scholarly journals Progressive Immunodeficiency with Gradual Depletion of B and CD4+ T Cells in Immunodeficiency, Centromeric Instability and Facial Anomalies Syndrome 2 (ICF2)

Diseases ◽  
2019 ◽  
Vol 7 (2) ◽  
pp. 34 ◽  
Author(s):  
Georgios Sogkas ◽  
Natalia Dubrowinskaja ◽  
Anke K. Bergmann ◽  
Jana Lentes ◽  
Tim Ripperger ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Late Onset ◽  
Epigenetic Modification ◽  
Primary Immunodeficiency Disorder ◽  
Hematopoietic Stem ◽  
Facial Anomalies ◽  
Immunodeficiency Disorder ◽  
Centromeric Instability ◽  
Tract Infections

Immunodeficiency, centromeric instability and facial anomalies syndrome 2 (ICF2) is a rare autosomal recessive primary immunodeficiency disorder. So far, 27 patients have been reported. Here, we present three siblings with ICF2 due to a homozygous ZBTB24 gene mutation (c.1222 T>G, p. (Cys408Gly)). Immune deficiency in these patients ranged from late-onset combined immunodeficiency (CID) with severe respiratory tract infections and recurrent shingles to asymptomatic selective antibody deficiency. Evident clinical heterogeneity manifested despite a common genetic background, suggesting the pathogenic relevance of epigenetic modification. Immunological follow-up reveals a previously unidentified gradual depletion of B and CD4+ T cells in all three presented patients with transition of a common variable immunodeficiency (CVID)-like disease to late-onset-CID in one of them. Considering all previously published cases with ICF2, we identify inadequate antibody responses to vaccines and reduction in CD27+ memory B cells as prevalent immunological traits. High mortality among ICF2 patients (20%) together with the progressive course of immunodeficiency suggest that hematopoietic stem cell transplantation (HSCT) should be considered as a treatment option in due time.

scholarly journals Epigenetic modification of the human CCR6 gene is associated with stable CCR6 expression in T cells

Blood ◽  
2011 ◽  
Vol 117 (10) ◽  
pp. 2839-2846 ◽  
Author(s):  
Svenja Steinfelder ◽  
Stefan Floess ◽  
Dirk Engelbert ◽  
Barbara Haeringer ◽  
Udo Baron ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd4 T Cells ◽  
Transcriptional Activity ◽  
Epigenetic Modification ◽  
Cell Receptor ◽  
Noncoding Region ◽  
Differentially Methylated Region

Abstract CCR6 is a chemokine receptor expressed on Th17 cells and regulatory T cells that is induced by T-cell priming with certain cytokines, but how its expression and stability are regulated at the molecular level is largely unknown. Here, we identified and characterized a noncoding region of the human CCR6 locus that displayed unmethylated CpG motifs (differentially methylated region [DMR]) selectively in CCR6+ lymphocytes. CCR6 expression on circulating CD4+ T cells was stable on cytokine-induced proliferation but partially down-regulated on T-cell receptor stimulation. However, CCR6 down-regulation was mostly transient, and the DMR within the CCR6 locus remained demethylated. Notably, in vitro induction of CCR6 expression with cytokines in T-cell receptor-activated naive CD4+ T cells was not associated with a demethylated DMR and resulted in unstable CCR6 expression. Conversely, treatment with the DNA methylation inhibitor 5′-azacytidine induced demethylation of the DMR and led to increased and stable CCR6 expression. Finally, when cloned into a reporter gene plasmid, the DMR displayed transcriptional activity in memory T cells that was suppressed by DNA methylation. In summary, we have identified a noncoding region of the human CCR6 gene with methylation-sensitive transcriptional activity in CCR6+ T cells that controls stable CCR6 expression via epigenetic mechanisms.

