Low-Dose Alemtuzumab Is Uniquely Effective in Refractory Leukemic Cutaneous T Cell Lymphoma (L-CTCL).

Abstract Abstract 3748 Poster Board III-684 Sezary Syndrome and other Cutaneous T Cell Lymphomas with peripheral blood involvement (collectively termed leukemic CTCL, or L-CTCL) are often refractory to multiple therapies. Alemtuzumab, an antibody directed against the pan-lymphocyte antigen CD52, has been used to treat multiple hematologic malignancies, including L-CTCL. However, conventional treatment regimens are associated with an increased risk of infections, and this is of particular concern in CTCL patients with compromised skin integrity. We have treated six L-CTCL patients with a modified regimen of alemtuzumab: 10mg/kg, subcutaneously, three times weekly for six weeks (1/3 of the conventional dose). All patients had had disease for >3 years duration, skin biopsies consistent with CTCL, elevated absolute CD4 counts, CD4/CD8 ratios of >10 and were refractory to at least two prior therapies, including extracorporeal photochemotherapy, interferon, and single and combination agent chemotherapy. In five of six patients, the malignant clone was identifiable using a Vb family specific monoclonal antibody; all cells of the malignant clonotype strongly expressed CD52. Within three weeks, all six patients achieved clearance of detectable T and B cells in peripheral blood, including loss of the malignant clone. In parallel, all patients developed clearing of erythroderma, relief of pruritus, and by the end of the six week treatment period, all patients had complete to near complete resolution of all clinical signs of disease. Isolation of T cells from a skin biopsy of one patient in complete remission revealed that <6% of T cells resident in skin expressed the malignant Vb subunit. At this reduced dose, no infectious complications were encountered. We conclude from that low-dose Aletuzumab is a well tolerated, safe, and highly effective therapy in a carefully selected population of patients with refractory L-CTCL. Disclosures: No relevant conflicts of interest to declare.

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Haploidentical Stem Cell Transplantation with CD3 Depleted Mobilized Peripheral Blood Stem Cell Grafts for Children with Hematologic Malignancies.

Blood ◽

10.1182/blood.v106.11.2910.2910 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2910-2910 ◽

Cited By ~ 9

Author(s):

Gregory A. Hale ◽

Kimberly A. Kasow ◽

Kwan Gan ◽

Edwin Horwitz ◽

Joseph P. Woodard ◽

...

Keyword(s):

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Graft Rejection ◽

Peripheral Blood ◽

Acute Gvhd ◽

Ex Vivo ◽

Hematologic Malignancies ◽

Infectious Complications ◽

Persistent Disease

Abstract Allogeneic hematopoietic stem cell transplantation is the only curative therapy for patients with high-risk or recurrent hematologic malignancies. As only 25% of patients have matched siblings and not all have unrelated donors, haploidentical HSCT using mismatched related donors is the only option for many patients. However, historically the risks of GVHD, graft rejection, and prolonged immunocompromise have made this donor option rather limited. More recently, highly purified CD34+ hematopoietic cells have been used with decreased GVHD rates, but at the risk of graft rejection and prolonged immunodsuppression with infectious complications. In an attempt to obtain a PBSC graft with higher T-cell content to maintain acceptable GVHD rates while promoting more rapid immune reconstitution, we initiated a prospective clinical trial for patients with hematologic malignancies who lacked a matched related donor or unrelated donor using a novel method of graft processing. The conditioning regimen consisted of TBI (12 Gy in 8 fractions over 4 days), cyclophosphamide (60 mg/kg/day for 2 days), thiotepa (10 mg/kg/day for 1 day), and rabbit ATG (10 mg/kg/course over 4 days). GVHD prophylaxis consisted of cyclosporine initiated at day -2. G-CSF mobilized PBSC grafts from mismatched related donors were infused after ex vivo T-cell depletion using OKT3 on the CliniMACS device. Patients had weekly peripheral blood analysis for evidence of EBV, CMV, or adenovirus DNA by PCR. If positive, pre-emptive therapy was administered. Twenty patients were enrolled with a median age of 11.9 yrs (range, 2.7–22.1). Diagnoses included ALL (2-CR1, 5-CR2, 3-CR3), AML (2-CR1, 1-CR2, 1-persistent disease), MDS (1-CR1, 2-persistent disease), CML (2- first chronic phase) and NHL (1-CR2). Donors and recipients were matched at 3 (n=11), 4 (n=8) or 5 (n=1) of 6 HLA loci. Of the 19 evaluable patients (one patient died prior to engraftment), the median time to attain ANC > 500/mm3 was 13 days (range, 10–19) and the median time to attain a transfusion-independent platelet count of 50,000/mm3 was 18 days (range, 8–37) post-HSCT. Only 3 patients developed grade 1–2 acute GVHD and none developed grade 3–4 acute GVHD. One patient developed limited chronic GVHD. Complications included post-transplant lymphoproliferative disorder (PT-LPD, n=3), VOD (n=2), BOOP (n=1), CMV retinitis (n=1), and adenovirus reactivation (n=7). No patient died of infectious complications or PT-LPD. 6 patients have died of regimen-related toxicities (n=4), or disease recurrence (n=2) at a median of 160 days (range, 4–208) post-HSCT. Fourteen patients remain alive in remission at a median of 162 days (range, 49–947) post-HSCT. OKT3 depleted PBSC grafts from haploidentical donors depleted of T-celss ex vivo results in favorable outcomes and acceptably low rates of GVHD and infectious complications for children undergoing HSCT from parental donors.

