Comparison and Incorporation of Reasoning and Learning Approaches for Cancer Therapy Research.

Representing knowledge in a comprehensible and maintainable way and transparently providing inferences thereof are important issues, especially in the context of applications related to artificial intelligence in medicine. This becomes even more obvious if the knowledge is dynamically growing and changing and when machine learning techniques are being involved. In this paper, we present an approach for representing knowledge about cancer therapies collected over two decades at St.-Johannes-Hospital in Dortmund, Germany. The presented approach makes use of InteKRator, a toolbox that combines knowledge representation and machine learning techniques, including the possibility of explaining inferences. An extended use of InteKRator's reasoning system will be introduced for being able to provide the required inferences. The presented approach is general enough to be transferred to other data, as well as to other domains. The approach will be evaluated, e. g., regarding comprehensibility, accuracy and reasoning efficiency.

Immunohistochemical markers for histopathological diagnosis and differentiation of acute cutaneous graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major complication following stem-cell or solid-organ transplantation. Accurate diagnosis of cutaneous GvHD is challenging, given that drug eruptions and viral rashes may present with similar clinical/histological manifestations. Specific markers are not available. We performed the histological examination of biopsy samples from acute GvHD (aGvHD; n = 54), Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN; n = 27), maculopapular drug eruption (MDE; n = 26) and healthy controls (n = 26). Samples of aGvHD showed a decrease in Langerhans cells (LC, p = 0.0001) and an increase in macrophages (MΦ, p = 0.0001) compared to healthy skin. Compared to SJS/TEN, MDE and healthy skin, aGvHD biopsies contained greater numbers of CD4+ and CD8+ T cells. The majority of CD4+ T-helper cells were localized in the upper dermis, whereas cytotoxic CD8+ T cells were found in the epidermis. Increased numbers of CD56+ natural killer (NK) cells in the upper dermis of aGvHD skin (p = 0.007) were not observed in controls or SJS/TEN and MDE. There were no differences in elafin staining between aGvHD and the latter two conditions. Acute GvHD appears to have a distinct inflammatory cell profile (T cells/NK cells) that may aid establishing in a more accurate diagnosis, especially when used to rule out differential diagnoses such as SJS/TEN or MDE.

Pharmacokinetics and safety results from the Phase 3 randomized, open-label, study of intravenous posaconazole in patients at risk of invasive fungal disease.

OBJECTIVES: A two-part (Phase 1B/3), sequential, open-label, multicentre study evaluated the pharmacokinetics (PK) and safety of intravenous (iv) posaconazole given as antifungal prophylaxis to neutropenic patients with AML or myelodysplastic syndrome (MDS) or to recipients at risk of invasive fungal disease (IFD) after allogeneic HSCT. METHODS: Patients (N = 237) received 300 mg of posaconazole iv twice daily on day 1, followed by 300 mg of posaconazole iv once daily for 4-28 days. After at least 5 days, patients were randomly assigned to receive posaconazole oral suspension, 400 mg twice daily or 200 mg three times daily, to complete a 28 day treatment course. Primary PK parameters were steady-state average concentration over the dosing interval (Cavg) and posaconazole trough levels (Cmin). RESULTS: Mean posaconazole Cmin was 1320 ng/mL (day 6) and 1297 ng/mL (day 8); steady-state Cmin was 1090 ng/mL (day 10). Mean steady-state posaconazole Cavg was 1500 ng/mL (day 10 or 14) and was similar in HSCT recipients (1560 ng/mL) and AML/MDS patients (1470 ng/mL). The most commonly reported treatment-related adverse events were diarrhoea (8%), nausea (5%) and rash (5%). IFD was reported in 3/237 patients (1%; 2 proven, 1 probable). CONCLUSIONS: Intravenous posaconazole at 300 mg was well tolerated, resulted in adequate steady-state systemic exposure and was associated with a low incidence of IFD in this population at high risk. TRIAL REGISTRY AND NUMBER: ClinicalTrials.gov, NCT01075984.

Rituximab combined with DexaBEAM followed by high dose therapy as salvage therapy in patients with relapsed or refractory B-cell lymphoma: mature results of a phase II multicentre study.

