Reconstitution of Regulatory T Cell Subpopulations After Allogeneic Hematopoietic Stem Cell Transplantation and Graft-Versus-Host-Disease

Abstract Abstract 1948 Previous studies on the reconstitution of regulatory T cells (Treg) after allogeneic hematopoietic stem cell transplantation (HSCT) have shown a delayed reconstitution in patients (pts) with acute graft-versus-host-disease (GvHD) (Magenau, 2010) and an association between impaired Treg reconstitution and the development of extensive chronic GvHD (Matsuoka, 2010). However, no studies have analyzed naive (nTreg) versus memory (mTreg) Treg reconstitution in a longitudinal cohort with large numbers of pts. From 2006 to 2009, 165 consecutive pts were prospectively analyzed in our center post-HSCT. Fresh whole blood samples were obtained 3 (n=155), 6 (n=162), 12 (n=165) and 24 (n=94) months after HSCT and analyzed by flow cytometry to quantify CD4 T cells, including naive, activated, central memory and effector memory subsets (Sallusto, 1999), as well as Treg (CD4+ CD25+ CD127neg/lo), including nTreg (CD45RA+) and mTreg (CD45RAneg). The results are presented as median values of circulating cells. Median age was 41 years (range: 6–68). The indication for HSCT was malignant disease in 92%. The conditioning regimen was reduced-intensity (RIC) in 51%. The donor was an HLA-identical sibling in 56%. The source of stem cells was peripheral blood (PBSC), bone marrow (BM) and cord blood (CB) in 65%, 28% and 7%, respectively. All pts received cyclosporine as GvHD prophylaxis. GvHD was defined as acute if occurring before day 100 and chronic thereafter. Total Treg (tTreg) increased from 13/μL at 3 months to 44/μL at 24 months, but always remained inferior to healthy controls (HC) (66/μL). nTreg increased from 1.8/μL at 3 months to 4.8/μL at 24 months (HC: 24/μL). mTreg increased from 10.7/μL at 3 months to 33.3/μL at 24 months (HC: 42/μL). The CD4/Treg ratio remained stable at 12.6 at 3 months and 11.6 at 24 months while the nCD4/nTreg ratio increased from 17.4 at 3 months to 42.7 at 24 months, showing a larger expansion of naive cells in the CD4 T cell compartment than in the Treg compartment (Figure 1) and a larger expansion of memory cells in the Treg than within the CD4 cells. At 3 months post-HSCT, tTreg, nTreg and mTreg were significantly higher in PBSC recipients (18.4, 2.7 and 14.5/μL) than in BM (8.1, 0.9 and 6.5/μL) and CB recipients (6.5, 0.6 and 5.3/μL) (p=0.0001), respectively. Pts transplanted after a RIC regimen had significantly more tTreg and mTreg than pts transplanted after a standard regimen (17 and 14/μL, compared with 9.8 and 8/μL, p=0.004 and 0.008 respectively). Pts transplanted for an aplastic anemia had significantly fewer nTreg than pts transplanted for a malignant disease (0.4 and 1.9/μL, p=0.001). At 6 months post-HSCT, tTreg, nTreg and mTreg were significantly higher (p=≤0.01) in pts transplanted from an HLA-identical sibling (19.5, 1.9 and 17.2/μL) compared with pts transplanted from an unrelated donor (13.2, 1.2 and 11/μL). At 12 and 24 months post-HSCT, younger pts (≤15 years) had significantly more nTreg than older pts (9.8 and 28.7/μL compared with 2.1 and 4.2, p=0.001). In pts with previous acute GvHD, tTreg and mTreg were significantly lower at 3 (8.5 and 7.7/μL) and 6 months (14.6 and 12.5/μL) compared with pts without (15.6 and 13.8/μL at 3 months, p=0.005 and 21.3 and 18.2/μL at 6 months, p≤0.007), respectively. Absolute numbers of tTreg, nTreg and mTreg, and the frequencies of Treg relative to activated, effector memory and central memory CD4 T cells at 3, 6 and 12 months post-HSCT did not predict the occurrence of a later episode of chronic GvHD up to 2 years post-HSCT. In our population, total, naive and memory Treg reconstitution was delayed post-HSCT and remained below the normal range up to 2 years after HSCT. tTreg reconstitution post-HSCT was mostly due to mTreg expansion. RIC regimen and PBSC as source of stem cells were associated with a better short-term reconstitution. At 6 months, pts transplanted from siblings had a better reconstitution while nTreg long-term reconstitution was mainly influenced by recipient age (better if ≤15 years). While previous acute GvHD impaired Treg reconstitution, Treg subsets (absolute numbers and frequencies relative to CD4 T cell subsets) at 3, 6 and 12 months post-HSCT were unable to predict chronic GvHD in this large cohort of patients. We believe these data are of particular interest regarding the recently increasing number of Treg interventional studies in humans in the context of HSCT. Disclosures: No relevant conflicts of interest to declare.

