Effect Of Donor Lymphocyte Infusion Research Articles

Detecting T-cell clonal expansions and quantifying clone survival using deep profiling of immune repertoires.

An individual's T-cell repertoire constantly changes under the influence of external and internal factors. Cells that do not receive a stimulatory signal die, while those that encounter and recognize a pathogen or receive a co-stimulatory signal divide, resulting in clonal expansions. T-cell clones can be traced by monitoring the presence of their unique T-cell receptor (TCR) sequence, which is assembled de novo through a process known as V(D)J rearrangement. Tracking T cells can provide valuable insights into the survival of cells after hematopoietic stem cell transplantation (HSCT) or cancer treatment response and can indicate the induction of protective immunity by vaccination. In this study, we report a bioinformatic method for quantifying the T-cell repertoire dynamics from TCR sequencing data. We demonstrate its utility by measuring the T-cell repertoire stability in healthy donors, by quantifying the effect of donor lymphocyte infusion (DLI), and by tracking the fate of the different T-cell subsets in HSCT patients and the expansion of pathogen-specific clones in vaccinated individuals.

Donor Lymphocyte Infusion and Molecular Monitoring for Relapsed Myelofibrosis After Hematopoietic Cell Transplantation

Hematopoietic cell transplantation (HCT) is a curative approach for myelofibrosis patients, but relapse is a major cause of treatment failure. We investigated the effect of donor lymphocyte infusion (DLI) in 37 patients with molecular (n = 17) or hematological relapse (n = 20) after HCT. Patients received median of 2 (range, 1–5) cumulative DLI (total of 91 infusions). Median starting dose was 1 × 106 cells/kg, escalated by half-log ≥6 weeks if no response nor graft-versus-host disease (GvHD) occurred. Median time to first DLI was 40 weeks for molecular relapse versus 145 weeks for hematological relapse. Overall molecular complete response (mCR) at any time was 73% (n = 27) and was significantly higher for initial molecular relapse (88%) versus hematological relapse (60%; P = 0.05). The 6-year overall survival was 77% versus 32% (P = 0.03). Acute GvHD 2–4 occurred in 22% and half of the patients achieved mCR without any GvHD. All patients who relapsed from mCR achieved after first DLI could be salvaged with subsequent DLI, showing long-term survival. No second HCT was needed for molecular relapse versus 6 for hematological relapse. This comprehensive and largest study to date suggests molecular monitoring together with DLI as standard of care and a crucial approach to achieve excellent outcomes in relapsed myelofibrosis.

Targeting Molecular Remission with Donor Lymphocyte Infusions Shows Excellent Outcomes for Persistent or Relapsed Myelofibrosis after Stem Cell Transplantation

About 10-30% of myelofibrosis patients who undergo allogeneic hematopoietic cell transplantation will eventually experience relapse within 5 years. While significant improvement has been achieved in stratifying patients according to their risk for posttransplant mortality, preventing and treating relapse remains an unmet clinical need. Notably, no standard of care exists for treatment of relapsed myelofibrosis. On option in the transplant setting (mainly established in CML and AML) is transferring donor T cells to reinforce a graft-versus-leukemia (GVL) effect targeting the recurring malignant cells. Previous small sample studies also indicated a strong graft-versus-myelofibrosis effect of donor lymphocyte infusion (DLI). Additionally, molecular monitoring posttransplant may further help to target outcomes and identify cohorts with distinct risk for and outcome after relapse receiving DLI. Here, we evaluated the largest cohort to date of myelofibrosis patients receiving DLI. The aim of this study was to induce molecular complete remission (CR) and to evaluate associations with overall outcomes. Patients were eligible to receive DLI if they were free of GVHD and immunosuppressive therapy. If no GVHD or response occurred within 6 weeks, another escalating dose (half-log) of DLI was administered. Highly sensitive chimerism and molecular monitoring with developed driver mutation-specific PCRs for JAK2, CALR and MPL was done every month (until day 360 posttranspant) and with routine visites thereafter We present results of 57 genetically annotated myelofibrosis patients who received donor lymphocyte infusion (DLI) for persistent disease (n=20), molecular relapse (n=17), or hematologic relapse (n=20), aiming to achieve molecular complete remission (CR). A total of 143 DLI procedures were given (range, 1-5 cumulative DLIs). Median CD3+ starting dose of first DLI was 1 x106 (range, 1 x105-1 x107), escalated by half-log after at least 6 weeks if no response nor graft-versus-host disease occurred. Overall molecular CR rate was 70% (n=38) and seemed to be higher for patients who received first DLI for molecular relapse (88%, 15 out of 17). For each DLI, molecular CR was achieved in 40% (23 out of 57) after first, in 44% (17 out of 39) after second, in 32% (8 out of 25) after third, in 470% (7 out of 15) after fourth, and in 43% (3 out of 7) after fifth DLI. In terms of acute GVHD, 15 overall cases occurred after a total number of 143 procedures (11%) and appeared to be associated with response. Of 22 patients who achieved initial molecular CR after first DLI, 11 patients (50%) received subsequent DLIs, of whom 4 patients initially received first DLI for hematological relapse, 4 patients for molecular relapse, and 3 patients for disease persistence. Relapses after first DLI were hematological in 8 patients (73%) and molecular in 3 patients (27%), and second DLI was given within a median of 10 weeks (range, 6-323 weeks) from first DLI. Of those, 9 patients could be eventually converted with subsequent DLIs into durable molecular CR (82%) with a median of 7 years after last DLI. Overall outcome was driven by response to first DLI. Five-year overall survival after first DLI was 85% (95% CI, 67-100%) for patients with molecular CR versus 52% (95% CI, 33-71%) for patients with less than molecular CR. Achievement of molecular CR after first DLI was associated with 78% reduced risk of death (P=0.02). All patients who relapsed after initial molecular CR after first DLI (50%, n=11) were alive at last follow-up. Multivariable analysis on overall survival and disease-free survival, adjusting for time-dependent and continuous effects of total number of DLIs, showed significantly reduced risk of death for patients with molecular CR after first DLI (with patients achieving less than molecular CR as reference), with hazard ratio of 0.22 (95% CI, 0.06-0.80; P=0.02). Similar results were retrieved for risk of death or relapse/disease persistence after last DLI, with a hazard ratio of 0.14 (95% CI, 0.05-0.39; P<0.001). No significant effect of number of DLIs or indication for first DLI were identified. Targeting molecular CR after DLI is essential to achieve excellent long-term survival in persistent or relapsed myelofibrosis, and DLI together with molecular monitoring should be standard of care for relapsed myelofibrosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effect of Donor Lymphocyte Infusion From Two Types of Donors on Mixed Chimerism With Secondary Graft Failure After Allogeneic Hematopoietic Stem Cell Transplantation

