Abstract 1270
After umbilical cord blood transplantation (CB-SCT), we have observed a new syndrome of culture-negative antibiotic-responsive diarrhea, with pathologic findings on biopsy suggestive of ...an infectious/inflammatory colitis, but distinct from graft-versus-host disease (GVHD). The clinical characteristics and epidemiology of this gastrointestinal syndrome have not been previously described.
We studied the entire CB-SCT cohort at our center from 3/2003 through 3/2010. Charts were reviewed in detail for all episodes of diarrheal illness after engraftment. Demographic, CB-SCT, and diarrheal illness characteristics, and gastrointestinal pathology were analyzed. Cord colitis syndrome (CCS) was defined as a persistent diarrheal illness in a CB-SCT recipient not due to GVHD, cytomegalovirus, Clostridium difficile or other identifiable etiology on extensive microbiologic and pathologic examination, with histopathological evidence of colitis, and who responded to empirical antibacterial treatment.
104 patients underwent CB-SCT during the study period; 101 were double cord recipients. 72 underwent reduced-intensity conditioning, most commonly with fludarabine, melphalan, and thymoglobulin; 32 underwent myeloablative conditioning with cyclophosphamide, fludarabine, total-body irradiation or other agents. GVHD prophylaxis with sirolimus and tacrolimus was used in 69 patients, cyclosporine and mycophenolate mofetil in 17, and other combinations in the rest of the cohort. Median follow up was 452 days (range, 1–2409).
Eleven (10.6%) patients met criteria for CCS; an additional patient had relapsing antibiotic-responsive diarrhea compatible with CCS, but was not biopsied and not included in the analysis. The 1-year cumulative probability of CCS was 0.159. Median time to onset of CCS was 131 days after CB-SCT (range, 88–314). Patients reported watery, non-bloody diarrhea, commonly associated with weight loss. 8 patients required hospital admission. 7 patients underwent abdominal imaging during their evaluation, 6 had colonic involvement (3 with pancolitis). All patients underwent colonoscopy a median of 18 days (range, 5–59) after the onset of CCS. On gross inspection, 9 had mucosal erythema and 7 had mucosal ulcerations. Pathologic review of the colorectal biopsy specimens revealed diffuse active colitis (6 cases) or chronic active colitis (5 cases); 7 of them demonstrated loose granuloma formation. No biopsies had evidence of active GVHD, pseudomembranous colitis, viral cytopathic changes, nor evidence of CMV or adenovirus on immunostains. 9 patients were treated with a fluoroquinolone and metronidazole, 2 others were treated with metronidazole alone. All patients responded to antibiotic treatment initially, but 4 relapsed, requiring further treatment courses. Patients were initially treated for a median of 14 days (range, 10–90 days) and relapsed patients were treated for a median of 120 days (range, 30–330).
There was no association between the occurrence of CCS and age, underlying disease, conditioning or GVHD prophylaxis agents used. There was no clustering of cases over time. CCS patients (5/11) were more likely to have a prior diagnosis of grade II or higher acute GVHD compared to the cohort (16/93; p=0.04), without organ-specific predominance. The median time from acute GVHD to CCS diagnosis was 184 days (range, 105–328). The crude mortality at the end of follow-up was lower among patients who developed CCS (27.3%) compared to the rest of the cohort (58.1%; Log-rank 0.04).
CCS is a distinct diarrheal illness that affects CB-SCT recipients that is antibiotic-responsive and differs from other common causes of diarrhea following SCT. Histologic findings mimic idiopathic inflammatory bowel disease and often include granulomatous colitis. This entity should be considered in CB-SCT patients with diarrhea in which other common causes of diarrhea have been ruled out. Further investigation is underway to determine whether there is an infectious etiology responsible for this syndrome or if it is an inflammatory colitis of alloimmune or autoimmune origin.
No relevant conflicts of interest to declare.
Abstract 1132
Poster Board I-154
Adenovirus infections are an important source of morbidity and mortality among stem cell transplant (SCT) recipients, with infections occurring in 5-21% of patients, ...with an associated mortality of up to 50%. Little is known about the clinical features and incidence of adenovirus infection after cord blood SCT (CB-SCT) in adults.
The DFCI/BWH umbilical CB-SCT cohort transplanted from 2003-2008 was analyzed. Adenovirus infection was diagnosed by blood PCR, culture, immunofluorescence, or immunohistochemistry. Baseline covariates included age, sex, malignancy being treated, conditioning, graft versus host disease (GVHD) prophylaxis, incident GVHD and its severity. Data was censored on 7/1/2009.