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Early Recovery of Thymus-Derived Naïve T Cells in Pediatric Patients (pts) Treated with Non-Myeloablative Allogeneic Peripheral Blood Stem Cell Transplantation (NMSCT) for Cancer.

Blood ◽

10.1182/blood.v108.11.310.310 ◽

2006 ◽

Vol 108 (11) ◽

pp. 310-310

Author(s):

Terry J. Fry ◽

Alison R. Rager ◽

Frances Hakim ◽

Cynthia Love ◽

Paula Layton ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

High Risk ◽

Peripheral Blood ◽

T Cell Subsets ◽

Immune Recovery ◽

Early Recovery ◽

Thymic Function ◽

Cell Counts ◽

Increased Risk

Abstract Background: Current SCT approaches consistently achieve rapid donor myeloid engraftment, but delayed immune recovery remains a significant obstacle and results in increased risk of infection and relapse. T cells are regenerated via 2 pathways, thymus-derived and peripheral expansion, processes for which IL-7 is critical. We postulated that non-myeloablative pre-transplant conditioning might preserve thymic function in pediatric SCT recipients thus enhancing thymus-derived naïve T cell regeneration. Methods: We analyzed T cell subsets, T cell receptor excision circles (TREC), and IL-7 levels in peripheral blood after SCT in 21 pediatric pts with high-risk malignancies (median age 14, range 4–21). Fludarabine-based induction chemotherapy was administered for disease control and targeted CD4 count reduction. Pre-transplant conditioning consisted of cyclophosphamide (1,200 mg/m2/day) and fludarabine (30 mg/m2/day) × 4 days plus melphalan (100 mg/m2 × 1 dose in sarcoma pts). Grafts consisted of G-CSF mobilized unmodified peripheral blood stem cells from 5–6/6 HLA-matched first-degree relatives (median CD34 dose 11.7 × 10E6/kg, range 4.4–19.1; median CD3 dose 416 × 10E6/kg, range 228–815). Cyclosporine was used for GVHD prophylaxis. Results: Donor-derived engraftment was rapid (absolute neutrophil count > 500/uL median day 9, range 8–11). Complete donor lymphoid chimerism (>95% by VNTR-PCR on CD3 sorted peripheral blood) was achieved in all by day 28. Immune recovery was brisk and sustained. Substantial numbers of naïve (CD45RA+/CD62L+) CD4+ and CD8+ T-cells were detected at day 28 (Fig 1). There was a steady increase in TREC from 3 to 12 months consistent with early, robust thymic-dependant T cell generation (Fig 2). This was not seen in adult pts treated on a parallel trial (data not shown). IL-7 levels were elevated and inversely correlated with T cell counts (r=−0.56, p<0.0001). Conclusions: Targeted immune depletion and NMSCT results in rapid, sustained immune reconstitution in pediatric pts with malignancy. Preserved thymic function appears to contribute to naïve T cell recovery in this setting. We postulate that non-myeloablative conditioning is thymus sparing and that this, in combination with immune depletion-induced IL-7 elevation, promotes early thymic-derived lymphoid recovery. This approach may serve as a strategy to overcome the prolonged immunodeficiency commonly encountered after allogeneic SCT in pediatrics and might be used as a platform to direct allogeneic anti-tumor immune responses in high-risk childhood cancers. Figure 1 Figure 1. Figure 2 Figure 2.