Mixed chimerism (MC) and secondary graft failure (SGF) with recipient- or donor-type chimerism is a major obstacle in allogeneic hematopoietic stem cell transplantation (HSCT). Donor lymphocyte infusion (DLI) can eradicate minimal residual disease or be used to rescue a hematologic relapse, being able to induce durable remissions after HSCT. This study aimed to analyze the efficacy and immune mechanism of DLI from the original and alternative donor for patients of mixed donor chimerism with SGF. The alternative donor refers to the candidate relative donor who did not initially provide stem cells and includes HLA-matched sibling donor or HLA-haploidentical donor. We conducted a retrospective study of 246 patients with a median age of 37 (9-58) years who had regularly detected MC, complete donor chimera (CC), and regulatory T cells (Treg). The median diagnosis time of SGF was 69 (39-141) days after transplantation. Sixteen patients with SGF received DLI from the alternative donor, including 3 patients who chose DLI from the original donor with no initial response and 13 patients who directly chose DLI from the alternative donor. Sixteen patients with SGF existed mixed chimerism synchronously and the rate calculated overall chimerism of MC was 63% (range 42%-85%) after transplantation. The proportion of Treg decreased significantly in SGF patients from a median of 2.66% ± 0.80% to 0.93% ± 0.57% at a time point after transplantation (P = .02). The DLI of the alternative donor in 14 patients achieved complete response and MC gradually convert to CC state, simultaneously there was significant increase in the Treg fraction (SGF versus complete response: 0.93% ± 0.57% versus 3.61% ± 0.82% [P = .01]). For the clinical nonresponders from 2 types of donors, there was no significant change in MC and Treg cells. The OS and disease-free survival at 2 years after DLI were 69.7% ± 3.19% and 61.3% ± 4.80%, respectively. DLI from the alternative donor may be an effective treatment for MC with SGF, and the mechanism is closely related to the activation of Treg cells level.

Differential effects of donor lymphocyte infusion upon treatment response and GVHD according to relapse level and donor sources in patients with myelodysplastic syndrome.

Introduction:Donor lymphocyte infusion (DLI) is one of the effective options for post-transplant disease control of myelodysplastic syndrome (MDS). Its success or failure depends on the induction of antitumor immune reactions, durability of clinical responses, and severity of unwanted toxicities mainly from graft-versus-host disease (GVHD).Methods:By analyzing 61 patients receiving DLI for post-transplant MDS relapse, we assessed treatment outcomes and affecting factors, especially focusing on the level of relapse (hematological, molecular, and imminent relapse).Results:The response rate (42.1%, 36.4%, 72.7%), and overall survival (OS) at 2 years (27.8%, 45.5%, 70.1%) were different for each relapse level with imminent relapse group showing the most promising results. For OS, response to DLI or pre-DLI chemotherapy, and time to relapse were independent prognostic factors. Meanwhile, post-DLI GVHD and time to relapse were independently predictive for DLI response; post-DLI GVHD was predictive for DLI response, but not for OS, suggesting a potential detrimental impact of GVHD on survival. The incidence of GVHD and GVHD-related deaths were 37.7% and 10.0%, respectively, and CD3+ cell doses triggering GVHD tended to be lower in cases with haploidentical donor or imminent relapse.Conclusion:Despite being limited by small number of cases and its retrospective nature, this study again demonstrated the therapeutic effects of DLI in relapsed MDS, and that earlier detection and intervention at lower level relapse might possibly be associated with better results. Furthermore, we propose that tailored cell dosing schedule based on relapse level and donor source may be helpful in minimizing fatal GVHD.

