Growth Factor-mobilized Peripheral Blood Research Articles

Cryopreservation of Growth Factor-Mobilized Peripheral Blood Stem Cells Does Not Compromise Major Outcomes after Allogeneic Hematopoietic Cell Transplantation: A Single-Center Experience

During the COVID-19 pandemic, cryopreservation of allogeneic donor stem cell products ensured availability of products at the start of conditioning for hematopoietic cell transplantation (HCT). Following recommendations from unrelated donor registries, including the National Marrow Donor Program (NMDP), many centers started to cryopreserve related donor peripheral blood stem cell (PBSC) products. Throughout the process, several centers have published outcomes with cryopreserved versus fresh products, some with conflicting results. Even though cryopreservation was initially considered only a temporary measure driven by the pandemic, potential advantages include greater flexibility of transplant timing. However, concern about detrimental effects of cryopreservation including increased risks of graft rejection, relapse and consequently mortality remained. The primary objective was to describe our center's experience comparing outcomes following PBSC transplantation with cryopreserved versus fresh grafts. This was an observational case study with retrospective review comparing cryopreserved grafts (N=213), to a recent historical cohort (controls) using fresh grafts (N=167). In multivariable analyses, the adjusted hazard ratio (fresh vs. cryo) we for overall mortality was HR= 1.20 (95% confidence interval (CI), 0.79-1.82; p=0.40), for non-relapse mortality HR=0.99 (95% CI, 0.55-1.77; p=0.98), and for relapse HR=0.94 (95% CI, 0.60 -1.48; p=0.80). The adjusted hazard ratio for platelet engraftment was HR=1.31 (95% CI, 1.05-1.63; p=.02) and the odds ratio of grades III-IV acute GVHD was OR=1.75 (95% CI 1.01-3.04; p=.05) with fresh compared to cryopreserved grafts. There was no demonstrable difference in the risk of chronic GHVD. Although longer-term follow-up is needed, these data provide preliminary reassurance that in the event of another pandemic or should the logistical need arise in individual patients, cryopreservation of PBSC products constitutes a reasonably safe alternative.

GVHD Prevention in 2018: Tried and True or Something New?

Graft-versus-host disease (GVHD) is a major contributor to early and late morbidity and mortality after allogeneic stem cell transplantation. Despite results from a randomized controlled trial demonstrating an increased risk of chronic GVHD with use of growth factor-mobilized peripheral blood stem cells (PBSC) compared with bone marrow, PBSCs are the most widely used graft source in allogeneic transplantation for hematologic neoplasms in the U.S. This lecture will review established, recent, and novel strategies for GVHD prevention in unrelated donor PBSC transplantation and will highlight ongoing clinical research at Fred Hutchinson Cancer Research Center. Clinical trials aimed at defining standard-of-care GVHD prophylaxis after myeloablative and nonmyelablative conditioning will be presented. In addition, novel pharmacologic agents and graft manipulation strategies under investigation will be discussed. (Presented at the 1962nd Meeting, May 12, 2018).

Comparison of Tacrolimus Versus Cyclosporine with Methotrexate for Immunosuppression after Allogeneic Hematopoietic Cell Transplantation for Severe Aplastic Anemia: A CIBMTR Analysis

