Immune Reconstitution After Antithymocyte Globulin (ATG)-Conditioned Hematopoietic Cell Transplantation (HCT)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1981-1981 ◽  
Author(s):  
Mark Bosch ◽  
Manveer Dhadda ◽  
Mette Hoegh-Petersen ◽  
Yiping Liu ◽  
Laura M Hagel ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Immune Reconstitution ◽  
Memory B Cells ◽  
Inkt Cells ◽  
Cell Counts

Abstract Abstract 1981 Introduction: Immune reconstitution after HCT is important for curbing infections and malignancy. ATG has been increasingly used to prevent graft-vs-host disease (GVHD), however, its impact on immune reconstitution has not been well studied. Here we studied (1) immune reconstitution after ATG-conditioned HCT, (2) compared it to non-ATG-conditioned HCT, and (3) determined factors influencing the immune reconstitution. Patients and Methods: Immune subset cell counts were determined on day 28, 56, 84, 180, 365 and 730 post transplant in 125 recipients of allogeneic filgrastim-mobilized blood stem cells who received ATG (Thymoglobulin, 4.5 mg/kg) during conditioning. The subset counts were also determined in 47 non-ATG-conditioned patients (otherwise similarly treated). Subset counts (in blood) and ATG levels (in serum) were quantified by flow cytometry. Mann-Whitney rank sum test was used to compare subset counts (1) in ATG-conditioned patients vs donors, (2) in ATG-conditioned patients vs non-ATG-conditioned patients, and (3) between subgroups of ATG-conditioned patients; Spearman rank correlation test was used to determine associations between subset counts and ordinal variables like ATG levels. Results: (1) After ATG-conditioned HCT, the counts of the following subsets normalized (became not significantly lower than in donors) by day 28: NK cells, monocytes, myeloid dendritic cells (MDCs), and plasmacytoid dendritic cells (PDCs). The counts of the following subsets normalized by day 84: memory/effector CD8 T cells, and CD4−CD8− T cells. The counts of naïve B cells normalized by day 180. The counts of the following subsets have not normalized by day 365 or 730: memory B cells (both isotype switched and unswitched), both naïve and memory/effector CD4 T cells, naïve CD8 T cells, CD4+CD8+ T cells, and invariant NKT (iNKT) cells. (2) Compared to non-ATG-conditioned HCT, counts of B cells, CD4 T cells and CD8 T cells were significantly lower after ATG-conditioned HCT on day 28. Thereafter, recovery of both naïve and memory B cells and memory/effector CD8 T cells was significantly faster in ATG-conditioned patients, leading to higher total B and higher total CD8 T cell counts on day 84 (Figure). On the contrary, recovery of naïve CD8 T cells and both naïve and memory/effector CD4 T cells was significantly slower, the latter leading to low total CD4 T cell counts throughout the first year (Figure). (3) Reconstitution after ATG-conditioned HCT was influenced by (a) the number of cells of the same subset transferred with the graft in case of increased memory B cells, naïve CD4 T cells, naïve CD8 T cells, iNKT cells and MDCs, (b) age of recipient in case of decreased naïve CD4 T cells and naïve CD8 T cells, (c) cytomegalovirus (CMV) serostatus of recipient in case of increased memory/effector T cells, (d) GVHD in case of increased naïve B cells, and (e) day 7 or 28 ATG levels in case of decreased T cell subsets. Conclusion: (1) Reconstitution after ATG conditioned HCT is very fast for NK cells, monocytes, MDCs and PDCs, fast for memory/effector CD8 T cells and CD4−CD8− T cells, slow for naïve B cells, and very slow for memory B cells, both naïve and memory/effector CD4 T cells, naïve CD8 T cells, CD4+CD8+ T cells and iNKT cells. (2) Compared to no ATG, the patients conditioned with ATG have lower counts of B and T cells on day 28. Thereafter, the ATG-conditioned patients have faster recovery of both naïve and memory B cells and memory/effector CD8 T cells, and slower recovery of both naïve and memory/effector CD4 T cells and naïve CD8 T cells. (3) Similar to what has been described for non-ATG-conditioned HCT, reconstitution after ATG-conditioned HCT is influenced by the number of the immune cells transferred with the graft, recipient age, recipient CMV serostatus and GVHD. Moreover, the reconstitution after ATG-conditioned HCT is influenced by ATG clearance. Disclosures: No relevant conflicts of interest to declare.