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The Effect of Costimulatory Moleculars on Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v110.11.4875.4875 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4875-4875

Author(s):

Zhenhua Qiao ◽

Fang Ye ◽

Lei Zu

Keyword(s):

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Hematopoietic Stem ◽

Graft Versus Host ◽

Group A ◽

Group B

Abstract Objective: To explore the effect of costimulatory molecular and CD25 expressed on peripheral CD4+ T lymphocytes on graft-versus-host disease(GVHD) after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Methods: 1. The 21 patients who suffered of hematology diseases or malignant solid tumors and were underwent allo-HSCT and 10 normal individuals were enrolled in the study.2. For the sake of difference conditioning regimens we divided the 21 patients into two groups: patients undergoing non-myeloablative stem cell transplantation(NST) belonged to group A, others undergoing traditional myeloablative stem cell transplantation belonged to group B; we divided them into five groups for with GVHD or without GVHD and types of GVHD: group 1(group A with acute GVHD), group 2(group A with chronic GVHD), group 3(group B with acute GVHD), group 4(group B without GVHD), group 5(group A without GVHD).3. The levels of CD28, CD80, CD152 and CD25 expressions on peripheral CD4+ T lymphocytes were detected by three colors flow cytometry (FCM)in different time(before allo-HSCT,7days,14days,21days,30days after allo-HSCT, the time of GVHD and the time after GVHD treated).4.STR-PCR for detecting micro-satellites chimeras forming. Results: 1. All 21 patients achieved engraftment. By STR-PCR assay,12 cases formed complete chimeras(CC) and 9 cases formed mixed chimeras(MC). In group A,3 cases developed acute GVHD and 4 cases developed chronic GVHD; in group B,4 cases developed aGVHD. The incidence of GVHD and infection rates between group A and B has no difference(X2=3.711, P=0.144).2. Among these 21 cases,5 cases died:2 cases died of multiple organs function failure due to primary disease relapse,1 case died of bleeding in brain and 2 cases died of liver function failure for the sake of complicated with acute GVHD; others survive with disease free till present.3. The results of multivariate logistic regression models and Kaplan-Meier survival curves analyses showed: age, sex, infection, HLA-type, blood type, conditioning regiment and the times of absolute neutrophil counts and platelets recovering to normal, had no association with the incidence of GVHD;A multivariate COX survival function model analysis showed CD4CD152 and CD4CD25 are independent prognostic factors for GVHD(X2=13.128, P<0.0001).4. Patients with GVHD demonstrated higher CD4+CD28+ and CD4+CD80+ T cell levels than those without GVHD(P<0.01);patients with aGVHD demonstrated higher than those with cGVHD(P<0.05) and without GVHD(P<0.05); Patients with GVHD demonstrated lower CD4+CD152+ and CD4+CD25+ T cell levels than those without GVHD(P<0.01); the same result occurs between aGVHD and cGVHD and without GVHD. After effective treatment, unnormal CD4+CD28+, CD4+CD80+, CD4+CD152+ and CD4+CD25+ T cell levels recovered to the levels before transplantation. Conclusions: The incidences of GVHD between NST and traditional myeloablative stem cell transplantation had no difference. B7-CD28/CD152 costimulatory pathway plays a critical role in developing of GVHD. Peripheral CD4+CD28+, CD4+CD80+, CD4+CD152+ and CD4+CD25+ T cell levels were relative to recipient GVHD, especially CD4+CD152+ and CD4+CD25+ T cell levels. Down-grade CD4+CD28+ and CD4+CD80+ T cell levels and up-grade CD4+CD152+ and CD4+CD25+T cell levels could reduce the incidence of GVHD.

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CD28-Directed T Cell Costimulation Blockade with Abatacept to Prevent GvHD During High-Risk Unrelated HSCT: A First-in-Disease Feasibility Trial,

Blood ◽

10.1182/blood.v118.21.4078.4078 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4078-4078

Author(s):

Leslie S. Kean ◽

Amelia Langston ◽

Muna Qayad ◽

H. Jean Khoury ◽

Divya Tiwari ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Hematologic Malignancies ◽

