Functional Comparison of Freshly Isolated and Ex-Vivo Expanded CD4+CD25+Foxp3+ Regulatory T Cells in Suppressing Murine Acute GvHD

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 812-812 ◽  
Author(s):  
Emanuela I Sega ◽  
Dennis Leveson-Gower ◽  
Vu H. Nguyen ◽  
Robert Negrin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Acute Gvhd ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Allogeneic Bone ◽  
Cell Reactivity ◽  
Hematopoietic Stem

Abstract Graft versus host disease (GVHD) is a major complication of hematopoietic stem cell transplantation resulting from donor T cell reactivity against host tissue antigens. CD4+CD25+Foxp3+ regulatory T cells (Treg) are known to be important in maintaining self tolerance and preventing autoimmunity. Using murine models of acute GVHD in which allogeneic bone marrow cells are transplanted into lethally irradiated hosts, we and others have shown that donor Treg are able to suppress GVHD induced by donor allogeneic T cells and dramatically improve survival. Treg are rare and suppression of GVHD requires adequate numbers of Treg in relation to the number of conventional T cells (Tcon). To overcome this problem, expansion of Treg has been performed, however there has not been a head to head comparison of the function of expanded vs fresh Treg. Highly purified CD4+CD25+Foxp3+ T cells (>98% purity) were expanded using anti-CD3/anti-CD28 dynabeads and 1000 U/ml IL-2. Under these conditions, after five days Treg expanded up to 13 fold while maintaining high Foxp3 expression levels (85–90%). Longer expansion periods result in more T cell expansion but an overgrowth of Foxp3 negative T cells. In a mixed lymphocyte reaction assay, the ex-vivo expanded Treg efficiently suppressed the proliferation of alloreactive T cells. The expanded Treg were evaluated in an in vivo acute GVHD mouse model in direct comparison with freshly isolated Treg using a novel bioluminescent imaging assay that allowed for assessment of Tcon proliferation in addition to traditional metrics of GVHD severity including weight gain, survival and GVHD score. Initial experiments show that, similar to freshly isolated Treg, the ex-vivo expanded Treg suppress GVHD symptoms and improve survival, although a greater number of expanded Treg were required comparable to freshly isolated Treg. The mean GVHD score for the Tcon alone group was 5.8±1.02. Fresh Treg added at 1:1 ratio decreased the GVHD score to 0.75±0.25 (p=0.0036). Ex-vivo expanded Treg demonstrated a dose-dependent decrease in GVHD score, although four times more expanded Treg were needed to obtain a similar reduction in GVHD score (0.50±0.5, p=0.0036). This observed difference in potency was not due to the ex-vivo expanded Treg being short-lived when infused in mice. Bioluminescence imaging of luciferase positive (luc+) cultured Treg showed the same in vivo persistence as freshly isolated Treg. The ability to expand ex-vivo generated Treg is greater than the difference in potency, making ex-vivo expanded Treg potentially a viable option for treatment of GVHD, however, increased ratios of Treg:Tcon are likely to be required.

Naive and Ex Vivo Activated Human T Cells Generate Consistent Engraftment and Lethal Graft-Versus-Host Disease (GvHD) in NOD SCID β 2M Null Mice: A New Xenogeneic Model for GvHD.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3106-3106
Author(s):  
Bruno Nervi ◽  
Michael P. Rettig ◽  
Julie K. Ritchey ◽  
Gerhard Bauer ◽  
Jon Walker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Conditioning Regimen ◽  
Activation Markers ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Human T Cells ◽  
The Impact