Comparison of Outcomes of Donor Lymphocyte Infusions with or without Lenalidomide in Patients with Hematological Malignancies Post Allogeneic Transplant

Comparison of Outcomes of Donor Lymphocyte Infusions with or without Lenalidomide in Patients with Hematological Malignancies Post Allogeneic Transplant

Outcomes and Impact of Donor Lymphocyte Infusion after Reduced Intensity Conditioned-Alemtuzumab Allogeneic Hematopoietic Stem Cell Transplantation for Patients with Mature Lymphoid Malignancies

Allogeneic hematopoetic stem cell transplantation (allo-HSCT) offers long-term remission for patients with lymphoid malignancies. In particular, reduced intensity conditioned (RIC) protocols with Alemtuzumab T-cell depletion lead to durable engraftment and reduced graft versus host disease (GvHD). However, such regimens may be associated with reduced graft-versus-lymphoma (GvL) effect and higher incidence of disease progression, leading to increased use of donor lymphocyte infusion (DLI). This study examined transplant outcomes and use of DLI in a large UK RIC-Alemtuzumab conditioned HSCT cohort.288 consecutive adult patients from three UK transplant centres (Birmingham, Glasgow, Oxford) undergoing first Alemtuzumab-based RIC HSCT for Hodgkin's lymphoma (57%), Non-Hodgkin's lymphoma (24%) and chronic lymphocytic leukemia (19%) between 2000 and 2012 were included. Overall survival (OS), current progression-free survival (CPFS), relapse incidence (RI), transplant-related mortality (TRM), incidence of GvHD and effects of DLI were analyzed.Median age was 49y (range 17-68) and 60% were male. Patients were heavily pre-treated with 58% having received ≥3 prior lines of treatment and 49% having failed a previous autograft. 55% received stem cells from an HLA-matched unrelated donor and 94% received a peripheral blood stem cell harvest. The most frequent conditioning regimen was Fludarabine, Melphalan and Alemtuzumab (FMC) (70%) and the median total Alemtuzumab dose was 50mg (range 30-100). Median duration of follow-up for survivors was 64 months.3y OS and CPFS were 53% and 50%, respectively. Factors negatively associated with OS in univariate (UV) analysis were previous autograft (p=0.01), no requirement for DLI (p=0.05), disease status not CR (p=0.05), previous treatment number ≥3 (p=0.007) and failure to engraft plts (p=0.001). On multivariate (MV) analysis, only previous autograft (HR 1.58 p=0.01) and failure to engraft plts (HR 2.3 p=0.0001) retained significance. Factors negatively associated with CPFS on UV analysis were no requirement for DLI (p=0.006), failure to engraft plts (p=0.0003) and previous treatment lines ≥3 (p=0.04). On MV analysis, no DLI remained a significant predictor of adverse CPFS (HR 1.8 p=0.01), as did failure to engraft plts (HR 1.77 p=0.005) and ≥3 treatment lines (HR 1.46 p=0.04).At 3y, the cumulative incidence of relapse and TRM was both 27%. The incidence of grade 2-4 acute GvHD (aGvHD) and extensive chronic GvHD (cGvHD) was 22% and 18%, respectively. UV predictors of RI were unrelated donor (p=0.0009), HLA mismatch (p=0.04), age > median (p=0.04) and lack of aGvHD (p=0.04). The presence of cGvHD may have been protective against relapse (p=0.08). No variables retained significance on MV modelling. UV predictors of TRM were failure to engraft plts (p=0.001), HLA mismatch (p=0.005), age > median (p=0.004), previous autograft (p=0.02), FMC conditioning (p=0.03), ≥3 previous lines of treatment (p=0.001) and development of aGvHD (p=0.04). On MV modelling, failure to engraft plt (p=0.001), HLA mismatch (p=0.03), age > median (p=0.003) and treatment number ≥3 (p=0.04) retained significance.62/288 patients received DLI; 37 receiving prophylactic DLI (pDLI) for mixed chimerism and 25 receiving therapeutic DLI (tDLI) for disease relapse. The median dose of DLI was 1x 10^6 CD3+ cells/kg (range 0.1 - 10 x 10^6). Median time from HSCT to first DLI was 10.5 months (range 3-30), to 2nd DLI 15 months (6-51) and to 3rd DLI 19 months (9-30). At 3y, OS after pDLI was 75% and after tDLI was 29%. 29/37 (78%) of pDLI and 14/25 (56%) of tDLI recipients achieved full or stable mixed chimerism. 3y RI after pDLI was 29% and after tDLI was 59% (p=0.02). Overall, the incidence of GvHD post-DLI was 32% (48% tDLI, 22% pDLI, p=ns). Median time to GvHD after last DLI was 46 days (range 17-249). There was no significant impact of post-DLI GvHD on RI (RI at 1y post DLI 18% in GvHD cohort vs 21% with no GvHD, p=ns), but GvHD increased TRM at 1y post DLI (17% in the GvHD cohort vs 3% with no GvHD, p=0.05).This is the largest series to date with long-term follow-up examining outcomes after Alemtuzumab-based RIC HSCT for mature lymphoid malignancy. Our data indicate that allo-HSCT is effective and is associated with good long term outcomes. Use of pDLI for was associated with excellent outcomes and supports use of DLI in patients developing mixed chimerism following allo-HSCT for lymphoid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Allogeneic haematopoietic stem cell transplantation in myelodysplastic syndromes.

Allogeneic haematopoietic stem cell transplantation remains the only curative treatment for myelodysplastic syndrome. We highlight the various issues to consider in the pretransplant, transplant and posttransplant periods with emphasis on the management of relapse following transplant. Cytogenetic and molecular characteristics are becoming more important in predicting transplant outcome. Hypomethylating agents are effective in the pretransplant setting to reduce disease burden. Haploidentical and umbilical cord blood donations may be valid transplant options for patients without human leukocyte antigen-identical sibling or match unrelated donor options. A preemptive management approach to patients at high risk of relapse is more effective. Adjusting the timing and dose of donor lymphocyte infusion reduces the risk of graft-versus-host disease without jeopardizing the graft-versus-leukaemia effect of donor lymphocyte infusion. Allogeneic haematopoietic stem cell transplantation is curative in up to 40% of myelodysplastic syndrome patients. Appropriate patient selection, modification of conditioning regimes and donor selection should be considered carefully. A preemptive approach for the management of patients at high risk of relapse should be employed following transplant, with the use of immune modulating therapies such as donor lymphocyte infusion and azacitidine.

