Cylindromatosis (Cyld) gene mutation in T cells promotes the development of an IL-9-dependent allergic phenotype in experimental asthma.

Cylindromatosis (CYLD) is a ubiquitously expressed deubiquitinating enzyme which removes activating ubiquitin residues from important signaling molecules of the NF-κB pathway. In CYLDex7/8 transgenic mice, a naturally occurring short isoform (sCYLD) is overexpressed in the absence of full length CYLD, leading to excessive NF-κB activity. Herein, we investigated the impact of the CYLDex7/8 mutation selectively in T cells on the development of experimental allergic airway disease induced by sensitization and challenge with ovalbumin. Compared with their wildtype littermates, mice bearing the T cell-specific mutation (CD4+CYLDex7/8) display stronger eosinophilia and mucus production in the lungs and higher IgE serum levels. The reason for these observations is excessive production of T cell-derived IL-9, a cytokine to whom allergy-promoting properties were ascribed. Consequently, blockade of IL-9 in CD4+CYLDex7/8 mice alleviates the development of disease symptoms. Thus, by polarization of the T cell cytokine response, sCYLD can favor the development of allergic airway disease.

Single-agent therapy with sorafenib or 5-FU is equally effective in human colorectal cancer xenograft--no benefit of combination therapy.

BACKGROUND: We initiated this preclinical study in order to analyze the impact of sorafenib single treatment versus combination treatment in human colorectal cancer. METHODS: The effect of increasing sorafenib doses on proliferation, apoptosis, migration, and activation of signal cascades was analyzed in vitro. The effect of sorafenib single treatment versus 5-fluorouracil (5-FU) single treatment and combination therapy on in vivo proliferation and target cytokine receptor/ligand expression was analyzed in a human colon cancer xenograft mouse model using HT29 tumor cells. RESULTS: In vitro, SW480 and HT29 cell lines were sensitive to sorafenib, as compared to Caco2 and SW620 cell lines, independent of the mutation status of K-ras, Raf, PTEN, or PI3K. The effect on migration was marginal, but distinct differences in caspases activation were seen. Combination strategies were beneficial in some settings (sorafenib + 5-FU; irinotecan) and disadvantageous in others (sorafenib + oxaliplatin), depending on the chemotherapeutic drug and cell line chosen. Sensitive cell lines revealed a downregulation of AKT and had a weak expression level of GADD45ß. In resistant cell lines, pp53 and GADD45ß levels decreased upon sorafenib exposure. In vivo, the combination treatment of sorafenib and 5-FU was equally effective as the respective monotherapy concerning tumor proliferation. Interestingly, treatment with either sorafenib or 5-FU resulted in a significant decrease of VEGFR1 and PDGFRß expression intensity. CONCLUSIONS: In colorectal cancer, a sensitivity towards sorafenib exists, which seems similarly effective as a 5-FU monotherapy. A combination therapy, in contrast, does not show any additional effect.

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.

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.

PDGFRalpha/beta expression correlates with the metastatic behavior of human colorectal cancer: a possible rationale for a molecular targeting strategy.