92 patients underwent CB-SCT during the period; 89 were double cord recipients. 68 underwent reduced-intensity conditioning consisting of fludarabine (180mg/m2), melphalan (100 mg/m2), and ATG (Thymoglobulin® 6 mg/kg). GVHD prophylaxis with sirolimus and tacrolimus was used in 57 patients, cyclosporine and mycophenolate mofetil in 17, and other combinations in the rest of the cohort. Median follow up was 363 days (range, 1-1848 days). Adenovirus testing was routinely done for persistent febrile illness, respiratory, gastrointestinal and hepatic syndromes. Adenovirus infection was diagnosed in 6/92 for a cumulative incidence of 6.5%. Three were female, median age was 51 years (range, 37-62). Underlying disease was MDS in 1, NHL in 2, AML in 2 and HD in 1. Median time to diagnosis was 152 days (range, 23-768) from CB-SCT. Three patients were diagnosed within 100 days post CB-SCT: all had diarrhea and fever. The other 3 patients were diagnosed between 8 months to 2 years after transplant: one patient each had respiratory, urinary and gastrointestinal infection. Three of the six patients developed concomitant CMV reactivation with their adenovirus infection: 2 of these occurred within 100 days of transplant, and one occurred 1 year after HSCT. Three of the 6 patients with adenovirus infection also developed grade II-IV acute GVHD and 2 developed chronic GHVD. The onset of acute GVHD was closely associated in time with the development of adenovirus infection; this was not the case of chronic GVHD. Two patients had blood adenovirus loads > 1 million copies/mL, and were treated with cidofovir with improvement in symptoms and decline in virus loads. Of the 6 patients, 3 died of relapsed disease, none died of complications attributable to adenovirus infection.
Since recipients of T-cell depleted transplants are at higher risk for viral infections compared to those receiving unmanipulated grafts, we hypothesized that umbilical cord recipients would be at an increased risk for adenovirus infection due to the limited number of immunocompetent T-cells in cord blood grafts. Surprisingly, we detected adenovirus infection in only 6.5% of patients in this cohort, and none died as a consequence of adenovirus infection. Further study of the immunity to adenoviruses in CB-SCT adult recipients is warranted.
Off Label Use: cidofovir for treatment of adenovirus infection.
Single-unit umbilical cord blood (CB) SCT is limited by low total nucleated cell (TNC) dose. Co-infusion of CD34+ cells from a third party HLA-mismatched donor, known as dual or haplo-cord ...transplant, reduces the period of post-transplant neutropenia and related complications. The aim of this study was to analyze the value of early post-transplant peripheral blood (PB) and T cell chimerism after 28 dual transplants regarding CB engraftment. Cumulative incidence of myeloid engraftment at 30 days was 93% with a median time to engraftment of 14 days (10-29). Patients who developed CB graft failure (n=5) showed very low percentages of CB cells on days +14, +21 and +28 with decreasing dynamics. On the other hand, percentages of CB cells in patients who achieved CB engraftment increased over time. Interestingly, such patients showed two distinct chimerism dynamics in PB, but all of them showed a predominance of CB T cells early after SCT with increasing dynamics over time. Early post-transplant chimerism dynamics in PB and T cells predicts CB graft failure enabling rapid therapeutic measures to be applied. On the other hand, early increasing percentages of CB T cells correlates with ultimate CB engraftment.
Introduction
Chronic expansion of large granular lymphocytes (LGL) has been reported after classical matched allogeneic hematopoietic stem cell transplantation (SCT) with bone marrow, peripheral (PB) ...or cord blood (CB) as source of graft. This proliferation is indolent and carries a favorable prognosis. Little is known of the incidence and features of LGL expansions in haplo-SCT with post-transplant cyclophosphamide (PTCy), where the impact of the duration of lymphopenia is also ill-documented.
Patients & methods
This study included 85 adult patients (pts) who received a haplo-SCT between 11/2013 and 03/2019 for hematological diseases. The 3 conditioning regimen used were i) Baltimore (n=28, 11/2013-05/2017) i.e. fludarabine 30 mg/m²/day (d), d -6 to -2, cyclophosphamide 14.5 mg/kg d -6, low dose total body irradiation (LDTBI) 2 Grays d-1, ii) Clo-Baltimore (n=28, 03/2014-04/2017), i.e. Clofarabine 30 mg/m²/d, d -6 to -2, cyclophosphamide 14.5 mg/kg d -6, LDTBI 2 Grays d -1, iii) CloB2A1 (n=29, 05/2017-03/2019) with Clofarabine 30 mg/m²/d,d -6 to -2, busulfan 3,4 mg/kg d -3 and -2, ATG 2,5 mg/kg d-1. All pts received mobilized PB as SC source on d 0 and PTCY 50 mg/kg/d on d +3 and +4 with cyclosporine and mycophenolate mofetyl as graft versus host disease (GVHD) prophylaxis. All pts provided informed consent for data collection.