Immunity of patients surviving 20 to 30 years after allogeneic or syngeneic bone marrow transplantation

Blood ◽  
2001 ◽  
Vol 98 (13) ◽  
pp. 3505-3512 ◽  
Author(s):  
Jan Storek ◽  
Ansamma Joseph ◽  
German Espino ◽  
Monja A. Dawson ◽  
Daniel C. Douek ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Marrow Transplantation ◽  
De Novo ◽  
Average Rate ◽  
Cell Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Transplant Recipients ◽  
Specific Igg ◽  
Tract Infections

Abstract The duration of immunodeficiency following marrow transplantation is not known. Questionnaires were used to study the infection rates in 72 patients surviving 20 to 30 years after marrow grafting. Furthermore, in 33 of the 72 patients and in 16 donors (siblings who originally donated the marrow) leukocyte subsets were assessed by flow cytometry. T-cell receptor excision circles (TRECs), markers of T cells generated de novo, were quantitated by real-time polymerase chain reaction. Immunoglobulin G2 (IgG2) and antigen-specific IgG levels were determined by enzyme-linked immunosorbent assay. Infections diagnosed 15 years after transplantation occurred rarely. The average rate was 0.07 infections per patient-year (one infection every 14 years), excluding respiratory tract infections, gastroenteritis, lip sores, and hepatitis C. The counts of circulating monocytes, natural killer cells, B cells, CD4 T cells, and CD8 T cells in the patients were not lower than in the donors. The counts of TREC+ CD4 T cells in transplant recipients younger than age 18 years (at the time of transplantation) were not different from the counts in their donors. In contrast, the counts of TREC+ CD4 T cells were lower in transplant recipients age 18 years or older, even in those with no history of clinical extensive chronic graft-versus-host disease, compared with their donors. The levels of total IgG2 and specific IgG against Haemophilus influenzae and Streptococcus pneumoniae were similar in patients and donors. Overall, the immunity of patients surviving 20 to 30 years after transplantation is normal or near normal. Patients who received transplants in adulthood have a clinically insignificant deficiency of de novo–generated CD4 T cells, suggesting that in these patients the posttransplantation thymic insufficiency may not be fully reversible.

Reconstitution of CD52-Negative CD4 T Cells after Alemtuzumab-Based T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1182-1182
Author(s):  
Eva M Wagner ◽  
Aline N Lay ◽  
Sina Wenzel ◽  
Timo Schmitt ◽  
Julia Hemmerling ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Lymphocytic Leukemia ◽  
Cell Depletion ◽  
Down Regulation ◽  
T Cell Depletion ◽  
Hematopoietic Stem

Abstract The human CD52 molecule is the target of the monoclonal antibody Alemtuzumab, which is used for treating patients with chemo-refractory chronic lymphocytic leukemia as well as for T cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT). The molecule is expressed on the surface of lymphocytes, dendritic cells and to a lesser extent on blood-derived monocytes. Previously, investigators have demonstrated that the surface expression of CD52 on T cells is down-regulated after in vitro incubation with Alemtuzumab. By treating purified human CD4 T cells over 4 hours with 10 μg/mL Alemtuzumab in medium supplemented with 10% human AB serum in vitro, we observed a strong decrease of CD52 expression by flow cytometry with a maximum 3–7 days after incubation. The CD52 down-regulation was also found at weaker intensity on CD8 T cells. From previous studies in chronic lymphocytic leukemia patients, it is known that Alemtuzumab treatment also leads to a down-regulation of CD52 on T cells in vivo. However, similar experiments have not been performed in allogeneic HSCT patients receiving Alemtuzumab in vivo for T cell depletion. We therefore analyzed the expression of CD52 on human peripheral blood mononuclear cells isolated at repeated time points from 22 allogeneic HSCT patients after reduced-intensity conditioning with fludarabine and melphalan and in vivo T cell depletion with Alemtuzumab (100 mg). Half of the patients received prophylactic CD8-depleted donor lymphocyte infusions (DLI) to promote immune reconstitution. By flow cytometry, we observed that the CD52 expression on monocytes, B cells, and natural killer cells remained unaltered after transplantation and was not influenced by the application of DLI. In contrast, the majority of CD4 T cells were CD52-negative (median, 72%) after transplantation and they remained CD52-negative in patients who did not receive DLI throughout the first year after HSCT. The permanent lack of CD52 expression could not be explained by a continuous effect of Alemtuzumab, because earlier studies have shown that the antibody is not present in active plasma concentrations beyond day +60 after HSCT. In contrast, patients receiving CD8-depleted DLI demonstrated a significant increase in the proportion of CD52-positive CD4 T cells. In three of our patients (DLI: n=2, non-DLI: n=1) we analyzed the donor chimerism of CD52-positive and CD52-negative CD4 T cells sorted with high purity by flow cytometry. Three months after HSCT (before DLI), the proportion of donor T cells was clearly higher among the CD52-negative compared to the small proportion of CD52-positive cells in all patients (44% vs. 10%, 83% vs. 0%, and 100% vs. 40%). In the patient who did not receive DLI, the donor T cell chimerism remained mixed in the CD52-negative and CD52-positive fractions on days 200 (CD52-negative: 95%; CD52-positive: 15%) and 350 (CD52-negative: 92%; CD52-positive: 65%). In contrast, the two patients receiving CD8-depleted DLI showed a strong increase in the proportion of CD52-positive CD4 T cells that were of complete donor origin. Altogether, CD52 is permanently down-regulated in reconstituting CD4 T cells following HSCT with an Alemtuzumab-based TCD regimen unless DLI are applied. Our data support the idea of an active mechanism for CD52 down-regulation in CD4 T cells that is not related to B cells and natural killer cells and that appears to differently affect donor and host T cells, respectively.