Salvage therapy followed by high-dose therapy (HDT) remains a mainstay for patients with relapsed lymphoma, however no optimal regimen has been defined. Here we report on the results of R-DexaBEAM (rituximab, dexamethasone, carmustine, etoposide, cytarabine, melphalan) followed by HDT. Patients aged 18-65 years, Eastern Cooperative Oncology Group performance score 0-2, with relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) were eligible. R-Dexa-BEAM was given for two cycles followed by stem cell mobilization and HDT. Primary endpoint of the trial was progression-free-survival (PFS). One hundred and three patients were included: aggressive NHL (aNHL): diffuse large B-cell lymphoma 55, mantle cell lymphoma 7, follicular lymphoma (FL) grade 3: 5, indolent Lymphoma (iNHL): FL grade 1-2: 29, marginal zone lymphoma 6, Immunocytoma 1. The overall response rate after salvage therapy was 62% for aNHL and 78% for iNHL patients. 66% of patients with aNHL and 86% with iNHL underwent HDT. Treatment-related mortality for HDT was 1·3%. For aNHL patients, the median PFS was 0·83 years with 44% alive at the median follow-up of 7·3 years. Corresponding figures for iNHL were: median PFS 3·7 years and 72% alive after 8 years. The combination of rituximab with DexaBEAM followed by HDT resulted in high response rates and sustained remissions in responders. R-DexaBEAM followed by HDT can be considered a valid salvage option for NHL.

Human CD8+ memory and EBV-specific T cells show low alloreactivity in vitro and in CD34+ stem cell-engrafted NOD/SCID/IL-2Rγc null mice.

Current strategies in cellular immunotherapy of cancer and viral infections include the adoptive transfer of T cell receptor (TCR) and chimeric antigen receptor engineered T cells. When using transient RNA expression systems in clinical studies, multiple infusions with receptor-redirected T cells appear necessary. However, in allogeneic hematopoietic stem-cell transplantation, repeated transfer of donor-derived T cells increases the risk of alloreactive graft-versus-host disease. We investigated naive-derived (TN), memory-derived (TM), and Epstein Barr virus-specific (TEBV) CD8(+) T cell subsets for alloreactivity upon redirection with RNA encoding a cytomegalovirus-specific model TCR. We observed that alloreactivity to human leukocyte antigen (HLA)-mismatched hematopoietic cells developed at much stronger levels in TN compared with TM or TEBV populations in cytokine-release and cytotoxicity assays. Cytomegalovirus-specific effector function was higher in TCR-transfected TEBV and TM over TN cells. To measure alloreactivity in vivo, we reconstituted NOD/SCID/IL-2Rγc(null) mice with human CD34(+) stem cells and adoptively transferred them with CD8(+) T cell subsets previously stimulated against cells of the HLA-mismatched stem-cell donor. TN cells showed a significant ability to eliminate CD34-derived hematopoietic cells, which was not found with TM and TEBV cells. This reduced alloreactive potential along with strong effector function upon receptor RNA engineering makes CD8(+) memory and EBV-specific T cells advantageous tools in adoptive immunotherapy after allogeneic transplantation.

Intronic promoters and their noncoding transcripts: a new source of cancer-associated genes.

Recent studies of mammalian genomes suggest that alternative promoters are associated with various disorders, including cancer. Here we present an intronic promoter of the murine proteinase 3 gene, which drives the expression of an alternative mRNA in intron 2 of the prtn3 gene. The proximal promoter sequences were identified and a series of promoter deletion constructs were used to identify the sequence elements that are required for basal promoter activity. Expression of the homeobox transcription factor CUX1 p75 isoform was found to suppress the activity of the alternative PR3 promoter. Data base analyses, multiple alignments and expression data showed that the intronic PR3 promoter is active in leukemia and other tumor cells as well as in mouse embryo, male mammary gland and bone marrow. In the spleen, the transcript is exclusively expressed by Gr-1(int) /CD11b(+) cells, which are also known as myeloid-derived suppressor cells (MDSCs). In humans, an alternative transcript of the PR3-gene could be detected in the bone marrow and in various cancer cell lines but not in primary leukemia cells, suggesting a species-overarching function of this kind of promoter. Therefore, the alternative PR3 promoter and its mRNA may be useful tools to investigate the fate of hematopoietic stem cells.

Second allograft for hematologic relapse of acute leukemia after first allogeneic stem-cell transplantation from related and unrelated donors: the role of donor change.