As new multi-target tyrosine kinase inhibitors are emerging in the therapy of various malignancies, our aim was to define the co-expression pattern of receptor-tyrosine-kinase platelet-derived growth factor receptors alpha and beta (PDGFRalpha/beta) in human colorectal cancer. The co-expression pattern of PDGFRalpha/beta was analyzed by RT-PCR in 99 histologically confirmed human colorectal carcinomas and five colorectal cancer cell lines. In addition, immunohistochemical (IHC) staining was applied for confirmation of expression and analysis of receptor tyrosine kinase (RTK) localisation. The colorectal cancer cell lines that were analysed revealed varying expression intensities of PDGFRalpha and PDGFRbeta. The majority of human colorectal cancer specimens revealed a PDGFRalpha (83%) or PDGFRbeta (60%) expression. While PDGFRalpha showed a predominantly cytoplasmic staining in tumor cells as well as in stromal pericytes, PDGFRbeta was restricted to stromal pericytes only. Furthermore, PDGFRalpha expression significantly correlated with lymph node metastasis (P=0.0082) and advanced UICC stages III/IV (P=0.018) in older patients (P=0.043). PDGFRbeta expression only revealed a trend towards lymphatic dissemination (P=0.099). Co-expression of PDGFRalpha/beta occurred in 57% of the colorectal cancer samples, whereas another 29% of the samples depicted mono-expression of PDGFRalpha or PDGFRbeta. Notably, PDGFRalpha/beta expression significantly correlated with lymphatic metastasis (P=0.007) and advanced UICC stages III/IV (P=0.017) in older patients (P=0.03). In summary, our results revealed that PDGFRalpha/beta expression significantly correlates with lymphatic dissemination and therefore encourages application of PDGFRalpha/beta RTK-inhibitors within a combination therapy.

Xenograft models for undifferentiated pleomorphic sarcoma not otherwise specified are essential for preclinical testing of therapeutic agents.

Undifferentiated pleomorphic sarcoma not otherwise specified belongs to the heterogeneous group of soft tissue tumors. It is preferentially located in the upper and lower extremities of the body, and surgical resection remains the only curative treatment. Preclinical animal models are crucial to improve the development of novel chemotherapeutic agents for the treatment of undifferentiated pleomorphic sarcoma. However, this approach has been hampered by the lack of reproducible animal models. The present study established two xenograft animal models generated from stable non-clonal cell cultures, and investigated the difference in chemotherapeutic effects on tumor growth between undifferentiated pleomorphic sarcoma in vivo and in vitro. The cell cultures were generated from freshly isolated tumor tissues of two patients with undifferentiated pleomorphic sarcoma. For the in vivo analysis, these cells were injected subcutaneously into immunodeficient mice. The mice were monitored for tumor appearance and treated with the most common or innovative chemotherapeutic agents available to date. Furthermore, the same drugs were administered to in vitro cell cultures. The most effective tumor growth inhibition in vitro was observed with doxorubicin and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA), also known as vorinostat. In the in vivo xenograft mouse model, the combination of doxorubicin and the tyrosine kinase inhibitor pazopanib induced a significant tumor reduction. By contrast, treatment with vorinostat did not reduce the tumor growth. Taken together, the results obtained from drug testing in vitro differed significantly from the in vivo results. Therefore, the novel and reproducible xenograft animal model established in the present study demonstrated that in vivo models are required to test potential chemotherapeutic agents for the treatment of undifferentiated pleomorphic sarcoma prior to clinical use, since animal models are more similar to humans, compared with in vitro cell cultures.

Analysis of the expression of SDF-1 splicing variants in human colorectal cancer and normal mucosa tissues.

C-X-C motif chemokine ligand 12 (CXCL12), also termed stromal cell-derived factor-1 (SDF-1) is a small protein 8-14 kDa in length that is expressed as six isoforms, consisting of SDF-1α, SDF-1ß, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1θ. All six isoforms are encoded by the single CXCL12 gene on chromosome 10. This gene regulates leukocyte trafficking and is variably expressed in a number of normal and cancer tissues. The potential role of the novel CXCL12 splice variants as components of the CXCR4 axis in cancer development is not fully understood. The present study aimed to analyze the expression profile of the various SDF-1 isoforms and SDF-1 polymorphisms, and the association with the clinicopathological features and overall survival of patients with colorectal cancer (CRC). SDF-1 polymorphism analysis was performed using restriction fragment length polymorphism (RFLP) analysis in 73 histologically confirmed human CRC tissue samples at various stages of disease. The expression pattern of the SDF-1 isoforms was analyzed by reverse transcription-polymerase chain reaction in 40 histologically confirmed human CRC tissue samples obtained at various stages of disease, as well as in matched adjacent normal mucosa samples. The presence of the CXCL12 gene polymorphism rs1801157 demonstrated an association with local progression of the primary tumor, as indicated by the T stage. The frequency of the GG genotype was slightly increased in patients with stage 3 and 4 tumors (78.0%) compared with the incidence of the GA/AA genotype (69.5%; P=0.067). The expression of SDF-1ß was associated with the presence of metastases (P=0.0656) and the expression of SDF-1γ was significantly associated with tumor size (P=0.0423). The present study is the first to analyze the association between the expression profile of the chemokine CXCL12 splice variants in human CRC tissues and their clinical relevance. The present results reveal that the CXCL12 G801A polymorphism is a low-penetrance risk factor for the development of CRC, and was associated with the T stage. All six isoforms of SDF-1 were expressed in CRC tissues. The expression of SDF-1ß was found to be associated with metastases and SDF-1γ appears to be a possible tumor marker for local tumor progression.