Feasibility Trial ◽

Effector Memory ◽

Unrelated Donor ◽

Gvhd Prophylaxis ◽

The Mean

Abstract Abstract 4078 Background: Acute GvHD remains the major cause of complications and death following unrelated-donor HSCT. In a non-human primate model, we have previously shown that in vivo costimulatory blockade of donor T-cells could provide effective protection against GVHD. To begin to explore its clinical utility, we are conducting a trial (Clinical Trials.Org # NCT01012492) to determine the feasibility of combining abatacept (CTLA4-Ig) with cyclosporine and methotrexate as acute GVHD prophylaxis for patients undergoing unrelated marrow and peripheral blood stem cell transplants for hematologic malignancies. Methods: Patients older than 12 with advanced hematologic malignancies, conditioned with either TBI/Cytoxan, Busulfan/Cytoxan or Fludarabine/Melphalan are eligible. Abatacept is administered IV on days −1, +5, +14, and +28 at 10 mg/kg in addition to standard GvHD prophylaxis consisting of cyclosporine (day −2 to day 100), and methotrexate (15 mg/m2 on day +1 and 10 mg/m2 on days +3, 6 and 11). Patients are then followed for clinical outcomes and immunologic reconstitution through day +365. Results: 9 patients (planned enrollment = 11 patients) have thus far been enrolled on the study of which 5 are evaluable for engraftment, toxicity and acute GvHD. The other four patients consist of 2 who are currently receiving abatacept, 1 who was discovered to have an ongoing viral infection at the start of the first abatacept infusion so was removed from the treatment regimen, and 1 who is awaiting transplant. The median age for the 5 evaluable patients is 47 years (17–74 years). 3 patients had AML and 2 had ALL. Patients were conditioned with Bu/Cy (n=1), TBI/Cy (n=2) and Flu/Melphalan (n=2). 4 donor-recipient pairs were allele matched at 9 of 10 loci (A, B, C, DRB1 and DQB1), while 1 was fully matched. Four of the 5 patients are currently alive and in remission and 1 relapsed at day +98 (and died on day +121 with refractory AML). The four other patients are surviving without relapse with a follow-up of 155–313 days. All 5 patients received the 4 scheduled abatacept doses. No infusional side effects were noted. All patients achieved neutrophil engraftment (median day +20 (11–47). 4 of 5 patients have achieved platelet engraftment (median day +27 (14–35). Donor engraftment (100% CD33 and 99–100% CD3 at Day +30) occurred in all cases. All patients have demonstrated rapid lymphocyte engraftment, with the mean ALC reconstituting to >500 cells/μL by day +21 post-transplant. At day +100, the mean CD3+ count was 673 +/− 251 cells/μL. Both CD8+ and CD4+ T cells reconstituted by day 100, with the mean CD8+ count = 384 +/− 148 cells/μL and the mean CD4+ count = 229 +/− 119 cells/μL. T cell reconstitution was accompanied by a shift away from naïve (Tn, CCR7+/CD45RA+) toward a CCR7-/CD45RA- effector memory (Tem)-predominant phenotype. Thus, the average proportion of CD4+ Tem cells in the recipient increased from 22 +/− 6% pre-transplant to 46 +/− 7% at day +100 with a concomitant loss of CD4+ Tn cells. Likewise, the proportion of CD8+ Tem also significantly increased, from an average of 15 +/− 4% pre-transplant to 32 +/− 7% at day +100, also with a reciprocal decrease in CD8+ Tn cells. One patient developed steroid responsive grade 3 acute GVHD involving the skin and the liver, followed by steroid responsive liver chronic GvHD. This patient is currently weaning corticosteroids. Another patient developed steroid responsive late-onset (day +217) acute GVHD (liver and GI) during cyclosporine weaning, which was also steroid responsive, and is also currently weaning corticosteroids. No other systemic acute or chronic GvHD has occurred. No unexpected complications or life-threatening infections were observed. 3 patients have experienced 5 episodes of CMV reactivation, all responsive to antiviral therapy. One patient developed polyclonal EBV-related PTLD (plasmacytic hyperplasia) in the absence of EBV viremia, which regressed without intervention. No other EBV-related disease has occurred. Conclusions: These preliminary data suggest that abatacept can be safely added to cyclosporine and methotrexate for GVHD prophylaxis in recipients of hematopoietic grafts from unrelated donors, with encouraging rates of acute GVHD. As such, they support the conduct of a larger, randomized phase 2 study. Disclosures: Off Label Use: Abatacept: It is an immunosuppressive agent that targets the CD28/B7 T cell costimulation pathway. It is approved for use in Rheumatoid arthritis.

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

Blood ◽

10.1182/blood.v126.23.1940.1940 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1940-1940 ◽

Cited By ~ 1

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.

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Improved Immune Reconstitution with Low Incidence of Severe Graft-Versus-Host Disease after Transplantation of CD34+ or CD133+ Enriched Stem Cells with Add-Back of Ten Million T-Cells Per Kg.

Blood ◽

10.1182/blood.v104.11.1239.1239 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1239-1239

Author(s):

Johann Greil ◽

Peter Lang ◽

Peter Bader ◽

Matthias Eyrich ◽

Paul G. Schlegel ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Acute Gvhd ◽

