Maturation and phenotypic heterogeneity of human CD4+ regulatory T cells from birth to adulthood and after allogeneic stem cell transplantation

Copyright © 2021 Matos, Hirakawa, Alho, Neleman, Graca and Ritz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.CD4+ Regulatory T cells (Treg) play a critical role in maintaining immune homeostasis. Various Treg subsets have been identified, however the heterogeneity of Treg subpopulations during development remains uncharacterized. Using mass cytometry we obtained single cell data on expression of 35 functional markers to examine the heterogeneity of Treg cells at birth and in adults. Unsupervised clustering algorithms FlowSOM and ACCENSE were used to quantify Treg heterogeneity. As expected, Treg in umbilical cord blood were predominately naïve while Treg in adult blood were predominately central memory and effector memory cells. Although umbilical cord blood Treg are mostly naïve cells, we observed multiple phenotypic Treg subsets in cord blood. Nevertheless, peripheral blood in adults contained higher percentages of Treg and the heterogeneity of Treg was significantly increased in adults. We also studied Treg heterogeneity throughout a 2-year period after allogeneic hematopoietic stem cell transplantation (alloHSCT) and in patients with chronic graft-versus-host disease (cGVHD). Treg heterogeneity recovered rapidly after alloHSCT and gradually increased in the first two years post-transplant. However, patients with cGVHD had significantly fewer distinct Treg subpopulations, proposing a correlation between a disrupted Treg heterogeneity and cGVHD. Our study is the first to compare human Treg heterogeneity at birth, in healthy adults and in patients after alloHSCT with and without cGVHD. This approach to characterize Treg heterogeneity based on expression of a large panel of functional markers may enable future studies to identify specific Treg defects that contribute to immune dysfunction.This work was supported by NIH grant P01CA229092, EADV Research Fellowship, Rene-Touraine Fellowship and a generous contribution from the Fundação para a Ciência e a Tecnologia (FCT), FCT SFRH/BD/98980/2013info:eu-repo/semantics/publishedVersio

Cell-based therapy in prophylaxis and treatment of chronic graft-versus-host disease

Copyright © 2022 Doglio, Crossland, Alho, Penack, Dickinson, Stary, Lacerda, Eissner and Inngjerdingen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Hematopoietic allogeneic stem cell transplantation (allo-SCT) is a curative option for patients with hematological malignancies. However, due to disparities in major and minor histocompatibility antigens between donor and recipient, severe inflammatory complications can occur, among which chronic graft-versus-host disease (cGVHD) can be life-threatening. A classical therapeutic approach to the prevention and treatment of cGVHD has been broad immunosuppression, but more recently adjuvant immunotherapies have been tested. This review summarizes and discusses immunomodulatory approaches with T cells, including chimeric antigen receptor (CAR) and regulatory T cells, with natural killer (NK) cells and innate lymphoid cells (ILCs), and finally with mesenchymal stromal cells (MSC) and extracellular vesicles thereof. Clinical studies and pre-clinical research results are presented likewise.This work was supported by COST (European Cooperation in Science and Technology). www.cost.eu - COST Action 17138 EUROGRAFT.info:eu-repo/semantics/publishedVersio

Maintenance and breeding of Thrichomys (Trouessart, 1880) (Rodentia: Echimyidae) in captivity

South American histricognath rodents Thrichomys apereoides laurentius and Thrichomys pachyurus are natural hosts of Trypanosoma cruzi, agent of Chagas disease. We established breeding colonies of these species to serve as experimental models in various parasitological studies. Both species of Thrichomys have all the requirements necessary to become excellent laboratory models: they can be easily maintained in the standard laboratory conditions and breed throughout the year and they do not have any special dietary demands and can be fed by standard food pellets designed for laboratory mice. Both species produce precocious offspring that have their eyes and ears open, teeth erupted, fur well developed, and can eat solid food in the first week of life. T. a. laurentius has larger litter sizes and lower body masses at birth and weaning than T. pachyurus. Moreover, females of T. a. laurentius reach puberty earlier and with lower body mass than T. pachyurus