Increased Functional T Cell Defects in Patients with low CD4 Counts after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4588-4588
Author(s):  
Udo Holtick ◽  
Lukas P. Frenzel ◽  
Shimabukuro-Vornhagen Alexander ◽  
Sebastian Theurich ◽  
Julia Claasen ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Hematopoietic Stem ◽  
Cd4 Counts ◽  
Graft Vs Host Disease

Background The recovery of the host immune system after allogeneic hematopoietic stem cell transplantation is pivotal to prevent infections, relapse and secondary malignancies. In particular, numerical CD4 T-cell reconstitution is delayed and CD4-helper cell function considered impaired as consequence of the transplant procedure and concommitant immunosuppressive medication. From HIV/AIDS patients it is known that numerical and functional CD4 defects increase the risk of opportunistic infections. Therefore, even in the absence of immunosuppressants and graft-vs-host disease, anti-infective prophylaxis is usually given for at least six months. We hypothesized that the numerical CD4 defect in patients may be reflected by immunosuppressive RNA fingerprints previously established for certain immuno-inhibitory molecules and tested whether the functional CD4 capacity was different according to the CD4 cell number. Methods RNA was separated from CD4 T-cells of 10 patients with CD4 counts >500/µl, 10 patients with CD4 counts <200/µl and four healthy controls. All patients had to be off immunosuppression and without any clinical signs of graft-vs-host disease. Transcriptional activity was assessed with regard to previously defined fingerprints motives for CTLA-4, IL-10, PD-1, TGF-β and PGE-2. CD4 T-cells from all groups were further tested for their proliferative capacity and cytokine production. Results Hierarchical clustering segregated the three groups. Applying the immunosuppressive fingerprints, patients with CD4 T-cells >500/µl were demonstrated to be under the influence of PGE2, whereas patients with CD4 T-cells <200/µl were demonstrated to be under the influence of PGE2 and CTLA-4. In normal controls, no association was found. The proliferative capacity of patient CD4 T-cells upon CD3-CD28-bead stimulation was not significantly different from healthy controls. The production of IL-2 by stimulated CD4 T-cells was significantly downregulated in patients with CD4 T-cells <200/µl, while there was no difference in IFN-ƴ and TNF-α secretion. Conclusion The severity of the CD4 numerical defect reflects the state of immunosuppression as demonstrated by RNA immuno-inhibitory fingerprint motives. This partially translates into functional differences as measured by decreased IL-2 secretion. In addition to time after transplant, CD4 T-cell numbers should be considered for the decision to stop or maintain anti-microbial prophylaxis in patients after allogeneic stem cell transplantation. (UH, LPF and CW, JMC contributed equally to this work.) Disclosures: No relevant conflicts of interest to declare.