CD4+ T Cell–Depleted Lymphocyte Infusion Impairs Neither the Recovery of Recipient Thymus nor the Development of Transplanted Thymus

Thymus transplantation, in conjunction with bone marrow transplantation (BMT), has been attracting attention for the treatment of various diseases. Recently, donor lymphocyte infusion (DLI) has been used as a helpful tool for establishing donor chimerism and preventing a relapse of leukemia/lymphoma. However, the effects of DLI on transplanted and recipient thymuses have not been explored. We therefore performed DLI in the intrabone marrow-BMT + thymus transplantation setting. We have found that DLI leads to derangements in both recipient thymuses and transplanted thymuses; by 2 wk after BMT, we saw a decrease in total cell number, a lower percentage of CD4(+)CD8(+) cells, and the obliteration of the thymic corticomedullary junction. Four weeks later, the thymic impairment became more serious. However, when we depleted the CD4(+) T cells (CD4(-)-DLI), the recipient thymic recovery and transplanted thymic development were significantly restored by the treatment. In addition, there were much greater levels of TNF-α and Fas ligand, and a lower percentage of regulatory T cells in the DLI group than in the CD4(-)-DLI group. These findings indicate that inflammation induced by DLI, especially by CD4(+) T cells, plays a crucial role in the thymic impairment.

Depletion of T regulatory cells through selection of CD127-positive cells results in a population enriched in memory T cells: implications for anti-tumor cell therapy

Donor lymphocyte infusions can induce remissions in patients with relapse after allogeneic hematopoietic stem cell transplantation. Nevertheless, some grafted patients never display any signs of alloreactivity, either following allogeneic hematopoietic stem cell transplantation or after donor lymphocyte infusions. Consequently, they do not develop graft-versus-host disease and frequently do not respond to donor lymphocyte infusions. In a recently published clinical trial, we observed that elimination of CD4(+)CD25(+)Foxp3(+) natural regulatory T cells from the donor lymphocyte product could improve alloreactivity and the associated anti-tumor effect in a small proportion of patients with relapsed hematologic malignancies. Here, we aimed to improve the effect of donor lymphocyte infusion by modifying the procedure for depletion of T regulatory cells. We directly compared depletion of regulatory T cells from human peripheral blood mononuclear cells achieved by selection of CD127-positive cells or by selection of CD25-negative cells. We tested the manipulated products (i) in vitro in mixed lymphocyte reactions and against pathogen-derived recall antigens and (ii) in vivo in experimental graft-versus-host disease. In vitro, we found that depletion of regulatory T cells through CD127 positive selection improved both alloreactive and pathogen-specific immune responses. In vivo, we observed accelerated donor T-cell division and enhanced graft-versus-host disease due to efficient regulatory T-cell depletion accompanied by enrichment in memory T cells. Our results show that the strategy of CD127 positive selection is an efficient way of eliminating regulatory T cells from donor lymphocyte infusions and improves alloreactivity. This supports the investigation of CD127 positive selection in place of elimination of CD25-positive cells for clinical applications.

Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity

Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft-versus-leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft-versus-host disease. We analyzed in detail CD8(+) T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft-versus-host disease of the skin. CD8(+) T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens. Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones. Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft-versus-leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.

Novel Therapy with Interferon-α in Combination with Donor Lymphocyte Infusion for High Risk Acute Leukemia Patients Who Relapsed After Allogeneic Hematopoietic Stem Cell Transplantation

Abstract 658 BackgroundRelapse remains a major cause of failure for allogeneic stem cell transplantation (allo-HSCT). Donor lymphocyte infusion (DLI) is routinely employed as salvage for patients who relapsed after allo-HSCT. In order to improve the graft versus leukemia (GVL) effect of DLI, we investigated the efficacy and safety of combining interferon-α with DLI (aDLI) in patients with high risk acute leukemia who relapsed after allo-HSCT, and compared the efficacy, toxicity and leukemia free survival (LFS) of aDLI and traditional donor lymphocyte infusion(tDLI)in our transplantation center. MethodsSixteen acute leukemia patients were enrolled in this study. All patients were treated with interferon-α-2b therapy at a dose of 3×106U/day subcutaneously for 5 days followed by G-CSF mobilized donor peripheral stem cell infusion. IFN-α treatment continued until complete remission or development of graft-versus-host disease (GVHD). We coined the term “aDLI” for this novel treatment protocol. The median duration of interferon-α treatment was 17 (range, 5–50) days, and the median CD3+ cells dose was 9.25×107/kg(range, 4–20×107/kg)of recipient weight. Bone marrow smear, real-time PCR, STR-PCR,FISH,cytogenetic analysis and flow cytometry were used to monitor engraftment and minimal residual disease. In parallel, we retrospectively analyzed the results of tDLI for 14 acute leukemia patients with hematological relapse post allo-HSCT treated in the same period in our transplantation center, and compared the efficacy, toxicity and LFS of tDLI with aDLI. ResultsPatients treated on the aDLI protocol included 9 ALL and 7 AML, with a median age of 26.5 years. Fourteen of 16 patients had high risk acute leukemia: 3 patients with Ph+ ALL, 6 patients relapsed after CR1, 4 patients had complicated chromosome abnormality, and 1 patient had testicular leukemia. The median relapse time was 5.5 (range, 1–25) months post transplant. Salvage chemotherapy was administrated in 7 patients before aDLI, with only 3 patients achieved CR. The overall complete remission (CR) rate for aDLI protocol was 75% (12/16), with CR rate of aDLI alone without chemotherapy at 66.7% (6/9). The median time from aDLI to bone marrow CR was 7(range, 6–14)days, and the median time to molecular complete remission (MCR) and full donor chemerism (median level was 96.3%) in responsive patients was 2 weeks post DLI. With a median follow-up of 5.5 (range, 1–34) months, 7 patients were alive with durable MCR. Two-year LFS was 50%. Treatment related toxicities included reversible episode of fever, GVHD and myelosuppression. The tDLI group had similar demographic characteristics with respect to age, disease subtypes, transplant and relapse history. Compared to tDLI, the aDLI protocol had higher CR rate (75.0% vs 14.3%, p =0.001), faster response (median time to obtain BM CR and full donor chimerism were 7 days vs 23 days, and 30 days vs 60 days respectively), higher incidence of acute GVHD (56.3 % vs 27.3%, p=0.05), and significant better 2-year LFS(50.0% vs 7.1%,p=0.05. Importantly, there was no significant difference between the two groups with respect to the incidence of pancytopenia (53.8% vs 75%, p>0.05) and treatment related mortality(18.8% vs 7.1%, p>0.05). ConclusionInterferon-α-2b in combination with donor lymphocyte infusion appears to induce rapid and durable remissions in high risk acute leukemia patients who relapsed following allo-HSCT, with acceptable treatment-related toxicity. This novel adoptive immunotherapeutic strategy warrants additional studies in allo-HSCT settings Disclosures:No relevant conflicts of interest to declare.

