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We reported that the clinical efficacy of dendritic cell-based vaccination is strongly associated with immunologic responses in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients. We have now investigated whether postvaccination antibodies from responders recognize novel shared NHL-restricted antigens. Immunohistochemistry and flow cytometry showed that they cross-react with allogeneic B-NHLs at significantly higher levels than their matched prevaccination samples or nonresponders' antibodies. Western blot analysis of DOHH-2 lymphoma proteome revealed a sharp band migrating at approximately 100 to 110 kDa only with postvaccine repertoires from responders. Mass spectrometry identified heat shock protein-105 (HSP105) in that molecular weight interval. Flow cytometry and immunohistochemistry disclosed HSP105 on the cell membrane and in the cytoplasm of B-NHL cell lines and 97 diagnostic specimens. A direct correlation between HSP105 expression and lymphoma aggressiveness was also apparent. Treatment of aggressive human B-NHL cell lines with an anti-HSP105 antibody had no direct effects on cell cycle or apoptosis but significantly reduced the tumor burden in xenotransplanted immunodeficient mice. In vivo antilymphoma activity of HSP105 engagement was associated with a significant local increase of Granzyme B(+) killer cells that very likely contributed to the tumor-restricted necrosis. Our study adds HSP105 to the list of nononcogenes that can be exploited as antilymphoma targets.

Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

Eighteen relapsed patients with measurable indolent non-Hodgkin lymphoma (NHL) were vaccinated with dendritic cells (DCs) loaded with killed autologous tumor cells. Six patients had objective clinical responses including 3 continuous complete responses (CRs) and 3 partial responses (PRs), with a median follow up of 50.5 months. Eight patients had stable disease, whereas 4 had progressive disease. Clinical responses were significantly associated with a reduction in CD4(+)CD25(+)FOXP3(+) regulatory T cells, an increase in CD3(-)CD56(dim)CD16(+) natural killer (NK) cells, and maturation of lymphocytes to the effector memory stage in either postvaccination peripheral blood or tumor specimen samples. In partial responding patients, vaccination significantly boosted the IFN-gamma-producing T-cell response to autologous tumor challenge. In one HLA-A*0201(+) patient who achieved CR, IL-4 release by circulating T cells in response to tumor-specific IgH-encoded peptides was also documented. Immunohistochemical analysis of tumor biopsies using biotin-conjugated autologous serum samples revealed a tumor-restricted humoral response only in the postvaccination serum from responding patients. Collectively these results demonstrate that vaccination with tumor-loaded DCs may induce both T- and B-cell responses and produces clinical benefits in indolent NHL patients with measurable disease. This study is registered with the Istituto Superiore di Sanità: http://www.iss.it with protocol number 7578-PRE 21-801.

The activity of recombinant human growth hormone (rhGH) in enhancing CD34(+) cell mobilization elicited by chemotherapy plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) was evaluated in 16 hard-to-mobilize patients, that is, those achieving a peak of circulating CD34+ cells 10/microL or less, or a collection of CD34(+) cells equal to or less than 2 x 10(6)/kg. Patients who had failed a first mobilization attempt with chemotherapy plus rhG-CSF (5 microg/kg/d) were remobilized with chemotherapy plus rhG-CSF and rhGH (100 microg/kg/d). As compared with rhG-CSF, the combined rhGH/rhG-CSF treatment induced significantly higher (P < or =.05) median peak values for CD34(+) cells/microL (7 versus 29), colony-forming cells (CFCs)/mL (2154 versus 28,510), and long-term culture-initiating cells (LTC-ICs)/mL (25 versus 511). Following rhG-CSF and rhGH/rhG-CSF, the median yields of CD34(+) cells per leukapheresis were 1.1 x 10(6)/kg and 2.3 x 10(6)/kg (P < or =.008), respectively; the median total collections of CD34(+) cells were 1.1 x 10(6)/kg and 6 x 10(6)/kg (P < or =.008), respectively. No specific side effect could be ascribed to rhGH, except a transient hyperglycemia occurring in 2 patients. Reinfusion of rhGH/rhG-CSF-mobilized cells following myeloablative therapy resulted in prompt hematopoietic recovery. In conclusion, our data demonstrate that in poor mobilizers addition of rhGH to rhG-CSF allows the patients to efficiently mobilize and collect CD34(+) cells with maintained functional properties.