Generation of Functional Regulatory T Cells By FOXP3 Gene Transfer into CD4 T Cells from Scurfy Mice and IPEX Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2526-2526 ◽  
Author(s):  
Marianne Delville ◽  
Emmanuelle Six ◽  
Florence Bellier ◽  
Nelly Sigrist ◽  
David Zemmour ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Regulatory T Cells ◽  
Immune Tolerance ◽  
Adoptive Transfer ◽  
Cd4 T Cells ◽  
Hematopoietic Stem ◽  
Foxp3 Gene ◽  
Human Pathology

Abstract IPEX (Immunodysregulation Polyendocrinopathy Enteropathy X-linked) syndrome is the prototype of primary immunodeficiency with prevailing autoimmunity. The disease is caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3), which leads to the loss of function of thymus-derived CD4+CD25+ regulatory T (tTreg) cells. In IPEX patients, the absence of a functional Treg cell compartment leads to the development of multiple autoimmune manifestations (including severe enteropathy, type 1 diabetes and eczema) usually in the first months or years of life. The current treatments for IPEX syndrome include immunosuppressive, hormone replacement therapies. Unfortunately, immunosuppressive treatments are usually only partially effective and their dose is often limited because of the occurrence of infectious complications and toxicity. Currently, the only curative treatment for IPEX syndrome is allogeneic hematopoietic stem cell transplantation (HSCT). The absence of an HLA-compatible donor for all patients and their poor clinical condition particularly expose them to a risk of mortality when HLA partially compatible donors are used. For all these reasons, effective alternative therapeutic approaches are urgently needed. Various preclinical studies have shown that partial donor chimerism is sufficient for complete remission meaning that a small number of functional natural Treg is sufficient to restore immune tolerance. This suggests that a gene therapy approach designed to selectively induce a Treg program in T cells by expressing FOXP3 could be a promising potential cure for IPEX. However, several issues might compromise the success of this strategy: (i) will the introduction of FOXP3 alone be sufficient to induce a stable Treg program or will it require additional transcription factors to lock the Treg function and sustain the stability of transduced cells? (ii) Targeting effector CD4+ T cells might be an issue in terms of T-cell receptor repertoire, since the TCR repertoire of nTregs is different from the one of effector CD4+ T cells, (iii) will FOXP3-transduced T cells be able to migrate to appropriate tissues to control auto-immune reactions?, (iv) infusion of nTreg prevents the appearance of some autoimmune manifestations in murine models, however the infusion was done in prophylaxis before the appearance of the symptoms. In order to address these questions, we have developed a mouse scurfy model to evaluate the functional and stability of the correction in vivo in parallel to the characterization of gene corrected human CD4 T cells from IPEX patients. Scurfy mice develop a disease very close to human pathology due to a spontaneous mutation of Foxp3 gene. We improved Scurfy mice model to improve animal production and increase the timeline of treatement. We demonstrated that FOXP3 gene transfer into murine CD4+ T cells enable the generation of potent regulatory T cells. Indeed we showed the functional suppressive properties of the generated CD4-FOXP3 cells in an optimized flow-cytometry-based in vitro suppression assay. The ability of CD4-FOXP3 to prevent Scurfy disease by adoptive transfer in the first days of life is currently under evaluation. Similarly in humans, we demonstrated that FOXP3 gene transfer into CD4+ T cells from IPEX patients enable the generation of potent regulatory T cells, as shown through the functional in vitro suppressive properties of the generated CD4IPEX-FOXP3. Moreover comparison of the transcriptional profile of these regulatory CD4IPEX-FOXP3 cells to natural Treg by RNA-seq analysis demonstrated a good repression of cytokine transcripts (IL4/5/13/CSF2, CD40L), a strong repression of IL7R, a strong induction of IL1R2, and a moderate activation of typical Treg genes (IL2RA, IKZF2, CTLA4). Therefore, the introduction of a functional copy of the FOXP3 gene into an IPEX patient's T cells may be enough to restore immune tolerance and thus avoid the complications of allogenic HSCT. We will also discuss the challenge of generating a large, homogenous and stable population of cells in vitro for adoptive transfer and whether it can ensure long-term disease correction without generating a context of generalized immunosuppression. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rapid Generation of Multivirus-Specific T Lymphocytes for the Prevention and Treatment of Respiratory Viral Infections