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CD19 Targeted Allogeneic EBV-Specific T Cells for the Treatment of Relapsed ALL in Pediatric Patients Post HSCT

Blood ◽

10.1182/blood.v120.21.353.353 ◽

2012 ◽

Vol 120 (21) ◽

pp. 353-353 ◽

Cited By ~ 2

Author(s):

Kevin J. Curran ◽

Nancy A. Kernan ◽

Xiuyan Wang ◽

Clare Taylor ◽

Ekaterina Doubrovina ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Dose Escalation ◽

Peripheral Blood ◽

Progressive Disease ◽

Pediatric Patients ◽

Conflicts Of Interest ◽

Limit Of Detection ◽

Topical Steroids ◽

Early Results

Abstract Abstract 353 T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR modified T cells targeting the CD19 antigen is a novel therapeutic approach for patients with relapse B-ALL following allo-HSCT. We have previously demonstrated donor derived Epstein-Barr virus specific cytotoxic T cells (EBV-CTLs) can be safely infused in patients following allo-HSCT. Following retroviral gene transfer with our CD19-specific CAR (19–28z), EBV-CTLs demonstrate in vitro cytotoxicity against CD19+ targets, retain EBV-specificity without allogeneic reactivity, and in vivo, eradicate systemic Burkitt lymphoma in a SCID/Beige murine model. Based on our preclinical studies we have initiated a phase I dose escalation clinical trial (NCT01430390) in pediatric patients with relapsed CD19+ B-ALL following allo-HSCT utilizing donor 19–28z+ EBV-CTLs. Three patients have received conditioning chemotherapy (fludarabine 25mg/m2 × 5 days) followed by infusion of CAR modified EBV-CTLs without infusion related toxicity. Subsequently, patient 1 and 3 safely received additional T cell infusions without (patient 1) and with (patient 3; cyclophosphamide) conditioning chemotherapy. Total CAR+ T cell dose for each infusion ranged from 1.4 – 4.1×105 cells/kg (1×106 total EBV-CTLs/kg per infusion). Patient 1 developed fever and biopsy proven skin GVHD (grade II) three days following his first infusion which resolved 2 weeks after starting topical steroids. Modified T cells were detected by Q-PCR in the bone marrow and peripheral blood up to 3 and 18 weeks respectively in patient 1 following first infusion, up to 2 weeks in the peripheral blood in patient 2, and have been below the limit of detection in patient 3. Patient 1 died from progressive disease 29 weeks following first infusion and patient 2 died from progressive disease 19 weeks post infusion. Patient 3, initially treated in remission following salvage chemotherapy, has no evidence of disease (NED) 15 months after last extramedullary relapse and 5 months post first infusion of 19–28z+ EBV-CTLs. These early results demonstrate the feasibility of allogeneic 1928z+ EBV-CTL infusions without the development of significant GVHD post allo-HSCT. Subsequent cohorts of patients will receive dose escalation of modified T cells and will be evaluated for safety, persistence of 19–28z+ EBV-CTLs, and for anti-tumor efficacy. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v120.21.4670.4670 ◽

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.

