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  • American Society of Hematology  (76)
  • 1
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    Membrane-Associated Proteinase 3 On Granulocytes and Myeloid Leukemia Mediates Reversible Inhibition of T Cell Proliferation Associated with Altered ZAP70 and ERK Signaling
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 1031-1031
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 1031-1031
    Abstract: Abstract 1031 Proteinase 3 (P3), a serine protease constitutively expressed in primary granules and on the membrane of some resting granulocytes, is the target of T cell-mediated autoimmunity in Wegener's granulomatosis (WG) and of anti-leukemia immunity mediated by PR1 (VLQELNVTV)-specific cytotoxic T lymphocytes (PR1-CTL). We have previously shown anti-CD3/CD28 induced proliferation of healthy donor T-cells to be significantly inhibited by peripheral blood polymorphonuclear neutrophils (PMNs) expressing membrane P3 (mP3) at a ratio of 3 PMNs to 1 PBMC. Our results indicate that mP3+ PMNs begin to exert inhibitory effects on T cell proliferation at a ratio of 2.2 mP3+ PMNs to 1 PBMC, a ratio greater than that seen in normal homeostatic conditions in peripheral blood. The inhibition was predominantly enzyme-independent and dose-dependent. Notably soluble P3 exerted similar effects on T cells as was seen with mP3. Additionally soluble P3 induced a G0 cell cycle arrest. Of significance, soluble P3 in acute myeloid leukemia (AML) patient serum can be up to 5-fold higher than that seen in healthy control serum. To confirm mP3 specificity, we FAC-sorted PMNs based on the mP3 co-expressed CD177 molecule to obtain highly purified ( 〉 98%) mP3+ and mP3− PMNs. Compared to activated PBMC alone, activated PBMC co-cultured at a ratio of 1:3 with mP3+ PMNs showed 58% and 57% inhibition of CD8+ and CD4+ T cells, respectively (CD8+ and CD4+: p 〈 0.003). PBMC co-cultured at that same ratio with mP3− PMNs showed less inhibition - only 29% and 26% inhibition of CD8+ and CD4+ T cells, respectively (CD8+: p 〈 0.05; CD4+: p=ns). Inhibition of T cell proliferation by both mP3+PMNs and soluble P3 was blocked by anti-P3 mAbs but not by isotype-matched mAb. Furthermore, P3-mediated inhibition of T-cell proliferation is reversible since removal of PMNs or soluble P3 restored the proliferative capacity of the T cells. Because P3 is over-expressed in AML and chronic myeloid leukemia (CML), we hypothesized that mP3+ leukemia may suppress T-cell proliferation. Healthy donor T cell proliferation was studied with CFSE after stimulation with anti-CD3/CD28 mAbs in the presence or absence of mP3+ AML for five days. AML mediated a dose-dependent inhibition of T-cell proliferation contingent upon the level of mP3 expression (p 〈 0.0001). Co-incubation of PBMC with AML displaying 91% mP3 positivity, reduced proliferation of CD8+ T cells to 29.6%, compared to 94.7% in the PBMC culture alone. This inhibition could be completely abrogated by addition of anti-P3 mAb, restoring the proliferation of CD8+ T cells to a level comparable to that seen in control. In contrast, no inhibition of CD8+ T cell proliferation was observed in co-cultures of T cells with AML in which only 6% of the AML cells expressed mP3. Thus, there is an inverse correlation between percent proliferation of T cells and the amount of mP3 on AML (R=0.4539, p 〈 0.0001). In addition, AML expressing 91% mP3+ induced apoptosis of 〉 70% of the T cells at a ratio of 10 AML: 1 PBMC as assessed by uptake of aqua dye. The association between the amount of mP3 on AML and percent of apoptosis was significant (R=0.7852, p 〈 0.0001), and apoptosis induced by mP3+AML appeared to be specific since T cells did not undergo apoptosis when anti-P3 mAb was added. The same correlation was not seen after PMNs from healthy donors (% mP3+: 62%±21.7, n=14) were co-incubated with PBMC. The percentage of cells undergoing apoptosis was less than 10%, regardless of the extent of PMN mP3 positivity. Of note, mP3 expression is significantly higher in bone marrow myeloid derived suppressor cells (MDSC) from leukemia patients compared to MDSC from healthy donors (79.4±5.23% (n=7), 22.4±11.55% (n=3), respectively; p= 0.0007). Because mP3 inhibited proliferation of T cells stimulated via the T cell receptor (TCR), i.e. anti-CD3/CD28, we compared effects of mP3 on ZAP70 and ERK signaling by phosphoflow cytometry. ZAP70 phosphorylation in CD8+ and CD4+ T cells was reduced by 78% and 80%, respectively, within 5 minutes of co-incubation with mP3+ PMNs compared with T cells stimulated with anti-CD3/CD28 mAbs alone, while ERK1/2 phosphorylation was completely blocked within 10 minutes, suggesting that P3-mediated inhibition of T cell proliferation involves downstream TCR signaling pathways. Taken together, these data support an important new function of membrane-bound P3 on PMN and leukemia in controlling adaptive T cell immunity. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.1031.1031