Mantle cell lymphoma (MCL) is rarely cured with standard-dose chemotherapy. From January 1997 to February 2000, 28 previously untreated advanced-stage MCL patients younger than 61 years of age were treated at 9 Italian hematologic departments with 3 cycles of standard-dose debulking chemotherapy followed by a high-dose rituximab-supplemented sequence (R-HDS) including intravenous administration of high-dose cyclophosphamide, high-dose cytarabine, high-dose melphalan, and high-dose mitoxantrone plus melphalan. Study end points included toxicity, clinical and molecular response rates, long-term event-free survival (EFS), and overall survival (OS) rates, as well as the ability to harvest tumor-free peripheral blood stem cells. Optimal amounts of polymerase chain reaction-negative (PCR-negative) CD34+ cells were collected from all 20 informative patients. One patient died of toxicity. All 27 patients assessable for response achieved a complete response (CR), of which 24 remain in continuous complete remission (CCR) after a median follow-up of 35 months. Three patients had transient evidence of PCR-detectable disease in the bone marrow. The OS and EFS rates at 54 months were 89% and 79%, respectively. These results compare with the 42% OS rate and the 18% EFS rate observed in 35 age-matched historic controls treated with standard-dose chemotherapy at the participating centers. The use of rituximab in combination with high-dose chemotherapy represents a very effective in vivo purging method. The R-HDS regimen can be safely applied in a multicenter hematology setting and leads to long-term EFS and OS in the majority of patients with an otherwise incurable disease.

CD2(+) T lymphocytes obtained from either the donor of bone marrow stromal cells (BMSCs) or a third party were cultured in mixed lymphocyte reactions (MLRs) with either allogeneic dendritic cells (DCs) or peripheral blood lymphocytes (PBLs). When autologous or allogeneic BMSCs were added back to T cells stimulated by DCs or PBLs, a significant and dose-dependent reduction of T-cell proliferation, ranging from 60% +/- 5% to 98% +/- 1%, was evident. Similarly, addition of BMSCs to T cells stimulated by polyclonal activators resulted in a 65% +/- 5% (P =.0001) suppression of proliferation. BMSC- induced T-cell suppression was still evident when BMSCs were added in culture as late as 5 days after starting of MLRs. BMSC-inhibited T lymphocytes were not apoptotic and efficiently proliferated on restimulation. BMSCs significantly suppressed both CD4(+) and CD8(+) T cells (65% +/- 5%, [P =.0005] and 75% +/- 15% [P =.0005], respectively). Transwell experiments, in which cell-cell contact between BMSCs and effector cells was prevented, resulted in a significant inhibition of T-lymphocyte proliferation, suggesting that soluble factors were involved in this phenomenon. By using neutralizing monoclonal antibodies, transforming growth factor beta1 and hepatocyte growth factor were identified as the mediators of BMSC effects. In conclusion, our data demonstrate that (1) autologous or allogeneic BMSCs strongly suppress T-lymphocyte proliferation, (2) this phenomenon that is triggered by both cellular as well as nonspecific mitogenic stimuli has no immunologic restriction, and (3) T-cell inhibition is not due to induction of apoptosis and is likely due to the production of soluble factors.