Abstract GvHD remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion. The human GvHD pathophysiology includes recipient tissue destruction and proinflammatory cytokine production associated with the conditioning regimen; donor T cells become allo-activated, proliferate, and mediate tissue injury in various organs, including the liver, skin, and gut. Modern therapeutic strategies to control GvHD while maintaining the beneficial graft-versus-leukemia effects require ex vivo T cell stimulation and expansion. Multiple studies have demonstrated that these ex vivo expanded T cells exhibit decreased survival and function in vivo, including reduced alloreactivity and GvHD potential. Unfortunately no in vivo models exist to consistently examine the impact of ex vivo manipulation of human T cells (HuT) on T cell function. Naive HuT were compared to HuT activated using CD3/28 beads (XcyteTMDynabeads) with 50 U/ml IL-2 for 4 days (Act). We initially evaluated the HuT engraftment and GvHD potential of naive and Act in RAG2γ null mice (n=22) conditioned with clodronate liposomes on day −1 and 350cGy on day 0, as previously described by others. We injected 107 and 1.5x107 naive or Act HuT intravenously (iv). All mice exhibited low HuT engraftment and no lethal GvHD. NOD SCIDβ 2M null mice (β 2M) were next conditioned with 250cGy on day −1 (n=34), or 300cGy on day 0 (n=21). 107 naive vs Act HuT were injected retroorbitaly (ro). Lower HuT doses or iv injection resulted in no expansion or GvHD. Engraftment of HuT in peripheral blood of recipient mice was evaluated weekly by FACS and euthanasia was performed if mice lost > 20% body weight. 60% of the mice conditioned with 250cGy that received naive HuT developed lethal GvHD, in comparison to 75% of mice that received 300cGy and nave HuT, and 100% of mice that received 300cGy and Act HuT. Table 1 250cGy 300cGy Naive (n=34) Naive (n=8) Activated (n=13) *p<0.02 PB engraftment (%HuT) 20%±15 33%±21 59%±19 Lethal GvHD 60% 75% 100% All mice receiving 300cGy had well preserved CD4/CD8 ratios (1–1.5). Tissue infiltration was greatest in mice that had received 300cGy and Act HuT (spleen, liver, lung, kidney: 50–70%). Of interest, serum levels of hu IFNγ dramatically increased over time in all mice who went on to develop lethal GvHD (day 3=270 ug/ml and day 15=36,000 ug/ml) compared to mice that did not develop lethal GvHD (day 10=40 ug/ml and day 17=1,020 ug/ml)(p<0.05). Interestingly, the up-regulation of the activation markers CD25 and CD30 in HuT, and IFNγ production predicted lethal GvHD in β 2M null mice. In summary, we developed a xenogeneic model of lethal GvHD where naive or ex vivo Act HuT injected ro in sublethaly irradiated β 2M not only engraft, expand in vivo, but also infiltrate and damage different mouse target organs. HuT are allo-activated against mouse antigens and damage the target tissues, sharing the major characteristics of human GvHD and causing the death of mice. This model will allow us to study the effects of specific ex vivo T cell manipulation including transduction, selection, expansion, and the depletion or addition of various T cells and other cellular subsets on the outcome of GvHD, to determine improved therapeutic interventions.

Repertoire Analysis of Ex Vivo Polyclonally Expanded Regulatory T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3222-3222
Author(s):  
Jenny Zilberberg ◽  
Kira Goldgirsh ◽  
Robert Korngold ◽  
Thea M. Friedman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Clinical Situation ◽  
Post Transplantation ◽  
Cell Repertoire ◽  
Hematopoietic Stem ◽  
Pcr Products

Abstract CD4+CD25+ regulatory T cells (Treg) are essential for the maintenance of self-tolerance and have also been implicated in the control of alloreactive immune responses. Several studies using murine models of graft-vs.-host disease (GVHD) have shown that addition of equivalent numbers of Treg to the donor T cell inoculum at time of hematopoietic stem cell transplantation can significantly reduce the incidence of GVHD. In addition, in an MHC-matched, minor histocompatibility disparate model, the infusion of Treg ten days post-transplantation was shown to ameliorate the progression of GVHD while permitting a graft-versus-leukemia effect. However, because Treg constitute <5% of peripheral CD4+ T cells in humans, the use of freshly isolated Treg to treat and/or prevent GVHD, as well as other diseases in the clinical situation, is limited. Therefore, much effort is now under way to expand Treg in order to have sufficient numbers for therapeutic use. There is little available information regarding the repertoire complexity of ex vivo, polyclonally expanded regulatory T cells. We hypothesize that like their CD4+CD25− T cell counterparts, the diversity of the Treg T cell receptor (TCR) repertoire will also be complex. To this end, CD4+CD25− and CD4+CD25+ T cells from B10.BR mice were purified using fluorescence activated cell sorting; both populations were polyclonally expanded using CD3/CD28 paramagnetic microbeads in combination with high levels (100 IU/ml) of hrIL-2. After achieving a greater than 50 fold expansion, RNA from 1–1.5×107 cells was isolated for RT-PCR. The complexity of the T cell repertoire of expanded CD4+CD25− and CD4+CD25+ was determined using TCR Vb CDR3-size spectratype analysis. The PCR products were run on a sequencing gel and analyzed by the GeneMapper Software from Applied Biosystems. This comparison revealed that the number of resolvable Vb families is more heterogeneous in the CD25− populations. Whether this reflected a lack of complexity in the regulatory repertoire warrants further investigation. However, for the resolvable Vb families there were no significant differences in the complexity indexes between these two groups.

