A.S.-positive substance was found, more numerous in chagasics with abnormal electrocardiogram. Further. We found such lymphocytes infiltrated in the chagasic heart. Here, we considered our hypothesis that those lymphocytes would be Interferon -gamma producer cells. Material and Methods: Heart samples of 8 patients deceased due to chagasic heart disease (ChHD). Cuts of 5 microns were submitted to monoclonal antibodies for human Interferon-gamma; to CD45RO for activated T lymphocytes; and to the classical Periodic acid Schiff reaction (P.A.S.), respectively. In blood smears from chagasic patients with ChHD, the reactivity for anti Interferon gamma and for the P.A.S. reaction was compared, regarding the respective positive cell number. The myocardium status was compared with clinical date. Results: In hearts, 65-75% of infiltrated lymphocytes were positive for IFN-gamma; similar values were found, in seriated cuts, of P.A.S.-positive lymphocytes, as well as of CD45RO+. In blood, there were 41 % ±9 of P.A.S.positive lymphocytes, similar to positive cells for IFN-gamma. Conclusions: The data indicates that the P.A.S.-positive lymphocytes from chagasic patients are producers of IFN gamma in blood, as well as when infiltrated into the cardiac tissues. Such fact explains also the great affluence of macrophages in cardiac tissues in ChHD. The data indicate a strong response of T helper 1 type in this severe advanced stage of Chagasdisease. Either Trypanosome cruzi parasites or intracellular forms were seen in these hearts. This favours the data showing autoimmune mechanisms in this process. We open a question: to which antigen/s respond in the chagasic hearts the lymphocytes producers of IFN gamma?(AU)

Cardiopatía chagasica: identificación y cuantificación de células infiltrantes, en corazones de pacientes que sufrieron muerte cardiaca a distintas edades / Human chagasic cardiopathy: identification and quantification of intracardiac infiltrating cells, in patients died by cardiac failure at different ages

Esta investigación fue realizada para obtener datos nuevos sobre factores que intervendrían en la producción y progresión de la cardiopatía chagasica humana (CChH) con muerte por fallo cardíaco. Estudiamos muestras cardíacas de pacientes que murieron a edad temprana y más tardía. METODOS: Caracterizamos y cuantificamos los tipos de células infiltrantes mediante anticuerpos monoclonales; y con métodos histológicos clássicos. RESULTADOS: Hallamos intensa infiltración intracardíaca por linfocitos que reaccionaron con el anticuerpo para CD45RO que muestra linfocitos T activados (más de 70). Similar número mostró reactividad para subtipo CD4. Dichos linfocitos mostraron substancia PAS+ en citoplasma. Macrófagos fueron detectados en número superior a 25. Pocos linfocitos reaccionaron para CD8. Hubo un número muy bajo de linfocitos B. Otras células infiltradas fueron mastocitos intramiocárdicos, en número elevado. Cualitativamente, esos hallazgos fueron similares en los dos grupos de pacientes. Cuantitativamente, el número de linfocitos T y el de mastocitos fue significativamente mayor en los casos que sufrieron muerte temprana (p<0.001). No se observó T.cruzi. Se observaron daños en cardiomiocitos, con linfocitos T y macrófagos adheridos. CONCLUSIONES: Los hallazgos sugieren una activa e intensa existencia de mecanismos inmunocelulares en la producción y evolución de la CChH grave, en los que intervienen linfocitos T CD4+, con substancias PAS+, macrófagos, y mastocitos. Dichas células, mostraron citoadherencia a cardiomiocitos con lesiones; y por su ubcación y número asociáronse también a arritmias cardíacas malignas. En base a dichos hechos sugerimos que serían causas de pobre perfomance cardíaca en la CChH, y merecerían considerarse con respecto a estrategias terapéuticas (AU)