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Ex vivo Fucosylation Enhances Regulatory T Cell Anti-Graft Versus Host Disease Potency in Mice

Blood ◽

10.1182/blood.v124.21.660.660 ◽

2014 ◽

Vol 124 (21) ◽

pp. 660-660

Author(s):

Simrit Parmar ◽

Xiaoying Liu ◽

Yvon Eric ◽

Patrick zweidler-mcCay ◽

Nina Shah ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Preventive Strategy ◽

Adoptive Therapy ◽

Host Disease ◽

Graft Versus Host

Abstract Emerging preclinical data indicate a potential therapeutic use of regulatory T cells (Tregs) to suppress or eliminate graft versus host disease (GVHD). Adoptive therapy with Tregs has been examined as a prophylactic strategy for GVHD. Thus far, 1:1 ratio of Tregs to conventional T cells (Tcons) has been required to prevent GVHD, and such high targeted cell doses are hard to achieve in a clinical setting due to variability in Treg frequency and expansion potential. As a result, novel strategies are needed to generate clinically-effective Treg products. Additionally, since the ability of Tregs to enter inflamed tissues has been shown to be critically dependent on their ability to bind E- and P-selectins, we sought to exploit this pathway to improve Treg homing. Incubation of culture expanded Cord Blood (CB) Tregswith fucosyltransferase-VI (FTVI) increased the degree of fucosylation from 8% to 64%. Importantly, fucosylated Tregs are able to suppress T cell proliferation in an in vitro allogeneic mixed lymphocyte assay and show preferential increase in the E-selectin binding ability when compared to untreated Tregs. We injected 10e6 Tregs vs. 10e6 fucosylated Tregs followed by 10e7 peripheral blood mononuclear cells in the sublethally irradiated NOD/SCID gamma null (NSG) xenogenic GVHD mouse model, and recipients of untreated Tregs showed weight loss as early as day 6 whereas fucosylated Treg recipients retained weight until day 12 (Fig 1). Fucosylated Tregs were detected in the peripheral blood of mice until day 31 as opposed to day 12 for untreated Tregs. As compared to recipients of untreated Tregs, lower numbers of GVH-inducing allogeneic T-cells were detected in the secondary lymphoid organs in the fucosylated Treg recipients (Table 1). The overall survival of fucosylated Treg recipients was significantly superior to that of untreated Treg recipients (Fig 2). We conclude that prophylactic treatment with fucosylated Tregs prevents GVHD and improves survival in a xenogenic mouse model at doses that are i) at less than 1:1 ratio with conventional T-cells (Tcon) and ii) are lower as compared to untreated Tregs. We believe that by overcoming the 1:1 dose requirement of Tregs: Tcons, we will be able to effectively translate Treg based adoptive therapy into the clinic for the prevention of GVHD. Establishing fucosylated Tregs as a preventive strategy for GVHD will result in a major breakthrough in the field of stem cell transplantation eliminating a major life threatening outcome of the therapy. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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Interleukin-15 Increases Effector Memory CD8+ T Cells and NK Cells in Simian Immunodeficiency Virus-Infected Macaques

Journal of Virology ◽

10.1128/jvi.79.8.4877-4885.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 4877-4885 ◽

Cited By ~ 72

Author(s):

Yvonne M. Mueller ◽

Constantinos Petrovas ◽

Paul M. Bojczuk ◽

Ioannis D. Dimitriou ◽

Brigitte Beer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Peripheral Blood ◽

Low Dose ◽

Effector Memory ◽

High Dose ◽

Immunodeficiency Virus ◽

Interleukin 15

ABSTRACT Interleukin-15 (IL-15) in vitro treatment of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected individuals specifically enhances the function and survival of HIV-specific CD8+ T cells, while in vivo IL-15 treatment of mice preferentially expands memory CD8+ T cells. In this study, we investigated the in vivo effect of IL-15 treatment in 9 SIVmac251-infected cynomolgus macaques (low dose of IL-15, 10 μg/kg of body weight, n = 3; high dose of IL-15, 100 μg/kg, n = 3; control [saline], n = 3; dose administered twice weekly for 4 weeks). IL-15 treatment induced a nearly threefold increase in peripheral blood CD8+CD3− NK cells. Furthermore, CD8+ T-cell numbers increased more than twofold, mainly due to an increase in the CD45RA−CD62L− and CD45RA+CD62L− effector memory CD8+ T cells. Expression of Ki-67 in the CD8+ T cells indicated expansion of CD8+ T cells and not redistribution. IL-15 did not affect CD4+ T-cell, B-cell, and CD14+ macrophage numbers. No statistically significant differences in changes from baseline in the viral load were observed when control-, low-dose-, and high-dose-treated animals were compared. No clinical adverse effects were observed in any of the animals studied. The selective expansion of effector memory CD8+ T cells and NK cells by IL-15 further supports IL-15's possible therapeutic use in viral infections such as HIV infection.