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
    detail.hit.zdb_id: 1468538-3
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  • 2
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    Regulatory and Naïve T Cells in Unmanipulated Donor Grafts Are Not Associated with Acute Graft Vs Host Disease in Matched Sibling Transplants for AML
    American Society of Hematology ; 2008
    In:  Blood Vol. 112, No. 11 ( 2008-11-16), p. 719-719
    Details
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 719-719
    Abstract: Acute graft-versus-host disease (aGVHD) occurs in approximately 20–50% of matched-related stem cell transplants (HCT). The presence of increased frequencies of CD4+CD25+ regulatory T cells (Treg) in murine donor grafts has been shown to ameliorate aGVHD. In similar models, several groups have demonstrated that the transfer of equivalent numbers of naïve T cells (vs. various memory subsets) confers a relatively greater risk for aGVHD. To test the hypothesis that natural variations in donor graft naïve and Treg content would be associated with the incidence of aGVHD in graft recipients, we conducted a detailed analysis of donor graft T cells within unmanipulated grafts of 70 SCT recipients transplanted for AML/MDS following the administration of a uniform fludarabine/busulfan conditioning regimen. Median follow up time was 38 months. 19% (n=13) of recipients experienced grades II–IV aGVHD (n=3 grades III–IV). Cryopreserved donor grafts samples were analyzed using 9-color flow cytometry (with a panel including a viability dye and MAbs recognizing CD4, CD8, CD25, CD127, CD45RA, CD27, Foxp3, and Ki-67). Tregs were defined as being CD4+CD25+CD127loFoxp3+. CD45RA and CD27 were used to measure naïve (CD45RA+CD27+) and memory conventional CD4+ and CD8+ T cells. In addition, the proliferating fraction of all cells was defined by Ki-67 nuclear-antigen expression. We conducted analyses based on donor graft T cells assessed both continuously and by quartiles, with similar results. No significant associations were found between aGVHD incidence and total Tregs (HR 1.01, 95%CI 0.9–1.2, p 0.8), nor with proliferating Tregs (HR 1.2, 95%CI 0.2–5.9, p 0.8). Since murine models typically fix the dose of Tregs vs. conventional T cells (Tcon), we also analyzed GVHD risk by donor graft Treg:Tcon ratio, finding no association (HR 1.03, 95%CI 0.8–2.1, p 0.3). Although patients in the highest Treg:Tcon quartile received Treg doses known in vitro to induce suppression ( 〉 1:21.5), there was no reduction in aGVHD (HR 1.5 Quartile 4 vs others, 95%CI 0.5–4.9, p 0.5). Additionally, no significant association with aGVHD was found with total naïve T cells (HR 1, 95%CI 0.9–1.0, p 0.9), naïve CD4+ (HR 1, 95%CI 0.9–1.03, p 0.6), or naïve CD8+ cells (HR 0.98, 95%CI 0.9–1.03, p 0.6). Because naïve:memory cell ratios were also fixed in murine models demonstrating the importance of naïve T cells in GVHD, we also assessed naïve:memory T cell ratios within donor grafts with respect to recipient aGVHD. No reduction in aGVHD was observed considering naïve:memory ratios analyzed continuously (HR 1.1, 95%CI 0.3–4.2, p 0.8) or in the quartile receiving the lowest naïve:memory ratio (HR 0.5 Quartile 1 vs others, 95%CI 0.1–2.2, p 0.3). Our results demonstrate that donor graft Treg and naïve T cell content, assessed in the context of unmanipulated PBSC transplantation in matched siblings, does not predict recipient aGVHD incidence. These data suggest that the therapeutic efficacy of Treg enrichment and/ or naïve T cell depletion may require grafts to be manipulated to supraphysiologic levels for maximal therapeutic benefit.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V112.11.719.719
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2008
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 3
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    Platelet-derived TLT-1 is a prognostic indicator in ALI/ARDS and prevents tissue damage in the lungs in a mouse model
    American Society of Hematology ; 2018
    In:  Blood Vol. 132, No. 23 ( 2018-12-06), p. 2495-2505