PURPOSE: To evaluate the role of a second allogeneic hematopoietic stem-cell transplantation (HSCT2) given for relapsed acute leukemia (AL) after related or unrelated first hematopoietic stem-cell transplantation (HSCT1) and to analyze the role of donor change for HSCT2 in both settings. PATIENTS AND METHODS: We performed a retrospective registry study on 179 HSCT2s given for relapse after HSCT1 from matched related donors (n = 75) or unrelated donors (n = 104), using identical or alternative donors for HSCT2. Separate analyses were performed according to donor at HSCT1. RESULTS: Independent of donor, 74% of patients achieved complete remission after HSCT2, and half of these patients experienced relapse again. Overall survival (OS) at 2 years was 25% ± 4% (39% ± 7% after related HSCT2; 19% ± 4% after unrelated HSCT2). Long-term survivors were observed even after two unrelated HSCT2s. Multivariate analysis for OS from HSCT2 confirmed established risk factors (remission duration after HSCT1: hazard ratio [HR], 2.37; 95% CI, 1.61 to 3.46; P < .001; stage at HSCT2: HR, 0.53; 95% CI, 0.34 to 0.83; P = .006). Outcome of HSCT2 was better after related HSCT1 than after unrelated HSCT1 (2-year OS: 37% ± 6% v 16% ± 4%, respectively; HR, 0.68; 95% CI, 0.47 to 0.98; P = .042, multivariate Cox regression). After both related and unrelated HSCT1, selecting a new donor for HSCT2 did not result in a relevant improvement in OS compared with HSCT2 from the original donor; however, donor change was not detrimental either. CONCLUSION: After relapse from allogeneic HSCT1, HSCT2 can induce 2-year OS in approximately 25% of patients. Unrelated HSCT2 is feasible after related and unrelated HSCT1. Donor change for HSCT2 is a valid option. However, a clear advantage in terms of OS could not be demonstrated.

Influence of molecular subgroups on outcome of acute myeloid leukemia with normal karyotype in 141 patients undergoing salvage allogeneic stem cell transplantation in primary induction failure or beyond first relapse.

Based on molecular aberrations, in particular the NPM1 mutation (NPM1(mut)) and the FLT3 internal tandem duplication (Flt3-ITD), prognostic subgroups have been defined among patients with acute myeloid leukemia with normal karyotype. Whereas these subgroups are known to play an important role in outcome in first complete remission, and also in the indication for allogeneic stem cell transplantation, data are limited on their role after transplantation in advanced disease. To evaluate the role of molecular subgroups of acute myeloid leukemia with normal karyotype after allogeneic stem cell transplantation beyond first complete remission, we analyzed the data from 141 consecutive adults (median age: 51.0 years, range 18.4-69.3 years) who had received an allogeneic transplant either in primary induction failure or beyond first complete remission. A sequential regimen of cytoreductive chemotherapy (fludarabine, high-dose AraC, amsacrine) followed by reduced intensity conditioning (FLAMSA-RIC), was uniformly used for conditioning. After a median follow up of three years, overall survival from transplantation was 64 ± 4%, 53 ± 4% and 44 ± 5% at one, two and four years, respectively. Forty patients transplanted in primary induction failure achieved an encouraging 2-year survival of 69%. Among 101 patients transplanted beyond first complete remission, 2-year survival was 81% among patients with the NPM1(mut)/FLT3(wt) genotype in contrast to 43% in other genotypes. Higher numbers of transfused CD34(+) cells (hazard ratio 2.155, 95% confidence interval 0.263-0.964, P=0.039) and favorable genotype (hazard ratio 0.142, 95% confidence interval: 0.19-0.898, P=0.048) were associated with superior overall survival in multivariate analysis. In conclusion, patients with acute myeloid leukemia with normal karyotype can frequently be rescued after primary induction failure by allogeneic transplantation following FLAMSA-RIC. The prognostic role of NPM1(mut)/FLT3-ITD based subgroups was carried through after allogeneic stem cell transplantation beyond first complete remission.

Varicella-zoster virus glycoproteins B and E are major targets of CD4+ and CD8+ T cells reconstituting during zoster after allogeneic transplantation.