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3332-3332
Author(s):  
Spyridoula Vasileiou ◽  
Annie Turney ◽  
Manik Kuvalekar ◽  
Shivani Mukhi ◽  
Ayumi Watanabe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Viral Antigen ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Cell Transplant ◽  
Activation Markers ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Tract Infections

Abstract Acute upper and lower respiratory tract infections (RTIs) due to community-acquired respiratory viruses (CARVs) including respiratory syncytial virus (RSV), influenza, parainfluenza virus (PIV) and human metapneumovirus (hMPV) are a leading cause of morbidity and mortality worldwide, with individuals whose immune systems are naïve (e.g. children) or compromised being most vulnerable. In allogeneic hematopoietic stem cell transplant (HSCT) recipients, the incidence of CARV-related respiratory viral infection reaches 29%. Most patients initially present with mild symptoms of upper RTI and in 50% of cases the infection progresses to a lower RTI with severe symptoms including bronchiolitis and pneumonia and mortality rates as high as 50%. Currently there are no approved vaccines nor antiviral drugs for hMPV and PIV, while the preventative vaccine for Influenza is not indicated earlier than 6 months post-HSCT. Aerosolized ribavirin is FDA-approved for the treatment of RSV infections, but it is logistically difficult to administer and comes at a considerable cost. Thus, the lack of approved antiviral agents combined with the high cost of antiviral therapy emphasize the need for alternative treatment strategies for CARVs. Our group has previously demonstrated the safety and clinical efficacy of using adoptive T-cell transfer for the treatment of both latent [Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), human herpesvirus 6 (HHV6)] and lytic [adenovirus (AdV)] viruses in recipients of allo-HSCT by generation of multivirus-specific T cell (VST) lines. Given that susceptibility to CARVs is highly associated with underlying immune deficiency, we wanted to explore the potential for extending this approach to Influenza, RSV, hMPV and PIV3 infections. In order to do so, we exposed PBMCs from healthy donors to a cocktail of pepmixes (overlapping peptide libraries) spanning immunogenic antigens derived from our target viruses [Influenza - NP1 and MP1; RSV - N and F; hMPV - F, N, M2-1 and M; PIV3 - M, HN, N and F] followed by expansion in the presence of activating cytokines in a G-Rex device. Over 10-13 days we achieved an average 8.5 fold expansion [increase from 0.25x107 PBMCs/cm2 to mean 1.9±0.2x107 cells/cm2; n=12). Cells were comprised almost exclusively of CD3+ T cells (96.2±0.6%; mean±SEM), with a mixture of cytotoxic (CD8+) and helper (CD4+) T cells and a phenotype consistent with immediate effector function and long term memory, as evidenced by upregulation of the activation markers CD25, CD69, and CD28 as well as expression of central (CD45RO+/CD62L+) and effector memory markers (CD45RO+/CD62L−), with minimal PD1 or Tim3 expression. Anti-viral specificity of multi-R-VSTs was tested in an IFNγ Elispot assay using each of the individual stimulating antigens as an immunogen and all 12 lines screened proved to be reactive against all 4 of the target viruses [Influenza: mean 735±75.6 SFC/2x105, RSV: 758±69.8, hMPV: 526±100.8, PIV3: 391±93.7]. As demonstrated by intracellular cytokine staining, the immune response was mediated by both CD4+ and CD8+ T cell subsets, and the majority of IFNγ-producing cells also produced TNFα. In addition, the cells secreted GM-CSF as measured by Luminex array, with baseline levels of Th2/suppressive cytokines. Furthermore, upon antigenic stimulation our VSTs produced the effector molecule Granzyme B suggesting the cytolytic potential of these expanded cells, which was confirmed in a standard Cr51-release assay against viral pepmix-loaded autologous PHA blasts. Viral antigen-loaded targets were specifically recognized and lysed by our VSTs, while there was no evidence of activity against non-infected autologous or allogeneic targets. In conclusion, we have shown that it is feasible to rapidly generate a single preparation of polyclonal multi-respiratory (multi-R)-VSTs with specificities directed to Influenza, RSV, hMPV and PIV3 and a total of 12 encoded antigens using GMP-compliant manufacturing methodologies. The expanded cells are Th1-polarized, polyfunctional and selectively able to react to and kill viral antigen-expressing targets with no auto- or alloreactivity, attesting to both their selectivity and their safety for clinical use in HSCT recipients. We anticipate such multi-R-VSTs will provide clinical benefit in preventing or treating CARV infections in the immunocompromised. Disclosures Vera: Viracyte: Equity Ownership. Tzannou:Viracyte: Consultancy, Equity Ownership. Leen:Viracyte: Equity Ownership.