Last line therapy with sorafenib in colorectal cancer: A retrospective analysis.

AIM: To analyze the efficacy of last line sorafenib treatment in colorectal cancer patients. METHODS: All patients receiving chemotherapy for colorectal cancer in the outpatient clinic of the University of Mainz since 2006 were retrospectively analyzed for last line sorafenib exposure. Charts of identified patients were analyzed for clinic-pathological parameters, like data on gender, age, date of initial diagnosis, UICC stage, number and kind of the pre-therapies, therapy start and end of sorafenib, sorafenib mediated treatment cessation, side effects, response rates, time to progression and overall survival. RESULTS: Ten patients with a median of 3.0 prior chemotherapy lines had received a last line sorafenib therapy either alone (10%) or in combination with 5-fluorouracil derivates (90%). All patients suffered from colorectal cancer stage UICC 4 and were routinely seen in 2-wk intervals in the oncology outpatient clinic. Median duration of treatment was 142.0 d. At 8 wk 80% of patients showed stable disease but we did not observe any remissions. Median time to progression was 140.5 d (4.7 mo), while median overall survival reached 176.5 d. One patient ceased treatment due to side effects. Reason for treatment stop was bleeding complication in one case and non-specified sorafenib intolerance in another case. Due to the retrospective approach we did not further quantify side effects. CONCLUSION: This retrospective analysis encourages further investigation of sorafenib in colorectal cancer last line therapy.

HDAC inhibitor-loaded bone cement for advanced local treatment of osteosarcoma and chondrosarcoma.

UNLABELLED: The treatment of osteosarcoma, especially wide resection, is challenging. An additional local drug therapy after resection using anti-neoplastic bone cement (Polymethylmethacrylate (PMMA)) could help improve the outcome of therapy. In this study, we evaluated the effects of PMMA loaded with valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) on the cell activity of a SaOs-2 cell culture, as well as the elution rate of the drugs out of the bone cement. MATERIALS AND METHODS: In our experiments, we used the SaOs-2 osteosarcoma and the SW1353 chondrosarcoma cell line. Bone cement clots (5 g) were prepared and loaded with different drug concentrations of VPA (25 mg and 50 mg) and SAHA (1 mg, 2.5 mg and 5 mg). Two control groups were established, one with a native cement clot, the other with human mesenchymal stem cells, in order to evaluate toxicity on non tumor-cells. Cell activity was measured using an Alamar Blue assay on days 1, 2, 3, 4 and 7. The cement clots were additionally examined in a material testing unit for biomechanical and structural changes. RESULTS: Tumor cells showed a significant and complete reduction of activity under therapy with VPA and SAHA. Drug release of VPA was extensive between days 0 and 3 and decreased progressively to day 7. Cumulative drug concentration in the medium continuously increased. Biomechanical testing of the cement clots showed no differences in stability and architecture compared to the control group. SaOs-2 and SW1353 cells with medium from native cement clots without drug therapy presented a cell activity of 100% in all groups and during all measurements. Human mesenchymal stem cells were not significantly affected during therapy with VPA and low concentrations of SAHA. In contrast, cell activity of human mesenchymal stem cells was significantly reduced under therapy with higher concentrations of SAHA, with an approximately linear decrease between days 0-3 and a rapidly decreasing activity between days 4-7. CONCLUSION: A local cytotoxic therapy in the treatment of osteosarcoma and chondrosarcoma might improve the rate of metastasis and survival of patients. Our results present an encouraging approach to loading PMMA with anti-neoplastic drugs.

