Tracking the progeny of adoptively transferred virus-specific T cells in patients posttransplant using TCR sequencing.

Adoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase 1/2 trial, we prophylactically administered multivirus-specific T-cell products to protect recipients of T-cell-depleted allogeneic stem cell grafts against viral reactivation. To establish treatment efficacy, it is important to determine the fate of the individual transferred T-cell populations. However, it is difficult to unequivocally distinguish progeny of the transferred T-cell products from recipient- or stem cell graft-derived T cells that survived T-cell depletion during conditioning or stem cell graft manipulation. Using messenger RNA sequencing of the T-cell receptor ß-chains of the individual virus-specific T-cell populations within these T-cell products, we were able to track the multiple clonal virus-specific subpopulations in peripheral blood and distinguish recipient- and stem cell graft-derived virus-specific T cells from the progeny of the infused T-cell products. We observed in vivo expansion of virus-specific T cells that were exclusively derived from the T-cell products with similar kinetics as the expansion of virus-specific T cells that could also be detected before the T-cell product infusion. In addition, we demonstrated persistence of virus-specific T cells derived from the T-cell products in most patients who did not show viral reactivation. This study demonstrates that virus-specific T cells from prophylactically infused multiantigen-specific T-cell products can expand in response to antigen encounter in vivo and even persist in the absence of early viral reactivation.

Occult Fractures in Children with a Radiographic Fat Pad Sign of the Elbow: A Meta-Analysis of 10 Published Studies.

BACKGROUND: A radiographic fat pad sign after an elbow injury in children may indicate an occult fracture. Different incidences and locations of occult fractures have been reported. The primary objective of this meta-analysis was to assess the overall rate of occult fractures in children with a positive fat pad sign from the data of original studies. Secondary objectives were to assess the fracture types and to identify risk factors for sustaining an occult fracture. METHODS: A systematic literature search of the Embase, MEDLINE, and Cochrane databases was performed according to PRISMA guidelines. Studies on pediatric populations with a positive fat pad sign identified using a lateral elbow radiograph and with follow-up imaging were included in this meta-analysis. Included studies were assessed for risk of bias with use of the MINORS (Methodological Index for NOn-Randomized Studies) instrument. RESULTS: Ten studies with a total of 250 patients, of whom 104 had an occult fracture, were included. Accounting for heterogeneity between the studies, the overall occult fracture rate was 44.6% (95% confidence interval: 30.4% to 59.7%). The most common fracture locations were the supracondylar humerus (43%), proximal ulna (19%), proximal radius (17%), and lateral humeral condyle (14%). Definitions of a positive pad fad sign were not uniform among studies, and the follow-up imaging modality also varied (radiography, magnetic resonance imaging, or computed tomography). The average MINORS score was 10.1 for the 7 noncomparative studies and 18.7 for the 3 comparative studies, with both averages classified as moderate quality. We were not able to identify risk factors for an occult fracture in the presence of a positive fat pad sign. CONCLUSIONS: The occult fracture rate was 44.6% in pediatric elbow injuries with a positive fat pad sign. Supracondylar humeral fractures were the most frequently encountered type. The findings of this meta-analysis underline the potential clinical relevance of a positive fat pad sign in children and denote the opportunity for future studies to create evidence-based guidelines. LEVEL OF EVIDENCE: Level II. See Instructions for Authors for a complete description of levels of evidence.

Evaluation and management of osteochondral lesions of the talus in skeletally immature patients.

Osteochondral lesions of the talus (OLTs) are defined as damage to the talar cartilage, with pathological changes in the underlying bone. They include a group of injuries that involve juvenile osteochondritis dissecans (JOCD) and osteochondral fractures of the talus. The etiology of OLT remains not fully clarified but is more common in young and active patients. Treatment strategies for OLTs in skeletally immature populations depend on the magnitude of symptoms, lesion morphology (stability and overlying cartilage integrity), size, nature of the lesion (traumatic versus JOCD), ankle stability, lower extremity alignment, and previous treatment. The aim of this review is to provide an overview of the current evidence for the diagnosis and treatment of OLTs in skeletally immature patients.