Chimeric Regulatory T cells Enhance HSC Engraftment in An Antigen-Specific Fashion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2424-2424
Author(s):  
Yiming Huang ◽  
Larry D Bozulic ◽  
Thomas Miller ◽  
Hong Xu ◽  
Yujie Wen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Third Party ◽  
T Cell Proliferation ◽  
Hematopoietic Stem ◽  
Mixed Chimeras

Abstract Abstract 2424 Poster Board II-401 We previously reported that CD8+TCR- facilitating cells (FC) induce the generation of chimeric regulatory T cells (Treg) in vivo. Transplantation of a mixture of CD8+/TCR- FC and hematopoietic stem cells (HSC) into ablated recipients results in chimerism and tolerance. Treg harvested from the spleen of chimeras (chimeric Treg) potently increase long-term donor chimerism in secondary NOD recipient mice. Here, we evaluated whether chimeric Treg enhance engraftment of hematopoietic stem cells (HSC) in an antigen-specific manner. To prepare mixed chimeras (B6 → NOD), NOD recipients were conditioned with 950 cGy TBI and transplanted with 10,000 B6 HSC and 1,000 NOD HSC plus 45,000 CD8+TCR- B6 FC. At 5 weeks, CD8-CD4+CD25bright chimeric Treg were sorted from spleens of the mixed chimeras (B6 → NOD). 100,000 chimeric Treg were then mixed with 10,000 B6 HSC (donor-specific) + 10,000 B10.BR HSC (third-party) and transplanted into conditioned NOD recipients in competitive repopulation assays. NOD mice given HSC plus nonchimeric naïve B6 Treg or HSC alone served as controls. Two of the four animals that received HSC alone engrafted and exhibited an average of 6.7% donor B6 chimerism at 30 days, 11.2% at 60 days, and 10.6% at 90 days. Three of five animals given HSC plus naïve B6 Treg engrafted with 21.3% donor B6 chimerism at 30 days, 28.8% at 60 days, and 28.9% at 90 days. In contrast, eight of nine recipients of HSC + chimeric Treg engrafted. These animals exhibited a significantly higher level of donor B6 chimerism, ranging from 56.3% at 30 days, 75.4% at 60 days to 85% at 90 days (P = 0.034). None of the recipients engrafted with the MHC-disparate third-party B10.BR HSC. We then assessed the suppressive function of chimeric Tregin vitro by using MLR suppressor cell assays. CD8-/CD4+/CD25bright Treg were sorted from chimeric spleens 5 wks to 12 wks after HSC + FC transplantation. As shown in the Figure 1, Treg from naïve B6 mice resulted in 1.9 fold; 1.3 fold and 1.1 fold inhibition of proliferation at 1:1, 1:0.25, 1:0.125 responder/Treg ratios (n = 3). In contrast, chimeric Treg potently suppressed T cell proliferation by 10.5 fold; 3.2 fold; and 1.7 fold at responder/Treg ratios of 1:1, 1:0.25, 1:0.125 (n = 4). Chimeric Treg significantly suppressed T cell proliferation at responder/Treg ratios of 1:1 and 1:0.25 compared with naïve B6 Treg (P < 0.05). NOD responder splenocytes remained hypoproliferative in response to B6 stimulator and chimeric Treg compared with stimulator plus B6 Treg, suggesting that chimeric Treg are significantly more potent than naïve B6 Treg in suppressing effector T cell proliferation in vitro. These data show that chimeric Treg enhance donor B6 HSC engraftment but not third-party B10.BR HSC, demonstrating that chimeric Treg function in vivo in an antigen-specific fashion. These data also show that the mechanism of FC function in vivo is associated with the establishment of an antigen-specific regulatory feedback loop. Figure 1 Figure 1. Disclosures: Bozulic: Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

Anti-Thymocyte Globulin (ATG) Differentially Depletes naïve and Memory T Cells and Permits Memory-Type Regulatory T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4670-4670
Author(s):  
Chang-Qing Xia ◽  
Anna Chernatynskaya ◽  
Clive Wasserfall ◽  
Benjamin Looney ◽  
Suigui Wan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Memory T Cells ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Hematopoietic Stem

