Dean G. Heimann

Rapid immunologic reconstitution following transplantation with mobilized peripheral blood stem cells as compared to bone marrow

A majority of patients with intermediate or high-grade non-Hodgkin’s lymphoma (NHL) who are treated with high-dose chemotherapy (HDT) and hematopoietic stem cell transplantation subsequently relapse. Until recently, transplantation was associated with high morbidity and mortality and the focus was on improving the safety of this procedure. However, the use of growth factors and other supportive measures has successfully reduced treatment mortality to less than 5%. Therefore, new strategies need to be developed to eliminate the growth of any occult tumor cells reinfused with the stem cell products and the tumor cells remaining in the patient. One approach is to improve the immune function of the patients by a more rapid immune reconstitution and augmentation of effector cell function. We report studies comparing immune recovery following HDT and autologous peripheral stem cell transplantation (PSCT) as compared to autologous bone marrow transplantation (ABMT). These studies examined patients with intermediate and high-grade non-Hodgkin’s lymphoma (NHL) who were treated with HDT and PSCT (n = 56) or ABMT (n = 60). The PSCT patients had a significantly faster recovery of circulating monocytes (CD14+ cells), natural killer ((NK) CD56+) cells, T helper (CD4+) cells, TCRγ/δ cells, and naive T lymphocytes (CD45RA+). Following ABMT there was a significantly more rapid increase in the frequency of T suppressor/effector (CD8+) cells, B (CD19+) cells, CD34+ cells, polymorphonuclear leukocytes (PMN) and memory T lymphocytes (CD45RO+). The CD4:CD8 and CD45RA:CD45RO ratios were consistently higher in the PSCT group as compared to ABMT suggesting an improved ratio of T helper to T effector/suppressor cells and naive T cells. The differences in cellular phenotype translated into improved T cell function (PHA mitogenesis) and T cell help (pokeweed mitogenesis). In addition, there was an accelerated reconstitution of NK cell activity following PSCT as compared to ABMT. The more rapid reconstitution of NK and T cells in patients rescued with PSCT as compared to ABMT may contribute to an improved clinical outcome. Further, patients receiving a PSCT may be more responsive to adjuvant immunotherapy following transplantation.

Immunoregulatory cytokines in bone marrow and peripheral blood stem cell products

In these studies, we compared the phenotype, function, and expression of type 1, type 2, and monocyte-associated cytokine mRNA transcripts in autologous bone marrow (BM) and growth factor-mobilized peripheral blood stem cell (PSC) products. These studies demonstrate that lymphocytes and monocytes in stem cell products are abnormally activated, expressing significantly higher levels of interleukin (IL)-2, 4 and 10, interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), but not IL-8, as compared to normal peripheral blood mononuclear cells (PBMC). In addition, the levels of IL-2, IL-10 and TNF-α are significantly higher in mobilized PSC as compared to BM products. The high cytokine levels are unexpected as T cell function in stem cell products is depressed. PSC products have high levels of T cell inhibitory activity, which directly correlates with IL-10 expression, both of which are mechanisms that might be involved in the immune dysfunction within stem cell products used for autologous stem cell transplantation. These data demonstrate that: (1) immune cells in autologous BM and PSC products are activated with the expression of high levels of type 1 and type 2 cytokines as well as monokines; (2) PSC products contain a high frequency of monocytes which mediate T cell inhibitory activity; and (3) despite the high levels of cytokine expression, T cell function in stem cell products is depressed. The significance of these immune abnormalities within stem cell products for myeloid and lymphoid recovery following autologous stem cell transplantation remains to be determined.

Relaxin decreases the severity of established hepatic fibrosis in mice

Hepatic fibrosis is characterized by excess collagen deposition, decreased extracellular matrix degradation and activation of the hepatic stellate cells. The hormone relaxin has shown promise in the treatment of fibrosis in a number of tissues, but the effect of relaxin on established hepatic fibrosis is unknown. The aim of this study was to determine the effect of relaxin on an in vivo model after establishing hepatic fibrosis

Restoration of T and NK cell function in GM-CSF mobilized stem cell products from breast cancer patients by monocyte depletion

Rapid immune reconstitution is observed following autologous peripheral blood stem cell transplantation (PSCT) as compared to autologous bone marrow transplantation (ABMT), although it is depressed compared to that observed in normal individuals. The immune dysfunction occurs despite the restoration of normal lymphoid cell numbers and may be associated with the immunologic characteristics of the infused peripheral blood stem cell (PSC) product. We report herein that the in vitro T cell proliferation and NK activity in PSC products of breast cancer patients are significantly increased following the removal of CD14+ monocytes (33 ± 2% of the PSC product) by carbonyl iron magnetic cell isolation (CI). In vitro expansion of PSC cells cultured for 7–21 days in the presence of interleukin-2 (IL-2) is also significantly increased by depletion of the phagocytic cells. The PHA and IL-2 mitogenic responses, as well as NK activity of the expanded cells, was also significantly increased by the depletion of the phagocytes. In summary, the depletion of phagocytic monocytes from PSC products restores the proliferative and functional properties of T and NK lymphocytes and may facilitate adoptive cellular therapy, as well as rapid immunologic reconstitution post-PSCT.

Monocyte activation by an oral immunomodulator (bestatin) in lymphoma patients following autologous bone marrow transplantation.