Mechanisms of cyclic nucleotide phosphodiesterases in modulating T cell responses in murine graft-versus-host disease.

Graft-versus-host disease (GvHD) is a key contributor to the morbidity and mortality after allogeneic hematopoetic stem cell transplantation (HSCT). Regulatory Foxp3(+) CD4(+) T cells (Treg) suppress conventional T cell activation and can control GvHD. In our previous work, we demonstrate that a basic mechanism of Treg mediated suppression occurs by the transfer of cyclic adenosine monophosphate (cAMP) to responder cells. Whether this mechanism is relevant for Treg mediated suppression of GvHD is currently unknown. To address this question, bone marrow and T cells from C57BL/6 mice were transferred into lethally irradiated BALB/c recipients, and the course of GvHD and survival were monitored. Transplanted recipients developed severe GvHD that was strongly ameliorated by the transfer of donor Treg cells. Towards the underlying mechanisms, in vitro studies revealed that Treg communicated with DCs via gap junctions, resulting in functional inactivation of DC by a metabolic pathway involving cAMP that is modulated by the phosphodiesterase (PDE) 4 inhibitor rolipram. PDE2 or PDE3 inhibitors as well as rolipram suppressed allogeneic T cell activation, indirectly by enhancing Treg mediated suppression of DC activation and directly by inhibiting responder T cell proliferation. In line with this, we observed a cooperative suppression of GvHD upon Treg transfer and additional rolipram treatment. In conclusion, we propose that an important pathway of Treg mediated control of GvHD is based on a cAMP dependent mechanism. These data provide the basis for future concepts to manipulate allogeneic T cell responses to prevent GvHD.

Cetuximab-induced skin exanthema: prophylactic and reactive skin therapy are equally effective.

PURPOSE: Treatment with cetuximab is accompanied by the development of an acneiform follicular skin exanthema in more than 80 % of patients. Severe exanthema (grade III/IV) develops in about 9-19 % of patients with the necessity of cetuximab dose reduction or cessation. METHODS: The study presented was a retrospective analysis of 50 gastrointestinal cancer patients treated with cetuximab in combination with either FOLFIRI or FOLFOX. One cohort of 15 patients received an in-house reactive skin protocol upon development of an exanthema. A second cohort of 15 patients received a skin prophylaxis starting with the first dose of cetuximab before clinical signs of toxicity. A third historic group of 20 patients had received no skin prophylaxis or reactive treatment. RESULTS: 19/20 patients of the historic group developed a skin exanthema. Grade III/IV exanthema was observed six times. Forty percent discontinued cetuximab therapy. The average time to exanthema onset was 14.7 days. Applying the reactive skin protocol after the first occurrence of an exanthema, the exanthema was downgraded as follows: No patients developed grade IV° exanthema, and two patients developed a grade II/III exanthema. In the majority of cases, the reactive skin protocol controlled the exanthema (grade 0-I°). No dose reductions in cetuximab were necessary. Applying the prophylactic skin protocol starting at the beginning of cetuximab application was not superior to the reactive skin protocol. CONCLUSIONS: Cetuximab-induced skin exanthema can be coped with a reactive protocol equally effective as compared to a prophylactic skin treatment. A prospective study with higher patient numbers is planned.

Combination therapies with oxaliplatin and oral capecitabine or intravenous 5-FU show similar toxicity profiles in gastrointestinal carcinoma patients if hand-food syndrome prophylaxis is performed continuously.

