Download pose: The essential role of CD4 T cells as helpers of anticancer immunity is indisputable. Little is n, however, about their capacity to serve as effector cells in cancer treatment. Therefore, we explored ficacy of immunotherapy with sole CD4 cytotoxic human T cells directed at a hematopoieticted minor histocompatibility antigen (mHag). erimental Design: In macrophage-depleted Rag2γc mice, which were also devoid of T, B, and l killer cells, mHag-specific native T cells or tetanus toxoid (TT)-specific T cells transduced with the -specific T-cell receptor (TCR) were injected to treat full-blown mHag human multiple myeloma s. ults: mHag-specific antitumor responses were achieved after injection of native or mHag-TCRuced T cells. Although the therapy completely eradicated the primary tumors in the bone marrow, d to control extramedullary relapses, even after repeated T-cell injections. Detailed analyses ruled Hag or MHC downregulation as mechanisms of extramedullary tumor escape. Impaired T-cell al in vivo or defective homing to the tumor site were also ruled out as mechanisms behind extrallary relapses, because injections of TT-loaded antigen presenting cells could facilitate homing of erm surviving T cells to s.c. tumor sites. Moreover, intratumoral treatment of extramedullary tumors AB11 was also ineffective. clusions: Taken together, these results for the first time show the feasibility of immunotherapy mary bone marrow tumors with sole CD4 human T cells directed to a tumor-associated mHag. edullary relapses, probably due to microenvironment-dependent inhibitory mechanisms, remain Extram a challenging issue towards effective cellular immunotherapy of hematologic malignancies. Clin Cancer Res;

Early immune reconstitution after allogeneic-haematopoietic stem cell transplantation (HSCT) is important for controlling infectious complications. We compared T-, B- and NK-cell reconstitution during the first 100 days after bone marrow (matched sibling: id-SIB, unrelated bone marrow: u-BM) or cord blood (u-CB) HSCT in a single-center prospective study among pediatric patients. Between 2006 and 2008, 103 patients were included with a median age of 5 years (range 0-21); 33 recipients received id-SIB, 33 u-BM and 37 u-CB grafts. All patients received myelo-ablative conditioning and in the unrelated donor setting thymoglobulin (10 mg/kg) was added from 4 to 2 days before SCT. The number of CD3+ cells in the graft graft was for uCB 8∗10e6 (range 8∗10e5 – 4∗10e7)/kg, for id-SIB 4∗10e7 (range 1 – 9∗10e7)/kg and for u-BM 5 ∗10e7 (range 1∗10e7 – 5∗10e8)/kg. Statistical analysis was performed by Kruskal-Wallis rank sum tests and Wilcoxon tests with continuity corrections. Median follow up was 16 months (range 1-28); overall survival was 73%. Probability of neutrophil at day 60 did not differ among id-SIB u-BM and u-CB recipients (median 23 days, range 3-60 after HSCT, p=0.092). Overall T-cell (CD3+) reconstitution (first 100 days) was similar between the 3 groups, but CD8+T-cell numbers were higher after id-SIB- and u-BM compared to u-CB HSCT (155 and 309 versus 52 CD8+T-cells/uL, p=0.002). Interestingly, NK-cell and B-cell numbers were significantly higher in u-CB compared to id-SIB (p=0.018 and p=0.0003, resp.) and u-BM recipients (p=0.036 and p=0.0003, resp.) within the first 100 days. On the long term, in u-CB and id-SIB recipients higher ratios of nave CD4+ and CD8 T-cells were found (u-CB: 62% and 87%, id-SIB: 55% and 57%) compared to u-BM recipients (30% and 17%) at 1 year after HSCT (p=0.050 and p=0.029). Higher NK- and B-cell numbers early after HSCT indicate a better proliferative capacity of these u-CB stem cells compared to id-SIB and u-BM stem cells. We speculate the observed delayed T-cell reconstitution after u-CB HSCT is caused by a deeper in vivo depletion using the same dose of thymoglobuline in a setting of u-CB grafting where the T-cell dose is 1 log lower than an u-BM graft. Insight in the early immune reconstitution is essential for the future development of immune-mediated therapies and the development of optimal graft specific conditioning regimens to prevent prolonged post-HSCT lymphopenia and the associated viral complications.