Background: Allogeneic hematopoietic cell transplantation (HCT) is a curative treatment for patients with severe aplastic anemia (SAA), but graft-versus-host disease (GVHD) has been a major impediment to success. Cyclosporine (CSP) has been widely used as GVHD prophylaxis after HCT for SAA, while both CSP and tacrolimus (TAC) have been used after HCT for hematological malignancies. The aim of this study was to compare transplant outcomes according to TAC+methotrexate (MTX) vs. CSP+MTX as GVHD prophylaxis after HCT for SAA using large registry data. Patients and methods: The retrospective cohort included 949 patients who had a first allogeneic bone marrow or growth factor-mobilized peripheral blood cell transplantation from HLA-identical siblings (SIB) or unrelated donors (URD) for treatment of SAA using GVHD prophylaxis with MTX in combination with either TAC or CSP from 2001 to 2011. Patients who had ex-vivo T cell depletion were excluded. Study endpoints included grades II-IV acute GVHD, grades III-IV acute GVHD, chronic GVHD, overall mortality, hematopoietic recovery and graft failure. Multivariate Cox regression models were used to evaluate hazard ratios for endpoints with TAC+MTX as compared with CSP+MTX. A stepwise procedure was used in developing models for each outcome, using a P value threshold of 0.05, with GVHD prophylaxis and year of HCT forced in all models. Analyses were performed separately in SIB (n=631) and URD (n=318) recipients. Results: The median age of patients was 19 (range, 0.5 to 70) years. TAC+MTX was used in 62 SIB recipients (10%) and in 120 (38%) URD recipients, and was used more frequently in older patents than in younger patients in both groups. Multivariate analysis showed a decreased risk of overall mortality with TAC+MTX in URD group and an increased probability of neutrophil recovery with TAC+MTX in SIB group, but other outcomes did not differ statistically between TAC and CSP prophylaxis (Table). Center effect was not statistically associated with overall mortality, and results were similar even after adjustment for center effect. Causes of death were similar between CSP and TAC prophylaxis in both SIB and URD recipients. Conclusion: Based on adjusted multivariate analyses, the use of TAC+MTX was associated with a decreased risk of mortality among URD recipients and with earlier neutrophil recovery among SIB recipients. Results showed no statistically significant differences in other outcomes with the 2 prophylaxis regimens, although power was limited by the number of patients treated with TAC. Our results support the use of either prophylaxis regimen for SIB recipients. TAC+MTX may be favored for URD recipients, but the interpretation of the survival difference remains uncertain, since TAC was not associated with a reduction in the risk of GVHD, causes of death were similar between the 2 prophylaxis regimens, and the actual difference in survival was small between the 2 prophylaxis regimens.TableMultivariate analysis of outcomes with TAC+MTX compared to CSP+MTXOutcomeIdentical Sibling (n=631)Unrelated (n=318)HR* (95% CI)PHR* (95% CI)PGrades II-IV acute GVHD1.79 (0.92-3.47)0.091.17 (0.76-1.80)0.48Grades III-IV acute GVHD0.79 (0.19-3.34)0.751.52 (0.81-2.86)0.19Chronic GVHD1.79 (0.92-3.47)0.091.23 (0.80-1.88)0.35Overall mortality1.38 (0.74-2.55)0.310.45 (0.25-0.81)0.008ANC recovery1.47 (1.04-2.08)0.031.29 (0.95-1.75)0.10Platelet recovery1.26 (0.93-1.71)0.130.89 (0.66-1.20)0.44Graft failure1.08 (0.45-2.56)0.870.48 (0.16-1.48)0.20*Adjusted for other covariates. DisclosuresNo relevant conflicts of interest to declare.

Engraftment and lineage potential of adult hematopoietic stem and progenitor cells is compromised following short-term culture in the presence of an aryl hydrocarbon receptor antagonist.