Abstract Abstract 4670 Anti-thymocyte globulin (ATG) has been used in clinic for the treatment of allograft rejection and autoimmune diseases. However, its mechanism of action is not fully understood. To our knowledge, how ATG therapy affects naïve and memory T cells has not been well investigated. In this study, we have employed nonobese diabetic mouse model to investigate how administration of anti-thymocyte globulin (ATG) affects memory and naïve T cells as well as CD4+CD25+Foxp3+ regulatory T cells in peripheral blood and lymphoid organs; We also investigate how ATG therapy affects antigen-experienced T cells. Kinetic studies of peripheral blood CD4+ and CD8+ T cells post-ATG therapy shows that both populations decline to their lowest levels at day 3, while CD4+ T cells return to normal levels more rapidly than CD8+ T cells. We find that ATG therapy fails to eliminate antigen-primed T cells, which is consistent with the results that ATG therapy preferentially depletes naïve T cells relative to memory T cells. CD4+ T cell responses post-ATG therapy skew to T helper type 2 (Th2) and IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) are less sensitive to ATG depletion and remain at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory-like immunophenotype is significantly increased in ATG-treated animals, which might play an important role in controlling effector T cells post ATG therapy. In summary, ATG therapy may modulate antigen-specific immune responses through modulation of naïve and memory T cell pools and more importantly through driving T cell subsets with regulatory activities. This study provides important data for guiding ATG therapy in allogenieic hematopoietic stem cell transplantation and other immune-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Prior Use of Mogamulizumab to Allogenic Hematopoietic Stem Cell Transplantation Induces Severe Acute Graft-Versus-Host Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1940-1940 ◽  
Author(s):  
Takeshi Sugio ◽  
Koji Kato ◽  
Takatoshi Aoki ◽  
Takanori Ota ◽  
Noriyuki Saito ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Regulatory T Cells ◽  
Research Funding ◽  
Acute Gvhd ◽  
Cell Lymphoma ◽  
Hematopoietic Stem ◽  
Graft Versus Host

Abstract [Introduction] Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma (PTCL) with a dismal prognosis. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment in ATL patients. Mogamulizumab, a humanized anti-CC chemokine receptor 4 (CCR4) monoclonal antibody, is a novel immunotherapeutic agent, effective in treating patients with PTCL such as ATL, PTCL-not specified, and cutaneous T-cell lymphoma. However, in allo-HSCT setting, we should be careful to use mogamulizumab because CCR4 is expressed in regulatory T cells: The mogamulizumab treatment may accelerate GVHD by eradicating regulatory T cells in allo-HSCT patients. Here, we retrospectively analyzed the effect of mogamulizumab on GVHD development in ATL patients treated with mogamulizumab prior to allo-HSCT. [Patients and Methods] Data from the Fukuoka Bone Marrow Transplantation Group were retrospectively analyzed after the approval of mogamulizumab use in Japan. [Results] A total of 24 patients with ATL received mogamulizumab prior to allo-HSCT between April 2012 and April 2015 in our group. The median age at allo-HSCT was 58.5 years (range, 32-72). The median intervals from the last administration of mogamulizumab to allo-HSCT were 25 days (range, 9-126). The median total dose of mogamulizumab was 3 mg/kg (range, 1-8 mg/kg). After treatment with mogamulizumab, 18 patients (75%) had achieved in remission (CR in 4 patients and PR in 14) at allo-HSCT. Ten patients received unrelated bone marrow, 5 received related peripheral blood, and 9 received cord blood as stem cell sources. Eleven patients were treated with full-intensity conditioning and 13 received reduced-intensity conditioning. Graft-versus-host disease (GVHD) prophylaxis consisted of calcineurin inhibitors (cyclosporine or tacrolimus) with short-term methotrexate in 14 patients and mycophenolate mofetil in 9. The cumulative incidence (CI) of acute GVHD at 100 days was 66.6% in grade 2-4 and 33.3% in grade 3-4. The involved organs of acute GVHD were skin in 14 patients, gut in 10, and liver in 4. Among 14 patients who developed grade 2-4 acute GVHD, 5 had severe fluid retention such as pleural effusion or ascites associated with GVHD. Chronic GVHD was observed in 6 patients, and 5 of them were extensive disease. The CI of transplant-related mortality (TRM) and relapse at 1-year were 53.2% (95%CI, 29.3-72.3%) and 29.6% (95%CI, 12.6-48.9%), respectively. The leading cause of death was GVHD (n = 7). The 1-year overall survival and progression-free survival were 19.2% (95%CI, 5.7-38.8%) and 17.2% (95%CI, 4.9-35.7%), respectively. [Discussion] Use of mogamulizumab prior to transplantation in allo-HSCT patients has a merit to decrease the burden of ATL cells. However, it was associated with an increase of TRM due to severe GVHD. Although most of ATL patients achieved better disease status at allo-HSCT through mogamulizumab and the survival rate was expected to be 50% based on the previous data, the survival in the present study was ~20%. These data suggest that mogamulizumab administered before transplantation may have retained until an early phase of post-transplantation, and the donor or host-derived regulatory T cells might be eliminated, allowing the GVHD T-cell clone to expand. Since mogalizumab is a potent anti-ATL agent, we need to develop new treatment protocols integrating mogalizumab at a suitable dose or administration timing, to minimize the unwanted GVHD development in future studies. Disclosures Akashi: Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