    Details
    In: Blood, American Society of Hematology, Vol. 132, No. 23 ( 2018-12-06), p. 2495-2505
    Abstract: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) affect & gt;200 000 individuals yearly with a 40% mortality rate. Although platelets are implicated in the progression of ALI/ARDS, their exact role remains undefined. Triggering receptor expressed in myeloid cells (TREM)–like transcript 1 (TLT-1) is found on platelets, binds fibrinogen, and mediates clot formation. We hypothesized that platelets use TLT-1 to manage the progression of ALI/ARDS. Here we retrospectively measure plasma levels of soluble TLT-1 (sTLT-1) from the ARDS Network clinical trial and show that patients whose sTLT-1 levels were & gt;1200 pg/mL had nearly twice the mortality risk as those with & lt;1200 pg/mL (P & lt; .001). After correcting for confounding factors such as creatinine levels, Acute Physiology And Chronic Health Evaluation III scores, age, platelet counts, and ventilation volume, sTLT-1 remains significant, suggesting that sTLT-1 is an independent prognostic factor (P & lt; .0001). These data point to a role for TLT-1 during the progression of ALI/ARDS. We use a murine lipopolysaccharide-induced ALI model and demonstrate increased alveolar bleeding, aberrant neutrophil transmigration and accumulation associated with decreased fibrinogen deposition, and increased pulmonary tissue damage in the absence of TLT-1. The loss of TLT-1 resulted in an increased proportion of platelet-neutrophil conjugates (43.73 ± 24.75% vs 8.92 ± 2.4% in wild-type mice), which correlated with increased neutrophil death. Infusion of sTLT-1 restores normal fibrinogen deposition and reduces pulmonary hemorrhage by 40% (P ≤ .001) and tissue damage by 25% (P ≤ .001) in vivo. Our findings suggest that TLT-1 uses fibrinogen to govern the transition between inflammation and hemostasis and facilitate controlled leukocyte transmigration during the progression of ARDS.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2018-03-841593
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
    detail.hit.zdb_id: 1468538-3
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  • 4
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    Demonstration of a Direct Immunomodulatory Role for Vitamin D in Human T Cells Using Flow Cytometry
    American Society of Hematology ; 2008
    In:  Blood Vol. 112, No. 11 ( 2008-11-16), p. 2577-2577
    Details
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 2577-2577
    Abstract: Clinical and epidemiological studies have demonstrated an increasingly stronger link between Vitamin D deficiency and a broad array of illnesses characterized by inflammation, including autoimmune diseases, coronary artery disease, and cancers. Vitamin D is a steroid hormone that exerts the majority of its biologic effects via the binding of the intracellular Vitamin D receptor (VDR). While upregulation of VDR has been demonstrated in activated bulk T cells using traditional approaches (e.g., western blotting), such assays cannot precisely define VDR distribution and kinetics. To overcome these limitations, we developed what we believe to be the first flow cytometric assay to quantify VDR expression at a single-cell level. We used a primary antibody against VDR (mouse monoclonal IgG2a to human VDR) in permeabilized T cells followed by a labeled secondary antibody. We detected a positive cell population using flow cytometry that was sharply increased following activation, consistent with upregulation of VDR confirmed by immunoblotting of sorted cells. We then applied this validated assay to define the kinetics of VDR upregulation in activated T cells. We stimulated PBMC with PMA:Ionomycin (P:I) for varying intervals and assessed intracellular VDR using flow cytometry. VDR is significantly upregulated by 15 min after stimulation, reaches a plateau after 6 hr, and may remain elevated for up to 7 d. We compared VDR to classical early and late T cell activation markers (CD69 and CD25, respectively), and we found that VDR was upregulated as consistently as (but even earlier than) CD69, and that VDR and CD25 were both consistently upregulated at later intervals (p & lt;0.0001). To examine the association between VDR expression and proliferation, we stimulated CFSE-labeled T cells with OKT3 (2mg/ml) for 5 d and found that proliferating T cells expressed a significantly higher level of VDR than resting T cells, which maintained baseline VDR expression (p & lt;0.0001). To assess the association between T cell cytokine