Median Number ◽

Control Group ◽

Graft Versus Host ◽

Short Course ◽

Unrelated Donors

Abstract Unrelated donors are commonly used for hematopoietic stem cell transplants, but graft-versus-host disease (GVHD) is a major problem. We investigated whether transplantation of CD34+ or CD133+ enriched stem cells with add-back of ten million T-cells per kg from unrelated donors would prevent acute GVHD in pediatric patients in combination with pharmacologic immunosuppression. Eighteen patients (1 CML in second chronic phase, 2 MDS, 4 ALL in CR1, 4 ALL in CR2, 1 JMML, 1 AML in CR1, 3 AML in CR2, 1 Wiskott-Aldrich syndrome and 1 NHL in CR2) were transplanted with G-CSF mobilized peripheral blood stem cells (PBSC) from HLA-matched unrelated donors (n = 18). Median of age was 8.9 years (0.5 to 18 years). Conditioning regimens were performed according to national therapy protocol guidelines. On the day of transplant patients received a median of 13.5 (4.5 to 30.0 x 106) CD34+ or CD133+ enriched stem cells and an aliquot of unmanipulated PBSC containing 10 x 106 T-cells per kg. GVHD prophylaxis consisted of cyclosporine A (CSA) and short course methotrexate (MTX) on day +1, +3 and +6. Engraftment was rapid with a median of 19.6 days in sixteen patients. Two patients failed to engraft at first. However, full donor chimerism and stable engraftment was achieved in both patients after cessation of CSA treatment and an additional stem cell boost without any reconditioning. One patient with ALL developed acute GVHD grade III (skin and gut) after cessation of CSA treatment, but responded well to treatment with CSA and steroids. None of the other 17 patients developed acute GVHD > grade I. Thirteen are alive and well with a median follow-up of 578 days (101 to 1095 days). Three patients died of severe infectious complications and two due to relapse (JMML, AML). Compared to a historical control group of patients transplanted with highly purified CD34+ selected cells, the group with add-back of 10 x 106 T-cells per kg showed significantly higher T-cell counts (p=0.008, Wilcoxon Rank sum test) on day 90 after transplantation with a median number of six T-cells/μl in the control group and a median number of 294 T-cells/μl in the study group. We conclude that add-back of 10 x 106 T-cells per kg in combination with CSA and short course MTX improves T-cell recovery and appears to be a safe T-cell dosage regarding acute GVHD in the setting of allogeneic peripheral blood stem cell transplantation from unrelated donors.

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Direct Intra-Bone Injection of Unrelated Cord Blood Cells Overcomes the Problem of Delayed Engraftment and Improves the Feasibility of Hematopoietic Transplant in Adult Patients.

Blood ◽

10.1182/blood.v110.11.334.334 ◽

2007 ◽

Vol 110 (11) ◽

pp. 334-334 ◽

Cited By ~ 1

Author(s):

Adalberto Ibatici ◽

AnnaMaria Raiola ◽

Marina Podesta ◽

Francesca Gualandi ◽

Nadia Sessarego ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Cord Blood ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Conditioning Regimen ◽

Significant Proportion ◽

Hla Antigen ◽

Hematopoietic Stem ◽

Seeding Efficiency

Abstract Background. Cord blood transplants (CBT) are associated with delayed or failed engraftment in a significant proportion of patients (pts). Two of our previous observations suggested (i) that, in the animal model, direct intra-bone (i.b.) injection improves seeding efficiency and (ii) that the delayed engraftment was not related to an insufficient number of hematopoietic stem cells but rather to some difficulties to differentiate and maturate. Methods. Unrelated CB cells were selected for 29 consecutive pts (18 CB units were 4/6 HLA antigen matched, 10 were 5/6 and one 3/6 antigen matched). Median cell dose infused was 2.3 x10^7/kg (range 1.4 – 4.2). CB cells were concentrated in 4 syringes of 5 ml each and injected in the supero-posterior iliac crest (SPIC) under rapid general anesthesia (10 min. with propofol). Pts’ median age was 38 years (18–63); 25 had acute leukaemia (21 with refractory or relapsed disease and 4 high risk first remission leukemia); 2 chronic myeloid leukemia in advanced phase; 2 refractory Hodgkin’s disease. Most pts (n=24) were prepared with conventional conditioning regimen (TBI-cyclophosphamide). Results. The infusion of cells i.b. in SPIC (11 pts bilaterally; 18 pts monolaterally) was uneventful. Five pts are not evaluable because they died within 14 days from transplant. All pts surviving more than 14 days engrafted (100%). Median time for PMN (>0.5x10^9/l) and platelets (>20x10^9/l) engraftment was day +23 (14–40) and +38 (range 22–60) respectivelly. Four pts died of infection; one patient died of PTLD on day +140. Four patients relasped and 3 died of relapse. Fifteen out of 16 alive patients are in hematologic or molecular remission at a median follow up of 7.5 months (range 2–17). From day +30 full donor chimerism was documented in CD3, bone marrow cells and progenitor cells from both the injected and in non-injected SPIC; from day +30, CFC progenitors had already reached the lower values of the range of normal individuals in bilateral sites. These findings document the colonization of the hematopoietc system and the recovery of stem cell reservoir possibly due to an improvement of seeding efficiency. Only 3 pts (8%) experienced acute GvHD (2 grade II and 1 grade I); 4 pts. have moderate chronic GVHD. It is known that lymphocyte trafficking is one of the crucial factor in immunity. Two combined factors might contribute to the low incidence of acute GvHD: few of the transplanted T cells do not reach/circulate primarily in the lymphatic organs, where they would be immediately confronted with host antigen presenting cells as probably occurs after i.v. injection; injected T cells come immediately in contact with mesenchymal stem cells (MSC) and osteoblasts, known to be potent immunosuppressants. Conclusion. Direct intra-bone transplant of CB cells overcomes the problem of graft failure and is associated with reduced incidence of acute GvHD even when low numbers of HLA mismatched CB cells are transplanted. Nearly all patients searching for a CB unit were able to undergo CBT. This approach may change our policy of hemopoietic cell transplants.