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Adverse Events Following Infusion of T Cells for Adoptive Immunotherapy: A 10 Year Experience.

Blood ◽

10.1182/blood.v114.22.3212.3212 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3212-3212

Author(s):

Conrad Russell Y. Cruz ◽

Patrick Hanley ◽

Hao Liu ◽

Vicky Torrano ◽

Yu-Feng Lin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Adverse Events ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Safe Procedure ◽

Cell Product ◽

Increased Risk ◽

Infusion Reactions ◽

Severe Adverse Events

Abstract Abstract 3212 Poster Board III-149 Following administration of ex-vivo expanded T cells, the FDA currently recommends at least 4 hours of recipient monitoring to detect early infusion reactions. Recent catastrophic reactions to “first in man” biological agents have emphasized the importance of this rule for initial studies of new products, but its value for longer established agents is less evident. We have therefore reviewed the incidence and nature of infusion-related adverse events (AEs) associated with administration of ex-vivo expanded T cell products (antigen specific CTLs, allodepleted T cells, and genetically modified T cells) on Investigational New Drug (IND) studies in our center over the last decade. From 1998 to 2008, we infused 381 T cell products to 180 recipients who were enrolled on 18 such studies, receiving T cells targeting malignancies or post-transplant viral infections. The age of these recipients ranged from 9 months to 80 years. Cell doses were protocol specific and ranged from 104/kg up to 3 ×108/m2. Patients were premedicated with diphenhydramine (0.5-1mg/kg) and acetaminophen (10mg/kg up to a maximum of 625mg) prior to infusion. All cellular products were cryopreserved and administered intravenously over 1-15 minutes immediately after thawing. There were no Grade 3-4 infusion reactions during initial monitoring or 24 hour follow-up. Twenty four grade 1-2, non-severe adverse events (AEs) occurred in 21 infusions either during or immediately following infusion (up to 6 hours). The most common AEs were nausea and vomiting (10/24; 41.6%), most likely due to DMSO used in cryopreservation of T cell products, and hypotension (20.8%), attributable to diphenhydramine pre-medication. An additional 22 non-severe events within 24 hours of infusion were reported, the most common of which were fever (with negative blood cultures)/chills/constitutional symptoms (6/22; 27.3%) and nausea/vomiting (4/22; 18.2%) Overall, a total of 46 non-severe adverse events were noted within 24 hours of T cell product infusion (12.56%). A Fisher's exact analysis of all T cell product infusions grouped by patient age, patient ethnicity, or cell source revealed no association with increased risk. T cells from both allogeneic and autologous sources produced similar adverse events in terms of type, frequency, and severity, and allogeneic cells that were mismatched at > 2 HLA antigens had the same AEs as donor T cells matched at 5/6 or 6/6 HLA loci. We further analyzed the data using Poisson regression and the general estimating equation (GEE) model for correlated counts, to seek associations that may have been missed because AEs within a subject may not be statistically independent. By this analysis, we found decreased risks of adverse events in older patients (IRR 0.98; 95% CI 0.96-1.00; p=0.05), and increased risks of immediate (defined as occurring during the monitoring period) infusion-related events in patients reporting allergies (IRR 2.72; 95% CI 1.00-7.40; p=0.05). We thus conclude that infusion of the ex-vivo expanded T cell products used in these studies is a safe procedure associated with no severe reactions, that it is safe in the outpatient setting and that monitoring can be limited to an hour after infusion. As many of the AEs observed were due to diphenhydramine premedication, a lower dose (0.25mg/kg) of this agent may be preferred. Disclosures No relevant conflicts of interest to declare.