production and VDR expression, we stimulated T cells with (P:I) for 6 hr in the presence of brefeldin A, and we confirmed that all cytokine-producing cells (TNFα, IL-2, IFNγ) were contained within the VDR-high population. We then assessed whether physiologic concentrations of Vitamin D could inhibit T cell proliferation in vitro. We stimulated CFSE-labeled PBMC with either OKT3 or irradiated allogeneic dendritic cells (DC) in the presence or absence of physiologic concentrations of calcitriol (50 nm) for 5 to 7 d. The presence of calcitriol during OKT3 stimulation resulted in significantly reduced cell division (p=0.004, n=5). Using a previously validated phenotype to demarcate activated alloreactive CD4+ T cells (CD4hiCD38+), we demonstrated that physiologic calcitriol supplementation decreased alloreactive activation following 7 d stimulation with allogeneic DC (p=0.0003, n=10). In conclusion, VDR is a consistent and specific early and late marker of T cell activation, suggesting a direct role for the Vitamin D axis in immunoregulation. Furthermore, physiological concentrations of Vitamin D can inhibit T cell proliferation induced by polyclonal stimuli, including allogeneic DC. These data provide confirmation for a direct immunoregulatory role for Vitamin D and suggest that further mechanistic and clinical studies may yield novel therapeutic strategies for inflammatory conditions, including graft-versus-host disease.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V112.11.2577.2577
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2008
    detail.hit.zdb_id: 1468538-3
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  • 5
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    Depletion of the C3 Component of Complement Enhances the Ability of Rituximab-Coated Target Cells to Activate Human NK Cells and Improves the Efficacy of Monoclonal Antibody Therapy in An in Vivo Model
    American Society of Hematology ; 2008
    In:  Blood Vol. 112, No. 11 ( 2008-11-16), p. 3620-3620
    Details
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 3620-3620
    Abstract: Antibody-dependent cellular cytotoxicity (ADCC) is felt to play an important role in mediating the anti-tumor effects of Rituximab (R). We previously reported that C3b deposition on R-coated target cells interferes with the binding of the Fc portion of R to NK cell CD16. This interaction inhibits the activation of NK cells and NK cell-mediated ADCC of 51Cr-labeled R-coated target cells. The current studies were designed to determine whether C3 depletion enhances the ability of mAb-coated targets to activate NK cells in vitro and improves mAb therapy in vivo. Normal human serum inhibited the ability of R-coated lymphoma cells to activate NK cells as previously reported. NK activation was increased when serum was pre-incubated with cobra venom factor (CVF) to deplete C3. Similar results were found when non-malignant ascites or transudative pleural fluid, as a surrogate for extravascular fluid, was used as the source of complement. For in vivo analysis, we utilized a syngeneic, immunocompetent murine model in which ADCC has been previously demonstrated to be a key mechanism of action. CVF or a human C3 derivative with CVF-like functions (HC3-1496) was used to deplete C3 in vivo. In this model, C3H/HeN mice were inoculated with murine 38C13 lymphoma cells (day 0) and treated with 2 doses of CVF or HC3-1496 (day 3 and day 5). Four hours after the initial dose of CVF or HC3-1496, mice were treated with a single dose of an anti-lymphoma mAb directed against the 38C13 idiotype (MS11G6). Untreated mice all developed tumor and died with a median survival of 28 days. All mice treated with mAb alone eventually developed tumor and died with a median survival of 42 days. Survival following treatment with CVF plus mAb was superior to that of mAb alone (Fig 1, p=0.0312) with 50% of mice remaining tumor free. Survival following treatment with HC3-1496 plus mAb was also superior to that of mAb alone (Fig 2, p=0.0002) with 80% of mice remaining tumor free. In summary, depletion of the C3 component of complement enhanced the ability of R-coated target cells to activate human NK cells, and improved the efficacy of mAb therapy in an in vivo model of lymphoma. Furthermore, these studies suggest the inhibitory effects of complement on NK activation and ADCC may be seen in the extravascular compartment such as within involved lymph nodes. We conclude that depletion of complement through use of agents such as CVF or HC3-1496 could be considered as an approach to enhancing the efficacy of R therapy. Figure Figure Figure Figure
