Thai M. Cao

Silencing Human NKG2D, DAP10, and DAP12 Reduces Cytotoxicity of Activated CD8+ T Cells and NK Cells

Human CD8+ T cells activated and expanded by TCR cross-linking and high-dose IL-2 acquire potent cytolytic ability against tumors and are a promising approach for immunotherapy of malignant diseases. We have recently reported that in vitro killing by these activated cells, which share phenotypic and functional characteristics with NK cells, is mediated principally by NKG2D. NKG2D is a surface receptor that is expressed by all NK cells and transmits an activating signal via the DAP10 adaptor molecule. Using stable RNA interference induced by lentiviral transduction, we show that NKG2D is required for cytolysis of tumor cells, including autologous tumor cells from patients with ovarian cancer. We also demonstrated that NKG2D is required for in vivo antitumor activity. Furthermore, both activated and expanded CD8+ T cells and NK cells use DAP10. In addition, direct killing was partially dependent on the DAP12 signaling pathway. This requirement by activated and expanded CD8+ T cells for DAP12, and hence stimulus from a putative DAP12-partnered activating surface receptor, persisted when assayed by anti-NKG2D Ab-mediated redirected cytolysis. These studies demonstrated the importance of NKG2D, DAP10, and DAP12 in human effector cell function.

Pulmonary toxicity syndrome in breast cancer patients undergoing BCNU-containing high-dose chemotherapy and autologous hematopoietic cell transplantation

We performed a retrospective review to investigate pulmonary toxicity syndrome (PTS) in a cohort of breast cancer patients undergoing BCNU-containing high-dose chemotherapy (HDC). Our aim was to characterize presentation, identify risk factors, determine outcome following therapy, and find any association with differences in survival. We reviewed the data of 152 patients with stage II or III or metastatic breast cancer treated with cyclophosphamide 5625 mg/m2, cisplatin 165 mg/m2, and BCNU 600 mg/m2 followed by autologous peripheral blood hematopoietic cell transplantation. During follow-up, PTS was diagnosed when the following criteria were met: (1) presentation with typical clinical symptoms of PTS, (2) an absolute carbon monoxide diffusion capacity (DLCO) decline of 10% compared with pre-HDC DLCO, and (3) no clinical evidence of active pulmonary infection. Patients were then treated with a course of corticosteroid therapy. The incidence of PTS for all 152 patients was 59%, with a median onset at 45 days (range, 21-149 days) post-HDC. The median absolute DLCO decrement was 26% (range, 10%-73%) at diagnosis of PTS. There was no significant correlation between patient age, stage of breast cancer, pre-HDC chemotherapy regimen, pre-HDC chest wall radiotherapy, tobacco use, prior lung disease, or baseline pulmonary function test results and the development of PTS. We did observe an interesting association between PTS and the development of a noncholestatic elevation of transaminases. Of PTS patients treated with prednisone therapy for a median of 105.5 days (range, 44-300 days), 91% achieved resolution of their PTS without pulmonary sequelae. At 3 years, the overall survival (OS) of stage II or III patients who developed PTS was 84% (95% confidence interval [CI], 73%-95%); of metastatic breast cancer patients with PTS, the OS was 58% (95% CI, 38%-78%). These values were not significantly different from those of patients who did not develop PTS (91% [95% confidence interval [CI], 81%-100%] and 53% [95% CI, 32%-74%], respectively). No significant differences in disease-free or event-free survival were observed between patients with and without PTS. The incidence of PTS in breast cancer patients treated with a BCNU-containing HDC regimen can be remarkably high. Treatment with a course of corticosteroid therapy is successful in the vast majority.

Salvage allogeneic hematopoietic cell transplantation with fludarabine and low-dose total body irradiation after rejection of first allografts.

We summarized results in 38 consecutive patients (median age=56 years) with hematologic malignancies (n=35), aplastic anemia (n=2), or renal cell carcinoma (n=1), who underwent salvage hematopoietic cell transplantation (HCT) for allograft rejection. In 14 patients, the original donors were used for salvage HCT, and, in 24 cases, different donors were used. Conditioning for salvage HCT consisted of fludarabine (Flu) and either 3 or 4 Gy total body irradiation (TBI). Sustained engraftment was achieved in 33 patients (87%). Grafts were rejected in 5 patients (13%), 4 of whom had myelofibrosis. With a median follow-up of 2 years (range: 0.3 to 7.8 years), the 2- and 4-year estimated survivals were 49% and 42%, respectively. The 2-year relapse rate and nonrelapse mortality (NRM) were 36% and 24%, respectively. The 2-year cumulative incidences of grades II-IV acute and moderate-severe chronic graft-versus-host disease (aGVHD, cGVHD) were 42% and 41%, respectively. In this cohort, TBI dose, grafts from original versus different donors, related versus unrelated donors, and HCT comorbidity scores did not have an impact on outcomes. We concluded that graft rejection after allogeneic HCT could be overcome by salvage transplantation using conditioning with Flu and low-dose TBI.