BACKGROUND: After allogeneic hematopoietic stem-cell transplantation patients are at increased risk for herpes zoster as long as varicella-zoster virus specific T-cell reconstitution is impaired. This study aimed to identify immunodominant varicella-zoster virus antigens that drive recovery of virus-specific T cells after transplantation. DESIGN AND METHODS: Antigens were purified from a varicella-zoster virus infected cell lysate by high-performance liquid chromatography and were identified by quantitative mass spectrometric analysis. To approximate in vivo immunogenicity for memory T cells, antigen preparations were consistently screened with ex vivo PBMC of varicella-zoster virus immune healthy individuals in sensitive interferon-γ ELISpot assays. Candidate virus antigens identified by the approach were genetically expressed in PBMC using electroporation of in vitro transcribed RNA encoding full-length proteins and were then analyzed for recognition by CD4(+) and CD8(+) memory T cells. RESULTS: Varicella-zoster virus encoded glycoproteins B and E, and immediate early protein 62 were identified in immunoreactive lysate material. Predominant CD4(+) T-cell reactivity to these proteins was observed in healthy virus carriers. Furthermore, longitudinal screening in allogeneic stem-cell transplantation patients showed strong expansions of memory T cells recognizing glycoproteins B and E after onset of herpes zoster, while immediate early protein 62 reactivity remained moderate. Reactivity to viral glycoproteins boosted by acute zoster was mediated by both CD4(+) and CD8(+) T cells. CONCLUSIONS: Our data demonstrate that glycoproteins B and E are major targets of varicella-zoster virus specific CD4(+) and CD8(+) T-cell reconstitution occurring during herpes zoster after allogeneic stem-cell transplantation. Varicella-zoster virus glycoproteins B and E might form the basis for novel non-hazardous zoster subunit vaccines suitable for immunocompromised transplant patients.

Human epidermal Langerhans cells replenish skin xenografts and are depleted by alloreactive T cells in vivo.

Epidermal Langerhans cells (LC) are potent APCs surveying the skin. They are crucial regulators of T cell activation in the context of inflammatory skin disease and graft-versus-host disease (GVHD). In contrast to other dendritic cell subtypes, murine LC are able to reconstitute after local depletion without the need of peripheral blood-derived precursors. In this study, we introduce an experimental model of human skin grafted to NOD-SCID IL2Rγ(null) mice. In this model, we demonstrate that xenografting leads to the transient loss of LC from the human skin grafts. Despite the lack of a human hematopoietic system, human LC repopulated the xenografts 6 to 9 wk after transplantation. By staining of LC with the proliferation marker Ki67, we show that one third of the replenishing LC exhibit proliferative activity in vivo. We further used the skin xenograft as an in vivo model for human GVHD. HLA-disparate third-party T cells stimulated with skin donor-derived dendritic cells were injected intravenously into NOD-SCID IL2Rγ(null) mice that had been transplanted with human skin. The application of alloreactive T cells led to erythema and was associated with histological signs of GVHD limited to the transplanted human skin. The inflammation also led to the depletion of LC from the epidermis. In summary, we provide evidence that human LC are able to repopulate the skin independent of blood-derived precursor cells and that this at least partly relates to their proliferative capacity. Our data also propose xeno-transplantation of human skin as a model system for studying the role of skin dendritic cells in the efferent arm of GVHD.

Myeloablative radioimmunotherapy in conditioning prior to haematological stem cell transplantation: closing the gap between benefit and toxicity?

High-dose radio-/chemotherapy in the context of autologous and allogeneic haematopoietic stem cell transplantation is a double-edged sword. The requirement for dose intensification is linked to an increase in toxicity to noninvolved organs. Particularly for older patients and patients with comorbidities, efficient but toxicity-reduced schemes are needed. Myeloablative radioimmunotherapy is a targeted, internal radiotherapy that uses radiolabelled monoclonal antibodies (mAb) with affinity to the bone marrow. It involves the administration of high radiation doses (up to 30 Gy) to the bone marrow and spleen but without exposing radiosensitive organs to doses higher than 1-7 Gy. Added to conventional or intensity-reduced conditioning, myeloablative radioimmunotherapy may achieve a pronounced antileukaemic effect with tolerable toxicities. A rational and individual design of the ideal nuclide-antibody combination optimizes therapy. The anti-CD33, anti-CD45 and anti-CD66 mAbs appear to be ideal tracers so far. The beta-emitter (90)Y is coupled by DTPA and is the best nuclide for myeloablation. Approval trials for DTPA anti-CD66 mAb are underway in Europe, and in the near future these therapies may become applicable in practice. This review gives an overview of current myeloablative conditioning radioimmunotherapy. We discuss the selection of the optimal radioimmunoconjugate and discuss how radioimmunotherapy might be optimized in the future by individualization of therapy protocols. We also highlight the potential advantages of combination therapies.

Rapid identification and sorting of viable virus-reactive CD4(+) and CD8(+) T cells based on antigen-triggered CD137 expression.