Elimination of tumor cells ("purging") from hematopoietic stem cell products is a major goal of bone marrow-supported high-dose cancer chemotherapy. We developed an in vivo purging method capable of providing tumor-free stem cell products from most patients with mantle cell or follicular lymphoma and bone marrow involvement. In a prospective study, 15 patients with CD20(+) mantle cell or follicular lymphoma, bone marrow involvement, and polymerase chain reaction (PCR)-detectable molecular rearrangement received 2 cycles of intensive chemotherapy, each of which was followed by infusion of a growth factor and 2 doses of the anti-CD20 monoclonal antibody rituximab. The role of rituximab was established by comparison with 10 control patients prospectively treated with an identical chemotherapy regimen but no rituximab. The CD34(+) cells harvested from the patients who received both chemotherapy and rituximab were PCR-negative in 93% of cases (versus 40% of controls; P =.007). Aside from providing PCR-negative harvests, the chemoimmunotherapy treatment produced complete clinical and molecular remission in all 14 evaluable patients, including all 6 with mantle cell lymphoma (versus 70% of controls). In vivo purging of hematopoietic progenitor cells can be successfully accomplished in most patients with CD20(+) lymphoma, including mantle cell lymphoma. The results depended on the activity of both chemotherapy and rituximab infusion and provide the proof of principle that in vivo purging is feasible and possibly superior to currently available ex vivo techniques. The high short-term complete-response rate observed suggests the presence of a more-than-additive antilymphoma effect of the chemoimmunotherapy combination used.

The identification of genes inducing resistance to anticancer chemotherapeutic agents and their introduction into hematopoietic cells represents a promising approach to overcome bone marrow toxicity, the limiting factor for most high-dose chemotherapy regimens. Because resistance to cyclophosphamide has been correlated with increased levels of expression of the aldehyde-dehydrogenase (ALDH1) gene in tumor cell lines in vitro, we tested whether ALDH1 overexpression could directly induce cyclophosphamide resistance. We have cloned a full-length human ALDH1 cDNA and used retroviral vectors to transduce it into human (U937) and murine (L1210) hematopoietic cell lines that were then tested for resistance to maphosphamide, an active analogue of cyclophosphamide. Overexpression of the ALDH1 gene resulted in a significant increases in cyclophosphamide resistance in transduced L1210 and U937 cells (50% inhibition concentration [IC50], approximately 13 mumol/L). The resistant phenotype was specifically caused by ALDH1 overexpression as shown by its reversion by disulfiram, a specific ALDH1 inhibitor. ALDH1 transduction into peripheral blood human hematopoietic progenitor cells also led to significant increases (4- to 10-fold; IC50, approximately 3 to 4 mumol/L) in cyclophosphamide resistance in an in vitro colony-forming assay. These findings indicate that ALDH1 overexpression is sufficient to induce cyclophosphamide resistance in vitro and provide a basis for testing the efficacy of ALDH1 gene transduction to protect bone marrow cells from high-dose cyclophosphamide in vivo.

Hematopoietic progenitor cells circulate in the peripheral blood (PB) of cancer patients during the recovery phase that follows treatment with high-dose cyclophosphamide followed by hematopoietic growth factor infusion. We report that when PB progenitors were exposed in vitro to filtered supernatant from cell line PA317-N2, producing amphotropic helper-free N2 vector at conventional titers, successful retroviral-mediated transfer of neomycin resistance gene was documented by polymerase chain reaction in 93% of day 14 myelomonocytic colonies. Under the same conditions, gene transfer was achieved in 22% of steady-state bone marrow-derived myelomonocytic colonies. Neo-resistance gene transfer was documented also in a CD34+/cyclophosphamide-resistant precursor to granulocyte-macrophage colonies, an undifferentiated progenitor close to the hematopoietic stem cell. Neither cocultivation with vector-producing cells nor high vector titer were stringent requisites for efficient gene transfer. The large-scale availability of PB hematopoietic progenitors in cancer patients, together with the high gene transfer rate achieved under safe and clinically feasible conditions, support an optimal approach for gene transfer procedures into the human hematopoietic system.