Hematopoietic stem cell gene therapy for HIV/AIDS is a promising alternative to lifelong antiretroviral therapy. One of the limitations of this approach is the number and quality of stem cells available for transplant following in vitro manipulations associated with stem cell isolation and genetic modification. The development of methods to increase the number of autologous, gene-modified stem cells available for transplantation would overcome this barrier. Hematopoietic stem and progenitor cells (HSPC) from adult growth factor-mobilized peripheral blood were cultured in the presence of an aryl hydrocarbon receptor antagonist (AhRA) previously shown to expand HSPC from umbilical cord blood. Qualitative and quantitative assessment of the hematopoietic potential of minimally cultured (MC-HSPC) or expanded HSPC (Exp-HSPC) was performed using an immunodeficient mouse model of transplantation. Our results demonstrate robust, multilineage engraftment of both MC-HSPC and Exp-HSPC although estimates of expansion based on stem cell phenotype were not supported by a corresponding increase in in vivo engrafting units. Bone marrow of animals transplanted with either MC-HSPC or Exp-HSPC contained secondary engrafting cells verifying the presence of primitive stem cells in both populations. However, the frequency of in vivo engrafting units among the more primitive CD34+/CD90+ HSPC population was significantly lower in Exp-HSPC compared with MC-HSPC. Exp-HSPC also produced fewer lymphoid progeny and more myeloid progeny than MC-HSPC. These results reveal that in vitro culture of adult HSPC in AhRA maintains but does not increase the number of in vivo engrafting cells and that HSPC expanded in vitro contain defects in lymphopoiesis as assessed in this model system. Further investigation is required before implementation of this approach in the clinical setting.

Optimized Processing of Growth Factor Mobilized Peripheral Blood CD34+ Products by Counterflow Centrifugal Elutriation

Cell separation by counterflow centrifugal elutriation has been described for the preparation of monocytes for vaccine applications, but its use in other current good manufacturing practice (cGMP) operations has been limited. In this study, growth factor-mobilized peripheral blood progenitor cell products were collected from healthy donors and processed by elutriation using a commercial cell washing device. Fractions were collected for each product as per the manufacturer's instructions or using a modified protocol developed in our laboratory. Each fraction was analyzed for cell count, viability, and blood cell differential. Our data demonstrate that, using standard elutriation procedures, >99% of red blood cells and platelets were removed from apheresis products with high recoveries of total white blood cells and enrichment of CD34+ cells in two of five fractions. With modification of the basic protocol, we were able to collect all of the CD34+ cells in a single fraction. The CD34-enriched fractions were formulated, labeled with a ferromagnetic antibody to CD34, washed using the Elutra device, and transferred directly to a magnetic bead selection device for further purification. CD34+ cell purities from the column were extremely high (98.7 ± 0.9%), and yields were typical for the device (55.7 ± 12.3%). The processes were highly automated and closed from receipt of the apheresis product through formulation of target-enriched cell fractions. Thus, elutriation is a feasible method for the initial manipulations associated with primary blood cell therapy products and supports cGMP and current good tissue practice-compliant cell processing.

Bone Marrow-Derived Cell Mobilization with G-CSF and GM-CSF in Patients with Acute Myocardial Infarction: A Pilot Study on Feasibility, Safety and Efficacy.

Introduction. Intracoronary or intramyocardiac injection of bone marrow stem cells or growth factor-mobilized peripheral blood stem cells (PBSC) may contribute to myocardial regeneration and neovascularization of the ischemic myocardium either in animal models or in human. This effect is probably due to a selective colonization of the ischemic area by PBSC. Very few studies were done in the acute setting of ischemic heart disease, with controversial results. Aims of this pilot study were: i) to verify feasibility and safety of PBSC mobilization in patients with acute myocardial infarction (AMI); ii) to monitor clinical effects of PBSC mobilization in terms of global and segmental myocardial perfusion and function.Patients and methods. Eight male patients (mean age: 51,7±5,6 years) were enrolled. All were treated with a primary percutaneous transluminal coronary angioplasty (PTCA) for an anterior (5 patients) or an inferior (3 patients) AMI. The mobilization regimen consisted of G-CSF 5 μg/kg/12h from day 1 to day 3 and GM-CSF 2.5 μg/kg/24h from day 1 to day 5 (starting within 24 hours from PTCA). Since transient severe hypotension was noticed in the first three patients, the protocol was amended by reducing and tapering GM-CSF administration (2.5 μg/kg/24h at days 3, 5, 7). All patients underwent coronary angiography, intracoronary doppler flow study, echocardiography, and nuclear Thallium scan before treatment and at 6 months apart.Results. WBC and PBSC peaked during the 3th day of mobilization. Mean WBC and PBSC peaks were 34960± 10794 leukocytes/μL and 29.71± 30.8 CD34+/μL. Five in hospital adverse events were recorded: severe hypotension in 3 patients (as previously mentioned), atrial fybrillation in 1 patient, recurrent ischemia in 1 patients. No death was observed. At present 7 patients completed a 6-months follow-up evaluation: target lesion revascularization rate was 14,3% (1 patient) and target vessel revascularization rate was 42,8% (3 patients). Angiographic mean ejection fraction increased from 49,8±11,9 to57,1±8,9 (p=NS), and mean coronary flow reserve raised from 1,63±0,42 to 2,45±0,36 (p=0,005). Perfusion improvement was observed by nuclear study in 66% of patients.Conclusions. We conclude that: a) cytokine-induced stem cell mobilization is feasible in AMI patients with adequate GM-CSF administration; b) myocardial perfusion appears to improve within the first six months of follow-up; c) the 3 cases of progressive coronary disease give a concern about a potential negative effect of hematopoietic cytokines (particularly GM-CSF) on silent coronary lesions. However, PBSC mobilization using G-CSF only is worthwhile of further investigation.