scholarly journals A Highly Efficient and GMP Compliant Protocol to Manufacture CCR5 Edited Cells to Treat HIV Infection

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5795-5795
Author(s):  
Marianna Romito ◽  
Emily Meyer ◽  
Sushmita Poddar ◽  
Helena Heinz ◽  
Julia Rositzka ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Transcription Activator ◽  
Ccr5 Gene ◽  
Hematopoietic Stem ◽  
Highly Efficient ◽  
Proliferation Capacity

Abstract Targeted genome editing in blood and immune cells enable new therapeutic applications, especially for infectious diseases. We present a GMP-compliant protocol to manufacture CCR5-edited CD34+ hematopoietic stem and precursor cells (HSPCs) with the goal to cure patients suffering from chronic infection with human immunodeficiency virus type 1 (HIV1). We hypothesize that genetic disruption of the CCR5 gene, which encodes the major HIV1 co-receptor, in HSPCs will give rise to an HIV-resistant immune system after transplantation. We have developed engineered nucleases based on transcription activator-like effector nucleases (TALENs) targeting CCR5. Electroporation of CD4+ T-cells and CD34+ HSPCs with mRNAs encoding TALENs revealed disruption of up to 80% of CCR5 alleles in CD4+ T-cells and over 90% of alleles in HSPCs. The high gene editing frequencies in T-cells and HSPCs were confirmed by deep sequencing, and no cleavage activity above background levels were detected at the top 20 predicted off-target sites. CCR5-edited CD4+ cells preserved their proliferation capacity and their biological function. Importantly, these cells showed significantly reduced CCR5 expression and became resistant to infection with the R5-tropic HIV-1JR-FL virus. The CCR5-edited HSPCs maintained their proliferation potential and their capacity to differentiate into the various blood lineages in vitro and in vivo, and clonal analysis revealed bi-allelic CCR5 disruption in more than 75% of cells. In summary, our developed protocol enables highly efficient and GMP-compliant knockout of the CCR5 locus in clinically relevant cells, so forming the foundation for a planned phase I/II clinical study. Disclosures Gautron: Cellectis SA: Employment. Busser:Cellectis: Employment, Patents & Royalties: Cellectis. Smith:Cellectis. Inc: Employment, Patents & Royalties. Duchateau:Cellectis: Employment, Patents & Royalties: Cellectis. Cathomen:TRACR Hematology: Consultancy; Cellectis: Research Funding; Miltenyi Biotec: Research Funding. Cornu:Cellectis: Research Funding; Miltenyi Biotec: Research Funding.