Donor-Lymphocyte Infusion in Patients With Persistent or Recurrent Multiple Myeloma After Allogeneic Hematopoietic Stem Cell Transplantation

The graft-versus-leukemia (GVL) effect of donor-lymphocyte infusions (DLI) is well documented in hematologic malignancies, including multiple myeloma (MM). We evaluated the role of DLI in persistent or recurrent MM after allogeneic hematopoietic stem cell transplantation (allo HCT).

CD4 + CD25 + Regulatory T Cell Depletion Improves the Graft-Versus-Tumor Effect of Donor Lymphocytes After Allogeneic Hematopoietic Stem Cell Transplantation

Donor T cells play a pivotal role in the graft-versus-tumor effect after allogeneic hematopoietic stem cell transplantation. Regulatory T cells (T(reg)s) may reduce alloreactivity, the major component of the graft-versus-tumor effect. In the setting of donor lymphocyte infusion after hematopoietic stem cell transplantation, we postulated that T(reg) depletion could improve alloreactivity and likewise the graft-versus-tumor effect of donor T cells. The safety and efficacy of T(reg)-depleted donor lymphocyte infusion was studied in 17 adult patients with malignancy relapse after hematopoietic stem cell transplantation. All but one had previously failed to respond to at least one standard donor lymphocyte infusion, and none had experienced graft-versus-host disease. Two of the 17 patients developed graft-versus-host disease after their first T(reg)-depleted donor lymphocyte infusion and experienced a long-term remission of their malignancy. Four of the 15 patients who did not respond after a first T(reg)-depleted donor lymphocyte infusion received a second T(reg)-depleted donor lymphocyte infusion combined with lymphodepleting chemotherapy aimed to also eliminate recipient T(reg)s. All four developed acute-like graft-versus-host disease that was associated with a partial or complete and durable remission. In the whole cohort, graft-versus-host disease induction through T(reg) depletion was associated with improved survival. These results suggest that T(reg)-depleted donor lymphocyte infusion is a safe, feasible approach that induces graft-versus-host or graft-versus-tumor effects in alloreactivity-resistant patients. In patients not responding to this approach, the combination of chemotherapy-induced lymphodepletion of the recipient synergizes with the effect of T(reg)-depleted donor lymphocyte infusion. These findings offer a rational therapeutic approach for cancer cellular immunotherapy.

Human T Cells with Distinct Specificities Mediate Graft-Versus-Leukemia Reactivity and Xenogeneic Graft-Versus-Host Disease in a NOD/Scid Mouse Model for Human Acute Leukemia.