In Vivo Expansion of CD4+ FOXP3+ Regulatory T Cells May Contribute To Control of Acute GvHD after HLA-Mismatched Alloanergized HSCT.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2978-2978 ◽  
Author(s):  
Jeff Davies ◽  
Dongin Yuk ◽  
Lisa Brennan ◽  
Lee Nadler ◽  
Eva Guinan
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Peripheral Blood ◽  
Immune Reconstitution ◽  
Acute Gvhd ◽  
Ex Vivo ◽  
Hla Dr ◽  
Donor T Cells ◽  
Mismatched Donor

Abstract Preventing Graft-versus-Host Disease (GvHD) without impairment of immune reconstitution is a major goal in HLA-mismatched hematopoietic stem cell transplantation (HSCT). Many experimental strategies to selectively destroy or remove alloreactive donor T cells after allostimulation prior to infusion have been explored. An alternative approach is costimulatory blockade (CSB) during ex vivo allostimulation of donor T cells, rendering allospecific T cells within the donor cell pool alloanergized (i.e hyporesponsive to subsequent alloantigenic challenge). Murine and human data suggest effective induction of alloanergy by ex vivo CSB may involve an active cell-mediated suppression process requiring the presence of CD4+ CD25+ regulatory T cells (Tregs). We conducted a pilot clinical study of haploidentical HSCT after allospecific CSB with anti-B7.1 and -B7.2 antibodies and measured reconstitution of Treg by intracellular flow cytometry. 5 patients (pts; 4 high risk acute lymphoblastic leukemia, one marrow failure) underwent cyclophosphamide/TBI-conditioned haploidentical HSCT with cyclosporine and methotrexate as GvHD prophylaxis. Donor bone marrow was incubated with irradiated recipient peripheral blood mononuclear cells and 10μg anti-B7.1/2 antibodies/106 cells for 48 hours to induce alloanergy, washed and infused. All pts engrafted. All evaluable patients had a marked relative increase in peripheral blood CD4+ FOXP3+ T cells at D+20-60 (Figure 1). CD4+ FOXP3+ cells were CD25+ CD45RO+ intracellular CTLA4+ CD127lo consistent with a memory Treg phenotype. Treg were predominantly negative for HLA DR differentiating them from activated T cells. Despite receiving high doses of mismatched donor T cells (median 1.8 (CD4) and 3.1 (CD8) x 107/kg) and all pts achieving 100% donor chimerism, only 2 pts developed acute GvHD, both Grade II, resolving after short courses of corticosteroids. All evaluable patients also had an increase in CD4+ T effector (Teff) cells with an activated phenotype (CD25+ HLA DR+ FOXP3-) at D+30-50. Pts had very rapid immune reconstitution (CD4, CD8, NK and CD8-CMV-tetramer+ cell numbers and immunoglobulin levels) and have had normal vaccination responses. 2 pts died, at D+35 (bacterial sepsis) and D+71 (multi-organ failure), both without GvHD. 3 pts survive (median follow up 5 years) with normal performance status with no chronic GvHD or disease relapse. Conditioning-related cytokine secretion may have led to reversal of anergy in vivo and expansion of alloreactive cells within the Teff cell population. The marked in vivo expansion of Treg may represent one mechanism of suppression of alloreactive Teff and subsequent immunological control of acute GvHD without impairing immune reconstitution in pts receiving HLA-mismatched donor T cells after ex vivo allospecific CSB. We are using a modification of this strategy in a clinical trial of delayed infusion of escalating doses of alloanergized donor T cells after CD34-selected haploidentical HSCT, to determine the optimal dose of alloanergized donor T cells that controls acute GvHD without impairment of immune reconstitution. Figure Figure

Adoptive Therapy with T-Cell Precursors for Immune Reconstitution After Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3525-3525
Author(s):  
Emanuela Burchielli ◽  
Antonella Tosti ◽  
Loredana Ruggeri ◽  
Katia Perruccio ◽  
Claudia De Angelis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Triple Negative ◽  
Ex Vivo ◽  
Double Positive ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Single Positive ◽  
Human Hscs