Preferential Th2 Polarization at the Onset of Thrombotic Microangiopathy (TMA): Immunological Comparison of TMA Versus Acute GVHD in Terms of Peripheral T Cell Subsets, Dendritic Cell Subsets and Cytokine Profiles.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2885-2885
Author(s):  
Ki-Ryang Koh ◽  
Hirohisa Nakamae ◽  
Kensuke Ohta ◽  
Hideo Koh ◽  
Takahiko Nakane ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dendritic Cell ◽  
Thrombotic Microangiopathy ◽  
Cd4 T Cells ◽  
Acute Gvhd ◽  
T Cell Subsets ◽  
Immunological Parameter ◽  
Hematopoietic Stem ◽  
And Control

Abstract Thrombotic microangiopathy (TMA) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a serious complication with a high mortality. Acute GVHD (aGVHD) is one of risk factors for TMA and often overlaps it. In particular, gastrointestinal endothelium is a common target of aGVHD and TMA, which makes clinical diagnosis of TMA difficult, leading to delay early and appropriate treatment for it. In this study, to gain more insight into differences between TMA and aGVHD, comprehensive immunological analysis was performed. Methods: We determined kinetics of peripheral T cell subsets (CD4, CD8, Th1, Th2, γδ-T, NKT) and dendritic cell (DC) subsets (CD11c+DC and CD123+DC), serum 17 different cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF-α, IFN-γ, G-CSF, GM-CSF, MIP-1β, MCP-1), and C-reactive protein (CRP) at the onsets of aGVHD or TMA in 25 patients undergoing allo-HSCT. T cell subsets including CD4 (CD3+CD4+CD8−), CD8 (CD3+CD4−CD8+), Th1 (CD4+CXCR3+CCR4−), Th2 (CD4+CXCR3−CCR4+), γδ-T (CD3+TCR-Vδ2+), and NKT (CD3+CD161+) and DC subsets were determined with a flow cytometer. TMA was diagnosed, following Iacopino’s criteria (Iacopino et al, Bone Marrow Tranplant. 24: 47, 1999). Data of aGVHD or TMA were compared with those of control without either aGVHD nor TMA between on 30 days and 60 days after allo-HSCT. Results: There was a significant decrease in the percentage of Th1 cells in CD4+T cells in TMA (9.1%, n=10), compared to in aGVHD (24.9%, n=9, p=0.003) or in control (21.6%, n=12, p=0.009). In contrast, the percentage of Th2 cells in CD4+T cells was higher in TMA (20.2%) than in aGVHD (9.3%, p<0.001) or in control (12.1%, p=0.003). Accordingly, a significant increase in Th2/Th1 ratio was observed in TMA (4.0), compared to in aGVHD (0.4, p<0.001) or in control (0.7, p<0.001). In addition, a significant increase in the percentage of CD4 cells in CD3+T cells in TMA (59.2%, n=9) was found, compared to in aGVHD (31.2%, n=8, p=0.005) or in control (34.8%, n=11, p=0.007). The percentage of CD8+ cells in CD3+T cells was lower in TMA (26.3%) than in aGVHD (45.8%, p=0.03) or in control (54.4%, p=0.002). On the other hand, there was a significant increase in the ratio of CD11c+DC/CD123+DC in aGVHD (3.6, n=8), compared to in TMA (1.4, n=5, p=0.02) or in control (n=1.8, p=0.02). γδ-T and NKT did not show any significant changes among aGVHD, TMA and control. Moreover, no significant changes were observed in either 17 different cytokines among aGVHD, TMA and control. Of note, in TMA but not in aGVHD nor in control, positive correlations of Th2/Th1 ratio were found with IL-6 (p=0.01, r=0.91, n=6), IL-10 (p=0.03, r=0.86, n=6), and CRP (p<0.001, r=0.93, n=9). Conclusion: Preferential Th2 and CD4 polarizations were observed at the onset of TMA. Thus, simultaneous monitoring of Th1, Th2, CD4 and CD8 was suggested to become a useful immunological parameter for differentiating TMA from aGVHD after allo-HSCT.