Abstract 1330Poster Board I-352Immunodeficient mice inoculated with human leukemia can be used as a model to investigate Graft-versus-Leukemia (GvL) effects of donor lymphocyte infusions (DLIs). In addition to GvL reactivity, treatment with DLI induces xenogeneic Graft-versus-Host Disease (GvHD) in mice, characterized by pancytopenia and weight loss. In patients treated with DLI for relapsed or residual leukemia after allogeneic stem cell transplantation, immune responses against non-leukemic cells may also cause GvHD. It has been suggested that GvL reactivity and GvHD, which co-develop in vivo, can be separated and that distinct T cells exist with the specific capacity to mediate GvL reactivity or GvHD. Since adoptive T cell transfer models that allow analysis of separation of GvL and GvHD are rare, we aimed to establish whether GvL reactivity and xenogeneic GvHD could be separated using our model of human leukemia-engrafted NOD/scid mouse after treatment with human donor T cells. In this study, non-conditioned NOD/scid mice engrafted with primary human acute lymphoblastic leukemic cells were treated with CD3+ DLI. Established tumors were effectively eliminated by emerging human T cells, but also induced xenogeneic GvHD. Flowcytometric analysis demonstrated that the majority of emerging CD8+ and CD4+ T cells were activated (HLA-DR+) and expressed an effector memory phenotype (CD45RA-CD45RO+CCR7-). To investigate whether GvL reactivity and xenogeneic GvHD were mediated by the same T cells showing reactivity against both human leukemic and murine cells, or displaying distinct reactivity against human leukemic and murine cells, we clonally isolated and characterized the T cells during the GvL response and xenogeneic GvHD. T cell clones were analyzed for reactivity against primary human leukemic cells and primary NOD/scid hematopoietic (BM and spleen cells) and non-hematopoietic (skin fibroblasts) cells in IFN-g ELISA. Isolated CD8+ and CD4+ T cell clones were shown to recognize either human leukemic or murine cells, indicating that GvL response and xenogeneic GvHD were mediated by different human T cells. Flowcytometric analysis demonstrated that all BM and spleen cells expressed MHC class I, whereas only 1-3 % of the cells were MHC class II +. Primary skin fibroblasts displayed low MHC class I and completely lacked MHC class II expression. Xeno-reactive CD8+ T cell clones were shown to recognize all MHC class I + target cells and xeno-reactive CD4+ T cells clones displayed reactivity only against MHC class II + target cells. To determine the MHC restriction of xeno-reactive T cell clones, NOD/scid bone marrow (BM) derived dendritic cells (DC) expressing high levels of murine MHC class I and class II were tested for T cell recognition in the presence or absence of murine MHC class I and class II monoclonal antibodies in IFN-g ELISA. Xeno-reactive CD8+ T cell clones were shown to be MHC class I (H-2Kd or H-2Db) restricted, whereas xeno-reactive CD4+ T cell clones were MHC class II (I-Ag7) restricted, indicating that xeno-reactivity reflects genuine human T cell response directed against allo-antigens present on murine cells. Despite production of high levels of IFN-gamma, xeno-reactive CD8+ and CD4+ T cell clones failed to exert cytolytic activity against murine DC, as determined in a 51Cr-release cytotoxicity assay. Absence of cytolysis by CD8+ T cell clones, which are generally considered as potent effector cells, may be explained by low avidity interaction between human T cells and murine DC, since flowcytometric analysis revealed sub-optimal activation of T cells as measured by CD137 expression and T cell receptor downregulation upon co-culture with murine DC, and therefore these results indicate that xenogeneic GvHD in this model is likely to be mediated by cytokines. In conclusion, in leukemia-engrafted NOD/scid mice treated with CD3+ DLI, we show that GvL reactivity and xenogeneic GvHD are mediated by separate human T cells with distinct specificities. All xeno-reactive T cell clones showed genuine recognition of MHC class I or class II associated allo-antigens on murine cells similar as GvHD-inducing human T cells. These data suggest that our NOD/scid mouse model of human acute leukemia may be valuable for studying the effectiveness and specificity of selectively enriched or depleted T cells for adoptive immunotherapy. Disclosures:No relevant conflicts of interest to declare.

Quantitation of WT1 Transcripts by Copy Number Provides a Sensitive Indication of Minimal Residual Disease (MRD) and Response to Adoptive Cell Therapy in Patients Transplanted for Ph(+) CML.

Abstract 4489▪The Wilm's tumor gene, WT-1, is markedly overexpressed in a high proportion of acute leukemias, advanced MDS and Ph(+) CML. Prior studies, employing semiquantitative PCR-based techniques, have suggested that elevated levels of WT-1 transcripts in bone marrow and PBMC are indicative of MRD, and predictive of relapse in AML and CML following chemotherapy. We have now developed a quantitative real time PCR-based assay for WT-1 transcripts and used it to evaluate the long term effects of donor lymphocyte infusions in a series of patients with Ph(+) CML treated for relapse or persistent MRD following allogeneic marrow transplants between 1993 and 2004 who are currently in molecular or cytogenetic remission.To quantitate WT-1 mRNA copy number in the marrow and PBMC, total RNA from patient samples received at different times that had been extracted with trizol reagent and stored at -20°C was transcribed into cDNA using MMLV reverse transcriptase. Real time PCR was performed with 1-2 μg of total RNA using the ABI 7300 instrument with a 25 base pair FAM labeled probe containing a sequence located at the boundary of exon 6 and 7 of the WT-1 gene in order to eliminate amplification of genomic DNA and insure measurement of all transcribed WT-1 isoforms. The forward and reverse primers were synthesized from sequences contained in exon 6 and 7 of the WT-1 gene respectively. Cloned plasmids containing defined copy numbers of WT-1 and human GAPDH were used to prepare standard curves using serial dilutions. Quantitation of WT-1 copy number expression was performed by calibration against these standard curves. The expression of WT-1 was normalized against the expression of the control GAPDH to adjust for variation in RNA and cDNA synthesis.Using this assay, we evaluated sequential samples from each patient for correlations between WT-1 copy number detected and the results of qualitative PCR assays for bcr/abl fusion gene transcripts previously performed on the same samples using nested primers as well as the results of clinical, hematologic and cytogenetic analyses of each patient obtained at each time of sampling. Blood samples from a group of 8 normal volunteers served as negative controls.The average WT-1 copy number in the blood of normal volunteers was 25 copies/ μg RNA (range 2-60). With one exception, blood and marrow samples from patients with CML in molecular remission (bcr/abl not detected) had WT-1 copy numbers < 50 copies/μg RNA. One sample of marrow from a patient who had been bcr/abl + until 6 months post transplant and had WT-1 copy numbers that had fallen from 12,932 one month post transplant to 1855 at that time, became bcr/abl negative at 11 months but still had a WT-1 copy number of 931/ μg RNA. In contrast, patients with molecular disease (+ bcr/abl) maintained WT-1 copy numbers ranging from 70-1000/ μg RNA. Samples from patients with cytogenetic relapse consistently had WT-1 copy numbers exceeding 1000/ μg RNA, with levels exceeding 10,000 in 4/6 such patients tested. In a subset of patients who have failed to achieve or sustain molecular remission following DLI, marrow and blood samples with detectable bcr/abl transcripts and WT-1 copy numbers ranging from 60-3000 have been repeatedly detected for periods of up to 6 years without additional treatment and without disease progression implying an ongoing mechanism of disease control. Therefore, quantitation of WT-1 copy number provides a useful technique for monitoring minimal residual disease and may be helpful in identification of patients at higher risk of cytogenetic relapse. Disclosures:No relevant conflicts of interest to declare.