The duration of lymphopenia (<1.5x109/L) as well as occurrence, duration and immunophenotype of LGL expansion (>4x109/L) were recorded. The patients with primary graft failure (n=6) or dead before 3 months (mo) post-SCT were excluded (n=7). Engraftment was monitored by qPCR on PB cells and sorted CD3+ T-cells. TCR-γ/β gene rearrangements of CD3+ collected during sustained lymphocytosis were assessed with the Biomed-2 PCR method (n=7). Data were analyzed considering viral reactivation episodes (CMV, EBV, HHV6 or BK virus), acute or chronic GVHD, relapse and survival.
Results
The study included 72 adults treated with haplo-SCT (43 males, median age: 59 yo (24-71)) with a median follow-up of 31 mo for alive patients. Most pts had a myeloid disease (64%) and 57% were in complete remission at the time of haplo-SCT. The median duration of lymphopenia was 6 mo (1-49), significantly shorter in pts with a CloB2A1 conditioning (151 d vs. Baltimore 293 d vs. Clo-Baltimore 387 d, p=0.003) or with CMV reactivation (138 d vs 361 d, p<0.0001).
Brisk LGL expansion was characterized morphologically in 10 pts (14%), of donor origin in the 9 pts tested. It occurred at a median of 5 mo (2-8), whatever the GVHD prophylaxis. These pts had a shorter duration of lymphopenia (4 vs 10 mo, p=0.0002). The median duration of LGL expansion was 6 mo (0.1-22) with a median lymphocyte count of 5.8x109/L (4.3-19.4). Immunophenotyping disclosed expansions of NK-cells (n=2), CD8+ CD4- T-cells (n=6) or CD4- CD8- TCR gd T-cells (n=2). They were oligoclonal (n=4) or monoclonal (n=3). A recipient CMV+ status was strongly associated with the onset of LGL expansion (89% vs 20%, p=0.0001), and with CMV reactivation (35% vs 4%, p=0.001) but not with that of other viruses. Grade 2-4 acute and chronic GVHD were not correlated with LGL expansion.
ROC curve analyses identified that pts with more than 216 d of lymphopenia (AUC=0.83, p<0.001) had a better 2y disease-free survival (DFS) (77% vs 38%, p=0.0008) and 2y overall survival (OS) (81% vs 45%, p =0.0006) (Fig. 1). LGL expansion was associated with a significantly lower incidence of relapse (10% vs 50%, p=0.03), better 2y-DFS (86% vs. 51%, p=0.05) and a trend towards a better 2y-OS (86% vs. 54%, p=0.1) (Fig. 2). Only 1 of these pts has relapsed and died of transplant-related mortality. Neither the recipient's CMV status nor CMV reactivation influenced DFS or OS. Multivariate analysis showed that the disease risk index score (Armand 2014), lymphopenia (>216 days) and LGL expansion, but not age (> 60yo), were independently associated with a better DFS and OS (p<0.0001).
Conclusion
A shorter duration of lymphopenia after haplo-SCT confers unexpectedly shorter survivals, suggesting the expansion of non-allo-reactive T-cells with a reduced graft versus leukemia effect. LGL expansion (14%) is not a rare event after haplo-SCT, mainly involves CD8+ T-cells, occurs preferably in CMV+ recipients or in pts with CMV reactivation. It is associated with a favorable outcome, similar to that observed in matched and CB SCT.
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Peterlin:AbbVie Inc: Consultancy; Jazz Pharma: Consultancy; Daiichi-Sankyo: Consultancy; Astellas: Consultancy. Le Gouill:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche-Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support. Chevallier:Incyte: Consultancy, Honoraria; Daiichi Sankyo: Honoraria; Jazz Pharmaceuticals: Honoraria.
Background: Allo-SCT is a curative option for patients with AML and MDS. It is unclear whether there is an upper age limit for Allo-SCT. CIBMTR showed similar outcomes for patients older than 65 ...years after Allo-SCT (McClune et al J Clin Oncol 28:1878-1887). A single center report of 54 patients (70 - 75 years) established the feasibility of Allo-SCT in patients over age 70 (Brunner et al Biol Blood Marrow Transplant 19:1374-1380). There is no published data on Allo-SCT in patients older than 75 years.