Fifth European Dirofilaria and Angiostrongylus Days (FiEDAD) 2016

Peer reviewe

Homeostasis of CD4+ regulatory T cells after allogeneic hematopoietic cell transplantation

Following allogeneic hematopoietic stem cell transplantation (allo-HSCT), the homeostasis of the T-cell pool is deeply challenged, requiring years to recover. Frequently, this recovery is dysfunctional, accompanied by loss of T-cell tolerance and subsequent development of chronic Graft-versus-Host Disease (cGvHD), leading to significant morbidity and mortality. With this work we investigated the mechanisms involved in the restoration of peripheral T-cell homeostasis following allo-HSCT, particularly the role of CD4+ regulatory T-cells (Tregs), critical mediators of peripheral T-cell tolerance. We performed a comprehensive analysis of the reconstitution of peripheral blood CD4+ and CD8+ T-cells in patients undergoing allo-HSCT for the treatment of hematologic malignancies. We took advantage of being able to study three distinct transplant settings, differing primarily in the conditioning regimen, GvHD prophylaxis and HLA (Human Leukocyte Antigen) matching. We consistently found a significantly lower number of Tregs at nine months post-transplant in patients developing cGvHD, coinciding with decreased Treg:Tcon (conventional T-cells) and Treg:CD8 ratios. This unbalance resulted from significantly delayed proliferation of naïve Tregs, and increased expansion of memory Tcon and CD8+ T-cell, observed in all cohorts. The expansion of memory subsets, likely involved in cGvHD pathogenesis, together with impaired thymic generation of de novo naïve CD4+ T-cells, resulted in significantly decreased diversity of the CD4+ TCR repertoire in cGvHD patients. Importantly, we were able study for the first time the contribution of true naïve and naïve-derived T memory stem cells (TSCM) to the development of cGvHD in humans. An early and transient increase of naïve T-cells was observed in two cohorts, anticipating the development of cGvHD, while the sustained increase of TSCM CD8 was observed in the cohort where allo-HSCT was performed with concomitant T-cell depletion . The heterogeneity of these results, reflect the effect of the different transplant settings, particularly in the recovery of donor-infused naïve T-cells and their progeny, in the early post-transplant period. In the final part of this work, we sought to determine the mechanisms involved in cGvHD amelioration after the administration of low-dose rIL-2 (interleukin 2) to patients with steroid dependent/refractory cGvHD. In this setting, the expansion of novel or low frequency Treg clones was the only variable associated to cGvHD improvement. This finding enhances the relevance of antigen specificity for adequate Treg suppression and induction of tolerance post allo-HSCT. Overall, the data obtained reiterates that achieving an adequate balance between effector and regulatory T-cells post allo-HSCT is of critical importance to avoid the development of cGvHD in the post transplantation period. Hence, we not only confirm the association between impaired Treg homeostasis and cGvHD, but further validate the contribution of naïve T-cells to the initiation of cGvHD. In addition, we describe for the first time, a role for TSCM in cGvHD pathogenesis in humans, and stress the importance to clarify the role of antigen-specificity to efficient Treg function

Seroprevalence of vector-borne pathogens and molecular detection of Borrelia afzelii in military dogs from Portugal