Requirements for Co-Stimulation in T-Cell Dependent and T-Cell Independent Re-Stimulation of FVIII-Specific Memory B Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 238-238 ◽  
Author(s):  
Aniko Ginta Pordes ◽  
Christina Hausl ◽  
Peter Allacher ◽  
Rafi Uddin Ahmad ◽  
Eva M Muchitsch ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Specific Memory ◽  
Stimulation Of

Abstract Memory B cells specific for factor VIII (FVIII) are critical for maintaining FVIII inhibitors in patients with hemophilia A. They are precursors of anti-FVIII antibody-producing plasma cells and are highly efficient antigen-presenting cells for the activation of T cells. The eradication of FVIII-specific memory B cells will be a prerequisite for any successful new approach to induce immune tolerance in patients with FVIII inhibitors. Little is known about the regulation of these cells. Previously we showed that ligands for toll-like receptors (TLR) 7 and 9 are able to re-stimulate FVIII-specific memory B cells in the absence of T-cell help. However, alternative “helper cells” such as dendritic cells are essential for providing help to memory B cells under such conditions. Based on these findings, we asked which co-stimulatory interactions are required for the restimulation of memory B cells in the presence of dendritic cells and ligands for TLR and whether these co-stimulatory interactions are the same as those required for the restimulation of memory B cells in the presence of activated T cells. We used spleen cells from hemophilic mice treated with human FVIII to generate highly purified populations of memory B cells, CD4+ T cells and dendritic cells. The required purity was achieved by a combination of magnetic bead separation and fluorescence-activated cell sorting. The memory B cell compartment was specified by the expression of CD19 together with IgG and the absence of surface IgM and IgD. Memory B cells were cultured in the presence of FVIII to stimulate their differentiation into anti-FVIII antibody-producing plasma cells. Different combinations of CD4+ T cells, ligands for TLR 7 and 9 and dendritic cells were added to the memory-B-cell cultures. Blocking antibodies and competitor proteins were used to specify the co-stimulatory interactions required for the re-stimulation of memory B cells in the presence of either CD4+ T cells or dendritic cells and ligands for TLR 7 and 9. Our results demonstrate that the blockade of B7-1 and B7-2 as well as the blockade of CD40L inhibit the re-stimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing plasma cells in the presence of T-cell help. Similar requirements apply for the re-stimulation of memory B cells in the presence of dendritic cells and ligands for TLR 7 or 9. Dendritic cells in the absence of ligands for TLR are not able to provide help for the re-stimulation of memory B cells, which indicates that dendritic cells need to be activated. Furthermore, ligands for TLR 7 or 9 were not able to re-stimulate memory B cells in the complete absence of dendritic cells. Based on these results we conclude that dendritic cells activated by ligands for TLR 7 or 9 can substitute for activated CD4+ T cells in providing co-stimulatory help for memory-B-cell re-stimulation. CD40-CD40L interactions seem to be the most important co-stimulatory interactions for the re-stimulation of memory B cells, not only in the presence of activated CD4+ T cells but also in the presence of ligands for TLR and dendritic cells.

scholarly journals Neuraminidase-treated macrophages stimulate allogenic CD8+ T cells in the presence of exogenous interleukin 2.

1988 ◽  
Vol 168 (4) ◽  
pp. 1443-1456 ◽  
Author(s):  
Y Hirayama ◽  
K Inaba ◽  
M Inaba ◽  
T Kato ◽  
M Kitaura ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Class I ◽  
Cd4 Cells ◽  
Class I Mhc ◽  
Neuraminidase Treatment

Prior work has shown that purified, resident, and inflammatory peritoneal macrophages are weak stimulators of the allogeneic MLR. We have identified conditions whereby thioglycollate-elicited macrophages become stimulatory, but primarily for the CD8+ T cell subset. The conditions were to treat the macrophages with neuraminidase and to supplement the MLR with rIL-2. These treatments together led to proliferative and cytotoxic responses by isolated CD8+ but not CD4+ T cells. Likewise when MHC-congenic strains were evaluated, an MLR was observed across isolated class I but not class II MHC barriers. Pretreatment of the macrophages with IFN-gamma further enhanced expression of class I MHC products and stimulatory activity, but did not seem essential. While these treatments did not render macrophages stimulatory for an MLR in purified CD4+ cells, blastogenesis of CD4+ cells was observed when the MLR involved bulk T cells. Small allogeneic B lymphocytes behaved similarly to macrophages, in the pretreatment with neuraminidase and supplementation with rIL-2 rendered B cells stimulatory for allogeneic, enriched, CD8+, but not CD4+, T cells. Spleen adherent cells, which are mixtures of macrophages and dendritic cells, stimulated both CD4+ and CD8+ T cells, and neither neuraminidase nor exogenous IL-2 was required. We think that these data suggest that most macrophages and small B cells lack three important functions of dendritic cells: a T cell-binding function that can be remedied by neuraminidase treatment, a T cell growth factor-inducing function that can be bypassed with exogenous IL-2, and an IL-2 responsiveness function that is required by CD4+ lymphocytes.