Dasatinib may not suppress the GVL effect of donor lymphocyte infusions for CML

Donor lymphocyte infusion (DLI) has been considered the standard management for disease relapse after hemopoietic SCT in CML. Responses have been shown to be dose dependent and affected by donor/recipient histocompatibility.1 Disease phase at relapse remains the most important predictor of DLI efficacy and response in the blast phase is typically poor.2 There is some suggestion that imatinib and DLI used together may have a synergistic effect,3 but there is evidence that both imatinib4 and dasatinib5 inhibit the activation and proliferation of normal T lymphocytes in vitro. Dasatinib has been shown to suppress a variety of immune responses in vitro.5, 6, 7, 8 We present here a case of a 32-year-old female with a relapse of CML in myeloid blast crisis who achieved a durable complete molecular remission after dasatinib and DLI used in combination.

Detailed Analysis of CD8+ T Cell Immunity and Identification of a Novel Minor Histocompatibility Antigen Contributing to Graft-Versus- Leukemia Reactivity.

Donor lymphocyte infusion (DLI) can be an effective cellular immunotherapy for patients with hematological malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). The effect of DLI is mediated by donor derived T cells recognizing minor histocompatibility antigens (mHags) on malignant cells of the recipient. These donor originated T cells may also induce Graft-versus-Host Disease (GvHD) when directed against mHags with broad expression on non-malignant tissues of the patient. In this study, we performed a detailed analysis and characterization of mHags recognized by CD8+ T cells contributing to Graft-versus-Leukemia (GvL) reactivity in a patient treated with DLI for relapsed chronic myeloid leukemia (CML) more than one year after HLA-matched alloSCT. The GvL effect in this patient was accompanied with only mild GvHD of the skin.To investigate the specificity of the CD8+ T cell response induced in this patient, activated (HLA-DR+) CD8+ T cells were single cell sorted from a bone marrow sample obtained five weeks after DLI by flowcytometry. A number of isolated CD8+ T cell clones were shown to be specific for mHags, as determined by differential recognition of patient and donor EBV-transformed B cells (EBV-LCL) in IFN-g ELISA and 51Crrelease assays. By screening a panel of third party EBV-LCL sharing one or more HLA class I restriction molecules with the patient, CD8+ T cell clones directed against 7 different mHags were identified, including the known hematopoiesis restricted mHags HA-1 and HA-2, which are presented in HLA-A*0201. Of the 5 remaining specificities, 4 mHags were presented in HLA-B*4001 (B60) and one mHag in HLA-B*0801. To determine the tissue distribution patterns in more detail, we tested recognition of selected non-malignant hematopoietic cells (monocytes, B cells, T cells), malignant CD34+ CML precursor cells, and skin-derived fibroblasts. One HLA-B*4001-restricted T cell clone (clone ZRZ16) failed to recognize all primary hematopoietic cells and skin fibroblasts. The four remaining T cell clones were all capable of recognizing and lysing (specific subsets of) non-malignant hematopoietic cells and malignant CD34+ CML precursor cells. Fibroblast recognition could be demonstrated for two of these four T cell clones. Since clone ZRZ16 failed to recognize all hematopoietic and non-hematopoietic cells, except for EBV-LCL, we tested whether this clone selectively recognizes antigen presenting cells (APC), which are known to be required for efficient induction of immune responses in vivo. Clone ZRZ16 showed indeed strong recognition of monocyte-derived dendritic cells as well as in vitro differentiated CD34+ CML cells with APC phenotype. To identify the mHag recognized by the CD8+ T cell clone, we screened a cDNA expression library constructed from EBV-LCL from the patient. One single cDNA was isolated as the target for B*4001 restricted CD8+ T cell clone ZRZ16. The epitope recognized by this clone was derived from the 3' untranslated region (UTR) of a cDNA encoding thyroid hormone receptor interactor 10 (TRIP10). The peptide epitope was translated in a reading frame different from the TRIP10 protein and comprises three single nucleotide polymorphisms, which were all different between patient and donor. Two of the three SNPs were shown to be important for recognition by clone ZRZ16. Despite ubiquitous tissue expression of the TRIP10 gene as determined by public microarray analysis, CD8+ T cells specific for the newly-identified LB-TRIP10-1EPC mHag selectively recognized APC and failed to recognize CD34+ CML precursor cells, suggesting a predominant role in the initiation, but not effector, phase of the anti-tumor response. In conclusion, our data show a detailed analysis of mHag specific CD8+ T cell immunity induced in a patient successfully treated with DLI for relapsed CML and provide evidence for differential involvement of HLA class I restricted mHags in the onset and execution of GvL reactivity.

