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Chimeric antigen receptor (CAR) T-cell therapy is a new, effective treatment for patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukaemia (ALL). Tisagenlecleucel achieved a complete remission (CR) rate and minimal residual disease (MRD) negativity of 81% at 3 months in the pivotal study; overall survival (OS) was 76% at 12 months (Maude et al, 2018). Real world data confirmed similar outcomes, with 1-year OS of 77% and event free survival (EFS) of 52% (Pasquini et al, 2020). Relapse can occur in the form of CD19 negative or CD19 positive ALL. The latter is associated with lack of persistence of the CAR T product. B-cell aplasia (BCA) is an indirect measure of CAR T presence. Early (<6 months from infusion) loss of BCA is associated with high relapse risk (Pillai et al, 2019); therefore, allogeneic stem cell transplantation (SCT) is often considered. However, SCT is associated with therapy-related morbidity and mortality and not all patients will find a suitable donor. Therefore, the optimal management of patients with loss of BCA is yet to be defined.

Introduction

The mechanisms of the requirement of glucose for steroidogenesis were investigated by monitoring the uptake of the glucose analogue 2-deoxy-D-glucose by rat testis and tumour Leydig cells. The characteristics of glucose transport in both of these cell types were found to resemble those of the facilitated-diffusion systems for glucose found in most other mammalian cells. The Leydig cells took up 2-deoxy-D-glucose but not L-glucose, and the uptake was inhibited by both cytochalasin B and forskolin. In the presence of luteinizing hormone, the rate of 2-deoxy-D-glucose uptake by both cell types was increased by approx. 50%. In addition to D-glucose, it was shown that the Leydig cells could also utilize 3-hydroxybutyrate or glutamine to maintain steroidogenesis.

High level expression of many eukaryotic genes is achieved through the action of distal regulatory sequences or enhancers. We have utilized the interaction between the erythroid-specific enhancer in hypersensitivity site 2 (HS2) of the human beta-globin locus control region and the globin gene promoters as a model to elucidate the mechanisms governing promoter/enhancer interactions. HS2 contains a 400-base pair core element consisting of tandem AP1/NF-E2 motifs flanked by binding sites for multiple ubiquitous and erythroid-specific factors. We have compared the enhancer activity of this core element with a synthetic enhancer lacking the factor binding sites flanking the AP1/NF-E2 motif (HS2(M)). In fetal/erythroid K562 cells, enhancement of a linked gamma-promoter was significantly greater with wild-type HS2 than with HS2(M). In contrast, the increase in beta-promoter activity in these cells was equivalent with either enhancer fragment. Truncation of the binding site for the fetal/erythroid-specific stage selector protein in the gamma-promoter abolished the additional enhancer activity of HS2. Similarly, insertion of the stage selector protein site into the beta-promoter boosted enhancer activity observed with HS2 but not HS2(M). In adult erythroid MEL cells, enhancement of a linked beta-promoter was significantly greater with HS2 than with HS2(M). This effect was dependent on the binding of the adult stage-specific factor, erythroid Kruppel-like factor, to the beta-promoter. Taken together, this data suggests that the stage-specific factors binding the proximal globin promoters and the factors flanking the AP1/NF-E2 motif of HS2 act in synergy.

Interaction between the stage selector element (SSE) in the proximal γ-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the β-globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5′-untranslated region of the γ-gene also contributes to this competitive silencing of the β-gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal γ-promoter in the absence of the SSE, completely silencing a linked β-promoter. Mutation of a 20-base pair sequence of the γ-gene 5′-untranslated region (UTR) led to derepression of β-promoter activity. A marked activation of γ-promoter activity was also observed with this mutation, suggesting the presence of a repressor. Fine mutagenesis dissected these activities to different regions of the 5′-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5′-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor GATA-1. These results indicate that the 5′-UTR of the γ-gene contains sequences that may be important for its transcriptional and developmental regulation.