Vaccine-elicited CD4 T cells prevent the deletion of antiviral B cells in chronic infection

2021 ◽  
Vol 118 (46) ◽  
pp. e2108157118
Author(s):  
Kerstin Narr ◽  
Yusuf I. Ertuna ◽  
Benedict Fallet ◽  
Karen Cornille ◽  
Mirela Dimitrova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Type I ◽  
Clonal Deletion ◽  
Cell Immunity ◽  
B Cell Immunity

Chronic viral infections subvert protective B cell immunity. An early type I interferon (IFN-I)–driven bias to short-lived plasmablast differentiation leads to clonal deletion, so-called “decimation,” of antiviral memory B cells. Therefore, prophylactic countermeasures against decimation remain an unmet need. We show that vaccination-induced CD4 T cells prevented the decimation of naïve and memory B cells in chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. Although these B cell responses were largely T independent when IFN-I was blocked, preexisting T help assured their sustainability under conditions of IFN-I–driven inflammation by instructing a germinal center B cell transcriptional program. Prevention of decimation depended on T cell–intrinsic Bcl6 and Tfh progeny formation. Antigen presentation by B cells, interactions with antigen-specific T helper cells, and costimulation by CD40 and ICOS were also required. Importantly, B cell–mediated virus control averted Th1-driven immunopathology in LCMV-challenged animals with preexisting CD4 T cell immunity. Our findings show that vaccination-induced Tfh cells represent a cornerstone of effective B cell immunity to chronic virus challenge, pointing the way toward more effective B cell–based vaccination against persistent viral diseases.

A Detailed Phenotypic Analysis Using Six Color Flow Cytometry of Lymphocyte Subsets Mobilized with AMD3100 Compared to G-CSF.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 408-408 ◽  
Author(s):  
Yoshiyuki Takahashi ◽  
S. Chakrabarti ◽  
R. Sriniivasan ◽  
A. Lundqvist ◽  
E.J. Read ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Lymphocyte Subsets ◽  
Phenotypic Analysis ◽  
Color Flow ◽  
Cd34 Cells

Abstract AMD3100 (AMD) is a bicyclam compound that rapidly mobilizes hematopoietic progenitor cells into circulation by inhibiting stromal cell derived factor-1 binding to its cognate receptor CXCR4 present on CD34+ cells. Preliminary data in healthy donors and cancer patients show large numbers of CD34+ cells are mobilized following a single injection of AMD3100. To determine whether AMD3100 mobilized cells would be suitable for allografting, we performed a detailed phenotypic analysis using 6 color flow cytometry (CYAN Cytometer MLE) of lymphocyte subsets mobilized following the administration of AMD3100, given as a single 240mcg/kg injection either alone (n=4) or in combination with G-CSF (n=2: G-CSF 10 mcg/kg/day x 5: AMD3100 given on day 4). Baseline peripheral blood (PB) was obtained immediately prior to mobilization; in recipients who received both agents, blood was analyzed 4 days following G-CSF administration as well as 12 hours following administration of AMD3100 and a 5th dose of G-CSF. AMD3100 alone significantly increased from baseline the PB WBC count (2.8 fold), Absolute lymphocyte count (ALC: 2.5 fold), absolute monocyte count (AMC: 3.4 fold), and absolute neutrophil count (ANC: 2.8 fold). Subset analysis showed AMD3100 preferentially increased from baseline PB CD34+ progenitor counts (5.8 fold), followed by CD19+ B-cells (3.7 fold), CD14+ monocytes (3.4 fold), CD8+ T-cells (2.5 fold), CD4+ T-cells (1.8 fold), with a smaller increase in CD3−/CD16+ or CD56+ NK cell counts (1.6 fold). There was no change from baseline in the % of CD4+ or CD8+ T-cell expressing CD45RA, CD45RO, or CD56, CD57, CD27, CD71 or HLA-DR. In contrast, there was a decline compared to baseline in the mean percentage of CD3+/CD4+ T-cells expressing CD25 (5.5% vs 14.8%), CD62L (12.1% vs 41.1%), CCR7 (2.1% vs 10.5%) and CXCR4 (0.5% vs 40.9%) after AMD3100 administration; similar declines in expression of the same 4 surface markers were also observed in CD3+/CD8+ T-cells. A synergistic effect on the mobilization of CD34+ progenitors, CD19+ B cells, CD3+ T-cells and CD14+ monocytes occurred when AMD3100 was combined with G-CSF (Figure). In those receiving both AMD3100 and G-CSF, a fall in the % of T-cells expressing CCR7 and CXCR4 occurred 12 hours after the administration of AMD3100 compared to PB collected after 4 days of G-CSF; no other differences in the expression of a variety activation and/or adhesion molecules on T-cell subsets were observed. Whether differences in lymphocyte subsets mobilized with AMD3100 alone or in combination with G-CSF will impact immune reconstitution or other either immune sequela (i.e. GVHD, graft-vs-tumor) associated with allogeneic HCT is currently being assessed in an animal model of allogeneic transplantation.