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GPI-AP Deficient Cells Detected Exclusively in T Cells in Patients with Aplastic Anemia: Evidence against An Escape Mechanism for the Initial Proliferation of PIG-AMutant Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.3197.3197 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3197-3197

Author(s):

Takamasa Katagiri ◽

Zhirong Qi ◽

Yu Kiyu ◽

Naomi Sugimori ◽

J. Luis Espinoza ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Immune System ◽

T Cell ◽

Hematopoietic Stem Cells ◽

Aplastic Anemia ◽

Effector Memory ◽

Hematopoietic Stem ◽

Escape Theory ◽

Effector Memory Cells

Abstract Abstract 3197 Poster Board III-134 Small populations of glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient blood cells are often detectable in the peripheral blood (PB) of patients with aplastic anemia (AA) and refractory anemia (RA) of myelodysplastic syndromes defined by the FAB classification. Such PNH-type cells are thought to be derived from PIG-A mutant hematopoietic stem cells (HSCs) that avoid the immunological attack against HSCs. Inefficient T cell responses to PNH-type cells were indeed demonstrated by a murine study. However, there is no direct evidence in support of the escape theory concerning the expansion of PIG-A mutant HSCs in such patients with bone marrow (BM) failure. If the escape theory is true, the PNH-type cells should be detected in myeloid cells derived from HSCs that are targeted by the immune system attack. The PB of 527 patients with BM failure was examined for the presence of GPI-AP deficient cells in various lineages of cells including granulocytes, erythrocytes, monocytes, T cells, B cells, and NK cells using high sensitivity flow cytometry to verify this hypothesis. PNH-type cells were detectable in at least one lineage of cells from 228 (43%) patients. Although most of the positive patients showed PNH-type cells in two or more lineages including granulocytes or monocytes, 14 patients (13 with AA and 1 with amegakaryocytic thrombocytopenia) displayed PNH-type CD48-CD55-CD59- cells only in T cells at a frequency of 0.003-0.3% of the total T cells (Figure). The PNH-type T cells were undetectable in any of 25 healthy individuals. The CD48-CD55-CD59- T cells consisted of predominantly effector memory and terminal effector memory cells with naïve phenotype cells. The phenotypic pattern of the PNH-type T cells was very similar to that of CD48-CD55-CD59- T cells from 11 patients with florid PNH but was different from that of CD48-CD55-CD59- T cells (central and effector memory cells alone) detected in 4 marrow transplant recipients who received anti-CD52 antibody (alemtuzumab) therapy as conditioning. PIG-A gene analyses of CD48-CD55-CD59- T cells revealed a single mutation in 2 patients with PNH-type T cells alone, while two different mutations were revealed in 2 patients treated with alemtuzumab. BM failure patients with PNH-type T cells alone and other BM failure patients possessing PNH-type granulocytes or monocytes showed similar clinical features characterized by predominant thrombocytopenia and good response to immunosuppressive therapy, thus suggesting an increase in the number of PNH-type cells in both groups to be associated with a similar immune pathophysiology. The escape theory cannot account for the presence of PNH-type cells exclusively in T cells in immune-mediated BM failure because T cell precursors are not the target of the immune system attack in AA. Therefore, mechanisms other than the escape theory must be considered for the initial proliferation of PIG-A mutant HSCs associated with the development of AA, such as preferential activation of dormant PIG-A mutant HSCs or T cell precursors due to the deficiency of GPI-APs that transmit negative signal Disclosures No relevant conflicts of interest to declare.

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Functionally Altered GPI-Anchor Negative Treg Following Alemtuzumab-Based T-Cell Depletion Are Associated with Acute Gvhd.

Blood ◽

10.1182/blood.v120.21.3059.3059 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3059-3059

Author(s):

Eva M Wagner ◽

Lukas A Schaefer ◽

Tobias Bopp ◽

Matthias Theobald ◽

Wolfgang Herr ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Cell Depletion ◽