Interaction between the stage selector element (SSE) in the proximal gamma-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the beta-globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5'-untranslated region of the gamma-gene also contributes to this competitive silencing of the beta-gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal gamma-promoter in the absence of the SSE, completely silencing a linked beta-promoter. Mutation of a 20-base pair sequence of the gamma-gene 5'-untranslated region (UTR) led to derepression of beta-promoter activity. A marked activation of gamma-promoter activity was also observed with this mutation, suggesting the presence of a repressor. Fine mutagenesis dissected these activities to different regions of the 5'-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5'-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor GATA-1. These results indicate that the 5'-UTR of the gamma-gene contains sequences that may be important for its transcriptional and developmental regulation.

We report serial transplantation procedures in 2 sets of brothers with X-linked primary immunodeficiency. The first boy in each family received a T-cell–depleted transplant from a mismatched donor. The recipients then acted as donors for T-replete transplantation of the “tolerized” graft into their HLA-identical brothers with the same disorder. Immune reconstitution was noted to occur at a significantly faster rate in the secondary recipients, and without the occurrence of graft-versus-host disease (GVHD), despite the presence of donor cells mismatched for 1 to 3 HLA antigens. This serial transplantation technique allows the primary recipient of HLA-mismatched donor cells to act as a functionally “HLA-matched” donor for subsequent affected siblings, and should be considered as a therapeutic option in families with congenital disorders.

Ghorashian: Celgene: Honoraria; novartis: Honoraria; UCLB: Patents & Royalties: UCLB. Kramer:UCLB: Patents & Royalties. Ciocarlie:Servier: Other: Financial Support. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Pule:Autolus: Employment, Equity Ownership, Patents & Royalties. Amrolia:UCLB: Patents & Royalties.

Karl S Peggs and Sarah J Albon contributed equally to the work and are joint first author

Ghorashian: novartis: Honoraria; UCLB: Patents & Royalties: UCLB; Celgene: Honoraria. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Thrasher:4BIOCapital: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Amrolia:UCLB: Patents & Royalties.

Introduction

Introduction

IntroductionCAR T-cell therapies directed against CD19 or CD22 antigens have shown significant activity in pediatric patients with r/r B-ALL. Whilst complete response (CR) rates of 70‒90% have been observed, relapse due to target antigen downregulation or loss is the major cause of treatment failure. This Phase I/II study evaluates the safety and efficacy of AUTO3, a CAR T-cell therapy designed to target CD19 and CD22 simultaneously in order to reduce the likelihood of relapse due to antigen loss.

Introduction

Introduction:

Universal gene-edited CAR19 T cells eliminate infant leukemia.

Qasim: Autolus: Consultancy, Equity Ownership, Research Funding; Cellectis: Research Funding; Calimmune: Research Funding; Catapult: Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.

Onuoha: Autolus Ltd: Employment, Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.

Introduction:

Qasim: CATAPULT: Research Funding; CELLMEDICA: Research Funding; CALIMMUNE: Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding; CELLECTIS: Research Funding. Off Label Use: UCART19 T Cells are an unlicensed investigational medicinal product and in this case were used under MHRA special licence arrangements. Stafford:CELLECTIS: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Thrasher:CATAPULT: Patents & Royalties, Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding. Pule:AUTOLUS: Employment, Equity Ownership, Research Funding; CELLECTIS: Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Pule: Cellectis: Martin Pule's laboratory receives funding for contract research from Cellectis Therapeutics Other.