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V112.11.3620.3620
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2008
    detail.hit.zdb_id: 1468538-3
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  • 6
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    CD25 expression on donor CD4+ or CD8+ T cells is associated with an increased risk for graft-versus-host disease after HLA-identical stem cell transplantation in humans
    American Society of Hematology ; 2004
    In:  Blood Vol. 103, No. 3 ( 2004-02-01), p. 1140-1146
    Details
    In: Blood, American Society of Hematology, Vol. 103, No. 3 ( 2004-02-01), p. 1140-1146
    Abstract: Graft-versus-host disease (GVHD) occurs in an unpredictable fashion after 30% to 50% of matched-related transplantations. The presence of increased frequencies of CD4+CD25+ regulatory T cells in donor grafts has been shown to ameliorate GVHD after allogeneic transplantation in murine models. To determine whether a similar relationship exists in humans, we quantitated the coexpression of CD25 on CD4+ and CD8+ T cells within 60 donor grafts infused into matched siblings and examined GVHD incidence in the respective recipients. Recipients in whom GVHD developed received donor grafts containing significantly higher frequencies of CD4+ T cells coexpressing CD25 than those who did not (median, 9.26% vs 2.22%; P = .004). Frequencies of donor graft CD8+ T cells coexpressing CD25 were also higher (0.65% vs 0.14%; P = .002). Furthermore, transplant recipients who received grafts containing fewer CD4+CD25+ and CD8+CD25+ T cells were less likely to acquire acute GVHD, even though these donor-recipient pairs were similar to others with respect to relevant clinical variables. These data suggest that the coexpression of CD4 and CD25 may be insufficient to identify regulatory T cells in humans and that increased frequencies and numbers of CD25+ T cells in donor grafts is associated with GVHD in transplant recipients. (Blood. 2004;103:1140-1146)
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2003-06-2085
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2004
    detail.hit.zdb_id: 1468538-3
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  • 7
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    Characterization of optimal T Cell/Dendritic Cell (DC) Co-Culture Conditions for Ex Vivo Expansion of Antigen-Specific Human T Cells.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 3654-3654
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 3654-3654
    Abstract: Adoptive T cell immunotherapy has demonstrated clinical efficacy in controlling reactivation of human herpesviruses (e.g., cytomegalovirus, CMV and Epstein-Barr Virus, EBV) that are known etiologic agents of life-threatening illness in immunocompromised populations. Early approaches utilized in the expansion of large numbers of human virus-specific CD4+ and CD8+ T cell often involved laborious culture techniques impractical for widespread clinical use in populations at risk, including stem cell transplant (SCT) recipients. Advances in our understanding of the biology of myeloid dendritic cells (DC) can now facilitate more practical approaches for ex vivo antigen-specific T cell expansion. These advances have included novel approaches to isolate human peripheral blood monocytes (e.g., CD14 selection) and the optimization of cytokine cocktails inducing DC maturation. To define the optimal culture conditions for human antigen-specific T cell expansion, we conducted an iterative series of pairwise CMV-specific T cell expansions, in which important determinants of the expansion process could be directly compared. T cells, derived from apheresis products obtained from CMV-seropositive healthy donors, were expanded under GLP-compliant conditions, following incubation with autologous myeloid DC pulsed with overlapping pentadecapeptide pools spanning the CMV pp65 protein. In each case, we assessed the expanded CMV-specific T cell populations by immunophenotyping of the generated DC, and by phenotypic analysis, cytokine flow cytometry (CFC), ELISPOT analysis and HLA-peptide tetramer staining of the expanded T cells. Specifically, we compared:DC generated from monocytes isolated via plastic adherence to those isolated via positive selection of CD14+ T cells using magnetic beads;DC matured using tumor necrosis factor-α (TNFα) alone or in combination with other cytokines (ITIP, containing TNFα, IL-1β, IL-6 and PGE2);the