Las lesiones osteocondrales del astrágalo (OLT) se definen como lesiones del cartílago talar, con cambios patológicos en el hueso subyacente. Incluyen un grupo de lesiones que implican osteocondritis disecante juvenil (JOCD) y fracturas osteocondrales del astrágalo. La etiología de las OLT aún no está totalmente aclarada, pero son más frecuentes en pacientes jóvenes y activos. Las estrategias de tratamiento de las OLT en poblaciones esqueléticamente inmaduras dependen de la magnitud de los síntomas, la morfología de la lesión (estabilidad e integridad del cartílago suprayacente), el tamaño, la naturaleza de la lesión (traumática frente a JOCD), la estabilidad del tobillo, la alineación de la extremidad inferior y el tratamiento previo. El objetivo de esta revisión es proporcionar una visión general de las pruebas actuales para el diagnóstico y el tratamiento de los OLT en pacientes esqueléticamente inmaduros.

Abduction treatment in stable hip dysplasia does not alter the acetabular growth: results of a randomized clinical trial.

Background The effect of bracing over natural history of stable dysplastic hips is not well known. This multicenter randomized trial aimed at objectifying the effect of abduction treatment versus active surveillance in infants of 3 to 4 months of age. Methods Patients were randomized to either Pavlik harness or active surveillance group. Ultrasound was repeated at 6 and 12 weeks post randomization. The primary outcome was the degree of dysplasia using the Graf α-angle at 6 months of age. The measurement of the acetabular index (AI) on plain pelvis X-rays was used to identify persistent dysplasia after 9 months and walking age (after 18 months). Findings The Pavlik harness group (n = 55) and active surveillance group (n = 49) were comparable for predictors of outcome. At 12 weeks follow-up the mean α-angle was 60.5° ± 3.8° in the Pavlik harness group and 60.0° ± 5.6° in the active surveillance group. (p = 0.30). Analysis of secondary outcomes (standard of care) showed no treatment differences for acetabular index at age 10 months (p = 0.82) and walking age (p = 0.35). Interpretation Pavlik harness treatment of stable but sonographic dysplastic hips has no effect on acetabular development. Eighty percent of the patients will have a normal development of the hip after twelve weeks. Therefore, we recommend observation rather than treatment for stable dysplastic hips.

Current trends in tendinopathy: consensus of the ESSKA basic science committee. Part II: treatment options.

The treatment of painful chronic tendinopathy is challenging. Multiple non-invasive and tendon-invasive methods are used. When traditional non-invasive treatments fail, the injections of platelet-rich plasma autologous blood or cortisone have become increasingly favored. However, there is little scientific evidence from human studies supporting injection treatment. As the last resort, intra- or peritendinous open or endoscopic surgery are employed even though these also show varying results. This ESSKA basic science committee current concepts review follows the first part on the biology, biomechanics and anatomy of tendinopathies, to provide a comprehensive overview of the latest treatment options for tendinopathy as reported in the literature.

Current trends in tendinopathy: consensus of the ESSKA basic science committee. Part I: biology, biomechanics, anatomy and an exercise-based approach.

Chronic tendinopathies represent a major problem in the clinical practice of sports orthopaedic surgeons, sports doctors and other health professionals involved in the treatment of athletes and patients that perform repetitive actions. The lack of consensus relative to the diagnostic tools and treatment modalities represents a management dilemma for these professionals. With this review, the purpose of the ESSKA Basic Science Committee is to establish guidelines for understanding, diagnosing and treating this complex pathology.

Return to sports after arthroscopic debridement and bone marrow stimulation of osteochondral talar defects: a 5- to 24-year follow-up study.

PURPOSE: Osteochondral defects (OCD) often have a severe impact on the quality of life due to deep ankle pain during and after weight bearing, which prevents young patients from leading an active life. Arthroscopic debridement and bone marrow stimulation are currently the gold standard treatment. The purpose of this study was to evaluate the number of patients that resume and maintain sports to their pre-injury activity level after arthroscopic debridement and bone marrow stimulation. METHODS: This retrospective study evaluated patients treated with arthroscopic debridement and bone marrow stimulation between 1989 and 2008. All patients who were participating in sports before injury were included. The Ankle Activity Scale (AAS) was used to determine activity levels during specific time points (before injury, before operation, after operation and at the time of final follow-up). RESULTS: Ninety-three patients were included. Fifty-seven (76%) patients continued participating in sports at final follow-up. The median AAS before injury of 8 (range 3-10) significantly decreased to 4 (range 2-10) at final follow-up. CONCLUSION: It is shown that 76% of the patients were able to return to sports at long-term follow-up after arthroscopic debridement and bone marrow stimulation of talar OCDs. The activity level decreased at long-term follow-up and never reached the level of that before injury. The data of our study can be of importance to inform future patients on expectations after debridement and bone marrow stimulation of a talar OCD. LEVEL OF EVIDENCE: Retrospective case series, Level IV.