Gpi Anchor ◽

Activation Markers ◽

T Cell Depletion ◽

Hematopoietic Stem

Abstract Abstract 3059 Introduction: The monoclonal anti-CD52antibody Alemtuzumab is frequently used for T-cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT) to prevent graft versus host disease (GVHD). We previously demonstrated the long term persistence of functionally impaired glycosylphosphatidylinositol (GPI)-anchor negative effector T-cells in patients receiving high dose (100mg) Alemtuzumab in combination with a dose reduced conditioning regimen (Fludarabin + Melpahlan) (Meyer, Wagner et al. BMT 2010). Despite of Alemtuzumab-mediated TCD, half of our patients developed acute GVHD. Since regulatory T cells (Treg) play a major role for controlling GVHD, we asked whether GPI-anchor negative Treg are present in patients with or without GVHD. Methods: We analyzed peripheral blood samples of 12 patients with acute GVHD (aGVHD), 7 patients with chronic GVHD (cGVHD), and 10 patients who never developed GVHD after Alemtuzumab-mediated TCD. To analyze Treg-subsets, we stained for CD3, CD4, CD25, CD127, FoxP3, CD52 as well as for the activation-markers GARP, HLA-DR and CD45RA. Treg were identified as CD3+CD4+CD25+CD127- or CD3+CD4+CD25+FoxP3+ cells and subdivided according to their CD52-expression. We used FLAER staining to confirm that the loss of CD52 on Treg resulted from the loss of the GPI-anchors themselves. We were able to study Treg subpopulations in the time course of patients who recovered from acute GVHD in comparison to patients with persisting late acute GVHD. In individual patients, we isolated GPI-anchor positive and negative Treg by FACS-Sort, expanded them and performed Treg suppression assays. Results: GPI-anchor negative Treg were observed in all patients, independent of the development of GVHD. However, the frequency of GPI-anchor negative Treg varied considerably between patients with acute GvHD and those with chronic GVHD or without GvHD. The percentage of GPI-anchor negative Treg was significantly elevated in patients with aGVHD: median 80.35% (range 56,2–96,8%) in comparison to 17,4% (range 0–57,8%) in patients with cGVHD or without GVHD. Activated Treg were almost exclusively detected among GPI-anchor positive Treg-subpopulation. Patients who resolved from aGVHD restored GPI-anchor positive Treg and the amount of activated Treg rose. The percentage of GPI-anchor negative Treg populations remained high in patients with ongoing aGVHD. In addition, these patients had no GARP-positive activated Treg even under long term immunosuppressive treatment. Preliminary experiments with sorted and expanded Treg populations suggest that GPI-anchor negative Treg were unable to suppress T-cell proliferation upon IL-2 stimulation. Summary: We demonstrate for the first time the reconstitution of GPI-anchor negative Treg in patients following Alemtuzumab-mediated TCD. These T cells were functionally altered and were less likely to exhibit an activated phenotype in vivo. Ongoing acute GVHD was associated with high percentages of GPI-negative Treg suggesting that their functional alteration might play a role in aGVHD pathophysiology. This is in line with the finding that only in patients who resolved aGVHD, the frequency of GPI-anchor positive Treg increased significantly. Further functional analyses are ongoing to estimate the cellular consequence of missing GPI-anchored proteins. In addition, correlating the reconstitution of GPI-anchor negative T-cell populations with further clinical events is ongoing. Disclosures: No relevant conflicts of interest to declare.

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Increased Effector CD4+ T-Cells Early Post Hematopoietic Stem Cell Transplantation Using an Alemtuzumab-Based Reduced Intensity Conditioning Regimen Correlate with Subsequent Development of Graft Versus Host Disease.

Blood ◽

10.1182/blood.v110.11.4999.4999 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4999-4999

Author(s):

Katie Matthews ◽

ZiYi Lim ◽

Laurence Pearce ◽

Khalid Tobal ◽

Alejandro Madrigal ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Cd4 T Cells ◽

Subsequent Development ◽

Effector T Cells ◽

Cd4 T Cell ◽

Hematopoietic Stem ◽

Graft Versus Host

Abstract Lymphocyte depletion using the anti CD52 monoclonal antibody alemtuzumab reduces the incidence of graft versus host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), but some patients still develop this potentially life-threatening complication. We previously reported that patients achieving rapid full donor T cell chimerism after fludarabine, busulphan and alemtuzumab (FBC) conditioned allo-HSCT have a significantly increased risk of GvHD compared to patients with prolonged mixed donor chimerism beyond day 100 (Lim et al. Br. J. Haematology 2007). We performed a prospective study of 29 patients who received allo-HSCT with FBC conditioning (median age: 53 years; range: 34 – 69 presenting with AML or MDS) to examine the kinetics of lymphocyte reconstitution in relation to T-cell chimerism patterns and incidence of GvHD. Naïve, memory, effector and terminally differentiated CD4+ and CD8+ T-cells, activated T-cells (CD25+ HLA-DR+); putative regulatory CD4+ CD25high Foxp3+ T-cells, B-cells and NK cells were enumerated in whole peripheral blood of patients at days 30, 60, 90, 180, 270 and 360 after HSCT. Chimerism analysis of purified T-cells was performed by genetic profiling of polymorphic short tandem repeat loci. Ten patients developed GvHD (acute or chronic). Although alemtuzumab induced profound depletion of all T-cell subsets, significantly higher numbers of CD4+ effector (CD45RO+ CD27−) T-cells were detected at day 30 post transplant in patients who later developed GvHD (24 cells/μl; range: 1 – 84 cells/μl) compared to patients without GvHD (5 cells/μl; range: 1 – 40 cells/μl) (p = 0.026). In contrast, there were no significant differences in the numbers or rate of reconstitution of CD8+ T-cell sub-populations, NK cells or B-cells in patients that developed GvHD and those who did not at any time points. T-cells present at day 30 in patients that subsequently developed GvHD were 100% donor whereas the majority of patients that did not develop GvHD exhibited mixed donor and recipient T cell chimerism. Development of GvHD pathology was associated with expansion of these donor effector CD4+ T-cells (at day 60: 35 cells/μl; range: 9 – 154 cells/μl compared to 7 cells/μl; range: 1 – 56 cells/μl for patients without GvHD, p = 0.04). Absolute numbers of CD4+ CD25high Foxp3+ T-cells at day 30 were similar in both groups of patients (p = 0.8). However, of note, a significant deficit of these putative regulatory T-cells in the group that developed GvHD was apparent when numbers were considered relative to CD4+ effector T cells at day 30 (41 CD4+ effector T-cells; range: 28 – 51 /per regulatory CD4+ T cell for the GvHD group compared to 12 CD4+ effector T-cells; range: 2 – 33 /per regulatory CD4+ T-cell for patients without GvHD, p = 0.03). We speculate the higher numbers of effector CD4+ T-cells detected in patients at day 30 post HSCT are donor-derived mature T cells that alemtuzumab fails to deplete. In the solid organ transplant setting, alemtuzumab has been shown to be relatively sparing of effector memory CD4+ T-cells. Our correlation of donor-derived effector CD4+ T-cells with subsequent development of GvHD suggests they are alloreactive and that a deficit of T-regs relative to CD4+ effector T-cells early post HSCT contributes to GvHD.