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Pre-Transplant Low Dose Cyclophosphamide Enhances Engraftment and Immune Reconstitution in Haploidentical Hematopoietic Stem Cell Transplant (HCT) Recipients Receiving Total Body Radiation (TBI) Free Reduced Intensity Conditioning with Post- Transplant Cyclophosphamide (PTCY)

Blood ◽

10.1182/blood.v128.22.3431.3431 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3431-3431

Author(s):

Yasser Khaled ◽

George Yaghmour ◽

Christy R Parks ◽

Deborah Lamontagne ◽

Joshua Boss ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Immune Reconstitution ◽

Low Dose ◽

Hematological Malignancies ◽

Conditioning Regimen ◽

Immune Recovery ◽

Activated T Cells ◽

Immunogenic Properties

Abstract The outcomes after Haploidentical HCT with Fludarabine (Flu)/Cyclophosphamide (Cy)/TBI and PTCY as pioneered by the Hopkins group have been associated with higher risk of relapse and delayed immune reconstitution. Multiple groups have explored the potential benefits of more intensive conditioning regimens with or without TBI. However, none of these regimens to our knowledge has exploited the immunogenic properties of pre-transplant low dose Cy while avoiding the immune-suppressive effect of TBI. Additionally, there is limited knowledge regarding immune reconstitution post haploidentical HCT with the use of mobilized peripheral blood stem cells. Accordingly, we designed a protocol modification of the Hopkins regimen by replacing TBI with Melphalan (Mel) without adding Thiotepa in contrast to the MD Anderson regimen (Ciurea et al). We hypothesized that the immunogenic properties of pre-transplant low dose Cy along with the immunomodulatory effects of Mel would enhance early post-transplant engraftment and early peripheral T cell recovery through enhanced cytokine release and antigen alloreactivity We examined the clinical outcomes and immune reconstitution recovery of 12 consecutive haploidentical HCT patients with high-risk hematological malignancies. Patient and graft characteristics are as shown in table-1. The conditioning regimen was low dose Cy 14.5 mg/m2 on day -6 & day -5, Flu 30 mg/m2 on days -6 to Days -2 and Mel 70 mg/m2 on day -3 & -2. Graft versus host disease prophylaxis (GVHD) was PTCY 50 mg/Kg on day +3 and day +4 along with Tacrolimus and Mycophenolate starting at day +5 as previously described by the Hopkins group. An immune reconstitution panel of absolute lymphocyte count (ALC), CD3, CD4, CD8, Activated T cell (CD3-HLA Dr + T cells) & NK cell (CD56/16) was performed by 4 color flow-cytometry on days +60, +120 and +180 post HCT. Chimerism studies were performed at day +30 and +100 post HCT by variable number tandem repeat PCR analysis of peripheral blood & bone marrow. All of the patients were treated according to an institutional protocol and records were reviewed retrospectively after IRB approval. All patients engrafted with a median time to engraftment of 17 days (range 12-23). Chimerism studies revealed enhanced engraftment with 100% donor in bone marrow (unsorted) and peripheral blood (CD3, CD33 & CD 56) at day +30 in all 12 patients. All patients with active disease at time of transplant (5 Pts) achieved complete remission at day +30 evaluation. Post HCT immune recovery is shown in table 2. There was significant early recovery at day + 60 of the median ALC and all T cell subsets. This recovery was most pronounced in activated T cells. While we have observed a progressive reduction in the median number of early activated T cells at day 120 & 180, the number of helper T cells (CD4) and NK cells (CD56/16) did not decline (Figure 1). With a median duration of follow up of 261 days (range 62-390), the overall survival at day 100 and projected one year survival is 96% and 81% respectively. Only one of 12 patients had relapsed. Acute GVHD grade 2-4 developed in six of 12 patients, two of whom were grade 3-4. Chronic GVHD developed in four patients (1 serositis, 1 pericardial effusion and 2 nephrotic range proteinuria). Cytokine storm developed in 5/12 patients after stem cell infusion and resolved after PTCY. BK cystitis developed in four patients but continuous bladder irrigation was only required in two patients. All cases of BK cystitis were transient and resolved with supportive measures. CMV reactivation occurred in 9/12 patients; no CMV disease or CMV mortality was observed. Aspergillus antigen positivity in serum occurred in 4 /12 but only two developed clinical fungal infection. Conclusion: In this limited series of patients with high- risk hematological malignancies who underwent haploidentical HCT with low dose Cy/Flu/Mel and PTCY, the regimen was well tolerated and resulted in effective disease control. The regimen has also demonstrated enhanced early engraftment and robust immune recovery in comparison to other studies, table 3. However, it is not clear if this enhanced immune recovery is related to the conditioning regimen modification or the use of mobilized stem cells. The results are intriguing and need further confirmation. Disclosures No relevant conflicts of interest to declare.