Anti-GvHD Effect of Antithymocyte Globulin Is Mediated by Antibodies Binding to T Cells and Regulatory NK Cells, and Less So or Not at All by Antibodies Binding to Other Mononuclear Cell Subsets

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2346-2346
Author(s):  
Mette Hoegh-Petersen ◽  
Minaa Amin ◽  
Yiping Liu ◽  
Alejandra Ugarte-Torres ◽  
Tyler S Williamson ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Antithymocyte Globulin ◽  
Chronic Gvhd ◽  
Wilcoxon Test ◽  
Effector Memory

Abstract Abstract 2346 Introduction: Polyclonal rabbit-anti-human T cell globulin may decrease the likelihood of graft-vs-host disease (GVHD) without increasing the likelihood of relapse. We have recently shown that high levels of antithymocyte globulin (ATG) capable of binding to total lymphocytes are associated with a low likelihood of acute GVHD grade 2–4 (aGVHD) as well as chronic GVHD needing systemic therapy (cGVHD) but not increased likelihood of relapse (Podgorny PJ et al, BBMT 16:915, 2010). ATG is polyclonal, composed of antibodies for antigens expressed on multiple cell subsets, including T cells, B cells, NK cells, monocytes and dendritic cells. These cell subsets may play a role in the pathogenesis of GVHD. The anti-GVHD effect of ATG may be mediated through killing/inhibition of one or several of these cell subsets (eg, T cells) or their subsets (eg, naïve T cells as based on mouse experiments naïve T cells are thought to play a major role in the pathogenesis of GVHD). To better understand the mechanism of action of ATG on GVHD, we set out to determine levels of which ATG fraction (capable of binding to which cell subset) are associated with subsequent development of GVHD. Patients and Methods: A total of 121 patients were studied, whose myeloablative conditioning included 4.5 mg/kg ATG (Thymoglobulin). Serum was collected on day 7. Using flow cytometry, levels of the following ATG fractions were determined: capable of binding to 1. naïve B cells, 2. memory B cells, 3. naïve CD4 T cells, 4. central memory (CM) CD4 T cells, 5. effector memory (EM) CD4 T cells, 6. naïve CD8 T cells, 7. CM CD8 T cells, 8. EM CD8 T cells not expressing CD45RA (EMRA-), 9. EM CD8 T cells expressing CD45RA (EMRA+), 10. cytolytic (CD16+CD56+) NK cells, 11. regulatory (CD16-CD56high) NK cells, 12. CD16+CD56− NK cells, 13. monocytes and 14. dendritic cells/dendritic cell precursors (DCs). For each ATG fraction, levels in patients with versus without aGVHD or cGVHD were compared using Mann-Whitney-Wilcoxon test. For each fraction for which the levels appeared to be significantly different (p<0.05), we determined whether patients with high fraction level had a significantly lower likelihood of aGVHD or cGVHD than patients with low fraction level (high/low cutoff level was determined from ROC curve, using the point with maximum sum of sensitivity and specificity). This was done using log-binomial regression models, ie, multivariate analysis adjusting for recipient age (continuous), stem cell source (marrow or cord blood versus blood stem cells), donor type (HLA-matched sibling versus other), donor/recipient sex (M/M versus other) and days of follow up (continuous). Results: In univariate analyses, patients developing aGVHD had significantly lower levels of the following ATG fractions: binding to naïve CD4 T cells, EM CD4 T cells, naïve CD8 T cells and regulatory NK cells. Patients developing cGVHD had significantly lower levels of the following ATG fractions: capable of binding to naïve CD4 T cells, CM CD4 T cells, EM CD4 T cells, naïve CD8 T cells and regulatory NK cells. Patients who did vs did not develop relapse had similar levels of all ATG fractions. In multivariate analyses, high levels of the following ATG fractions were significantly associated with a low likelihood of aGVHD: capable of binding to naïve CD4 T cells (relative risk=.33, p=.001), EM CD4 T cells (RR=.30, p<.001), naïve CD8 T cells (RR=.33, p=.002) and regulatory NK cells (RR=.36, p=.001). High levels of the following ATG fractions were significantly associated with a low likelihood of cGVHD: capable of binding to naïve CD4 T cells (RR=.59, p=.028), CM CD4 T cells (RR=.49, p=.009), EM CD4 T cells (RR=.51, p=.006), naïve CD8 T cells (RR=.46, p=.005) and regulatory NK cells (RR=.55, p=.036). Conclusion: For both aGVHD and cGVHD, the anti-GVHD effect with relapse-neutral effect of ATG appears to be mediated by antibodies to antigens expressed on naïve T cells (both CD4 and CD8), EM CD4 T cells and regulatory NK cells, and to a lesser degree or not at all by antibodies binding to antigens expressed on B cells, cytolytic NK cells, monocytes or DCs. This is the first step towards identifying the antibody(ies) within ATG important for the anti-GVHD effect without impacting relapse. If such antibody(ies) is (are) found in the future, it should be explored whether such antibody(ies) alone or ATG enriched for such antibody(ies) could further decrease GVHD without impacting relapse. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immune Tolerance Developed in Platelet-Targeted FVIII Gene Therapy in Hemophilia Mice Is CD4+ T Cell-Mediated