Abstract Abstract 3525 Poster Board III-462 Recipients of allogeneic hematopoietic transplantation experience a slow reconstitution of donor-derived B and T cell number and function. This post-transplant period of immunodeficiency is associated with an increased risk of infection and malignant relapse. The developement of these complications notably correlates with the recovery of CD4+T cell subset. We proposed a strategy to enhances in vivo reconstitution by promoting donor-derived T cell development in the recipient's thymus. Recently Notch1-based ex-vivo system have been established to mature cord blood- or bone marrow-derived human HSCs into committed T-cell precursors. We used this system for the generation of T-cell precursors starting from G-CSF mobilized human HSCs. We cultured mobilized human CD34+ hematopoietic stem cells (HSCs) (2.5 × 105) in vitro on OP9 mouse stromal cells expressing the Notch 1 ligand Delta-like-1 (OP9-DL1) in the presence of rhFLT3-ligand (5ng/ml) and rhIL7 (5 ng/ml). After 6 weeks of co-culture we obtained a 3 log increase of human T-linage precursors of CD45RA+CD7high phenotype. Further co-colture (7-9 weeks) leed to the generation of CD4+ and CD8+ double-positive (DP) T cells and even mature CD4+ and CD8+ single positive (SP) ab-TCR lymphocytes. Experiments were designed in order to evaluate whether human CD45RA+CD7high T cell precursors could 1) engraft into NOD-SCID IL2 rg-/− mice 2) leed to in vivo expansion and maturation along T cell developmental pathway. Control mice were irradiated and transplanted with G-CSF-mobilized human CD34+ (dose 5×106 i.v.). 4 weeks after transplant more than 20% human CD45 positive cells engrafted in the bone marrow. Thymic engraftment occured at 8 weeks after transplant, with 80% human CD45 positive cells (thymic cellularity: 2.7×105 cells), mostly with T cell-immature phenotype of CD3-CD4-CD8 triple negative (95%) (TN) and CD4+CD8+double positive (5%) (DP). Co-transplant of CD45RA+CD7high T cell precursors (106 cells i.v.) along with CD34+HSC leed to an accelerated thymic engraftment (95% human CD45 positive cells; thymic cellularity 2.5 × 106 cells) already at 6 wks after transplant. Thymocytes were CD3-CD4-CD8 triple negative (51%) (TN) and CD4+CD8+double positive (DP) (42%) cells and at 8 weeks after transplant matured into CD3+CD4+ and CD3+CD8+ single positive (SP) T cells. Spectratyping analyses revealed a broad diversity of the T-cell receptor (TCR) repertoire. This occured in the complete absence of Graft versus Host Disease (GvHD) suggesting that adoptively transferred ex vivo-generated T-cell precursors developed into host-tolerant mature T cells. Ongonig experiment are needed to clarify the beneficial effect of adoptive immunotherapy with human T cell precursors on peripheral T cell reconstitution and control of infection in the humanized mouse system. We conclude that ex-vivo generation of human T-linage precursors is feasible from the G-CSF-mobilized HSCs and that can be succesfully tranfered in-vivo as a new strategie to enhance T-cell reconstitution after allogeneic HSCT with no risk of GvHD. Disclosures: No relevant conflicts of interest to declare.

Overexpression of the Notch ligand, Jagged-1, induces alloantigen-specific human regulatory T cells

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3815-3821 ◽  
Author(s):  
Eric S. Yvon ◽  
Stephane Vigouroux ◽  
Raphael F. Rousseau ◽  
Ettore Biagi ◽  
Persis Amrolia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Notch Pathway ◽  
Cell Phenotype ◽  
Hematopoietic Stem ◽  
Notch Ligand