BACKGROUND: Canine vector-borne diseases (CVBDs) are increasingly being reported worldwide and represent a serious threat to both animal and public health. Military dogs may constitute a risk group for the agents causing these diseases, as they frequently work outdoors in different areas and are thus exposed to vector arthropods. In order to assess the risk of exposure of this type of dogs, a serological and molecular survey was conducted in military working dogs in Portugal. One hundred apparently healthy dogs were surveyed. Serum samples were tested for antigens of Angiostrongylus vasorum and Dirofilaria immitis; and for antibodies to A. vasorum, Anaplasma spp., Babesia spp., Ehrlichia canis, Leishmania infantum, Rickettsia spp. and Toscana virus. Serum was tested by polymerase chain reaction for Borrelia burgdorferi (sensu lato), with sequencing of the DNA products. RESULTS: Forty-nine per cent of the dogs were seropositive for antibodies against Rickettsia spp., 16 % for Anaplasma spp., 13 % for L. infantum, 7 % for E. canis, 5 % for A. vasorum (including 1 % positive for both antibodies and circulating antigens), 3 % for Babesia spp. and 1 % positive for Toscana virus. B. burgdorferi (s.l.) was detected in eight out of 94 dogs tested (8.5 %) and in three cases (3.2 %) nucleotide sequence analysis showed identity with the genospecies Borrelia afzelii. No positive cases were recorded for D. immitis. Overall, 66 % of the dogs were positive for at least one out of the eight tested CVBD agents, six of which are zoonotic (i.e. Anaplasma spp., Borrelia spp., E. canis, L. infantum, Rickettsia spp. and Toscana virus). Serological specific antibody detection against more than one CVBD agent (including molecular detection of Borrelia spp.) was recorded in 25 % of the dogs, comprising 19 % with positive reaction to two agents, 5 % to three agents and 1 % to four agents. CONCLUSIONS: These results reveal a high occurrence of CVBD agents in military working dogs in Portugal and highlight the need to maintain a comprehensive and regular prophylaxis to reduce the contact between working dogs and those pathogens. For the first time in Portugal, B. afzelii DNA was identified in dogs and a dog was found seropositive for antibodies against Toscana virus

Eumycetoma due to Parathyridaria percutânea in an immunosuppressed patient - an imported case in Portugal

Abstract publicado em: J Fungi (Basel). 2021 Nov; 7(11):916. pp. 223-24. (P262). doi: 10.3390/jof7110916Objectives: Eumycetoma is a chronic subcutaneous fungal infection characterized by swelling, fistulization and discharge of fungal granules. These infections occur after traumatic inoculation and progress slowly over months or even years into a chronic form. Parathyridaria percutanea is a rare cause of subcutaneous phaeohyphomycoses and there are no published reports of discharging sinus or granuloma formation due to this fungus. We present a rare case of eumycetoma due to Parathyridaria percutanea in a 10-years-old patient from Angola, presenting after allogeneic hematopoietic stem cell transplant (alloHSCT) for severe aplastic anemia. Material and methods: A 10-year-old boy from Angola with severe aplastic anemia was transferred to Portugal and submitted to alloHSCT from a mismatched unrelated donor at a single HLA-locus (9/10). After secondary graft failure due to post-transplant hemophagocytic lymphohistiocytosis, he underwent a second alloHSCT, without engraftment. Twenty-three days after the 3rd alloHSCT from the same donor and seven days under prophylaxis with posaconazole, the patient presented with febrile neutropenia with no clear focus. He complained of painless swelling of the right knee, with slight fluctuation. An ultrasound was performed revealing a heterogeneous hypoechogenic area with imprecise contours, inside of which there was a nodular formation of approximately 10 mm. Two weeks later, a spontaneous drainage of a grossly spherical grain from the right knee occurred. It was characterized as extra-articular, subcutaneous with filamentous aspect. The material was sent to the laboratory where its macroscopic observation allowed the identification of a granule of possible mycotic etiology, consistent with the diagnosis of eumycetoma. This sample was promptly processed for bacteriology (aerobic and anaerobic) and mycology studies. Results: Direct microscopic examination of the grain revealed septate hyphae with irregular hyphal swellings. Cultures for bacteria (aerobic and anaerobic) were sterile. After 1 week, slow growing colonies appeared on Sabouraud dextrose agar (SDA) incubated at 25 °C. Those colonies were flat, spreading with sparse aerial hyphae, become creamy after 5 to 10 days and with orange reverse and then turned dark brown after 4 weeks. Lactophenol cotton blue mount revealed nonsporulating dematiceous hyphae with clamidospores. Phenotypic identification of the organism was uncertain but sequencing of the internal transcribed spacer (ITS) region of ribosomal DNA identified as Parathyridaria percutanea (100% homology, 99% coverage). The isolate showed in vitro resistance to itraconazole, fluconazole and anidulofungin and susceptibility to posaconazole, voriconazole and amphotericin B. As this pathogen is extremely rare, no antifungal guidelines have been proposed so far. The patient received dual coverage with posaconazole and liposomal amphotericin-B but died 2 weeks later of complications of the underlying disease. Conclusions: The presented case maybe the first described case of eumycetoma caused by Parathyridaria percutanea associated with fistulae presentation. Molecular identification of rare fungi is essential, especially when they are poorly sporulated, as in this case. Moreover, rare fungi can present with odd clinical signs, as in this case, emphasizing the relevance of a polyphasic approach to identify the etiological agent.info:eu-repo/semantics/publishedVersio