quality of expansions derived from a starting lymphocyte population selected by non-adherence to plastic vs. the CD14-negative fraction in the monocyte selection process.From this series of experiments, we can conclude that while CD14 selection of monocytes results in a more phenotypically homogeneous population of DC, that this does not improve the quantity or quality of expanded virus-specific T cells. When we compared TNFα to ITIP maturation of DC, we found that while ITIP induced significantly higher CD83 expression on DC, TNFα-matured DC favored the maintenance of CMV-specific CD4+ T cells in culture resulting in ultimately greater numbers and functional proportions of tetramer-stained CD8+ T cells. Finally, we consistently found that the use of non-adherent PBMC resulted in dramatically better quantitative expansions of CMV-specific CD4+ and CD8+ T cells, vs. a lymphocyte population obtained following depletion of monocytes using CD14 positive selection. These results suggest a set of optimal T cell/DC co-culture conditions that lead to the generation of the largest numbers of expanded, functional CD4+ and CD8+ T cells, and are likely to have relevance for human trials wherein adoptive immunotherapy and/or DC immunization is planned.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.3654.3654
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
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  • 8
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    Functional assessment and specific depletion of alloreactive human T cells using flow cytometry
    American Society of Hematology ; 2004
    In:  Blood Vol. 104, No. 12 ( 2004-12-01), p. 3429-3436
    Details
    In: Blood, American Society of Hematology, Vol. 104, No. 12 ( 2004-12-01), p. 3429-3436
    Abstract: Human T-cell alloreactivity plays an important role in many disease processes, including the rejection of solid organ grafts and graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. To develop a better understanding of the T cells involved in alloreactivity in humans, we developed a cytokine flow cytometry (CFC) assay that enabled us to characterize the phenotypic and functional characteristic of T cells responding to allogeneic stimuli. Using this approach, we determined that most T-cell alloreactivity resided within the CD4+ T-cell subset, as assessed by activation marker expression and the production of effector cytokines (eg, tumor necrosis factor α [TNF]α) implicated in human GVHD. Following prolonged stimulation in vitro using either allogeneic stimulator cells or viral antigens, we found that coexpression of activation markers within the CD4+ T-cell subset occurred exclusively within a subpopulation of T cells that significantly increased their surface expression of CD4. We then developed a simple sorting strategy that exploited these phenotypic characteristics to specifically deplete alloreactive T cells while retaining broad specificity for other stimuli, including viral antigens and third-party alloantigens. This approach also was applied to specifically enrich or deplete human virus-specific T cells.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2004-05-1918
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2004
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 9
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    Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T-cell skewing
    American Society of Hematology ; 2007
    In:  Blood Vol. 110, No. 13 ( 2007-12-15), p. 4543-4551
    Details
    In: Blood, American Society of Hematology, Vol. 110, No. 13 ( 2007-12-15), p. 4543-4551
    Abstract: Advances in immune assessment, including the development of T-cell receptor excision circle (TREC) assays of thymopoiesis, cytokine-flow cytometry assays of T-cell function, and higher-order phenotyping of T-cell maturation subsets have improved our understanding of T-cell homeostasis. Limited data exist using these methods to characterize immune recovery in adult cord blood (CB) transplant recipients, in whom infection is a leading cause of mortality. We now report the results of a single-center prospective study of T-cell immune recovery after cord blood transplantation (CBT) in a predominantly adult population. Our primary findings include the following: (1) Prolonged T lymphopenia and compensatory expansion of B and natural killer (NK) cells was evident; (2) CB transplant recipients had impaired functional recovery, although we did observe posttransplantation de novo T-cell responses to cytomegalovirus (CMV) in a subset of patients; (3) Thymopoietic failure characterized post-CBT immune reconstitution, in marked contrast to results in other transplant recipients; and (4) Thymopoietic failure was associated with late memory T-cell skewing. Our data suggest that efforts to improve outcomes in adult CB transplant recipients should be aimed at optimizing T-cell immune recovery. Strategies that improve the engraftment of lymphoid precursors, protect the thymus during pretransplant conditioning, and/or augment the recovery of thymopoiesis may improve outcomes after CBT.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2007-05-092130