Methods: Retrospective analysis was performed on all patients with diagnosis of AML or MDS who underwent an Allo-SCT after their 75th birthday at UMass Memorial Medical Center on an IRB approved protocol.
Results: 14 patients were identified from the database. Median age at transplant was 78.4 years (range 75.2 - 83.6). Diagnosis was AML (N=10) and MDS (N=4). Disease status at SCT for AML patients was CR1 (N=6), ≥ CR2 (N=2) and relapse (N=2). Source of stem cell was peripheral blood (PB) (N=11) and cord blood (CB) (N=3). Median time from diagnosis to SCT was 7 months (range 3 - 98). Karnofsky score (KS) at SCT was 90 (N=5) and 80 (N=9). Three patients had prior autologous SCT. Source of stem cells was peripheral blood (PB) (N=11) and cord blood (CB) (N=3). Donors were mostly unrelated (N=13) with one being mismatched related (N=1). For PB recipients the HLA matching was 10/10 in 7 patients and 9-11/12 in 4 patients. CMV serostatus was neg/neg (N=2), pos/pos(N=4) and pos/neg (N=7). ABO mismatch was major (N=1) and minor (N=6).
Conditioning regimen was reduced intensity in 13/14 recipients. All three CB recipients received Melphalan (Mel) based conditioning (Thio/Flu/Mel 100-140mg/m2). Conditioning for PB recipients was Busulfan(Bu) based (N=6) and Mel (dose 50 - 140mg/m2)based (N=5). Graft versus Host Disease (gvhd) prophylaxis was Tacrolimus (Tac) with Mycophenolate mofetil (MMF) for all CB-SCT recipients Flu/Bu-2 recipients. All Mel based PB-SCT recipients received post-transplant Cyclophosphamide (days 3, 4) with Sirolimus. HLA mismatched patients also receive Tac. 11/14 patients received ATG.
All PB recipients engrafted their neutrophils at a median of 16 days (range 13-21). 10/11 patients engrafted their platelets at a median of 18 days (range 0-32). One CB recipient died on day 10 before engrafting. Other two CB recipients engrafted their neutrophils on day 14 and platelets on days 33 &37. Day 100, 1- year and 5 - year overall survival for the entire cohort was 70.7% (95% CI 39.4-87.9), 53% (95% CI 23.3-75.9) and 22.1% (95% CI 3.8 -49.9) respectively. Two patients developed grade III and 3 patients developed grade II acute gvhd. Chronic (limited) gvhd was seen in one patient only. 9 patients died at a median of 264 days (range 10 - 802) post SCT. There were no deaths due to disease progression. Three patient are currently surviving beyond 2 years. Two of these patients are immune suppression free, gvhd free and live alone with a KS of 100. 3 out of 4 patients over age 80 years survived more than 2 years.
Conclusion: Allo-SCT is feasible in selected patients over the age of 75.
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No relevant conflicts of interest to declare.
The safety and usefulness of unrelated umbilical cord blood (CB) stem cell transplantation (SCT) in patients candidates for allogeneic SCT and HIV infection is unknown. Single CB with the co-infusion ...of CD34+ cells from a third party HLA-mismatched donor (TPD), or Haplo-cord SCT, has shown to reduce the period of post-transplant neutropenia and related early complications associated with single CB transplantation. This platform could potentially reduce the risk of early infections in this particular group of patients. On the other hand, the use of a cell source homozygous for the CCR5 delta32 allele mutation, which confers high resistance against HIV-1 acquisition, is of particular interest in HIV+ patients.
We report here on the first two patients with HIV infection and high-risk haematological malignancies who underwent haplo-cord SCT in two different centers: (Case 1) a 53 year-old male patient with high-risk MDS; (Case 2) a 34 year-old male with Burkitt lymphoma in CR2. Both CB transplants were performed using the haplo-cord platform with the co-infusion of mobilized and selected CD34+ cells from a TPD. Conditioning regimen were myeloablative in both cases. Case 1 had the additional potential benefit from the use of a CB unit with the homozygous CCR5delta32 mutation. Both cases achieved neutrophil and platelet engraftment similarly to previous haplo-cord SCT performed in HIV negative patients, as well as full CB chimerism. However, case 1 developed beside a severe lung infection, relapse of the underlying malignancy 2 months after SCT and died consequently. Case 2 presented one episode of bacterial sepsis with good response to antibiotics, and one episode of CMV reactivation controlled with valgancyclovir. Thirteen months after SCT, this patient is alive and in CR. Extensive analyses of viral and immunological compartments were performed showing the early viral dynamics in both patients.