Abstract Graft-versus-host disease (GVHD) represents one of the major complications of allogeneic hematopoietic stem cell transplantation. Techniques to prevent GVHD have included ex vivo T-cell depletion of the graft or prolonged in vivo immunosuppression. Both reduce the frequency and severity of GVHD but also reduce T-cell-mediated graft-versus-malignancy effect, and increase the risk of infection. A major goal in transplantation is to prevent alloreactivity while preserving activity against tumors and infectious agents. We have used activation of the Notch pathway to try to generate T cells able to specifically regulate alloantigen responses. We used allogeneic Epstein-Barr virus lymphoblastoid B cells (EBV-LCLs) as stimulator cells. Such LCLs are excellent (allo) antigen-presenting cells and can be obtained in large numbers even from donors who have received extensive chemo/radiotherapy. We overexpressed a Notch ligand, Jagged-1, in these cells by adenoviral vector transduction. Stimulation of CD45RA+ naive T cells by Jagged-1 EBV-LCL reduces production of interferon-γ, interleukin-2, and interleukin-5, but up-regulates transforming growth factor-β1 synthesis, consistent with induction of a regulatory T-cell phenotype. Transfer of these T cells to fresh lymphocyte cultures inhibits proliferative and cytotoxic immune responses to the priming alloantigens while sparing responses to third-party stimulator cells. Notch activation in the presence of alloantigen-presenting cells may therefore be a means of inducing specific regulatory T cells while preserving other T-cell functionality. (Blood. 2003;102:3815-3821)

Regulatory T Cells Do Not Affect Human Hematopoietic Stem Cell Engraftment and Prevent T Cell Alloreactivity Against CD34+ Cells: A Preclinical Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4870-4870
Author(s):  
Dolores Mahmud ◽  
Sandeep Chunduri ◽  
Nadim Mahmud ◽  
Lennert Van Den Dries ◽  
John J. Maciejewski ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Regulatory T Cells ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Intracellular Expression

Abstract We have previously demonstrated that allogeneic blood T cells stimulate cord blood (CB) CD34+ cell differentiation into professional antigen presenting cells (APC) in-vitro and in-vivo (Abbasian J, Blood2006:108:203–208). In this study we immunomagnetically selected human CD4+CD25+ regulatory T cells (Tregs) and showed that >80% of these cells were positive for FoxP3 intracellular expression. Then we tested whether Tregs may affect CB CD34+ cell clonogenic activity in-vitro an in-vivo, and if co-incubation of Tregs and CD34+ cells may modify the phenotype and function of Tregs. A colony-forming cells (CFU-C) assay performed with CD34+ cells mixed with allogeneic Tregs at 1:2 ratio resulted in comparable numbers of Granulocyte- Macrophage CFU (CFU-GM), burst-forming unit-erythroid (BFU-E) and CFU-Mix as compared to cultures with CD34+ cells alone (p=0.2, p=0.5 and p=0.5, respectively)(n=3 exps). Human CD34+ cells were co-transplanted with human CD4+CD25+ allogeneic Tregs into NOD/SCID mice at 1:1 and 1:2 ratio. After 6 weeks mice marrow was harvested and showed a 1.3±1.1% (n=3 mice) and 1.6±0.8% (n=4 mice) engraftment of huCD45+ cells, respectively, which was comparable to the engraftment observed in control animals transplanted with CD34+ cells alone (1.4±0.4). In addition, among the engrafted huCD45+ cells similar proportion of CD33+ myeloid cells, CD14+ monocytes and CD1c+ dendritic cells were observed in the three groups of animals. Mixed lymphocyte culture (MLC) experiments showed that irradiated CD34+ cells stimulated brisk proliferative responses of CD4+CD25- cells (S:R=1:2), but did not induce any proliferation of Tregs (n=3 exps). After incubation with CD34+ cells in the presence of IL2, on average >80% CD4+CD25+ cells maintained the intracellular expression of FoxP3 and surface expression of CD62L and CD152 (n=3 exps). Then, Tregs autologous to CD34+ cells were isolated from the CB CD34- cell fraction while allogeneic Tregs were isolated from healthy individuals’ peripheral blood. When 2.5 x 104 autologous or allogeneic Tregs were added to an MLC with 2.5 x 104 irradiated CD34+ stimulator cells and allogeneic responders at 1:2 ratio, they suppressed T cell alloreactivity to CD34+ cells on average by 68±14% and 41±16%, respectively (n=3 exps). Our findings suggest that co-transplantation of CD34+ cells and autologous or allogeneic Tregs may allow normal stem cell engraftment while limiting T cell alloreactivity. These results will prompt the design of new strategies in allogeneic hematopoietic stem cell transplantation, particularly in an HLA incompatible setting.