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Increased Proliferation of FoxP3 Regulatory T Cells after Allogeneic Hematopoietic Stem Cell Transplantation Is Counterbalanced by Increased Susceptibility to Apoptosis

Blood ◽

10.1182/blood.v112.11.720.720 ◽

2008 ◽

Vol 112 (11) ◽

pp. 720-720

Author(s):

Ken-ichi Matsuoka ◽

Corey Cutler ◽

John Koreth ◽

Joseph H Antin ◽

Robert J Soiffer ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Chronic Gvhd ◽

T Cell Subsets ◽

Cd4 T Cell ◽

Ki 67 ◽

Hematopoietic Stem ◽

Healthy Donors

Abstract CD4+FoxP3+ Regulatory T cells (Treg) play a critical role in the maintenance of tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). We previously demonstrated that patients with active chronic graft-versus-host disease (cGVHD) have a reduced frequency of Treg. However, the mechanisms responsible for inadequate Treg reconstitution in patients with cGVHD have not been characterized. We therefore examined phenotypic and functional characteristics of Treg in 16 patients 2–41 months (median 10 months) post-HSCT to elucidate these mechanisms. Treg were compared to conventional CD4+FoxP3-T cells (Tcon) within individual patient samples and to healthy donors. All patients received TBI-based myeloablative conditioning, peripheral blood stem cells from HLA-matched donors (12 MRD; 4 URD) and acute GVHD prophylaxis (11 tacrolimus and sirolimus; 5 tacrolimus and methotrexate). At the time of analysis, 9 patients had no chronic GVHD, 5 had active chronic GVHD (1 limited disease; 4 extensive disease) and 2 had inactive chronic GVHD. Total CD4 counts were relatively low after HSCT compared to healthy donors (median CD4 273/ul vs 756/ul). After HSCT, patient Treg exhibited a predominant CD45RA(−)CCR7(−) effector/memory phenotype. Expression of CD31 on CD45RA+ Tcon and Treg was used to identify cells within these subsets that were recent thymic emigrants (RTE). In patient samples, 16.5% of Tcon and 2.8% of Treg expressed CD31+CD45RA+. In healthy donors, 22.9% of Tcon and 5.4% of Treg were CD31+CD45RA+. The lower fraction of RTE within the Treg population after transplant suggests that Treg primarily reconstitute through peripheral proliferation rather than through thymic generation. The proliferative capacity of both Tcon and Treg was examined by evaluating expression of Ki-67 in these subsets. After transplant, Ki-67 expression was significantly higher in Treg (5.2%) than in Tcon (1.5%) (p<0.001). This was significantly higher in both populations compared to healthy donors where 2.5% of Treg (p<0.05) and 0.2% of Tcon (p<0.01) expressed Ki-67. In both patients and healthy donors, Ki-67 expression was found almost entirely in cells that were CD45RA-indicating that proliferation was primarily occurring within the memory subsets of Tcon and Treg. Increased expression of Ki-67 on Treg was associated with low CD4 T cell counts (p<0.001), but not with time after HSCT (p=0.21) and chronic GVHD status (p=0.35). Treg Ki-67 expression after HSCT showed a strong positive correlation with CD95 (FAS) expression (p<0.01), but this association was not present in Tcon post-HSCT or in Treg from healthy donors. To determine whether increased expression of CD95 results in apoptosis of Treg, we purified 4 different CD4+ T cell subsets by cell sorting (CD45RA+ Tcon, CD45RA− Tcon, CD45RA+ Treg and CD45RA− Treg) from healthy donors and HSCT patients. Purified cells were cultured with or without agonistic FAS antibody (anti-FAS) and apoptosis was measured using Annexin-V staining. Anti-FAS rapidly induced apoptosis of CD45RA− memory-like Treg from HSCT patients while all other Treg and Tcon subsets were relatively resistant to apoptosis. In summary, these results indicate that Treg reconstitution post-HSCT is characterized by high levels of peripheral proliferation, which appear to be driven primarily by persistent CD4 T lymphopenia. However, post-HSCT Treg are also highly sensitive to FAS-mediated apoptosis. This process does not affect the survival of other CD4 T cell subsets. In the absence of thymic generation of Treg from hematopoietic precursors, this dynamic process results in a relative deficiency of Treg post-HSCT. Our findings provide important information for developing strategies aimed at monitoring and modulating Treg to promote immune tolerance following HSCT.