Computed tomography analysis of osteochondral defects of the talus after arthroscopic debridement and microfracture.

PURPOSE: The primary surgical treatment of osteochondral defects (OCD) of the talus is arthroscopic debridement and microfracture. Healing of the subchondral bone is important because it affects cartilage repair and thus plays a role in pathogenesis of osteoarthritis. The purpose of this study was to evaluate the dimensional changes and bony healing of talar OCDs after arthroscopic debridement and microfracture. METHODS: Fifty-eight patients with a talar OCD were treated with arthroscopic debridement and microfracture. Computed tomography (CT) scans were obtained at baseline, 2 weeks postoperatively, and 1 year postoperatively. Three-dimensional changes and bony healing were analysed on CT scans. Additionally, clinical outcome was measured with the American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot score and numeric rating scales (NRS) for pain. RESULTS: Average OCD size increased significantly (p < 0.001) in all directions from 8.6 (SD 3.6) × 6.3 (SD 2.6) × 4.8 (SD 2.3) mm (anterior-posterior × medial-lateral × depth) preoperatively to 11.3 (SD 3.4) × 7.9 (SD 2.8) × 5.8 (SD 2.3) mm 2 weeks postoperatively. At 1-year follow-up, average defect size was 8.3 (SD 4.2) × 5.7 (SD 3.0) × 3.6 (SD 2.4) mm. Only average defect depth decreased significantly (p < 0.001) from preoperative to 1 year postoperative. Fourteen of the 58 OCDs were well healed. No significant differences in the AOFAS and NRS-pain were found between the well and poorly healed OCDs. CONCLUSION: Arthroscopic debridement and microfracture of a talar OCD leads to an increased defect size on the direct postoperative CT scan but restores at 1-year follow-up. Only fourteen of the 58 OCDs were filled up completely, but no differences were found between the clinical outcomes and defect healing at 1-year follow-up. LEVEL OF EVIDENCE: IV.

A rare case of adductor longus muscle rupture.

An adductor longus muscle rupture is a rare injury. This case report describes a 32-year-old patient with an adductor longus rupture. The trauma mechanism was a hyperabduction movement during a soccer game. Nonoperative treatment was initiated. After a follow-up of 4 years, the patient was without pain but a small swelling was still visible. This report describes the anatomy, pathophysiology, and evidence-based treatment of adductor longus rupture.

Pain experience and functional outcome of inpatient versus outpatient anterior cruciate ligament reconstruction, an equivalence randomized controlled trial with 12 months follow-up.

BACKGROUND: Arthroscopic reconstruction of the anterior cruciate ligament (ACL) has traditionally been performed in an inpatient setting. Outpatient treatment may offer the advantages of cost reduction and higher patient satisfaction. HYPOTHESIS/PURPOSE: We investigated whether ACL reconstruction in an outpatient setting is equally safe as in an inpatient setting and whether comparable functional outcomes can be achieved. We hypothesized that the outcomes of outpatient ACL reconstruction result in similar outcomes as inpatient ACL reconstruction. STUDY DESIGN: A prospective randomized controlled trial was conducted at one centre. METHODS: Forty-six patients were randomized to outpatient treatment or a 2-day admission after ACL reconstruction. The functional outcome was evaluated with the Lysholm, Tegner and International Knee Documentation Committee scores. Safety of the procedures was judged according to pain experience and readmission rate. The duration of follow-up was 1 year after ACL reconstruction. The patients were provided with a simple postoperative analgesic protocol. The linear mixed effect model for repeated measures was used for testing the differences between the study groups. RESULTS: No significant differences were found between the study groups in all the outcome measures. No readmissions were recorded related to pain. One complication was recorded in the outpatient group versus three in the inpatient group. CONCLUSION: This study indicates that outpatient care after ACL reconstruction yields comparable functional results and postoperative pain experience as inpatient care and is a safe option. A simple analgesic protocol provides adequate pain relief during the postoperative phase. LEVEL OF EVIDENCE: I.

Validation of the Dutch language version of the Foot and Ankle Outcome Score.