In vivo T-cell-depletion generated by the use of Thymoglobulin (ATG) within the conditioning regimen might contribute to the delayed immune-reconstitution observed after unrelated umbilical cord blood transplantation (UCBT). We studied the impact of early, late and no ATG on immune reconstitution and clinical outcome.127 Children receiving an unrelated UCBT in London or Utrecht between 2006 and 2011 were included and divided in 3 groups: late ATG (day -5 to 0, n=48), early ATG (day -9 to -5, n= 33) and no ATG(n=46). The no ATG group received MMF and CsA as GVHD prophylaxis, while the ATG groups both received CSA and prednison as prohylaxis, Endpoints studied were survival, early and late immune-recovery, infections and GvHD. Subset analysis CD3+, CD4+ and CD4+naïve, B and NK cell numbers were prospectively measured during post-transplant follow-up at 1, 2, 3, 6 and 12 months post-UCBT.The probability of survival was not significantly different between the groups: 71% +/- 8% (no ATG), 68% +/- 9% (early ATG) and 61% +/- 7% (late ATG). There were no significant differences in engraftment and primary or secondary graft failure. Immune reconstitution as defined by CD3+, CD4+ and CD4+ naïve T-cell counts were significantly higher (p<0.001) in the no ATG group at 1, 2, 3, 6, and 12 months post-UCBT with a log more T cells than in the ATG groups. The median CD3+ T-cell count in the no ATG group at 1, 2, 3, 6 and 12 months post UCBT was 340 x10e6/L, 720 x 10e6/L, 535 x 10e6/L, 940 ± 194 x 10e6/L, and 1860 x 10e6/L respectively. In the no ATG group significantly less viral reactivations (p=0.021) were noted. A higher probability (p<0,001) of severe acute GvHD (31%+/-9%) was found in the no ATG group compared to 18% +/- 9% for the early ATG and 5% +/-4% for the late ATGgroup. This was not associated with a higher probability of chronic GvHD or non-infectious lung injury in the no ATG group.Cord blood transplantation without Thymoglobulin (ATG), in the context of MMF and CsA post-transplant prophylaxis, is associated with higher GVHD risk but with a surprisingly good immune reconstitution and with an engraftment and survival similar to the groups with ATG. The findings of improved immune-reconstitution, associated with lower viral reactivations, albeit at the cost of increased rates of acute GvHD (but not chronic GvHD), suggest that omitting ATG in cord blood transplantation may be important to prevent viral reactivations, especially in those at high risk for these.No relevant conflicts of interest to declare.

EBV associated lymphoproliferative disease (PTLD) remains a major cause of morbidity and mortality after stem cell (SCT) or solid organ (SOT) transplant. Adoptive transfer of ex vivo-derived EBV-specific cytotoxic T lymphocytes (EBV-CTL) to reconstitute immunity to the oncogenic virus EBV has been highly effective for both the prophylaxis and treatment of PTLD after SCT, where immunosuppression could be withdrawn before transfer of EBV-CTLs. In contrast, the application of this approach for the treatment of PTLD in SOT patients, although feasible, has been challenging with restricted expansion, persistence and efficacy of the adoptive transferred T cells. This difference is likely to reflect the need for the on-going immunosuppression to prevent graft-rejection post SOT which inhibits the virus-specific T cell responses. Our group has previously genetically engineered EBV-CTL to enable them to function in vitro in the presence of the calcineurin inhibitor Tacrolimus (FK506) through retroviral transfer of a calcineurin mutant (CNA12).To examine the ability of genetically engineered EBV-CTLs resistant to FK506 to control EBV+ B cell lymphoma progression in vivoin a xenogeneic mouse model in the presence of FK506.NOD/SCID/IL2rgnull (NSG) mice were inoculated subcutaneously with 5×106 EBV-transformed lymphoblastoid B cell lines (LCL) labelled with F-Luc to develop human EBV+ lymphoma. To evaluate in vivo antitumor activity, 5×106 CTLs retrovirally transduced with CNA12 or eGFP control CTLs were injected intravenously after 7 days in the presence or absence of FK506 (10mg/kg/day). Tumour growth was analysed using the Xenogen-IVIS system.Adoptive transfer of autologous CNA12 transduced CTLs induced EBV+ lymphoma regression in the presence of FK506, as assessed both by IVIS and tumour size, whereas FK506 treated mice receiving eGFP control CTLs had tumour progression (P<0.05). This resulted in significantly improved survival of mice treated with CNA12-CTL in the presence of FK506 than eGFP-CTLs treated animals (P<0.0001). CNA12 transduced EBV CTLs persisted longer and expanded more (P<0.0001) than eGFP-CTLs in peripheral blood of mice treated with FK506 demonstrating a selective growth advantage and enrichment of CTLs resistant to immunosuppression. Immunohistochemical staining showed that adoptively transferred CTLs home to the tumour and an increase of tumour infiltrating T cells in CNA12-CTL compared with eGFP-CTLs treated mice in the presence of FK506 was observed.Our results demonstrate that CNA12 modified EBV-CTL can induce regression of EBV-associated tumours in vivo in the face of on-going immunosuppression with FK506. Clinical application of this novel approach may enhance the efficacy of adoptive transfer of EBV-CTL in SOT patients developing PTLD without the need for reduction in immunosuppressive therapy.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