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Improving Immune Reconstitution After Cord Blood Transplantation Using Ex Vivo Expanded Virus-Specific T Cells: A Phase I Clinical Study

Blood ◽

10.1182/blood.v120.21.224.224 ◽

2012 ◽

Vol 120 (21) ◽

pp. 224-224 ◽

Cited By ~ 1

Author(s):

Patrick J Hanley ◽

Caridad Martinez ◽

Kathryn Leung ◽

Barbara Savoldo ◽

Gianpietro Dotti ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Dose Level ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Cord Blood Transplantation ◽

Viral Reactivation ◽

Blood Transplantation

Abstract Abstract 224 Adenovirus(Ad), Cytomegalovirus(CMV) and Epstein-Barr virus(EBV) frequently cause severe morbidity and mortality in patients(pts) after stem cell transplantation (SCT) and cord blood transplantation(CBT). We have shown that adoptive immunotherapy with peripheral blood(PB) donor derived multivirus-specific Cytotoxic T Lymphocytes directed against Ad, CMV and EBV can effectively prevent and treat the clinical manifestations of these viruses after SCT. CBT, while less likely to cause GvHD than conventional SCT, is unlikely to provide passive transfer of virus-specific CTL, since CBTs come from virus-naïve donors. Here we report for the first time the transfer of CB-derived multivirus-specific CTL(cbmCTL) to CBT recipients to restore cellular immunity to Ad, CMV and EBV. The development of cbmCTLs for pts undergoing CBT requires the priming and extensive expansion of naïve T cells rather than the more limited and simple direct expansion of pre-existing memory T cell populations from virus-exposed donors. We hypothesize that cbmCTL, derived from naïve T cells, will be efficacious and persist in vivo. Our protocol uses an initial round of stimulation with autologous CB-derived dendritic cells transduced with a recombinant Ad5f35 vector containing a transgene for the immunodominant CMV antigen, pp65 (Ad5f35pp65) in the presence of IL-7, IL-12 (CTEP-NCI) and IL-15. This is followed by 2 rounds of weekly stimulation with autologous Ad5f35pp65-transduced EBV-LCL in the presence of IL-15 or IL-2. Seven cbmCTL cultures generated for clinical use contained a mean of 48% CD8+, and 36% CD4+ cells with a mean of 33% CD45RA-/CD62L+ central memory T cells. In 51Cr release and/or IFNg ELISPOT assays, cbmCTL lines showed specific activity against all viruses. We have treated 7 pts who received the 80% fraction of a fractionated CB unit followed by cbmCTLs generated from the remaining 20% fraction; two pts were treated on each dose level;5×106/m2; 1×107/m2; and 1.5×107/m2 while one pt has been treated with 2.5×107/m2 – dose level 4. Pts received cbmCTLs on days 63–146 after CBT (median: day 83). No early infusion-related toxicities or subsequent GvHD was observed. All pts engrafted neutrophils by day 30 (median: day 20) despite receiving only 80% of the CB unit. Five of 7 pts had no initial infection or reactivation episodes, remaining free of CMV, EBV, and Ad from 2 months to 2 years post-CBT. Of the two remaining pts, pt 1 was transiently viremic for CMV pre-infusion and became highly viremic 4-weeks post-cbmCTL. The pt received a 2nd dose of cbmCTLs and CMV DNA/antigen became undetectable in the PB within 16 days of the 2nd dose and remains asymptomatic and virus free >2 yr post-CBT. Analysis of this pt's PB showed a rise in CMV-T cells even prior to cbmCTL #2, with a 31-fold expansion of CMV-T cells by 4 weeks after the initial CTLs. This pt also had AdV in his stool, which resolved without additional therapy. Shortly after CTL infusion, pt 4 had detectable EBV DNA in the PB that was controlled without additional antiviral therapy. The transferred cells had long-term persistence, since T cell receptor(TCR) deep-sequencing (ImmunoSEQ) allowed us to track infused T cell clones (i.e. clones present in the infused cbmCTL but absent in peripheral blood before cbmCTL infusion) up to 1 year post-CBT in 5/5 pts tested. In summary, none of the recipients of cbmCTL developed viral disease; in two pts with viral infections, the infections resolved without progression to disease, coinciding with the appearance of virus-specific T cells in peripheral blood. Hence, administration of cbmCTL to pts after CBT has so far been safe and can facilitate reconstitution of virus-specific T cells and control viral reactivation/infection in vivo. Disclosures: No relevant conflicts of interest to declare.