PURPOSE: The aim of this study was to develop a Dutch language version of the Foot and Ankle Outcome Score (FAOS-DLV) and evaluate its measurement properties according to the definitions of the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN). METHODS: After a standard forward-backward translation procedure, the Dutch version of the FAOS was evaluated for reliability and validity in 110 patients with various hind foot and ankle complaints. Reliability was evaluated by calculation of intraclass correlation coefficients (ICC), Cronbach's alpha for internal consistency, and the smallest detectable change (SDC). Construct validity of the FAOS was assessed by calculation of Spearman's correlation coefficients with similar and dissimilar domains of the SF-36 health survey, American Orthopedic Foot and Ankle Society Ankle and Hindfoot Scale, and visual analogue scales for pain and disability. Dimensionality was tested with confirmatory factor analysis. RESULTS: Reliability of the FAOS-DLV was good. The ICC of the subscales ranged from 0.83 to 0.88. The minimal value of Cronbach's alpha was 0.76. The SDC at individual level ranged from 18 to 21 and at group level between 2.1 and 2.5. Construct validity was supported by confirmation of 85 % of the hypothesized correlations. Unidimensionality of the FAOS-DLV domains was moderate. CONCLUSION: The Dutch version of the FAOS seems to have acceptable measurement properties. The questionnaire can be used for functional assessment of patients with varying hindfoot and ankle symptoms. It is, however, more suitable for clinical evaluation at group level than for monitoring a specific patient. LEVEL OF EVIDENCE: Diagnostic study, Level I.

Translation and validation of the German version of the foot and ankle outcome score.

PURPOSE: Outcome assessment is critical in evaluating the efficacy of orthopaedic procedures. The Foot and Ankle Outcome Score (FAOS) is a 42-item questionnaire divided into five subscales, which has been validated in several languages. Germany has no validated outcome score for general foot and ankle pathology. The aim of this study was to develop a German version of the FAOS and to investigate its psychometric properties. MATERIALS AND METHODS: Forward and backward translation was executed according to official guidelines. The final version of the FAOS was investigated in 150 patients with various foot and ankle disorders. All patients completed the FAOS, Short Form-36, numeric rating scales for pain and disability, and the Hannover questionnaire. The FAOS was re-administered after 1 week. Test-retest reliability, internal consistency, minimal detectable change, construct validity, and floor and ceiling effects were analyzed. RESULTS: Test-retest reliability and internal consistency of each subscale were excellent (intraclass correlation coefficient, 0.88-0.95; Cronbach's α, 0.94-0.98). The minimal detectable changes of each subscale were 17.1-20.8 at the individual level and 2.0-2.4 at group level. There were moderate to strong correlations between FAOS subscales and physical outcomes and low to moderate correlations between FAOS subscales and mental outcomes. Floor and ceiling effects were not present. CONCLUSION: The German version of the FAOS is a reliable and valid instrument for use in foot and ankle patients.

Treatment of osteochondral defects of the talus with a metal resurfacing inlay implant after failed previous surgery: a prospective study.

We have evaluated the clinical effectiveness of a metal resurfacing inlay implant for osteochondral defects of the medial talar dome after failed previous surgical treatment. We prospectively studied 20 consecutive patients with a mean age of 38 years (20 to 60), for a mean of three years (2 to 5) post-surgery. There was statistically significant reduction of pain in each of four situations (i.e., rest, walking, stair climbing and running; p ≤ 0.01). The median American Orthopaedic Foot and Ankle Society ankle-hindfoot score improved from 62 (interquartile range (IQR) 46 to 72) pre-operatively to 87 (IQR 75 to 95) at final follow-up (p < 0.001). The Foot and Ankle Outcome Score improved on all subscales (p ≤ 0.03). The mean Short-Form 36 physical component scale improved from 36 (23 to 50) pre-operatively to 45 (29 to 55) at final follow-up (p = 0.001); the mental component scale did not change significantly. On radiographs, progressive degenerative changes of the opposing tibial plafond were observed in two patients. One patient required additional surgery for the osteochondral defect. This study shows that a metal implant is a promising treatment for osteochondral defects of the medial talar dome after failed previous surgery.

Demineralized bone matrix and platelet-rich plasma do not improve healing of osteochondral defects of the talus: an experimental goat study.