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1071-1071
Author(s):  
Yingyu Chen ◽  
Xiaofeng Luo ◽  
Juan Chen ◽  
Jocelyn Schroeder ◽  
Christina K Baumgartner ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Cd4 T Cell ◽  
Control Group

Abstract Immune response to factor VIII (FVIII) is not only a severe complication in protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Our previous studies have demonstrated that platelet-targeted FVIII (2bF8) gene therapy together with in vivo drug-selection of transduced cells can not only rescue the bleeding diathesis but also induce anti-FVIII specific immune tolerance in FVIIInull mice. In the current study, we investigated 1) whether our non-selectable lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and 2) which cell compartment is tolerized after platelet gene therapy. Platelet-specific FVIII expression was introduced by 2bF8LV-transduction of hematopoietic stem cells followed by syngeneic transplantation into FVIIInull mice preconditioned with 660 cGy total body irradiation (TBI) or Busulfan (Bu) plus ATG (anti-thymocyte globulin). After bone marrow transplantation and reconstitution, animals were analyzed by PCR, qPCR, FVIII:C assay, and tail clipping test to confirm the success of 2bF8 gene therapy. Sixteen weeks after transplantation, animals were challenged with recombinant human FVIII (rhF8) via retro-orbital venous administration at a dose of 50 U/kg weekly for 4 weeks. The titers of anti-FVIII inhibitory antibodies (inhibitors) were determined by Bethesda assay. The CFSE-labeled CD4 T cell proliferation assay and ELISPOT-based memory B cell maturation assay were used to determine which cell compartment is tolerized to FVIII after 2bF8 gene therapy. The level of platelet-FVIII expression was 1.44 ± 0.39 mU/108 platelets (n = 6) in the 660 cGy group, which is not significantly different from the level obtained from the Bu+ATG group [3.04 ± 1.19 mU/108 platelets (n = 6)]. Even after rhF8 challenge, no antibodies were detected in 2bF8LV-transduced recipients in either group. In contrast, all animals in the control group that did not undergo gene therapy developed various levels of inhibitors (204±97 BU/ml, n=7). The frequency of regulatory T cells in both 660 cGy TBI (11.01±0.52%) and Bu+ATG (11.02±0.68%) groups were significantly higher than the control group (8.05±0.57%). T cell proliferation assay demonstrated that CD4+ T cells from 2bF8 LV-transduced recipients that had been challenged with rhF8 did not proliferate when restimulated with rhF8 in vitro. The daughter CD4+ T cells in the group with 10 U/ml of rhF8 were 5.84 ± 2.49% (n = 6), which was not significantly different from the control group without no rhF8 stimulation (0 U/ml) (5.33 ± 1.72%). CD4+ T cells from primed FVIIInull mice did proliferate after rhF8 restimulation. The proliferated daughter cell was 13.12 ± 6.76% (n = 7) in the group with rhF8 (10 U/ml) re-stimulation, which is significantly higher than the group without rhF8 co-culture (4.99 ± 1.16%). Since FVIII-specific memory B cell maturation is CD4+ T cell dependent, we isolated CD4+ T and memory B cells from 2bF8LV-transduced or FVIIInull mice after rhF8 immunization and co-cultured with rhF8 followed by ELISPOT assay to examine the antibody secreting cells. No spots were detected when memory B cells from rhF8-primed FVIIInull mice were co-cultured with CD4+ T cells from 2bF8LV-transduced recipients. In contrast, when memory B cells from either rhF8 immunized 2bF8LV-transduced or untreated FVIIInull mice were cultured with CD4+ T cells from rhF8-primed FVIIInull mice, there were 142 and 205 anti-FVIII antibody secreting cells, respectively, detected per 106 cells seeded. These results indicate that CD4+ T cells from 2bF8LV-transduced mice are tolerized to rhF8 stimulation. In conclusion, 2bF8 lentiviral gene transfer without in vivo selection of genetically manipulated cells can introduce FVIII-specific immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell-mediated. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