High Avidity HLA-A2-Restricted CD8+ T Cells against the Wilms Tumor Protein (WT1) Can Be Isolated Only from HLA-A2 Negative Donors Not Subjected to HLA-A2-Mediated Thymic Deletion

The anti-tumor effect of donor lymphocyte infusions (DLI) after HLA-matched allogeneic stem cell transplantation (allo-SCT) can be mediated by donor T cells recognizing minor histocompatibility antigens (mHag) in the context of self HLA on the malignant cells of the recipient. However, T cells recognizing self antigens (Ag) that are overexpressed in malignant cells like the Wilms tumor protein (WT1) have also been proposed to contribute to the anti-tumor reactivity both after allo- and autologous SCT. WT1 is expressed in various types of cancer, including hematological malignancies at higher levels compared to their non-malignant counterparts. Different groups have isolated CD8+ HLA-A2 restricted WT1-specific T cells after HLA-matched transplantation and/or WT1 vaccination. Although these T cells stain with HLA-A2/WT1 peptide tetrameric complexes and are capable of recognizing peptide loaded HLA-A2+ target cells, recognition of primary leukemic cells could hardly be demonstrated. Stauss et al demonstrated recognition of endogenously processed Ag by WT1 specific T cells. However, these CD8 T cells were isolated in a situation where responder and stimulator cells were mismatched for HLA-A2. Based on these data we hypothesized that high avidity HLA-A2 restricted WT1 specific T cells may be deleted during thymic selection in HLA-A2+ donors. As a result, it is likely that only low avidity HLA-A2 restricted WT1 specific CD8+ T cells can be isolated from HLA-A2 positive donors, whereas it might be possible to isolate high avidity HLA-A2 restricted WT1 specific T cells from HLA-A2 negative donors due to the absence of the HLA restriction element in the thymus irrespective of the local WT1 expression. To test this hypothesis, we induced immune responses against the HLA-A2 binding WT1-derived peptide RMFPNAPYL using either HLA-A2+ or A2- donor T cells as responder cells. In case of HLA-A2+ donors (n=3) we stimulated CD45RO depleted donor T cells with autologous monocyte-derived antigen-presenting cells (APCs) loaded with 1E-6M WT1 peptide in the presence of 5ng/mL IL-7. At day 10 we either enriched tetramer-positive cells using immunomagnetic beads (MACS) or restimulated the responses with peptide-loaded autologous PBMC. At day 20 tetramer positive cells were isolated in bulk cultures and single cell/well by flowcytometric cell sorting. When using HLA-A2 negative donor T cells as responder cells, we used HLA-A2+ stimulator cells from an unrelated donor matched for all other class I alleles. The similar protocol was used for the induction of the immune response. At day 10 CD4+ T cells were depleted and the WT1 specific T cells were either enriched using tetramers and MACS or restimulated with peptide loaded HLA-A2+ PBMC. Using these approaches we were capable of isolating CD8+ T cell clones staining brightly with the A2/WT1 tetramer and expressing different T cell receptors (TCRs). HLA-A2+ WT1 specific T cells recognized only HLA-A2+ target cells loaded with 1E-8-1E-6M peptide both in IFNg release assays and cytotoxicity assays. Especially peptide-loaded target cells with an APC phenotype like EBVs were recognized efficiently, whereas normal peripheral blood cells were only recognized when loaded with 1E-6M peptide. In contrast, the HLA-A2 negative WT1-specific T cells efficiently recognized normal peripheral blood cells upon loading of <1E-9M WT1 peptide, indicating that these T cells express TCRs with a higher affinity for the WT1 peptide compared to the HLA-A2+ WT1-specific T cells. Strikingly, EBV-LCL as well as TAP-deficient T2 cells were recognized by the HLA-A2 negative T cells even in the absence of exogenously loaded WT1 peptide. This might be due to the efficient recognition by the high avidity T cells of low levels of WT1 presented in HLA-A2 processed from the endogenous WT1 expression. However, due to the absence of HLA-A2 mediated thymic selection in HLA-A2 negative donors, cross-recognition of other peptides in HLA-A2 may also occur. In conclusion, high avidity WT1 specific CD8+ T cells could be exclusively isolated from HLA-A2 negative donor cells. These high avidity T cells are capable of recognizing target cells expressing very low levels of WT1. These T cell responses will gain more insight into the correlation of WT1 expression in different normal and malignant cell types and the recognition by high avidity WT1 T cells, and the clinical applicability of T cells directed against self antigens.

Viewpoint: What is the role of allogeneic haematopoietic cell transplantation in the era of reduced-intensity conditioning – is there still an upper age limit? A focus on myeloid neoplasia

Allogeneic haematopoietic cell transplantation (HCT) is the most effective curative therapy in acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS). Incidence of AML and MDS increases with age, peaking in the seventh decade. Despite improved Ara-C and anthracyclin-based chemotherapy regimens, the prognosis of AML in patients beyond 60 years of age is dismal. The introduction of peripheral blood-derived stem cell grafts into allogeneic HCT and the known anti-leukaemic effect of donor lymphocyte infusions paved the way for reduced-intensity conditioning (RIC) allogeneic stem-cell transplantation, which makes transplant in advanced age possible and significantly reduces transplant-related organ toxicity and mortality. The success of RIC HCT relies on the alloreactivity of the donor immune system and the graft-versus-leukaemia effect. We try to answer the following questions in this paper: who should receive RIC HCT? when and how should the transplant be performed? is there an upper age limit and what is the future of RIC HCT?