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
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  • 10
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    Delayed Immune Recovery after Umbilical Cord Blood Transplantation (UCBT) Is Characterized by Thymic Regeneration Failure.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 312-312
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 312-312
    Abstract: UCBT is a potentially curative modality for many patients with relapsed or high-risk hematological malignancies who lack suitably matched hematopoietic progenitor cell donors. The advent of transplant approaches that combine multiple CB units to facilitate transplantation into adult recipients, as well as advances in conditioning regimens and supportive care, have significantly improved outcomes following UCBT. Despite these advances, infections and/or relapse associated with impaired T cell reconstitution remains an important source of morbidity and mortality. We prospectively assessed the quantitative and functional recovery of CD4+ and CD8+ T cells in 34 consecutively transplanted UCBT recipients. Functional studies of CD4+ and CD8+ T cells stimulated with a superantigen (staphylococcal enterotoxin B, SEB) and cytomegalovirus (CMV) antigens revealed that SEB-stimulated IFNγ responses by cytokine flow cytometry (CFC) were significantly lower in the UCBT recipients than in control bone marrow or peripheral blood stem cell transplant (SCT) recipients (median values of 0.99% (n=12) vs. 24.8% (n=24) in the CD4 compartment and 0.62% (n=14) vs. 6.37% (n=9) in the CD8 compartment, respectively). Despite this, many UCBT recipients had measurable donor-derived CMV-specific CD4+ and CD8+ T cells by 3 months (4 of 11, 44% in CD4+ T cells and 6 of 12, 50% amongst CD8+ T cells), indicating that primary immune responses were derived from the naïve CB graft precursors. Immunophenotypic studies demonstrated that a higher proportion of effector and late-effector memory cells (i.e., CCR7-CD45RA− and CCR7-CD45RA+ T cells) were present in UCBT recipients than SCT recipients. The median fraction of CCR7-negative T cells was 65% (n=12, UCBT recipients) vs. 30% (n=21, other SCT recipients) in the CD4 compartment, and 76% vs. 31%, respectively, in the CD8 compartment. Furthermore, the subgroup of UCBT recipients with a ratio of CCR7+ to CCR7-negative CD4+ T cells above the median at day 30 after SCT survived significantly longer (666 days vs. 224 days, p=0.03). Finally, we compared thymic regeneration (using a δ-chain T cell receptor excision, δTREC assay) in UCBT recipients with three other groups of SCT recipients, including longitudinally assessed autologous and allogeneic SCT recipients, as well another group of allogeneic SCT recipients assessed cross-sectionally. Thymic recovery was reduced at one and three months after UCBT, relative to all other SCT recipient groups. Median δTREC values (reported as δTRECs/million PBMC) at three months were 0.0 in UCBT recipients (n=12), 12 in autologous SCT recipients (n=11), 124 in allogeneic SCT recipients (n=27), and 2150 in a second group of allogeneic SCT recipients (n=31) (p 〈 0.04 for all observations). These results demonstrate that T cell immune recovery in UCBT recipients is characterized by delayed functional recovery and a relative predominance of late memory (CCR7−) T cells, relative to other SCT recipients. However, naïve donor-derived CB cells are capable of primary pathogen-specific expansion in vivo. Finally, these data suggest that efforts to improve T cell immune reconstitution after UCBT should be directed at improving thymic regeneration, which is likely to be important in facilitating the recovery of naïve and early memory T cells.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.312.312
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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