Flagellin, a TLR5 Agonist, Down-Regulate CD62L on Donor T Cells and Limit GvHD in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3521-3521
Author(s):  
Mohammad Hossain ◽  
Andrew T Gewirtz ◽  
John D Roback ◽  
Edmund K. Waller
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Immune Reconstitution ◽  
Major Complication ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Post Transplant ◽  
Donor T Cells

Abstract Bacground: Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Flagellin is a bacterial protein and a TLR5 agonist that showed diverse immunological responses in both human and animal including both activation of dendritic cells and immuno-suppression. We recently observed that prophylactic use of flagellin protected allogeneic HSCT recipient from GvHD without affecting host immune reconstitution. Acute GvHD has been reported to be mediated by allo-reactive CD62L+ T cells, and over 80% of murine naïve splenic CD4+ and CD8+ T cells express CD62L. In order to test the effect of flagellin on GvHD mediated by the CD62L+ CD4+ and CD62L+CD8+ donor T cells, we investigated clinical manifestation of GvHD as well as the in vivo expression of CD62L on donor T cells in flagellin treated versus control treated allogeneic HSCT recipients. Methods: We established a parent →F1 MHC major mismatched model (C57BL/6 → C57BL/6 × BALB/c) for allogeneic HSCT for which GvHD is the major complication. Recipient mice received 5 × 10^6 T cell depleted (TCD) bone marrow cells and 5×10^6 or 10×10^6 CFSE labeled donor splenocytes from naïve C57Bl/6 congenic donors. 50 μg flagellin per recipient was administered intraperitoneally 3 hours before irradiation and 24 hours after allogeneic HSCT (treated). CB6F1 recipients that received no flagellin (untreated) and recipients of syngeneic HSCT were used as control. Recipients were sacrificed on day 66+ transplant and the numbers of CD62L+ T cells and foxp3+CD4+CD25+ T cells were determined by FACS. Recipients of CFSE treated donor splenocytes were sacrificed on day 4 post HSCT, splenocytes were harvested and analyzed for CD62L expression on T cell subsets undergone in vivo cell division by Flow cytometry. 5 mice were used per group. Results: Flagellin treated recipients did not have GvHD and had no mortality. Untreated control recipients had 87% survival at 30 days post transplant and had signs of chronic GvHD. While total cell number and also donor spleen- and BM-derived CD4+ and CD8+ T cells per spleen in untreated recipients were significantly lower compared to flagellin treated recipients (p=0.0006) on day 66 post transplant, persistent of donor spleen-derived CD62L+CD4+ T cells and CD62L+CD8+ T cells per spleen were not significantly different (p=0.13 and p=0.07, respectively). Moreover, higher number of foxp3+CD25+CD4+ regulatory T cells were found in the spleen and thymus in treated recipients compared to untreated recipients. Within day 4 post transplant, the number of CD4+ T cells per spleen of treated and untreated recipients increased significantly compared to syngeneic recipients (p=0.001 and p=0.03, respectively). Although equivalent numbers of CD62L+CD4+ T cells were observed in both treated and untreated recipients (p=0.3), significantly increased numbers of CD62L+CD8+ T cells was found in treated recipients compare to untreated recipients (p=0.02). Moreover, significantly higher numbers of divided (far left CFSE staining population) CD62L+CD4+ and CD62L+CD8+ T cells were found in recipients of treated splenocytes within day 4 post transplant followed by down regulation of CD62L surface marker compared to untreated recipients (p=0.02 and p=0.01, respectively). Conclusion: Flagellin treated recipients had limited GvHD and had rapid increased divided CD4+CD62L+ T cells followed by CD62L-ve activated CD4+ T cells per spleen in treated recipients compared to untreated recipients may be one of the major affect mediated by flagellin. Flagellin-TLR5 receptor agonistic effect may reduce production of biological factor(s) essential to generate allo-reactive T cells or directly stimulate CD62L+CD4+ and CD62L+CD8+ T cells in different activation status other than allo-reactive T cells; maintain a balanced immune reconstitution in lymphoid organs by producing regulatory T cells through their thymus. Therefore, use of flagellin may be a novel therapeutic approach to treat blood cancer patients with allogeneic HSCT without GvHD and toxicity.

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