Current methods for the detection and isolation of antigen-specific CD4(+) and CD8(+) T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. Here we show that de novo cell surface expression of the TNF-receptor family member CD137 (4-1BB) identifies recently activated, but not resting, human CD4(+) and CD8(+) memory T cells. Maximum CD137 expression level is uniformly observed in both T-cell subsets at 24h after stimulation with antigen. In experiments with CMV and EBV-reactive T cells, we confirmed the specificity of CD137 expression by co-staining with peptide/HLA tetramers. Substantial proportions of CD137(+) T cells did not produce IFN-gamma, suggesting that CD137 detects a broader repertoire of antigen-specific T cells. Activated CD137(+) T cells could be easily purified by MACS and expanded in vitro thereafter. This CD137-based enrichment method was capable of isolating 2-fold higher numbers of anti-viral CD4(+) and CD8(+) T cells compared to the IFN-gamma secretion assay. In conclusion, antigen-triggered CD137 expression allows the rapid detection and sorting of virus-reactive CD4(+) and CD8(+) T cells. The CD137 assay is most attractive for the simultaneous targeting of anti-viral T helper and effector cells in monitoring studies and adoptive immunotherapy trials.

Acute myeloid leukemia (AML)-reactive cytotoxic T lymphocyte clones rapidly expanded from CD8(+) CD62L((high)+) T cells of healthy donors prevent AML engraftment in NOD/SCID IL2Rgamma(null) mice.

OBJECTIVE: Current in vitro techniques for isolating leukemia-reactive cytotoxic T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield responder populations with unknown biological significance. This study aimed at the development of a more reliable approach, allowing generation and expansion of acute myeloid leukemia (AML)-reactive CTLs using primary in vitro stimulation. MATERIALS AND METHODS: We established allogeneic mini-mixed lymphocyte-leukemia cultures (mini-MLLCs) by stimulating donor CD8(+) T cells with human leukocyte antigen (HLA) class I-matched AML blasts in microtiter plates. Before culture, CD8(+) T cells were separated into CD62L((high)+) and CD62L((low)+/neg) subsets enriched for naive/central memory and effector memory cells, respectively. RESULTS: In eight different related and unrelated donor/AML pairs, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs expressed T-cell receptors of single Vbeta-chain families, indicating their clonal origin. The majority of CTL clones were obtained from mini-MLLCs initiated with CD62L((high)+) cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10(8)-10(10) cells within 6 to 8 weeks. Three of four representative CTL clones were capable of completely preventing engraftment of human primary AML blasts in nonobese diabetic/severe combined immune deficient IL2Rgamma(null) mice. CONCLUSION: The mini-MLLC approach allows the efficient in vitro expansion of AML-reactive CTL clones from CD8(+)CD62L((high)+) precursors of healthy donors. These CTLs can inhibit leukemia engraftment in immunodeficient mice, suggesting their potential biological relevance.

Sustained remissions and low rate of BCR-ABL resistance mutations with imatinib treatment chronic myelogenous leukemia in patients treated in late chronic phase: a 5-year follow up.

The introduction of Imatinib (IM) has significantly altered the treatment for CML, although only limited follow-up results are available. As failure of Interferon-alpha had been associated with poor prognosis and results of IM-treatment in this patient group may allow earlier estimation of long-term benefits for early chronic phase patients. Therefore we prospectively analyzed the quality and duration of remissions and the rate of BCR-ABL resistance mutations occurring in patients treated with IM, if they were intolerant or refractory to interferon. Fifty-nine patients were included and median follow up is 4.75 years. Haematologic remission rate was 92% and 62% of patients achieved at least major cytogenetic remission. There were no major differences between patients failing or being refractory to IFN. Major molecular response was observed in 67% of patients evaluable. For the entire group, median PFS was 4.3 years and OS was 82% at 4.75 years. Haematologic and complete cytogenetic remissions, but not molecular responses were correlated with improved PFS and OS. In patients with progression or inadequate response, BCR-ABL kinase domain mutations were detected in 3/28 patients. IM resulted in prolonged remission rates in a substantial proportion after IFN-failure/intolerance, with no differences in these patient groups. Quality of hematologic and cytogenetic, but not molecular response was associated with duration of remissions and survival. A low rate of resistance-mutations was detected, suggesting that alternative mechanism may play an important role in disease progression.

An open-label, prospective phase I/II study evaluating the immunogenicity and safety of a ras peptide vaccine plus GM-CSF in patients with non-small cell lung cancer.