Bone marrow transplantation from α1,3‐galactosyltransferase gene‐knockout pigs in baboons

Successful hematopoietic cell allotransplantation results in donor-specific tolerance, but this approach has been unsuccessful in the wild-type pig-to-baboon xenotransplantation model, as pig cells were lost from the circulation within 5 days. However, after cessation of immunosuppressive therapy on day 28, all baboons demonstrated non-specific unresponsiveness on mixed leukocyte reaction (MLR) for at least 30 days. We have now investigated the transplantation of bone marrow (BM) cells from miniature swine homozygous for alpha1,3-galactosyltransferase gene-knockout (GalT-KO). Baboons (n = 3) were pre-treated with whole body and thymic irradiation, anti-thymocyte globulin, and splenectomy, and received immunosuppressive and supportive therapy for 28 days. BM was harvested from GalT-KO swine (n = 3). The baboons were monitored for the presence of pig cells by flow cytometry and colony-forming units (CFUs), and for cellular reactivity by MLR. A mean of 11 x 10(8) BM cells/kg was infused into each baboon. The mean absolute numbers and percentages of pig cells detected in the blood at 2 h and on days 1, 2 and 4, respectively, were 641/microl (9.5%), 132/microl (3.4%), 242/microl (3.9%), and 156/microl (2.9%). One baboon died (from accidental hemorrhage) on day 6, at which time chimerism was present in the blood (2.0%) and BM (6.4%); pig cell engraftment in the BM was confirmed by polymerase chain reaction (PCR) of CFUs. In the two other baboons, blood chimerism was lost after day 5 but returned at low levels (<1%) between days 9 to 16 and 7 to 17, respectively, indicating transient BM engraftment. Both surviving baboons showed non-specific unresponsiveness on MLR until they were euthanized on days 85 and 110, respectively. By using BM cells from GalT-KO pigs, chimerism was detected at levels comparable with previous studies when 30-fold more growth factor-mobilized peripheral blood progenitor cells had been transplanted. In addition, cellular hyporesponsiveness was prolonged. However, long-term engraftment and chimerism were not achieved.

Male DNA in female donor apheresis and CD34-enriched products

AbstractIncreased risk of graft-versus-host disease (GVHD) has been described in recipients of hematopoietic stem cell transplantations when the donor is a parous woman. Cells from prior pregnancies are now known to persist in women and could contribute to GVHD. We asked whether male DNA (presumed fetal microchimerism) is present in apheresis products of female donors. A total of 50 samples were studied by using real-time quantitative polymerase chain reaction (PCR) for the Y chromosome-specific sequence DYS14. Among 29 growth factor-mobilized peripheral blood mononuclear cell (G-PBMC) products, 34% were positive for male DNA. Quantitative results, expressed as DNA genome equivalent of male cells per million host cells (gEq/mil), ranged from 0 to 35 gEq/mil. Among 21 CD34-enriched cell fractions, 48% were positive with a range of 0 to 357 gEq/mil. In summary, male DNA was frequently detected in G-PBMC and CD34-enriched products from female donors. Whether fetal microchimerism contributes to GVHD merits further investigation. (Blood. 2003;102: 3845-3847)

Inhibition of baboon platelet aggregation in vitro and in vivo by the garlic derivative, ajoene.