CB SCT is feasible in patients eligible for allogeneic SCT and HIV infection. The haplo-cord strategy with the co-infusion of auxiliary cells from a TPD seems to offer a fast neutrophil engraftment similarly to HIV negative patients. Therefore, this strategy should be considered in patients with HIV infection and indication for allogeneic SCT, whenever CB is the source of choice and especially if a CCR5 mutated unit is available.
Kuball:Miltenyi: GMP product development Other.
Allogeneic stem cell transplantation (SCT) from cord blood (CB) as a stem cell source is a promising alternative when no human leukocyte antigen-matched donor is found. Donor lymphocyte infusion ...(DLI) is a possible treatment modality for threatening graft failure or relapse of an underlying malignancy after transplantation. Ethical and logistical reasons limit the possibility of DLI in the setting of CB SCT. To remedy this restriction, we performed expansion of donor T cells in vitro from CB grafts in a clinical setting for use as future DLI and characterized the expanded cells in comparison to T cells from CB acquired ex vivo and adult peripheral blood. T cells were expanded from grafts used for transplantation, upon CD3/CD28 crosslinking and culture in interleukin-2. Phenotype and function of T cells were assessed by flow cytometry and mixed lymphocyte culture assays. T-cell receptor repertoire distribution was evaluated with polymerase chain reaction-based spectratyping. We were able to amplify T cells to sufficient amounts for DLI in 13 out of 13 initiated expansions. Expanded T cells presented with an activated phenotype and could be induced to produce cytokines by a nonspecific stimulus. When exposed to allogeneic targets, expanded CB T cells proliferated at comparable levels to their ex vivo and adult blood counterparts. In summary, clinical expansion of CB T cells for DLI is feasible and may be a future modality for treatment of graft failure or relapse after SCT.
Abstract 3240
Analysis of donor chimerism is a well established technique to monitor engraftment and detect pending relapse in patients after allogeneic hematopoietic stem cell transplantation ...(HSCT). Over the last decade, use of unrelated and/or mismatched donors as well as alternative grafts like cord blood (CB) has increased, and, in addition reduced intensity conditioning regimens are widely applied. Thus, recipients are increasingly being exposed to both persistent mixed chimerism and infectious complications because of delayed immune reconstitution.
Donor chimerism is analyzed routinely from peripheral blood cells in all recipients. In addition, in a prospective study to evaluate usefulness of subset chimerism, T cell chimerism is analyzed in selected patients since 2007. Reconstitution of CMV-specific CD8 T cells (CMV-CTL) is monitored by multimers (multimeric dye-labeled recombinant-MHC-I-peptide-complexes) since 2006 to evaluate CMV-specific immune reconstitution post HSCT. Using this method, HLA-restriction of the multimers enables detection of residual recipient CMV-CTLs in mismatched transplantations.
Interestingly, we found that CMV reactivation is accompanied by a decline in donor chimerism in some patients and that recipient CMV-CTLs persisting post HSCT expand upon CMV reactivation. Table 1 summarizes first data of this analysis in patients transplanted between 2007 and 2011 in our centre.
Table 1:Data from patients transplanted between 2007 and 2011 at MHH#underlying diseaseR/D genderDonorConditioning regimenGvHD prophylaxisGraft1AMLf/fMMUDFlamsa(TBI)/ATGCSA/MMFPBPC2AMLf/fMMRDFlu/Melph/Thiotepa/ATGTCDPBPC3AMLm/mMMUDFlamsa(TBI)/ThymoCSA/MMFPBPC4NHLm/fMUDFlu/Cy/ThymoCSA/MMFPBPC5ALLm/mMMUDTBI/Cy/ATGCSA/MMFPBPC6AAf/mhla-ident sibl.Flu/Cy/TBI/ThymoCSA/MTXBM7MDSm/mMMUDFlu/Cy/TBI/ATGCSA/MMFcord blood8NHLm/mMMUDFlu/Cy/ATGCSA/MTXPBPC#R/D CMV-serostatusaGvHDCMV reactivation (CMV-R)leukocyte chimerism decline post CMV-RT cell chimerism decline post CMV-Rpersisting recipient CMV-CTLs1R+D-yes40, 61yesyes2R+D-no27, 90, 188noyes3R+D+yesnonoyes4R+D+no18yesyesyes5R+D-suspected34, 90nonoyes6R+D-no55yesyes7R+D-yes85, 113yesyesyes*8R+D-no (HvG)33yesyesyes**confirmed by chimerism analysis in enriched CMV-CTL
Patient 7 received a double mismatched cord-blood graft. As expected the recipient-CMV-CTLs declined after HSCT and by day +50 post-HSCT no CMV-CTLs (A*0201-NLVP multimer) could be detected anymore. The patient had a CMV reactivation on day +85 as shown by pp65 antigenemia assay. On day +90, 72 CMV-CTLs/μl were detected, further increasing to over 200/μl by day +152. To further analyze the origin and functionality, CMV-CTLs detected on day +90 were enriched by MACS to a purity of 97% in the CD3+CD8+ T cells. Donor chimerism was only 4%. After reconstitution of autologous CMV-CTLs, the patient experienced an additional subclinical CMV reactivation on day +113, not requiring any treatment at this time.