Abstract Bestatin (ubenimex), an inhibitor of aminopeptidase, is an oral immunomodulator that binds to CD13 (aminopeptidase N) on macrophages/monocytes. To examine its immunomodulatory effect after high-dose therapy and autologous bone marrow transplantation (BMT), a dose-finding phase Ib trial was conducted with 30 Hodgkin’s disease and non-Hodgkin’s lymphoma patients who received no drug (control), 10 and 30 mg (low dose), or 90 and 180 mg (high dose) of bestatin daily for 60 days following autologous BMT. Bestatin administration was initiated when the absolute neutrophil count was greater than 250/mm3 on 2 consecutive days. The serum neopterin levels, an indicator of monocyte/macrophage activation, increased in the high-dose group compared to the control group (not significantly) and the low-dose group (significantly). Similarly, the colony-stimulating activity in the sera was significantly increased in the high-dose group compared to the control and low-dose groups. We also examined the expression of cell-surface markers on monocytes in these patients by fluorescent cytometry analysis. There was no significant difference either in the frequency or absolute number of monocytes (CD14+) among the three groups at any time. However, a significant increase in the frequency of CD16(FcgRIII)-positive monocytes (a marker of activation) was observed in the high-dose group compared to controls from day 14 to day 60 after the start of bestatin administration. Further, the frequency of HLA-DR+ monocytes (another marker of activation) was significantly increased in the high-dose group. These results indicate that bestatin at higher doses (90 and 180 mg daily), but not lower doses, activates macrophages/monocytes, as demonstrated by phenotypic marker (HLA-DR and CD16) up-regulation, and this provides augmentation of neopterin and colony-stimulating activity in the serum of patients following autologous BMT.

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).

Degradation of Relaxin Family Peptides by Insulin-degrading Enzyme

Insulin‐degrading enzyme (IDE) is a ubiquitously expressed metalloproteinase responsible for the intracellular degradation of insulin. IDE also interacts with other members of the insulin superfamily, including relaxin, but no studies have been reported regarding the interaction of other relaxin‐like peptides with IDE. In this study, we determined that relaxin, relaxin‐3, and InsL3 all competitively inhibited the degradation of insulin by IDE to different degrees, and all inhibited covalent cross‐linking of insulin to IDE. Each of the peptides was degraded by IDE to various degrees (insulin > relaxin > InsL3 = relaxin‐3). In summary, relaxin, InsL3, and relaxin‐3 all bound to IDE, competed for the binding and degradation of insulin, and were all substrates for the proteolytic activity of IDE. Therefore, it is possible that in addition to insulin, IDE may be important for the cellular proteolysis of relaxin, InsL3, and relaxin‐3.

Relaxin Reduces Fibrosis in Models of Progressive and Established Hepatic Fibrosis

The effect of relaxin administration before (prevention) or after (treatment) the establishment of hepatic fibrosis in a mouse model was examined. In the prevention study, relaxin reduced collagen and smooth muscle actin content and significantly reduced serum levels of the liver enzymes alanine aminotransferase and aspartate aminotransferase. In the treatment study, administration of relaxin for 1 week reduced collagen and smooth muscle actin but not liver enzyme levels. Relaxin administered for 2 weeks had no significant effect. In conclusion, the data suggest that relaxin treatment before fibrosis can reduce collagen and improve liver function but that there is little effect of short‐term relaxin treatment after fibrosis is established.

Enhancement of Adenovirus-Mediated Gene Transfer to Human Bone Marrow Cells

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.

Growth Factor Mobilization and Modulation of Progenitor Cell Adhesion to Stromal Cells: Role of VLA-4

Cellular interactions between hematopoietic progenitor cells and bone marrow (BM) stromal cells are mediated by cell adhesion molecules (CAM). In agreement with previous studies, our flow cytometric analysis of isolated CD34+ cells showed that VLA-4 expression was significantly (p < 0.001) higher on steady-state BM than on CD34+ cells from growth factor-mobilized peripheral stem cell (PSC) products. To determine whether the expression of VLA-4 on progenitor cells plays a role in their adhesion to stromal cells, we examined the binding of isolated CD34+ progenitor cells from BM (n = 14) and PSC (n = 10) products to BM stromal cells in the presence or absence of a neutralizing antibody to VLA-4. In these studies, similar kinetics of BM and PSC CD34+ cell adhesion to BM stromal cells were observed. However, neutralizing antibody to VLA-4 significantly inhibited BM CD34+ but not PSC CD34+ cell adherence to stromal cells, suggesting a role for alternative CAM in cell binding. Further, in long-term co-cultures of BM CD34+ cells with BM stroma, we observed a significantly higher number of colony-forming units granulocyte-macrophage (CFU-GM) released into the media following treatment with neutralizing antibody to VLA-4 than in untreated control cultures. In contrast, no difference in the frequency of nonadherent CFU-GM between antibody-treated and control long-term co-cultures of PSC CD34+ cells with BM stromal cells was observed. This suggests that VLA-4 expression on mobilized PSC versus BM CD34+ cells has biologic relevance for at least 2 weeks based on the long-term BM culture results. In summary, these data suggest that the decreased expression of VLA-4 may have a role in the mobilization of progenitor cells, in part, by regulating their adherence to stromal cells, although additional mediators of adhesion are also involved.