The infusion of pig growth factor-mobilized peripheral blood leukocytes (containing 1 to 2% progenitor cells) (pPBPC) into baboons is associated with a thrombotic microangiopathy, which results from a direct effect of these pig cells on platelet aggregation. Ajoene is a synthetic derivative of garlic that inhibits aggregation of human platelets induced by all known agents. To assess its potential use in models of xenotransplantation, this agent was tested for its effect on baboon platelet aggregation in vitro and in vivo. Baboon platelet aggregation assays, using adenosine diphosphate (ADP) (20 or 40 microm) or collagen (12.5 microg/ml), were performed after incubation with ajoene (0 to 150 microg/ml) or dipyridamole (0 to 200 microg/ml). Platelets were also incubated with pPBPC (5 x 10(6) cells) without or with ajoene in the absence of a known agonist. In vivo studies: Baboons received either a single intravenous dose of ajoene (10 to 25 mg/kg) or dipyridamole (0.8 mg/kg), or repeated doses of both agents at 2 to 3 h intervals. Platelet-rich plasma was obtained for platelet aggregation assays at time points up to 4 h post-drug administration. In vitro, platelet aggregation was inhibited by 95% (ADP assay) and 89% (collagen assay) by ajoene at concentrations of > or =75 microg/ml. Dipyridamole had no effect at concentrations of <100 microg/ml, but inhibited aggregation almost completely at higher concentrations. Ajoene inhibited the aggregation caused by pPBPC by 33 to 50%. In vivo, platelet aggregation was completely inhibited for 2 h by ajoene at 25 mg/kg. Dipyridamole at 0.8 mg/kg reduced aggregation by 20% for 15 min, but the effect was lost by 60 min. In combination, the two agents prolonged inhibition marginally. Repeated doses of both agents at 2 h intervals maintained complete inhibition of aggregation, but did not do so when the interval between doses was extended to 2.5 or 3 h. Combined therapy was not associated with any bleeding complications. Although ajoene is a powerful inhibitor of platelet aggregation, the need for repeated administration and its partial effect on pPBPC-induced platelet aggregation would suggest that it may be of only limited value in preventing the thrombotic microangiopathy that develops when pPBPC are infused into baboons. However, it would seem worthy of further investigation when used in combination with other agents.

Impaired T and NK cell response of bone marrow and peripheral blood stem cell products to interleukin (IL)-2

The function of steady-state and interleukin (IL)-2-co-cultured mononuclear cells differs significantly between bone marrow (BM) products, growth factor-mobilized peripheral blood stem cell (PSC) products and normal peripheral blood mononuclear cells (PBMC). The natural killer (NK) cell activity and T cell proliferative response of PSC products from non-Hodgkin’s lymphoma (NHL) patients are significantly higher than that of BM products and similar to normal PBMC. However, following a five-day co-culture with IL-2 (100 IU/ml), the NK activity of PSC, PBMC, and BM products (lytic units) was increased 176-, 40-, and 14-fold, respectively, compared to that observed prior to IL-2 culture. In contrast, lymphokine activated killer (LAK) cytotoxicity prior to IL-2 culture was low in PSC and BM products and normal PBMC, but was significantly increased in PSC products and PBMC following IL-2 co-culture. The proliferative response of PSC and BM products to the T cell mitogen phytohemagglutinin (PHA) was significantly lower than that observed with normal PBMC; however, PSC had a significantly higher response than cells from BM products. Similar patterns of T cell PHA mitogenic response were observed after IL-2 co-culture. In addition, the IL-2 mitogenic responses of IL-2-co-cultured PSC and BM products were also significantly lower than that observed with PBMC co-cultured with IL-2. The IL-2 mitogenic response of PBMC was also significantly increased compared to prior to IL-2 co-culture; whereas, the IL-2 mitogenic responses from PSC and BM cells were not. In summary, co-culture with IL-2 can increase the NK and LAK cell cytotoxicity of PSC and BM products from NHL patients, but IL-2 co-culture does not improve T cell function within either BM or PSC products.