OBJECTIVE: The purpose of this study was to evaluate the effectiveness of demineralized bone matrix (DBM) with and without platelet-rich plasma (PRP) in the treatment of osteochondral defects (OCDs) of the talus. We hypothesized that treatment with DBM would result in more bone formation than no treatment in control OCDs, and that PRP would further enhance the regenerative capacity of DBM. METHOD: A standardized 6-mm OCD was created in each talus of 16 adult goats. According to a randomization scheme, one OCD of each goat was treated with allogeneic DBM hydrated with normal saline (n = 8) or hydrated with autologous PRP (n = 8). The contralateral OCD (n = 16) served as control. After 24 weeks, the animals were euthanized and the tali excised. Various outcome parameters were analyzed with use of macroscopic evaluation, micro-computed tomography (µCT), histology, histomorphometry, and fluorescence microscopy. RESULTS: None of the analyses revealed statistically significant differences between the groups for any of the parameters analyzed in any volume of interest. For example, the mean bone volume fraction (BV/TV) of the defect, as measured by µCT, was 0.56 (95% confidence interval [CI], 0.44-0.68) for DBM hydrated with normal saline and 0.52 (95% CI, 0.40-0.65) for DBM hydrated with PRP, compared to 0.53 (95% CI, 0.45-0.61) and 0.54 (95% CI, 0.44-0.64) for the internal controls, respectively (P > 0.05). CONCLUSION: In contrast to our hypotheses, no beneficial treatment effect of DBM with or without PRP was found for OCDs of the caprine talus.

The role of the fibula in varus and valgus deformity of the tibia: a biomechanical study.

It has been suggested that a supramalleolar osteotomy can return the load distribution in the ankle joint to normal. However, due to the lack of biomechanical data, this supposition remains empirical. The purpose of this biomechanical study was to determine the effect of simulated supramalleolar varus and valgus alignment on the tibiotalar joint pressure, in order to investigate its relationship to the development of osteoarthritis. We also wished to establish the rationale behind corrective osteotomy of the distal tibia. We studied 17 cadaveric lower legs and quantified the changes in pressure and force transfer across the tibiotalar joint for various degrees of varus and valgus deformity in the supramalleolar area. We assumed that a supramalleolar osteotomy which created a varus deformity of the ankle would result in medial overload of the tibiotalar joint. Similarly, we thought that creating a supramalleolar valgus deformity would cause a shift in contact towards the lateral side of the tibiotalar joint. The opposite was observed. The restricting role of the fibula was revealed by carrying out an osteotomy directly above the syndesmosis. In end-stage ankle osteoarthritis with either a valgus or varus deformity, the role of the fibula should be appreciated and its effect addressed where appropriate.

Treatment of osteochondral defects of the talus.

This review article provides a current concepts overview of osteochondral defects of the talus, with special emphasis on treatment options, their indications and future developments. Osteochondral defects of the talar dome are mostly caused by a traumatic event. They may lead to deep ankle pain on weight-bearing, prolonged swelling, diminished range of motion and synovitis. Plain radiographs may disclose the lesion. For further diagnostic evaluation, computed tomography (CT) and magnetic resonance imaging (MRI) have demonstrated similar accuracy. Computed tomography is preferred for preoperative planning. Treatment options are diverse and up to the present there is no consensus. Based on the current literature, we present a treatment algorithm that is mainly guided by the size of the lesion. Asymptomatic or low-symptomatic lesions are treated nonoperatively. The primary surgical treatment of defects up to 15 mm in diameter consists of arthroscopic debridement and bone marrow stimulation. For large cystic talar lesions, retrograde drilling combined with a bone graft is an important alternative. In adolescents or in (sub)acute situations, in which the fragment is 15 mm or larger, fixation of the fragment is preferred. Osteochondral autograft transfer and autologous chondrocyte implantation (ACI), with or without a cancellous bone graft, are recommended for secondary cases as well as large lesions.

The progenitor cell inhibition assay to measure the anti-leukemic reactivity of T cell clones against acute and chronic myeloid leukemia.

Adoptive immunotherapy with donor T lymphocytes may be used as a treatment for relapsed leukemia after allogeneic hematopoietic stem cell transplantation (SCT). In vitro selected and expanded anti-leukemic T cells may be more effective in inducing a response in vivo. To identify the anti-leukemic reactivity of in vitro generated T cells, standard target cell read-out assays like the 51Cr-release assay are not always appropriate. We developed an assay in which the ability of T cells to antigen specifically inhibit the in vitro growth of leukemic progenitor cells in the presence of cytokines can be measured. This assay allows the evaluation of the cytolytic or suppressive potential of leukemia reactive T cells for prolonged periods of time. The assay is based on inhibition of [3H]thymidine incorporation by the leukemic progenitor cells induced by multiple hematopoietic growth factors. T cell clones with a known specificity were used to compare the analytic potential of the new assay with those of other cytotoxicity assays. Based on the results of the T cell clones, a modification of a limiting dilution assay was developed to identify anti-leukemic allogeneic T cells in HLA identical donor-recipient combinations selected on their ability to inhibit the in vitro growth of CML or AML progenitor cells, to be used for the generation of leukemia-reactive CTL lines for clinical use.