In patient 8 a subclinical CMV reactivation on day +33 led to proliferation of CMV-CTLs and HLA-A*24 restricted and -B*35 restricted CMV-CTLs rose from 0 cells/μl (A*24 0.05%, B*35 0.04% of CD3CD8 T-cells) to 1 cell/μl and 21 cells/μl (A*24 0.28%, B*35 4.05% of CD3CD8 T-cells), respectively. Donor chimerism decreased from 51% on day +33 to 0% by day +62. Chimerism analysis of T-cell subsets on day +62 and of CMV-CTLs on day +69 revealed a 0% donor chimerism in these subsets. We speculate that in this patient CMV reactivation led to an inflammatory environment, which might have promoted loss of the graft.
Our data indicate that T cell-subset chimerism analyses may contribute to a better understanding of chimerism kinetics. Furthermore, recipient-derived CMV-CTLs may be able to control CMV reactivation, especially after reduced intensity conditioning but also after standard conditioning regimens (i.e. in patient 5), but can severely influence donor chimerism and thus might have negative effects as well. We are currently investigating donor chimerism in T cell subsets and CMV-CTL reconstitution to gain insight into the complex immune responses and reconstitution processes occurring after allogeneic HSCT or CB-SCT.
No relevant conflicts of interest to declare.
For patients lacking a human leukocyte antigen (HLA)-matched donor, umbilical cord blood (UCB) is a promising source of hematopoetic stem cells. Greater HLA disparity can be tolerated between the ...recipient and donor UCB compared with bone marrow or peripheral blood stem cells because of the naive and immature phenotype of UCB derived T cells. The risk of rejection is increased after UCB transplantation. After HLA-identical sibling or matched unrelated SCT the graft-versus-leukemia (GVL) effect may be increased by donor lymphocyte infusion (DLI) after SCT. However, after UCB transplantation DLI is not possible. This raised the question of whether ex vivo expanded CB lymphocytes (CBL) also can be used as a tool for adoptive immunotherapy after CB SCT. We have managed to establish a protocol for massive expansion of CB derived T cells making them usable in the clinic suitable for DLI after CB transplantation. We have further been able to show that the expansion protocol doesnt skew the T cell population regarding the phenotype, CD4:CD8 ratio as well as TCR usage profile measured by spectratyping. By activating the cells we have further investigated and confirmed the expanded T cells capacity to efficiently produce pro-inflammatory cytokines and respond in an allogeneic setting. We have now tried expanded CBLs in an adult patient with AML with threatening rejection after double UCB transplantation. The patient received Bu/Cy and ATG as conditioning therapy. He was transplanted with double UCB with a total nucleated cell dose of 5 x 10.sup.7/kg. Both UCB units were 5/6 matched for HLA-A,-B and -DRB1 with low resolution HLA-typing. Chimerism analysis one month after transplant showed mixed chimerism in T-, B- and myeloid cells of both donor units and recipient. At four months after transplant the patient had 85% recipient cells in both T cells and myeloid cells indicating a threatening rejection. However, in bone marrow CD34+ cells were 90% of donor origin. Due to anticipated rejection the patient was treated with expanded CBLs, 5 x 10.sup.3/kg at 4 months, 1 x 10.sup.4/kg at 5 months and 1 x 10.sup.5/kg at six months. Immunusuppression was tapered at six months. The immunemodulatory treatment was well tolerated with no development of GVHD. So far no change in chimeric pattern has been shown. The immunemodulatory treatment will be continued with increasing doses of CBLs.
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