Expression of interleukin-10 in isolated CD8+ T cells and monocytes from growth factor-mobilized peripheral blood stem cell products: a mechanism of immune dysfunction.

Previous reports showed the abnormal activation of immune cells in growth factor-mobilized peripheral blood stem cell (PBSC) products, which might be responsible for depressed T cell responsiveness to mitogens compared with normal peripheral blood mononuclear cells (PBMC). In the present study, the mRNA expression levels of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma (IFN-gamma) were significantly higher in CD4+ and CD8+ T cells from mobilized PBSC products compared with CD4+ and CD8+ cells from normal peripheral blood (PB). The mRNA expression levels of IL-4 and IL-10 were significantly higher in CD8+ compared with CD4+ cells from PBSC products. However, the expression of IL-2 and IFN-gamma mRNA transcripts was similar in the CD4+ and CD8+ T cells from PBSC products. The levels of IL-10, IL-8, and tumor necrosis factor (TNF)-alpha mRNA were also significantly higher in monocytes isolated from PBSC products compared with monocytes isolated from normal PB. Expression of IL-10-specific mRNA in monocytes also was significantly higher than the levels observed in CD8+ cells from PBSC products. We suggest that both CD4+ and CD8+ cells in the PBSC products are highly activated. However, their response to phytohemagglutinin (PHA) mitogenesis is depressed in part because of IL-10 expression by CD8+ cells and monocytes in addition to the higher levels of monocyte-dependent T cell inhibitory activity. These data demonstrate that aberrant IL-10 expression in the CD8+ T cells and monocytes present in PBSC products may represent a possible mechanism of immune dysfunction in patients after high-dose chemotherapy (HDT) and peripheral blood stem cell transplantation (PBSCT).

Negative selection of autologous peripheral blood stem cells

The use of chemotherapy and/or haematopoietic growth factor-mobilized peripheral blood stem cells (PBSC) has been shown to induce a more rapid haematopoietic recovery than the re-infusion of bone marrow (BM)-derived haematopoietic cells, thus reducing the morbidity and mortality of autologous stem cell transplantation (ASCT). PBSC collections were initially believed to have a lower incidence of tumour cells involvement than BM harvests. However, recent studies have shown that mobilized blood cell products of cancer patients eligible for autografting are frequently contaminated with tumour cells. Whereas positive selection of haematopoietic CD34+stem cells has been largely used as a means of indirect purging of circulating CD34−neoplastic cells, few groups have addressed the issue of tumour cell removal by direct targeting of cancer cells using physical or pharmacological strategies. In this chapter we review the available data concerning the contamination of tumour cells in PBSC collections from cancer patients, the functional and kinetic characteristics of primed CD34+cells which may affect the haematopoietic toxicity of purging procedures developed to eliminate the minimal residual disease (MRD) from BM samples, and the preclinical and clinical results of the selective killing of residual tumour cells from leukaphereses.The limited amount of data published so far do not allow any firm conclusion on the clinical usefulness of purging protocols. Nonetheless, the successful extension of ex vivo purging to PBSC collections may improve the feasibility of randomized studies aimed at determining the importance of tumour-free autografts.

Immune reconstitution following allogeneic peripheral blood stem cell transplants.