Low Blood Counts of Memory/Effector CD8 T Cells, Gamma/Delta T Cells, Memory B Cells and Plasmacytoid Dendritic Cells and High Counts of Th2 Cells in Systemic Sclerosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4917-4917
Author(s):  
Jan Storek ◽  
Rob Woolson ◽  
Paul K. Wallace ◽  
Gregory Sempowski ◽  
Peter A. McSweeney ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Systemic Sclerosis ◽  
B Cells ◽  
Cd8 T Cells ◽  
Plasmacytoid Dendritic Cells ◽  
Memory B Cells ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Memory Cd8 T Cells

Abstract Abstract 4917 Introduction: Systemic sclerosis (SSc) is presumed to result from aberrant activation of autoreactive T cells. However, the exact pathogenesis of SSc is not known. Patients and Methods: To contribute to the understanding of the immunopathology of systemic sclerosis (SSc), we compared blood counts of multiple lymphocyte subsets between 20 adult SSc patients not treated with immunomodulatory drugs and healthy controls. The patients had to fit entry criteria for SCOT trial (Scleroderma – Cyclophosphamide or Transplantation?, www.sclerodermatrial.org), i.e, 1. symptoms for no longer than 5 years (except for Raynaud's phenomenon), 2. diffuse scleroderma, and 3. either moderate lung involvement (forced vital capacity (FVC) or diffusion of carbon monoxide (DLCO) between 45 and 70% predicted) or moderate kidney involvement (history of hypertensive renal crisis, but normal renal function at study entry). Multiparameter flow cytometry was used for the determination of the lymphocyte subset counts. Results: Counts of the following subsets were significantly lower in the patients compared to the controls: total T cells (median 1316 vs 2088/ul, p=0.015), total CD8 T cells (273 vs 580/ul, p<0.001), central memory CD8 T cells (23 vs 87/ul, p<0.001), effector memory CD8 T cells (17 vs 39/ul, p=0.015), effector CD8 T cells (28 vs 68/ul, p=0.001), gamma/delta T cells (31 vs 77/ul, p<0.001), switched (IgM/DàIgG/A isotype switched) memory B cells (6 vs 26/ul, p<0.001), non-switched memory B cells (7 vs 17/ul, p=0.004), and plasmacytoid dendritic cells (2 vs 6/ul, p=0.002). Counts of Th2-biased (producing interleukin-4 upon polyclonal stimulation) CD4 as well as CD8 T cells were significantly higher in the patients compared to the controls (248 vs 139/ul for CD4, p=0.002, and 259 vs 164/ul for CD8, p<0.001). Conclusion: Immunopathology of SSc is complex. Low blood counts of memory/effector CD8 T cells, gamma/delta T cells, memory B cells and plasmacytoid dendritic cells and Th2-biased T cells may play a role in the pathogenesis of SSc. However, cause and effect relations need to be established. Given previous reports of increased numbers of CD8 and gamma/delta T cells in the affected tissues of patients with systemic sclerosis and increased numbers of plasmacytoid dendritic cells in the affected tissues of patients with autoimmune diseases (compared to healthy individuals) (Prescott RJ et al: J Pathol 166 (1992) 255–63, Atamas SP et al: Arthritis Rheum 42 (1999) 1168–78, Giacomelli R et al: Arthritis Rheum 41 (1998) 327–34, Yurovski VV et al: J Immunol 153 (1994) 881–91, Nestle FO et al: J Exp Med 202 (2005) 35–43, Farkas L et al: Am J Pathol 159 (2001) 237–43), it is possible that the low blood counts of CD8 T cells, gamma/delta T cells and plasmacytoid dendritic cells result from redistribution of these cells from blood to affected tissues. Disclosures: No relevant conflicts of interest to declare.