The use of anticancer drugs in palliative settings is often limited by their severe toxic effects. In gastrointestinal carcinomas the 5-fluorouracil-based palliative regimen FOLFOX-4 is often preferred to the equally effective, but more convenient oral capecitabine-based regimen XELOX. This preference is mainly based on the fact that the highly effective oral agent capecitabine induces hand-foot syndrome (HFS). In this study, we investigated whether the continuous administration of skin prophylaxis (10% urea, panthenol, bisabolol, vitamin A, C and E) is capable of protecting against capecitabine-induced HFS and allowing a more convenient oral therapeutic option. In this retrospective analysis, the toxicity profiles, according to NCI CTCAE 3.0 criteria, of 54 patients with gastrointestinal cancer who received either XELOX (34 patients) or FOLFOX-4 (20 patients) were compared using Fisher tests. The treatment protocols that were compared, herein, did not differ significantly in the majority of the analyzed items, with the exception of increased nausea (XELOX-70), fatigue (XELOX-130) and tumor pain (XELOX-70 and XELOX-130). No significant differences were observed among the various groups with regard to emesis, diarrhea, mucositis, exanthema, alopecia, loss of weight and the incidence of infections. In particular, no significant differences in toxicity levels occurred in terms of dose, and HFS was limited if skin prophylaxis was performed continuously. XELOX-based palliative regimens provide an equally effective and comparably toxic therapeutic alternative to FOLFOX-4 if HFS prophylaxis is performed continuously. Since the oral administration of capecitabine is a more convenient method of application, it provides patients with a quality of life-preserving therapeutic alternative.

Liver transplanted patients with preoperative autoimmune hepatitis and immunological disorders are at increased risk for Post-Transplant Lymphoproliferative Disease (PTLD).

BACKGROUND: Long term immunosuppression and therapy of acute rejections result in a 20-120-fold increased risk to develop Non Hodgkin lymphoma (NHL). Since immunosuppressive therapy and immunological disorders are major risk factors for the development of NHL in the non-transplant population we aimed to analyze risk factors for PTLD in our cohort of liver transplanted (LT) patients. METHODS: We analyzed retrospectively 431 patients liver transplanted between 1998 and 2008. RESULTS: PTLD was diagnosed in eleven of 431 patients (2.6%). PTLD, especially late PTLD, was significantly more frequent in patients who received steroids before LT (Kaplan-Meier: p<0.001). Moreover PTLD in immunocompromised patients with preoperative steroid treatment occurred at a significantly younger age (49.5+/-4.7 years) compared to patients without steroids (60.6+/-5.1 years; p=0.006). Multivariate analysis revealed pretransplant steroid treatment and liver transplantation for autoimmune hepatitis as main risk factors for the development of PTLD after liver transplantation (p<0.001). CONCLUSION: Liver transplanted patients who received steroids before LT due to immunological disorders and patients with autoimmune hepatitis seem to be at particular high risk to develop PTLD. Prospective cohort studies including immunoepidemiologic investigations of abnormalities of cellular, humoral and innate immunity should be carried out to identify predictive factors and patients at risk.

Strong expression of chemokine receptor CXCR4 by renal cell carcinoma correlates with advanced disease.

Diverse chemokines and their receptors have been associated with tumor growth, tumor dissemination, and local immune escape. In different tumor entities, the level of chemokine receptor CXCR4 expression has been linked with tumor progression and decreased survival. The aim of this study was to evaluate the influence of CXCR4 expression on the progression of human renal cell carcinoma. CXCR4 expression of renal cell carcinoma was assessed by immunohistochemistry in 113 patients. Intensity of CXCR4 expression was correlated with both tumor and patient characteristics. Human renal cell carcinoma revealed variable intensities of CXCR4 expression. Strong CXCR4 expression of renal cell carcinoma was significantly associated with advanced T-status (P = .039), tumor dedifferentiation (P = .0005), and low hemoglobin (P = .039). In summary, strong CXCR4 expression was significantly associated with advanced dedifferentiated renal cell carcinoma.

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.

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.