Growth factor-mobilized peripheral blood stem cells (PBSCs) engraft rapidly in myeloablated recipients compared to conventional BM, but this procedure also mobilizes mature lymphocytes and monocytes which can impact immune reconstitution and GVHD. Hence, we serially evaluated immune reconstitution and cytokine expression in PBSCT recipients in the first year. Engraftment of neutrophils and monocytes stabilized early but NK cells, B cells and CD4+ T cell numbers were significantly (P < 0.05) low with persistently reversed CD4:CD8 ratios. NK function remained low throughout the first year. The quantitative decrease in CD4+ T cells resulted in significantly decreased proliferation in response to mitogens and alloHLA antigens. Yet, a qualitative analysis of T cell function measured by Ca++ influx after T cell activation with antiCD3 as well as T-dependent polyclonal Ig secretion by mitogen-stimulated B cells was preserved even early post transplant. TNF alpha mRNA was detected in almost all recipients in the first year. IL-10 mRNA was detected in 77%, IL-2 in 22% and IFN gamma in 44% of recipients in the first 6 months. Only 30% expressed IL-10 in the second 6 months post transplant while expression of IL-2 and IFN gamma was detected in 38% and 46% respectively. Thirty-seven percent of PBSCT recipients developed grades II-IV acute GVHD but 72% went on to develop chronic extensive GVHD at a median of 120 days. Sixty-two percent developed CMV viremia and 5.4% developed overt CMV disease in the first year post PBSCT. Lymphocyte engraftment is quantitatively delayed but CD4 functions are preserved while NK numbers and function are compromised post PBSCT. IL-10 expression decreases after the first 6 months post transplant while TNF alpha is continually expressed. The balance between quantitative lymphocyte reconstitution and qualitative lymphocyte functions as well as changes in lymphokine patterns may influence infection and GVHD and thus the clinical outcome post PBSCT.

IL-2 expansion of T and NK cells from growth factor-mobilized peripheral blood stem cell products: monocyte inhibition.

The expansion of T and natural killer (NK) cells in growth factor-mobilized peripheral blood stem cell (PSC) products with interleukin-2 (IL-2) requires a reduction in monocyte frequency. Monocytes are enriched with stem cells during apheresis and, in this series of growth factor-mobilized PSC products from breast cancer patients, represented 36 +/- 6% of the cells in the product. Immunophenotyping studies revealed that monocytes inhibited the proliferation of NK cells (CD56+ and CD3- CD8+ CD56+ cells) and T cells (CD3+, CD4+, and CD8+ cells) during IL-2 co-culture for 7, 14, or 21 days. A reduction in monocytes resulted in 61-fold expansion of CD3- CD8+ CD56+ cells compared with a 3.7-fold increase of CD3+ cells by day 21. In addition, following IL-2 co-culture, cells from PSC products with a reduced frequency of monocytes had a significantly increased T cell mitogenic response and NK cell activity in PSC products compared with intact products. We suggest that monocytes inhibit the IL-2-dependent proliferation and augmented function of NK and T cells from growth factor-mobilized PSC products.

Detection of stromal cells in peripheral blood progenitor cell collections from breast cancer patients.

Transplantation of growth factor-mobilized peripheral blood progenitor cells (PBPC) is widely used in the treatment of several neoplastic diseases. While in PBPC harvests the presence of several accessory immune and tumor cells has been documented, that of stromal cells has not been reported. In the present study, we investigated for the presence of stromal cells in growth factor-mobilized PBPC harvests from breast cancer patients. Low-density cells from PBCP harvests in culture gave rise to an adherent layer containing fibroblast-like and large flat round cells. These cells express positive immunofluorescence staining for collagen I, collagen III, fibronectin, VCAM-1 (CD106), ICAM-1 (CD54) and mesenchymal antigens recognized by monoclonal antibodies, SH2 and SH3. PBPC-derived stromal cells do not express antigens CD34, CD45 and CD14. Stromal cells were detected in the PBPC harvests of 11/14 patients (median 0.63%; range 0.02-2.32) and their concentration correlates with the number of CD34+ cells in PBPC.