Pharmacologic Co-Blockade of IFNγR and IL6R Pathways to Prevent and Treat GvHD

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3353-3353 ◽  
Author(s):  
Jaebok Choi ◽  
Matthew L Cooper ◽  
Kiran R. Vij ◽  
Bing Wang ◽  
Julie Ritchey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Hematopoietic Stem ◽  
Therapeutic Benefits ◽  
In Vivo Administration ◽  
Jak2 Inhibitors

Abstract The therapeutic benefits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematologic malignancies are primarily derived from an anti-leukemia effect that is mediated by T cells in donor grafts. Unfortunately, these T cells also mediate graft-versus-host disease (GvHD), the major complication of allo-HSCT. We have previously published that in vivo administration of JAK1/JAK2 inhibitors to murine allo-HSCT recipients of interferon gamma receptor deficient (IFNγR-/-) T cells results in 100% survival in a fully MHC-mismatched B6 to Balb/c allo-HSCT model (Choi et al., 2014, PLoS ONE). Since the infusion of IFNγR-/- T cells alone is associated with only ~70% survival, we hypothesize that JAK1/JAK2 inhibitors have either additional off-target effects or are inhibiting other non-IFNγR signaling pathways which are themselves dependent on JAK1/JAK2. The major other cytokine receptor signaling pathway mediated via both JAK1 and JAK2 is the interleukin 6 receptor (IL6R) signaling pathway. Thus, it is possible that JAK1/JAK2 inhibitors also block signaling through IL6R in addition to IFNγR. In addition, Alam et al. have recently reported that single nucleotide polymorphisms of donor IFNγ and IL6 are closely linked to gastrointestinal GvHD in patients (2015, BMT). Therefore, we examined if blockade of both IFNγR and IL6R signaling results in complete elimination of GvHD after a fully MHC-mismatched allo-HSCT in which B6 (H-2b) T cell-depleted bone marrow cells (5x106) along with B6 pan T cells (5x105) are intravenously injected into lethally irradiated Balb/c mice (H-2d). As shown in Fig. 1, we have found that blocking both IFNγR (IFNγR-/- T cells) and IL6R (α-IL6R Ab) signaling dramatically reduces GvHD and results in >95% survival. In addition, we found that blocking both IFNγR and IL6R signaling significantly increased regulatory T cells (Tregs) in peripheral blood (23.2% Foxp3+ in CD4+ T cells (n=17) vs 2.5% in WT T cell control (n=16) at day 27 after allo-HSCT), suggesting that increase in Tregs might be a potential mechanism underlying the reduced GvHD after dual blockade of IFNγR and IL6R signaling. Baricitinib is a potent and balanced JAK1/JAK2 inhibitor currently being clinically developed by Eli Lilly for the treatment of inflammatory diseases. We hypothesize that baricitinib will optimally block both IFNγR and IL6R signaling pathways and prevent GvHD. We found that that baricitinib is a potent suppressor of GvHD in B6 to Balb/c allo-HSCT models (100% survival), superior to ruxolitinib and similar to blockade of both IFNγR and IL6R signaling (Fig. 2A). Baricitinib increases Tregs in vivo (Fig. 2B) and reduces the ratio of IL5 (Th2 cytokine) to IL2 (cytokine for Treg induction) in plasma (p=0.0046), a potential diagnostic marker for GvHD (Fujii et al., 2006, Int J Mol Med), significantly better than ruxolitinib. Lastly, we found that baricitinib inhibits the expression of T-bet (Fig. 2C), which is the master transcription factor of Th1 cells, that are primary effector T cells in inducing GvHD. These data suggest that the suppression of GvHD by baricitinib results from increased Tregs and decreased Th1 and Th2 cells. We next examined if the prevention of GvHD by baricitinib is dependent on natural regulatory T cells (Tregs) in donor grafts. Tregs were depleted from donor pan T cells before allo-HSCT (B6 to Balb/c). We found that in vivo administration of baricitinib resulted in 70% survival (0% control, p<0.0001; 100% Treg-replete T cells + baricitinib. In addition, based on clinical GvHD score when recipients of Treg-replete T cells were compared with those of Treg-deplete T cells, the beneficial effect of Tregs in the donor grafts for the prevention of GvHD was observed only for the first two weeks after allo-HSCT (p≤0.01). Lastly, we examined whether baricitinib can cure ongoing GvHD by administering baricitinib starting at day 10 after allo-HSCT when GvHD is established (B6 to Balb/c). We found that baricitinib treatment results in a significant reduction of GvHD and 100% survival (10% control, p<0.0001). All of these data suggest that pharmacologic co-blockade of IFNγR and IL6R pathways is a promising therapeutic strategy to prevent and effectively treat established GvHD. The inhibitory effect of baricitinib, ruxolitinib, and blockade of IFNγR and IL6R on JAK-STAT signaling using JAK/STAT phosphorylation antibody arrays is currently being investigated and will be presented. Disclosures DiPersio: Incyte Corporation: Research Funding.

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