Effect of biologic disease-modifying anti-rheumatic drugs targeting remission in axial spondyloarthritis : systematic review and meta-analysis

© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.Objectives: To assess the efficacy of biologic DMARDs (bDMARDs) in achieving Assessment of Spondyloarthritis International Society partial remission (ASAS-PR) and/or Ankylosing Spondylitis Disease Activity Score inactive disease (ASDAS-ID), as remission-like surrogates, in axial SpA (axSpA). Methods: Data from randomized controlled trials (RCTs), including long-term extensions, were included. A systematic literature review was performed using the MEDLINE database (first search May 2018, updated February 2020) and PICO criteria according to Patients—adults with radiographic or non-radiographic axSpA; Intervention—any bDMARD; Comparator—placebo and/or any different drug; Outcomes—ASAS-PR and/or ASDAS-ID as primary or secondary endpoints. Meta-analysis was performed after assessment of the homogeneity of study designs, populations and outcomes. Results: After screening 155 references, a total of 22 RCTs and 28 long-term extensions were retrieved. ASAS-PR was the dominant remission-like definition used. Concerning TNF inhibitors, 14/17 RCTs provided evidence of efficacy in reaching remission at different time points: 12, 16, 24 and 28 weeks (ASAS-PR in 16–62% of patients and ASDAS-ID in 24–40% of patients). With a limited number of studies available, IL-17A inhibitors exhibited remission rates of 15–21% for ASAS-PR and 11–16% for ASDAS-ID at week 16. A meta-analysis regarding ASAS-PR was performed considering RCTs with a similar duration (12, 16 or 24 weeks). The relative risk for achieving remission was 3.864 (95% CI 2.937, 5.085). Conclusion: bDMARDs have a clear impact in axSpA remission evaluated by ASAS-PR. Nevertheless, these data show an unmet need for improved reporting of remission-like outcomes.info:eu-repo/semantics/publishedVersio

Unbalanced recovery of regulatory and effector T cells after allogeneic stem cell transplantation contributes to chronic GVHD

© 2016 by The American Society of HematologyThe development and maintenance of immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT) requires the balanced reconstitution of donor-derived CD4 regulatory T cells (CD4Tregs) as well as effector CD4 (conventional CD4 T cells [CD4Tcons]) and CD8 T cells. To characterize the complex mechanisms that lead to unbalanced recovery of these distinct T-cell populations, we studied 107 adult patients who received T-replete stem cell grafts after reduced-intensity conditioning. Immune reconstitution of CD4Treg, CD4Tcon, and CD8 T cells was monitored for a 2-year period. CD3 T-cell counts gradually recovered to normal levels during this period but CD8 T cells recovered more rapidly than either CD4Tregs or CD4Tcons. Reconstituting CD4Tregs and CD4Tcons were predominantly central memory (CM) and effector memory (EM) cells and CD8 T cells were predominantly terminal EM cells. Thymic generation of naive CD4Tcon and CD8 T cells was maintained but thymic production of CD4Tregs was markedly decreased with little recovery during the 2-year study. T-cell proliferation was skewed in favor of CM and EM CD4Tcon and CD8 T cells, especially 6 to 12 months after HSCT. Intracellular expression of BCL2 was increased in CD4Tcon and CD8 T cells in the first 3 to 6 months after HSCT. Early recovery of naive and CM fractions within each T-cell population 3 months after transplant was also strongly correlated with the subsequent development of chronic graft-versus-host disease (GVHD). These dynamic imbalances favor the production, expansion, and persistence of effector T cells over CD4Tregs and were associated with the development of chronic GVHD.This work was supported by a Collaborative Research Grant from the Harvard Medical School-Portugal Program in Translational Research HMSP-ICT/0001/201, and National Institutes of Health, National Cancer Institute grants CA183559, CA183560, and CA142106.info:eu-repo/semantics/publishedVersio