Activation of DR3 Signaling Expands Recipient Derived Regulatory T Cells and Enhances Donor Immune Reconstitution Derived from Allogeneic Hematopoietic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1087-1087
Author(s):  
Hidekazu Nishikii ◽  
Byung Su Kim ◽  
Antonio Pierini ◽  
Jeanette Baker ◽  
Dominik Schneidawind ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
B Cells ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Immune Reconstitution ◽  
Hematopoietic Stem ◽  
Hsc Transplantation

Abstract CD4+ Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells which regulate the immune system, maintain the tolerance of self-antigens and enhance immune tolerance after transplantation. It was also reported that recipient derived Treg could provide immune privilege to allogeneic stem cells (HSC) after transplantation. However, the precise interaction with Treg and HSC has not been fully elucidated. In this study, we investigated the role of recipient derived Treg in the engraftment and immune reconstitution following transplantation of purified allogeneic HSC and the effectiveness of Treg expansion following activation of DR3 (Death receptor 3, also called as TNFRSF25) signaling in this model. 　We first tested the effect of Treg depletion using Foxp3-DTR mice in allogeneic HSC transplantation. In this system, FACS-sorted purified HSC (Lin-cKit+Sca1+ population) derived from WT-FVB mice (CD45.1+/H2kq+) were injected into lethally irradiated B6-Foxp3-DTR mice (CD45.2+/H2kb+) with or without pre-treatment of diphtheria toxin (DT). On day 0 and day 28 after transplantation decreased frequencies of Foxp3+ cells in residual recipient derived CD4+ T cells were observed in peripheral blood from the DT treated mice (P<0.001 on day 0, P<0.002 on day 28). Although total myeloid chimerism was comparable between control and DT-treated mice, the frequency of donor derived immune cells including CD4+ T cells (P<0.01 on day 56), CD8+ T cells (P<0.01 on day 56), and B220+ B cells (P<0.001 on day 56) was significantly decreased in DT-treated mice. These data suggested that recipient derived Treg play an important role in allogeneic immune reconstitution after transplantation. DR3 is a member of the TNF receptor superfamily and we previously reported the expansion of Treg by the activation of this signaling pathway (Kim et al, ASH abstract 2013). We next tested whether activation of DR3 signaling by its agonistic antibody would affect the donor immune reconstitution after allogeneic HSC transplantation. The frequencies of Foxp3+ cells in CD4+ T cells were significantly increased in thymus, spleen, peripheral blood, and bone marrow 4 days after antibody injection (P<0.01). Isolated Treg derived from antibody treated mice showed stronger suppressive function in the mixed lymphoid reaction compared with those from isotype antibody treated mice. The mice treated with antibody on day -4 were transplanted with purified allogeneic HSC on day 0. Antibody treated mice showed a higher frequency of donor derived CD4+ T cells (P<0.001 on day 28), CD8+ T cells (P<0.05 on day 28), and B220+ B cells (P<0.05 on day 28) in this allogeneic HSC transplantation model. In summary, our data suggest that recipient derived CD4+Foxp3+ Treg play an important role in donor immune reconstitution and the activation of DR3 signaling in recipient mice enhances donor immune reconstitution by expansion of recipient derived Treg. H.N and BS-K contributed equally to this work. Disclosures No relevant conflicts of interest to declare.

scholarly journals Resistance of Natural Killer and T Cells to Venetoclax Allows for Combination Treatment with Cancer Immunotherapy Agents

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1118-1118 ◽  
Author(s):  
Elisabeth A Lasater ◽  
An D Do ◽  
Luciana Burton ◽  
Yijin Li ◽  
Erin Williams ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Immune Cell ◽  
Single Agent ◽  
Employment Equity ◽  
Equity Ownership