EBV-associated Post Transplant Lymphoproliferative Disease (PTLD) is a major complication of solid organ transplantation (SOT), arising from suppression of normal EBV-specific T-cell responses by immunosuppressive drugs, chiefly the calcineurin inhibitors Cyclosporin (CsA) and Tacrolimus (FK506). Previous studies in the SCT setting have established that adoptive immunotherapy with EBV-specific cytotoxic T-cells (CTL) is highly effective in preventing/treating PTLD, but results with this approach in the SOT setting have been less impressive, reflecting the ongoing need for immunosuppression to prevent rejection in SOT patients. We have addressed the critical issue of how to enable infused CTL to proliferate and function in the face of immunosuppression with calcineurin inhibitors. We report the generation of Calcineurin A (CnA) and Calcineurin B (CnB) mutants which render CTL resistant to CsA and FK506. Based on structural data, we designed 22 CnA mutants and 32 CnB mutants predicted to dephosphorylate NFAT but not to bind to FKBP12-FK506/cyclophilin-CsA. Using transient assays with an NFAT-luciferase reporter in PMA/ionomycin stimulated 293T cells, we identified 6 CnA/4 CnB mutants which gave >15% resistance to FK506, and 5 CnA/5CnB mutants giving resistance to CsA. 4 CnB mutants resulted in resistance to both FK506 and CsA. Transduction of Jurkat T-cells with retroviral vectors coding for these Cn mutants resulted in high level expression, as determined by Western blotting/flow cytometry. The ability of these mutants to confer resistance to FK506 and CsA in Jurkat cells was then determined by measuring IL-2 secretion in response to PMA/ionomycin stimulation, in the presence of increasing concentrations of immunosuppressive drugs. Resistance was found to correlate well with that seen in 293T cells. Significantly, mutants CnA12 and B30 enabled equivalent secretion of IL-2 to untransduced Jurkats without immunosuppression despite supra-therapeutic doses of FK506 (40ng/ml) and likewise CnA22 and B30 despite supra-therapeutic CsA (800ng/ml). Some mutants rendered Jurkat T-cells more responsive to stimulation with increased output of IL-2 in the absence of IS. Next, EBV-CTLs from normal donors were retrovirally transduced with the 3 Cn mutants shown to confer greatest resistance in the Jurkat IL-2 secretion assay. In the absence of Cn inhibitors, the proportion of transduced cells, as determined by eGFP positivity, remained constant throughout subsequent culture. In contrast, in the presence of CsA/FK506, the proportion of transduced cells increased from a mean of 60% to >90% after 3 stimulations with autologous LCLs, demonstrating a selective advantage for transduced CTL. More importantly, transduced CTL were able to proliferate in response to stimulation with autologous LCL in the presence of immunosuppression, whereas non-transduced cells did not expand with FK506 or CsA. As shown in Fig 1, CTLs transduced with CnB30 proliferated at a comparable rate in the face of either FK506 (mean 30-fold expansion after 3 stimulations) or CsA (mean 47-fold expansion) to untransduced CTL not exposed to Cn inhibitors (mean 50 fold). CTL transduced with CnA22 were resistant to CsA whilst remaining sensitive to FK506. Further, transduced CTL secreted normal levels of IFN-g following stimulation with LCL in the presence of either FK506 or CsA: (CnB30 transduced CTL mean 1485 pg/ml IFN-g without immunosuppression, 1563 pg/ml in 10 ng/ml FK506 and 1600 pg/ml in 200 ng/ml CsA). In contrast, IFN-g secretion from untransduced CTL was abrogated by these drugs (mean 1538 pg/ml IFN-g without immunosuppression and 0 pg/ml with either FK506 or CsA). In summary, we have developed a strategy that effectively renders EBV-CTLs resistant to either FK506 or CsA. Potentially this may be of significant benefit to SOT patients developing PTLD, by allowing effective immunotherapy with EBV CTL without the need for reduction of calcineurin inhibitors and the attendant risk of graft rejection. More broadly, this may represent a generic approach to enabling adoptively transferred T-cells function in the face of ongoing immunosuppression. Figure 1. Proliferation of CTL in the presence of Tacrolimus or Cyclosporin A. CnB30 allows proliferation in the presence of both calcineurin inhibitors, CnA22 allows proliferation only in CsA. Figure 1. Proliferation of CTL in the presence of Tacrolimus or Cyclosporin A. CnB30 allows proliferation in the presence of both calcineurin inhibitors, CnA22 allows proliferation only in CsA.