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GvHD Prophylaxis with Everolimus and Mycophenolate in Allogeneic Hematopoietic Cell Transplantation - A Phase I/II Study.

Blood ◽

10.1182/blood.v114.22.4642.4642 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4642-4642

Author(s):

Reinhard Marks ◽

Alf Zerweck ◽

Razvan George Racila ◽

Hartmut Bertz ◽

Jürgen Finke

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Malignant Disease ◽

Acute Gvhd ◽

Hematopoietic Cell Transplantation ◽

Chronic Gvhd ◽

Tolerance Induction ◽

Treg Function ◽

Gvhd Prophylaxis

Abstract Abstract 4642 Next to the control of malignant disease, tolerance induction of the grafted cells remain to be a critical issue for longterm survivors after allogeneic hematopoietic cell transplantation (HCT). Regulatory T cells (Tregs) are believed to be involved in the process of tolerance induction to solid organ grafts and in the regulation of alloreactivity, e.g. graft versus host disease (GvHD) and graft versus leukemia effect (GvL), after allogeneic HCT. Since GvHD causes substantial morbidity, medication with calcineurin inhibitors (CI) like cyclosporine are established prophylactic measures for the prevention of GvHD after HCT. Next to the substantial renal toxicity of CI, tolerance induction might be hampered in HCT patients due to deteriorated Treg function. In contrast, data from in vitro and animal experiments suggest that inhibition of the mammalian target of rapamycin (mTOR) has not only an antiproliferative effect on many malignant cell lines but also results in an inhibition of proliferation of alloreactive T cells with sustained Treg function in a murine HCT model. Therefore we initiated a phase I/II, monocenter trial using everolimus and mycophenolate sodium (MMF-Na) as GvHD prophylaxis in patients undergoing allogeneic HCT with peripheral stem cell (PBSC) grafts after conditioning with fludarabine, melphalan, and BCNU (FBM). No additional T cell depleting agents were used for conditioning/GvHD prophylaxis. Enrolment was started in april 2008, and up to august 2009 10 patients were included (median age: 50.7 years, range: 26-64). The diagnoses included de novo AML (n=3), sAML (n=4), RAEB II (n=1), CML (n=1), T-PLL (n=1). 6/10 patients were regarded as high risk (not in CR1) for early relapse. PBSC grafts were obtained from unrelated (n=5) and related (n=5) HLA-matched donors. With no graft failures, engraftment kinetics for myeloid cells were normal, and reconstitution of the T cell compartment reached median cell counts of 251 CD4+ cells/μl and 163 CD8+ cells/μl at day +30. No grade IV/V toxicities (according to CTC criteria) were observed due to the study medication. After a median follow-up of 6 month two patients have died. The causes were acute GvHD, refractory to several lines of treatment, in a patient with CML, and severe pulmonary toxicity/BOOP in a patient with sAML. Out of 9 patients reaching CR after HCT, only one high risk patient relapsed after 6 month. In total 6 patients are alive and show complete donor chimerism for time periods of 1-14 months post transplant. The observed early recovery of T cell immunity correlated in 8/10 patients with an early brief period of acute GvHD, with 4 patients experiencing grade III/IV severities. Most of the cases could be controlled with steroids alone. Chronic GvHD could be observed in 6/7 patients, with mild to moderate forms in 5 cases, mainly involving skin, mucosa and liver. Interestingly, while early tapering of MMF-Na did not cause any problems, reduction of everolimus earlier as 6 month after HCT resulted in an induction of GvHD symptoms. Although viral reactivation (CMV, HHV6) did occur in patients receiving additional immunosuppression with steroids, no severe bacterial or fungal infections were observed even in cases with prolonged everolimus treatment. In conclusion, GvHD prophylaxis with everolimus and MMF-Na is feasible but results in an increased frequency of mild to moderate chronic GvHD. Since this sustained mild alloreactivity might reduce the risk of relapse, this GvHD prophylaxis could well be suited for patients undergoing HCT with advanced or uncontrolled malignant disease. Disclosures: Marks: Novartis: Research Funding. Off Label Use: Everolimus for prophylaxis of GvHD. Finke:Novartis: Research Funding.

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