Stilbene derivatives that are colchicine site microtubule inhibitors have antileukemic activity and minimal systemic toxicity

Stilbenes are a group of natural compounds with many biological activities. Two highly potent stilbenes, cis‐3,4′,5‐trimethoxy‐3′‐aminostilbene (stilbene 5c) and cis‐3,4′,5‐trimethoxy‐3′‐hydroxystilbene (stilbene 6c) induce G2/M cell‐cycle arrest and leukemic cell death in nanomolarity range without affecting normal bone marrow progenitor cells. The mechanism of stilbenes is mediated by interfering with microtubule polymerization through the colchicine‐binding site. Docking of the stilbenes into tubulin structure confirms that stilbenes fit into the colchicine‐binding pocket. Animal studies show that stilbenes are well tolerated in mice and are capable of inducing more than 50% leukemic cell death by a single dose injection. A 5‐day treatment with low‐dose stilbenes suppresses tumor growth in mice with established tumor xenografts. No major organ damage was detected by histological section. Our results indicate that stilbene 5c is a microtubule‐interfering agent and can be potentially useful in leukemic therapy. Am. J. Hematol., 2008.

Genomic expression profiling of TNF-α-treated BDC2.5 diabetogenic CD4+ T cells

TNF-α plays an important role in immune regulation, inflammation, and autoimmunity. Chronic TNF exposure has been shown to down-modulate T cell responses. In a mouse T cell hybridoma model, TNF attenuated T cell receptor (TCR) signaling. We have confirmed that chronic TNF and anti-TNF exposure suppressed and increased T cell responses, respectively. In adult TCR (BDC2.5) transgenic nonobese diabetic mice, DNA microarray analysis of global gene expression in BDC2.5 CD4+ T cells in response to chronic TNF or anti-TNF exposure showed that genes involved in functional categories including T cell signaling, cell cycle, proliferation, ubiquitination, cytokine synthesis, calcium signaling, and apoptosis were modulated. Genes such as ubiquitin family genes, cytokine inducible Src homology 2-containing genes, cyclin-dependent kinase inhibitors p21, p57, calmodulin family genes (calmodulin-1, -2, and -3) and calcium channel voltage-dependent, N type α1B subunit (CaV2.2) were induced by TNF, whereas Vav2, Rho GTPase-activating protein, calcium channel voltage-dependent, L type α1C subunit (CaV1.2), IL-1 receptor-associated kinase-1 and -2, and IL enhancer binding factor 3 were reduced by TNF. Genes such as CaV1.2 and proliferating cell nuclear antigen, repressed by TNF, were induced by anti-TNF treatment. Further, we showed that chronic TNF exposure impaired NF-κB and adaptor protein 1 transactivation activity, leading to T cell unresponsiveness. Thus, our results present a detailed picture of transcriptional programs affected by chronic TNF exposure and provide candidate target genes that may function to mediate TNF-induced T cell unresponsiveness.

Variable hematopoietic graft rejection and graft-versus-host disease in MHC-matched strains of mice

MHC typing for human hematopoietic cell transplantation (HCT) from unrelated donors is currently performed by using a combination of serologic and molecular techniques. It has been determined that allelic differences in human MHC molecules, revealed by nucleotide sequencing but not by serologic typing, substantially influence graft rejection and graft-versus-host disease, two serious complications of clinical HCT. We studied transplantation of purified hematopoietic stem cells in a series of mouse strains that were matched at the MHC but had different background genes, and we observed striking differences in engraftment resistance and graft-versus-host disease severity, both factors depending on the donor–recipient strain combination. The individual mouse lines studied here were established nearly a century ago, and their MHC types were determined exclusively by serologic techniques. We considered the possibility that serologically silent MHC polymorphisms could account for our observations and, therefore, we performed DNA sequencing of the class I and II MHC alleles of our mouse strains. At each locus, exact homology was found between serologically MHC-matched strains. Our results likely extend to all serologically MHC-matched mouse strains used in modern research and highlight the profound and variable influence that non-MHC genetic determinants can have in dictating outcome after HCT.