Immunotherapy with allodepleted donor T-cells generated using a CD25 immunotoxin improves immune reconstitution after haplo-SCT, but leukaemic relapse remains a problem. To develop a rational approach to refining allodepletion, we characterised the phenotype of proliferating alloreactive T cells flow cytometrically. CFSE-labelled T cells were co-cultured with HLA-mismatched dendritic cells and serial FACS analyses performed to determine expression of CD25, CD69, CD71, HLA-DR, OX40, ICOS, CD95, CD45RA, CCR7, IFNγ, TNFα, and IL–2. By staining each sample with CD3, CFSE and CD25 we were able to determine the phenotype of proliferating CD25 negative alloreactive T cells. In 5 donors, after a 3 day culture, CD25 was expressed in a mean of 83% (range 67–89%) of the proliferating (CFSE-dim) alloreactive T cells, confirming CD25 as an excellent target for allodepletion. 70% (39–81%) of the proliferating alloreactive CD25 –ve population expressed CD71, and 62% (50–74%) expressed CD45RA, identifying these as markers to target alloreactive T-cells that would persist after CD25 based allodepletion. To optimize our CD25 based allodepletion, we compared allodepletion using a CD25 immunotoxin or immunomagnetic beads in 6 HLA mismatched donor-recipient pairs. There was no significant difference between residual alloreactivity to host in primary and secondary MLRs and IFNγ ELISPOT assays. We then compared CD25 vs CD25/71 immunomagnetic depletion in 8 HLA-mismatched donor-recipient pairs. Rested allodepleted cells were restimulated with host or 3rd party stimulators in 2° proliferation or IFNγ ELISPOT assays. The median residual reactivity to host in IFNγ ELISPOT assays was significantly lower after combined CD25/71 than CD25 allodepletion (14.1% vs 54.6%, p < 0.05). Likewise, in 2° MLRs, CD25/71 depletion resulted in significantly lower residual proliferative response to host than CD25 depletion (median 4.8% of the response of unmanipulated PBMC vs 9.9%, p < 0.01). Third party responses after CD25/71 allodepletion were equivalent to unmanipulated PBMCs from the same donors in both assays. CD25/71 allodepletion also gave lower residual responses to host in both assays than combined CD25/45RA allodepletion. In IFNγ ELISPOT assays, anti-viral responses to CMV and EBV were preserved after combined CD25/71 allodepletion (CMV: unmanipulated 151 spots vs.CD25/71 123 spots, EBV unmanipulated 100 spots vs. CD25/71 142 spots).Similarly, the frequency of CD8 +CMV and EBV–specific T-cells assessed by pentamer analysis was not significantly reduced compared to unmanipulated PBMCs. We conclude that CD25/71 allodepletion will selectively delete 94% of the proliferating alloreactive T cells and enhances the depletion of alloreactivity compared with CD25-based methods. This strategy may facilitate immunotherapy with larger doses of allodepleted donor T-cells after haplo-SCT, enhancing graft versus leukaemia and antiviral effects.