Abstract Introduction: Intrinsic apoptosis is regulated by the BCL-2 family of proteins, which consists of both anti-apoptotic (BCL-2, BCL-XL, MCL-1) and pro-apoptotic (BIM, BAX, BAK, BAD) proteins. Interaction between these proteins, as well as stringent regulation of their expression, mediates cell survival and can rapidly induce cell death. A shift in balance and overexpression of anti-apoptotic proteins is a hallmark of cancer. Venetoclax (ABT-199/GDC-0199) is a potent, selective small molecule BCL-2 inhibitor that has shown preclinical and clinical activity across hematologic malignancies and is approved for the treatment of chronic lymphocytic leukemia with 17p deletion as monotherapy and in combination with rituximab. Objective: To investigate the effects of BCL-2 inhibition by venetoclax on viability and function of immune-cell subsets to inform combinability with cancer immunotherapies, such as anti-PD-L1. Methods and Results: B cells, natural killer (NK) cells, CD4+ T cells, and CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from healthy donors (n=3) were exposed to increasing concentrations of venetoclax that are clinically achievable in patients, and percentage of live cells was assessed by flow-cytometry using Near-IR cell staining. B cells were more sensitive to venetoclax (IC50 of ~1nM) than CD8+ T cells (IC50 ~100nM), NK cells (IC50 ~200nM), and CD4+ T cells (IC50 ~500nM) (Figure A). CD8+ T-cell subset analysis showed that unstimulated naive, but not memory cells, were sensitive to venetoclax treatment (IC50 ~30nM and 240nM, respectively). Resistance to venetoclax frequently involves compensation by other BCL-2 family proteins (BCL-XL and MCL-1). As assessed by western blot in PBMCs isolated from healthy donors (n=6), BCL-XL expression was higher in NK cells (~8-fold) and CD4+ and CD8+ T cells (~2.5-fold) than in B cells (1X). MCL-1 protein expression was higher only in CD4+ T cells (1.8-fold) relative to B cells. To evaluate the effect of venetoclax on T-cell function, CD8+ T cells were stimulated ex vivo with CD3/CD28 beads, and cytokine production and proliferation were assessed. Venetoclax treatment with 400nM drug had minimal impact on cytokine production, including interferon gamma (IFNg), tumor necrosis factor alpha (TNFa), and IL-2, in CD8+ effector, effector memory, central memory, and naïve subsets (Figure B). CD8+ T-cell proliferation was similarly resistant to venetoclax, as subsets demonstrated an IC50 >1000nM for venetoclax. Taken together, these data suggest that survival of resting NK and T cells in not impaired by venetoclax, possibly due to increased levels of BCL-XL and MCL-1, and that T-cell activation is largely independent of BCL-2 inhibition. To evaluate dual BCL-2 inhibition and PD-L1 blockade, the syngeneic A20 murine lymphoma model that is responsive to anti-PD-L1 treatment was used. Immune-competent mice bearing A20 subcutaneous tumors were treated with clinically relevant doses of venetoclax, murine specific anti-PD-L1, or both agents. Single-agent anti-PD-L1 therapy resulted in robust tumor regression, while single-agent venetoclax had no effect. The combination of venetoclax and anti-PD-L1 resulted in efficacy comparable with single-agent anti-PD-L1 (Figure C), suggesting that BCL-2 inhibition does not impact immune-cell responses to checkpoint inhibition in vivo. These data support that venetoclax does not antagonize immune-cell function and can be combined with immunotherapy targets. Conclusions: Our data demonstrate that significant venetoclax-induced cell death at clinically relevant drug concentrations is limited to the B-cell subset and that BCL-2 inhibition is not detrimental to survival or activation of NK- or T-cell subsets. Importantly, preclinical mouse models confirm the combinability of BCL-2 and PD-L1 inhibitors. These data support the combined use of venetoclax and cancer immunotherapy agents in the treatment of patients with hematologic and solid tumor malignancies. Figure Figure. Disclosures Lasater: Genentech Inc: Employment. Do:Genentech Inc: Employment. Burton:Genentech Inc: Employment. Li:Genentech Inc: Employment. Oeh:Genentech Inc: Employment. Molinero:Genentech Inc: Employment, Equity Ownership, Patents & Royalties: Genentech Inc. Penuel:Genentech Inc: Employment. Sampath:Genentech Inc: Employment. Dail:Genentech: Employment, Equity Ownership. Belvin:CytomX Therapeutics: Equity Ownership. Sumiyoshi:Genentech Inc: Employment, Equity Ownership. Punnoose:Roche: Equity Ownership; Genentech Inc: Employment. Venstrom:Genentech Inc: Employment. Raval:Genentech Inc: Consultancy, Employment, Equity Ownership.

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