Mutations of the ras gene have been reported in 20-40% of NSCLC patients. If present, they are critical for the malignant phenotype of these tumors. Therefore, targeting them by specific vaccination is a promising therapeutic approach. In a clinical trial we screened for ras mutations in patients with NSCLC. Patients with ras-positive tumors were immunized six times intradermally with a mixture of seven peptides representing the most common ras mutations. Objectives of the study were the feasibility, efficacy and safety of the vaccination. In addition, the induction of a specific immune reaction was investigated by DTH tests, and the induction of peptide-specific T cells was tested in ex vivo IFN-gamma-ELISPOT assays. Five of 18 patients had ras mutations at codon 12. Four of these patients, all with adenocarcinomas (stage I: n=3, stage IV: n=1) entered the study. The patient with stage IV disease withdrew prematurely after the third application because of disease progression associated with pulmonary embolism. Ras-specific T cells were not detected ex vivo. However, one patient developed a positive DTH reaction after the fifth vaccination that increased after the sixth vaccination. Our results are in line with earlier trials reporting ras mutations in 20-40% of NSCLC patients. Vaccination with mutated ras peptides is feasible and well tolerated. One patient revealed a positive DTH test. An ex vivo detectable T cell response was not induced in any of the patients.

Rapid identification of an HLA-B*1501-restricted vaccinia peptide antigen.

We identified a modified vaccinia virus Ankara (MVA)-peptide recognized by T cells of a vaccinated patient, applying a modified expression-based procedure. CD8(+) T cells reactive with MVA were expanded from peripheral blood of a patient after MVA-vaccination. The restricting HLA-elements and the antigen were determined applying stably HLA-class I-transfected K562 cells that were either infected wit MVA or transfected with a panel of MVA open reading frames (ORF). The T cells recognized the ORF 28R (K7R) gene product in association with HLA-B*1501 and the peptide-coding region was localized applying truncated in vitro transcribed mRNA. The 9-mer peptide encoded (ORF 28(25-33), SIIDLIDEY) was identified that was also recognized by ex vivo CD8(+) T cells from the patient and further healthy individuals.

Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell-depleted reduced-intensity transplantation.

Allogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with hematologic malignancies, 11 received a total of 21 CD8-depleted DLIs. Five patients developed transient grade I acute GVHD following transfer. Only 2 patients with HLA-C-mismatched donors showed grade II and III acute GVHD and subsequently progressed to limited chronic GVHD. Following DLIs, 4 patients with declining hematopoietic donor chimerism converted to full chimeras. A 2.1-fold median increase of circulating CD4 T cells was observed within 2 weeks after infusion. Non-DLI patients did not show a comparable rise in CD4 counts. Four patients demonstrated enhanced frequencies of cytomegalovirus-specific CD4 and CD8 T cells following transfer. Our results suggest that prophylactic CD8-depleted DLIs accelerate immune reconstitution after lymphodepleted HLA-matched SCT and carry a low risk of inducing severe GVHD.

Targeting the activation-induced antigen CD137 can selectively deplete alloreactive T cells from antileukemic and antitumor donor T-cell lines.

In HLA-incompatible hematopoietic stem cell transplantation, alloreactive donor T cells recognizing recipient mismatch HLA cause severe graft-versus-host disease (GVHD). Strategies allowing the selective depletion of alloreactive T cells as well as the enhancement of graft-versus-malignancy immunity would be beneficial. We generated donor CD8 T-cell lines in vitro using allogeneic recipient cells mismatched at a single HLA class I allele or haplotype as stimulators. Recipient cells were obtained from acute myeloid leukemias, renal-cell carcinomas, and CD40L-induced B lymphoblasts. Resulting alloreactive T cells were activated by incubating day 21 T-cell cultures with HLA-mismatch transfected K562 cells or recipient-derived fibroblasts. Selective allodepletion (SAD) was subsequently performed by a newly developed immunomagnetic depletion approach targeting the tumor necrosis factor receptor molecule CD137 (4-1BB). Compared with other activation-induced antigens, CD137 showed a superior performance based on a consistently low baseline expression and a rapid up-regulation following alloantigen stimulation. In 15 different SAD experiments, the frequency of alloreactive CD8 T cells was reduced to a median of 9.5% compared with undepleted control populations. The allodepleted T-cell subsets maintained significant antitumor and antiviral CD8 responses. In vitro expansion of tumor-reactive T cells followed by CD137-mediated SAD might enhance the antitumor efficacy of T-cell allografts with lower risk of inducing GVHD.