Childhood leukaemia which is refractory to chemotherapy or which relapses early following stem cell transplantation (SCT) has a very poor prognosis, with few patients becoming long-term survivors. Establishing limited graft-versus-host disease (GVHD) in an attempt to exert a graft-versus-leukaemia (GVL) response in these patients remains the only curative option. Second SCT procedures and/or donor lymphocyte infusions (DLI) have achieved some success but the pace of acute leukaemia is often too fast and the occurrence of GVHD/GVL too unpredictable to achieve this goal. We postulated that the addition of alpha interferon (alphaIFN) might induce/augment and control GVHD/GVL in this group of patients and so improve outcome. Thirteen patients underwent initial SCT, for cALL CR2 (n=1), Philadelphia positive ALL CR1 (5), MDS/AML (2), refractory AML (2), and CML (3), with unrelated (9) or sibling donors (4). Eight patients subsequently had a frank haematological relapse and 4 patients a molecular relapse of their leukaemia at a median of 10 months (2–18 months) from 1st SCT. Six patients underwent a 2nd SCT from the same donor, all with reduced intensity conditioned regimes; all received alphaIFN when GVHD did not occur after early withdrawal of immunosuppression. One (ALL CR2) received DLI 2 months after 2nd SCT for mixed chimerism. A further six patients received DLI plus alphaIFN, 3/6 together with low dose DLI (<106/kg); 3/6 had previously received108/kg DLI alone without response. One patient with refractory AML received alphaIFN in conjunction with the initial transplant. The dose of alphaIFN was escalated from 3–5 megaunits/m2 subcutaneously 3–5 days per week as tolerated and continued in the absence of GVHD for up to one year. 3 patients (CML) received concomitant therapy with Glivec, which has now been stopped. Following therapy, 9/13 (69%) patients developed aGVHD: Grade I (1), grade II (4), grade III (4); and 7/11 evaluable patients cGVHD: limited (4), extensive (3). 8 of 13 (62%) remain well and in remission, a median of 23 months (range 12 to 100) from alphaIFN therapy. Five have died: 2 from relapse, one from lung GVHD, one from idiopathic pneumonitis and one from aspergillus infection. Two of 8 surviving patients have ongoing cGVHD requiring immunosuppressive therapy. This data shows promising results for the use of alphaIFN to augment GVL responses in patients with high-risk leukaemia not cured with standard transplant procedures with 62% disease free survival and minimal residual symptoms. Further analysis is warranted.

Children with severe organ toxicities, DNA repair disorders and infants <1 year tolerate existing conditioning regimens, including reduced intensity conditioning, poorly. To reduce TRM in such patients, we used a minimal intensity conditioning regimen utilising 2 rat anti-CD45 monoclonal antibodies (Mabs) YTH24.5/YTH 54.12 for myelosuppression and Alemtuzumab (anti-CD52) for immunosuppression. As CD45 is expressed selectively on haemopoietic cells, the Mabs provide immunosuppression and transient myeloablation without the systemic toxicity associated with chemotherapy. 16 children (7 SCID, 5 CID, 2 dyskeratosis congenita, 1 reticular dysgenesis, 1 HLH) were transplanted at a median age of 11 months (5 months-11 years). Inclusion criteria were primary immunodeficiency (PID) in children age <1 (n=9), severe organ toxicity (n=14) and DNA repair defects/radiation sensitivity (n=4). Donors were MSD (n=5), MUD (n=9) and MMUD (n=2). Severe organ toxicity was defined as

Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality in patients undergoing allogeneic stem cell transplantation (SCT). This is particularly true in patients transplanted using volunteer unrelated donors (VUDs) where despite improvements in antiviral therapy CMV positive serostatus remains an adverse prognostic indicator. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones reduces the rate of viral reactivation but the complexity of this approach severely limits its clinical application. We have previously reported the purification of CMV-specific CD8+ cytotoxic T lymphocytes (CMV-CTL) from the blood of stem cell transplant donors using HLA-peptide tetramers and their subsequent transfer. Six patients undergoing stem cell transplantation from HLA identical sibling donors were studied. CMV-CTL were infused on a pre-emptive basis in patients who developed evidence of CMV viremia. All patients showed prompt and durable CMV-specific immune reconstitution demonstrating durable CMV-specific immune reconstitution. CMV-CTL constituted up to 21% of the patient lymphocyte CD8+ T cell compartment and were detectable up to 24 months in all patients. There was a reduced requirement for anti-viral agents in treated patients (33%) compared with a control group (82%) p=0.11. No secondary reactivation was seen in any patient in the treatment group. No infusion-related toxicity was seen in any treated patients. We have now extended these studies to recipients of VUD transplants in whom no CMV-CTL are detectable within the first 100 days of stem cell infusion. Tetramer-selected CMV-CTL have been adoptively transferred into six patients undergoing transplantation from an unrelated donor. CMV-CTL were given either for treatment of refractory CMV infection (n=5) or prophylactically 21 days post SCT (n=1). A cell dose of between 6x102/kg and 1.25x105/kg was administered with median purity of 95% of the infused T cell dose. No infusional toxicity or acute graft-versus-host disease was noted. CMV-CTL were not present in any patient prior to therapy. Within 7 days of adoptive transfer CMV-CTL were detectable in all patients comprising 0.6% – 20% of CD8+ T cell compartment. All patients demonstrated a reduction in the viral load and 5/6 cleared their viremia. In the patient who received prophylactic therapy durable expansion of CMV-CTL were observed up to 20% of CD8+ T cell pool. Taken together these data are consistent with the hypothesis that antigen-specific CMV specific CTL can be safely infused to recipients of VUD SCT and expand directly in vivo. Such an approach has considerable potential in the treatment or prophylaxis of CMV infection in patients undergoing VUD SCT.