Complete remission of accelerated phase chronic myeloid leukemia by treatment with leukemia-reactive cytotoxic T lymphocytes.

Relapse of chronic myeloid leukemia (CML) in chronic phase after allogeneic stem cell transplantation (SCT) can be successfully treated by donor lymphocyte infusion (DLI). However, relapse of accelerated phase CML, blast crisis, or acute leukemia after allogeneic SCT are resistant to DLI in the majority of cases. In vitro-selected and expanded leukemia-reactive T-cell lines may be more effective in inducing an antileukemic response in vivo. To treat a patient with accelerated phase CML after allogeneic SCT, leukemia-reactive cytotoxic T-lymphocyte (CTL) lines were generated from her HLA-identical donor. Using a modification of a limiting dilution assay, T cells were isolated from the donor, selected based on their ability to inhibit the in vitro growth of CML progenitor cells, and subsequently expanded in vitro to generate CTL lines. Three CTL lines were generated that lysed the leukemic cells from the patient and inhibited the growth of leukemic progenitor cells. The CTL did not react with lymphocytes from donor or recipient and did not affect donor hematopoietic progenitor cells. The 3 leukemia-reactive CTL lines were infused at 5-week intervals at a cumulative dose of 3.2 x 10(9) CTL. Shortly after the third infusion, complete eradication of the leukemic cells was observed, as shown by cytogenetic analysis, fluorescence in situ hybridization, molecular analysis of BCR/ABL-mRNA, and chimerism studies. These results show that in vitro cultured leukemia-reactive CTL lines selected on their ability to inhibit the proliferation of leukemic progenitor cells in vitro can be successfully applied to treat accelerated phase CML after allogeneic SCT.

T cells recognizing leukemic CD34(+) progenitor cells mediate the antileukemic effect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation.

Adoptive immunotherapy with donor lymphocyte infusions (DLI) is an effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. To identify the effector and target cell populations responsible for the elimination of the leukemic cells in vivo we developed an assay to measure the frequency of T lymphocyte precursor cells capable of suppressing leukemic progenitor cells. Target cells in this assay were CML cells that were cultured in the presence of stem cell factor, interleukin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. [3H]thymidine incorporation at day 7 represented the proliferation of the progeny of the CD34(+) CML progenitor cells, and not of the more mature CD34(-) CML cells. Effector cells were mononuclear cells, which were used in a limiting dilution analysis to measure the frequencies of CML progenitor cell-inhibitory lymphocyte precursors (PCILp) in peripheral blood of seven patients before and after DLI for relapsed CML. In the six patients who entered complete remission, a 5- to 100-fold increase of PCILp was found during the clinical response. In the patient with resistant relapse the frequency of PCILp was <10 per ml before and after DLI. Leukemia-reactive helper T lymphocyte precursor frequencies remained unchanged after DLI. A significant increase in cytotoxic T lymphocyte precursor frequency against more mature leukemic cells was found in only two responding patients. These results indicate that T cells specifically directed against CD34(+) CML progenitor cells mediate the antileukemic effect of DLI.

Generation of leukemia-reactive cytotoxic T lymphocytes from HLA-identical donors of patients with chronic myeloid leukemia using modifications of a limiting dilution assay.

Donor leukocyte transfusions (DLT) have an anti-leukemic effect in most patients with a relapse of chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. However, DLT are often complicated by graft-versus-host disease. Selection of donor lymphocytes with a relative specificity for leukemic cells is desirable. The generation of leukemia-reactive cytotoxic T lymphocyte (CTL) responses between HLA-identical donors and patients in bulk cultures showed major variations, and false negative results were observed. In a modification of a limiting dilution analysis (LDA) two-fold serial dilutions of HLA-identical donor mononuclear cells (MNC) were cultured in the presence of CML cells. The anti-leukemic CTL precursor frequencies in these donors varied between <1 and 9 per 106 MNC. HLA-restricted CD4+ or CD8+ lymphocytes as well as MHC non-restricted gammadelta T cells were responsible for the anti-leukemic responses. A positive correlation between cytotoxicity in the various wells after 3, 4 and 5 weeks of culture could be found. The LDA may be superior to bulk cultures in selecting stable immune responses and in separating multiple different anti-leukemic T cell responses in each donor-patient combination.