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Recruitment Of T Cells and Macrophages To The Eyes In Recipients Of Allogeneic Hematopoietic Stem Cell Transplants Correlate With Inflammatory Cytokine Presence In Ocular Gvhd

Blood ◽

10.1182/blood.v122.21.2012.2012 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2012-2012

Author(s):

Samantha Herretes ◽

Juan C. Murillo ◽

Duncan Ross ◽

Tan Yaohong ◽

Henry Barreras ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell ◽

Ocular Surface ◽

Conflicts Of Interest ◽

Fluorescent Microscopy ◽

Clinical Signs ◽

Corneal Tissue ◽

Hematopoietic Stem

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) has become the standard of care for the treatment of several life-threatening hematologic malignancies as well as certain immunodeficiency disorders. Unfortunately, as the survival rate of patients with these diseases is improved, the quality of life is negatively impacted by the development of Graft vs. Host Disease (GVHD). GVHD is a complex, multi-organ disorder arising from an immunological attack by donor allo-reactive T cells that results in damage to vital organs including the liver, skin, hematopoietic compartment and the ocular surface of the eye. Ocular GVHD occurs in >60% of these patients and is characterized by dry eye, conjunctiva damage, punctate keratopathy, corneal ulceration and perforation. Despite the high frequency of eye involvement in patients undergoing GVHD, little is known regarding the underlying immune mechanisms responsible for ocular GVHD, limiting the ophthalmic care of these patients to palliative therapies and global anti-inflammatory drugs. In this study, we examined the ocular and immunological changes occurring in recipients of MHC-matched, minor antigen mis-matched donor HSCT. C3H.SW (H-2b, Ly9.1+) mice transplanted with EGFP+ B6 (H-2b, Ly9.1-) T cell depleted bone marrow cells (TCD-BM) supplemented with T cells: a) underwent weight loss and began exhibiting clinical signs of GVHD ∼3wks post-HSCT, b) contained damaged thymuses, c) expressed an inverted CD4/CD8 ratio in the peripheral lymphoid compartments, d) contained activated effector cells and e) low CD19 levels. Importantly, these mice also developed ocular surface disease evidenced by progression of ocular surface damage characterized by increased corneal fluorescein staining and ulceration by week 6 (Figure). Furthermore, histological analyses demonstrated that only mice that developed systemic GVHD exhibited corneal thickening and epithelial irregularity. Ocular pathology was also associated with conjunctiva involvement indicated by significant goblet cell destruction as well as dense inflammatory cell infiltrates identified by intra-vital fluorescent microscopy (Figure). IHC and flow analyses demonstrated donor EGFP+ Ly9.1- CD4+ and CD8+ T cells. Notably, significant levels of Ly9.1- EGFP-CD11b+ macrophages had also infiltrated the ocular surface. In contrast to the systemic CD8>CD4 GVHD associated phenotype, the T cell infiltrate in the ocular compartment was reversed; i.e. CD8<CD4. We detected IFNγ and TNFα mRNA from corneal tissue, which is consistent with Th1 effector allo-reactive cells and M1 inflammatory macrophages involvement in ocular GVHD. In total, the present findings have identified alterations and pathology in the eye and adnexa reflective of ocular GVHD and unequivocally demonstrate the presence of donor T cells in the ocular surface. We hypothesize that T cell–macrophage interactions underlie the pathology detected in this pre-clinical model and studies are underway to develop local therapeutic modalities targeting these infiltrative populations. Disclosures: No relevant conflicts of interest to declare.

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