T-cell Prolymphocytic Leukemia: Update and Focus on Alemtuzumab (Campath-1H)

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-cell lymphoproliferative disorder. While the etiology of T-PLL is unknown, recent progress in unraveling the molecular basis of leukemogenesis has been substantial and may yield novel therapeutic targets. T-PLL is a distinct disease entity and the diagnosis can be readily made based on characteristic clinical features and laboratory findings. Prior to the appearance of pentostatin and alemtuzumab in clinical protocols, outcome for T-PLL patients was exceedingly poor with median survival measured in months. While the use of alemtuzumab in particular has improved remissions, the disease remains incurable. Future collaborative efforts investigating novel treatment approaches will be crucial to improving survival for patients with this disease.

A chromosome 16 quantitative trait locus regulates allogeneic bone marrow engraftment in nonmyeloablated mice.

Identifying genes that regulate bone marrow (BM) engraftment may reveal molecular targets for overcoming engraftment barriers. To achieve this aim, we applied a forward genetic approach in a mouse model of nonmyeloablative BM transplantation. We evaluated engraftment of allogeneic and syngeneic BM in BALB.K and B10.BR recipients. This allowed us to partition engraftment resistance into its intermediate phenotypes, which are firstly the immune-mediated resistance and secondly the nonimmune rejection of donor BM cells. We observed that BALB.K and B10.BR mice differed with regard to each of these resistance mechanisms, thereby providing evidence that both are under genetic control. We then generated a segregating backcross (n = 200) between the BALB.K and B10.BR strains to analyze for genetic linkage to the allogeneic BM engraftment phenotype using a 127-marker genome scan. This analysis identified a novel quantitative trait locus (QTL) on chromosome 16, termed Bmgr5 (logarithm of odds 6.4, at 11.1 cM). The QTL encodes susceptibility alleles, from the BALB.K strain, that are permissive for allogeneic BM engraftment. Further identification of Bmgr5 genes by positional cloning may reveal new and effective approaches for overcoming BM engraftment obstacles.

Pathways analysis of differential gene expression induced by engrafting doses of total body irradiation for allogeneic bone marrow transplantation in mice

A major challenge in allogeneic bone marrow (BM) transplantation is overcoming engraftment resistance to avoid the clinical problem of graft rejection. Identifying gene pathways that regulate BM engraftment may reveal molecular targets for overcoming engraftment barriers. Previously, we developed a mouse model of BM transplantation that utilizes recipient conditioning with non-myeloablative total body irradiation (TBI). We defined TBI doses that lead to graft rejection, that conversely are permissive for engraftment, and mouse strain variation with regards to the permissive TBI dose. We now report gene expression analysis, using Agilent Mouse 8x60K microarrays, in spleens of mice conditioned with varied TBI doses for correlation to the expected engraftment phenotype. The spleens of mice given engrafting doses of TBI, compared with non-engrafting TBI doses, demonstrated substantially broader gene expression changes, significant at the multiple testing-corrected P <0.05 level and with fold change ≥2. Functional analysis revealed significant enrichment for a down-regulated canonical pathway involving B-cell development. Genes enriched in this pathway suggest that suppressing donor antigen processing and presentation may be pivotal effects conferred by TBI to enable engraftment. Regardless of TBI dose and recipient mouse strain, pervasive genomic changes related to inflammation was observed and reflected by significant enrichment for canonical pathways and association with upstream regulators. These gene expression changes suggest that macrophage and complement pathways may be targeted to overcome engraftment barriers. These exploratory results highlight gene pathways that may be important in mediating BM engraftment resistance.

Identification of a Major Susceptibility Locus for Lethal Graft-versus-Host Disease in MHC-Matched Mice

Graft-vs-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hemopoietic cell transplantation. From a genetic perspective, GVHD is a complex phenotypic trait. Although it is understood that susceptibility results from interacting polymorphisms of genes encoding histocompatibility Ags and immune regulatory molecules, a detailed and integrative understanding of the genetic background underlying GVHD remains lacking. To gain insight regarding these issues, we performed a forward genetic study. A MHC-matched mouse model was used in which irradiated recipient BALB.K and B10.BR mice demonstrate differential susceptibility to lethal GHVD when transplanted using AKR/J donors. Assessment of GVHD in (B10.BR × BALB.K)F1 mice revealed that susceptibility is a dominant trait and conferred by deleterious alleles from the BALB.K strain. To identify the alleles responsible for GVHD susceptibility, a genome-scanning approach was taken using (B10.BR × BALB.K)F1 × B10.BR backcross mice as recipients. A major susceptibility locus, termed the Gvh1 locus, was identified on chromosome 16 using linkage analysis (logarithm of the odds, 9.1). A second locus was found on chromosome 13, named Gvh2, which had additive but protective effects. Further identification of Gvh genes by positional cloning may yield new insight into genetic control mechanisms regulating GVHD and potentially reveal novel approaches for effective GVHD therapy.