Objective: Haematopoietic stem cell transplantation (HSCT) is frequently complicated by early Human herpesvirus type 6 (HHV6) reactivation and is associated with poor survival and severe acute Graft-versus-host-disease (aGvHD). We hypothesized that HHV6 may be a trigger for immunedysregulation, resulting in alloreactivity. We investigated total T-cell numbers and HHV6-specific Interferon-γ (IFNγ) producing T-cells in children with or without HHV6-reactivation after HSCT using a newly developed enzyme-linked immunospot (ELISPOT) technique. Methods: Prospectively, HHV6, Cytomegalovirus, Adenovirus and Epstein Barr virus DNA-loads were weekly monitored by quantitative realtime-PCR and clinical data were collected. HHV6 reactivation was defined as HHV6 DNA-load >250cp/mL. T-cell reconstitution was prospectively measured every other week by immunophenotyping (markers CD3, CD4 and CD8). Numbers of IFNγ-producing T-cells in PBMCs were retrospectively determined by ELISPOT after overnight stimulation with HHV6-virus lysate (ABI, Columbia, Maryland, USA) 2 months after HSCT. Results: Twenty-one HSCT patients were analyzed (median age 4.4 years; range 1–16.5yrs). Within the first two months, 13/21 (62%) patients developed HHV6 reactivation; median time of reactivation was 14 (range 1–41) days. The development of other virusreactivations did not differ between the two groups; 4/13 versus 3/8 respectively. The median number of IFNγ-producing specific HHV6 T-cells 2 months after HSCT was significantly increased in the patients with HHV6-reactivation; 40 (0–362) versus 0 (0–25) specific T-cells per million PBMCs (p = 0.006). Additionally, the median CD3+ T-cell numbers were significantly increased in these patients; 393 (32–5514) versus 93 (0–641) T-cells/uL (p = 0.03), including median CD8+ T-cells; 79% (6–87 %) versus 33% (0–83%) (p = 0.03). Conclusions: Patients with HHV6 reactivation had significantly higher numbers of IFNγ-producing HHV6-specific T-cells and more CD3+T-cells/uL, among which mainly CD8+T-cells. Given the association of HHV6 reactivation and aGvHD, these T-cells may be alloreactive.

Preclinical testing of new therapeutical strategies for the treatment of multiple myeloma (MM) requires animal models that closely resemble human disease and allow quantitative evaluation of the applied therapy. Models that meet both requirements have thus far not been described. Here we present a novel in vivo MM model by engraftment of MM U266 or RPMI-8226/S cells, both of human origin, into RAG2γc double knock-out mice. These mice are totally immune deficient because they lack T-, B and NK cells and the mice easily accept human cells (van Rijn et al., Blood 2003, Rozemuller et al., 2004). After intravenous injection of 2x106 MM cells engraftment and outgrowth occurred in all mice but was limited to the bone marrow compartment only. Flow cytometry (FCM) confirmed the presence of human CD45/38/138 positive MM cells in femur, spine, tibia and sternum bone specimens. Infiltration into other organs was not observed. In a next step MM cells were stably transduced using a retroviral vector encoding both the Green Fluorescent Protein (GFP) and firefly Luciferase (fLuc) marker genes. Technical advances in recent years in optical imaging by Bioluminescence Imaging (BLI) techniques allow visualization and quantification of bioluminescent light by detecting photons that are transmitted through mammalian tissue. When luciferase converts the substrate luciferin, photons are emitted that can be registered by using sensitive CCCD cameras. The absolute number of photons that are produced correlates, in our application, with local tumor mass. Mice were injected i.v. with 2x106 GFP-fLuc transduced MM cells (U266 or RPMI8226/S) and imaged weekly using BLI. Within 2 weeks after injection significant BLI signals were detectable. Per mouse 5-10 foci showed luciferase activity, predominantly in the pelvic region, skull, limbs, sternum, ribs and the spine. This low frequency of engraftment is in line with earlier reports on RPMI8226/S (Mitsiades et al., Cancer Res 2003). At 9 weeks the first mice developed hind leg paralysis which could be attributed to tumor associated spinal lesions. After 12 weeks the last mouse was sacrificed. BLI revealed that the intensity of light production at the various sites of tumor growth within individual mice as well as between mice showed a similar increase. This reflects an increase in tumor mass. Quantitative analysis of subsequent BLI images allowed construction of tumor growth curves of the total tumor mass per mouse as well as for the individual foci of MM growth in individual mice. We typically observed exponential growth, with growth curves running parallel with an average population doubling time of approximately 4–5 days. The range in which tumor growth could be monitored (and as a consequence also the response to treatment) spans 3–4 decades. In contrast with previously reported murine models for human MM where -next to bone marrow homing- also extra-skeletal tumors were observed our model almost exclusively shows homing of MM cells to the BM and is therefore more consistent with the clinical manifestation in myeloma patients. The major advantage of the model is the option for quantitative evaluation of the effect of a given treatment on the tumorload. Currently we are studying the efficacy of newly developed geranyl-geranyl-transferase inhibitors (GGTI).