Primary hemophagocytic lymphohistiocytosis (HLH) is a disease characterized by hypercytokinaemia and T cell dysregulation, due to mutations of the perforin, SAP and other so far uncharacterized genes. Patients may achieve a remission with immunomodulation consisting of epipodophyllin/steroids +/− cyclosporin A, but the only curative option is allogeneic stem cell transplant (SCT). Patients often have significant pre-transplant morbidity with lung, liver and central nervous system disease and are hence at high risk of transplant related morbidity and mortality. Because HLH is caused by immune dysregulation, we hypothesized that a non-myeloablative conditioning regime might be sufficient for cure, by restoring normal immune regulation, while decreasing the transplant related morbidity and mortality. We report the outcome of 11 children with HLH transplanted with a reduced intensity regime (RI) from an unrelated (fully matched, n=4, 1 antigen-mismatched, n=2) or haploidentical (n=5) donor. Of 7 patients assessed, 1 patient had absent SAP expression and 2 had a perforin gene mutation. 8 patients had significant morbidity of at least one other organ (including lung, cardiac, liver and CNS) pre-SCT. All had previously received HLH 94 protocol: 7/11 were in CR at the time of SCT and 4/11 in good PR. The median age at transplant was 16 months (range 3 –151 months). SCT was performed at a median of 7 months from diagnosis (3–14 months). Patients were conditioned with fludarabine 30mg/kg x 5 and melphalan 125 to 140mg/m2. Unrelated donor transplants received serotherapy with Campath 1H 0.2mg/kg x 5 and haplos received busulphan 4mg/kg x 2 and ATG 5mg/kg x 5. One unrelated donor recipient received TBI 2Gy single fraction with fludarabine. Results: All patients engrafted at a median of 13 days post SCT. 8 of 11 (73%) children are alive and in CR a median of 19 months from transplant (range 5 to 61 months). There were 3 transplant related mortalities (2 UD, 1 Haplo) between 3 and 4 months post-SCT from CMV pneumonitis (n=1), multifactorial pneumonitis following previous HLH lung disease (n=1), and hepatic rupture post- transjugular liver biopsy (n=1). 4 patients had CMV reactivation and 2 EBV reactivation. 3 patients developed acute GVHD (2 Grade 2 skin/gut, 1 Grade 1 gut/liver) and 1 had an engraftment syndrome with T-cell sequestration in the lungs. 3/8 evaluable patients have developed limited chronic skin GVHD 6– 15 months post-SCT. 3 patients had early and continuing mixed chimerism with 20%, 84% and 82% donor cells in the peripheral blood at 61, 52 and 24 months respectively from transplant. At a median follow-up of 19 months, no patient has relapsed, including the 3 patients with mixed chimerism. In summary, mixed chimerism appears curative for HLH and our results with SCT using reduced intensity conditioning compare favourably to those seen with conventional BMT, where a 3 year EFS of 62% has been reported in the HLH94 study. We believe this approach may be of major benefit in those patients with severe pre-SCT organ toxicity and merits further study in standard risk patients.