The cumulative incidence of malignant transformation was studied in 88 patients with monoclonal gammopathy of undetermined significance (MGUS) that had a complete prospective follow-up. At a median follow-up of 6.75 years, 10 patients developed multiple myeloma (MM) (11.4%) and 2 developed immunocytoma (2.3%). The cumulative incidence of malignant transformation was 9.1, 21.3, 38 and 48.3% at 5, 10, 15 and 20 years, respectively. In univariate analysis on 102 MGUS patients, M-component level, bone marrow plasma cell percentage and kappa light chain correlated significantly with the development of a malignancy (p=0.0289, 0.0265 and 0.0013, respectively). In multivariate analysis, light chain type of M-component and plasma cell percentage had independent prognostic significance. A high-risk (M-component level > 10 g/l and/or plasma cell percentage > 2%) and a low-risk group ( M-component level < 10 g/l and/or plasma cell percentage < 2%) of MGUS patients was identified, which differed significantly in the cumulative incidence of developing a malignancy (p<0.001 for M-component level and p=0.007 for plasma cell percentage). These results imply that high-risk patients should receive a more frequent follow-up, in comparison to low-risk patients.

Recently the Belgium-Dutch Hematology-Oncology group initiated a multicenter study to evaluate whether myeloma patients treated with intensive chemotherapy benefit from additional peripheral stem cell transplantation. To determine treatment response accurately, we decided to quantitate malignant cells. To test a consensus quantitation strategy, 5 centers independently determined the immunoglobulin heavy chain sequences of patient tumor cells and developed allele-specific oligonucleotides (ASO) and ASO-polymerase chain reaction (PCR). We compared the reproducibility of real-time quantitation with quantitation using limiting dilutions. We distributed DNA samples with a 4-log range of tumor cell concentrations and found average quantitation values deviating 74% and 42% from the input values with real-time PCR (1 center) and limiting dilutions (4 centers), respectively. Within single centers we found an average variation coefficient of 0.74, with limiting dilutions not significantly different from the average 0.82 center-to-center variation coefficient. Within a single center, real-time quantitation proved more reproducible (average variation coefficient, 0.36). Quantification was confirmed in 3 patients during treatment in the protocol. This report shows that real-time PCR or limiting dilution assays can be used for quantitation in a single multicenter trial. We present a consensus strategy that allows an accurate comparison of quantitation data generated in independent centers.

The anti-ICAM-1 monoclonal antibody F10.2 was conjugated to liposomes to target to cells expressing the cell adhesion molecule ICAM-1. We demonstrate that F10.2 immunoliposomes bind to human bronchial epithelial cells (BEAS-2B) and human umbilical vein endothelial cells (HUVEC) in a specific, dose- and time-dependent manner. It appears that the degree of ICAM-1 expression is the limiting factor in the degree of immunoliposome binding to the cells. These results are a first step in the strategy for specific drug delivery to target sites characterised by increased expression of adhesion molecules.