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Population size is governed through cells reacting to a variety of intrinsic and extrinsic cues. Tumors, while liberated from many of the homeostatic constraints placed on physiologic counterparts, can nonetheless remain subject to both social and environmental control. Burkitt lymphoma cells faithful to the biopsy phenotype were used to model the reliance of the colony, if any, on an inbuilt population sensor. Below a normally suicidal threshold number of cells, low picomolar quantities of exogenous CD40 ligand (CD40L/CD154) were found to sustain the clone without the discernible shift in phenotype that accompanies high CD40L encounter. Although CD154 was undetectable in populous cultures, message was induced as numbers became limiting. Correspondingly, attempts to neutralize endogenous CD40L activity failed to perturb cells at optimal densities but resulted in their marked decline as the critical threshold was approached. These data reveal an auto-inducible survival mechanism seemingly regulated through the monitoring of population size, a process somewhat akin to that of “quorum sensing” among gram-negative bacteria in which diffusible molecules provide a means of communication to coordinate gene expression with population density. This process could be activated as cells discern depletions in their community or when deprived of signals otherwise furnished within an appropriate environmental niche.

Group I Burkitt lymphoma (BL) cell lines (L3055, Elijah, Louckes, BL2, and BL29) retaining the original biopsy phenotype were found to undergo prolonged phenotypic, functional, and molecular change on short-term exposure to soluble recombinant CD40L trimer. Sensitivity to, extent of, and duration of change appeared to reflect passage number in that the earliest passaged lines, L3055 and BL29, were generally the most susceptible. Culture of group I BL lines with CD40L resulted in significant growth arrest (without apoptosis) that, for L3055 cells, was sustained for 7 to 9 days after 72 hours of exposure. This was accompanied by the formation of large homotypic aggregates together with gross changes in individual cell morphology and a concomitant upregulation of CD54 (ICAM-1). Three of the five group I BL lines exhibited rapid downregulation of the hallmark CD77 surface antigen, which, for L3055 cells, was maintained for at least 12 days after 72 hours of incubation with CD40L. With the exception of BL2, all group I BL lines were induced to express CD40 homodimers on CD40-stimulation, whereas only monomers were detected in unstimulated cells. Experiments using CD40-transfected Rat-1 fibroblasts showed that the ability to signal for dimer formation required Thr234of CD40. For L3055 and BL29 cells, an initial 72 hours of exposure to CD40L resulted in the maintenance of homodimers for up to 14 and 10 days, respectively. There was a close correlation between the induction and duration of CD40 homodimers and the appearance of Bcl-2. For L3055 cells, which are sensitive to apoptosis-induction on BCR-engagement, exposure to CD40L for 72 hours was found to provide considerable protection from anti-IgM, which was still significant to 20 days. The implications of such sustained effects on relatively short-term exposure of tumor B cells to CD40L are discussed. © 1998 by The American Society of Hematology.

To probe the pathophysiologic mechanisms underlying neutropenia in patients with chronic idiopathic neutropenia (CIN) with hypoplastic and left-shifted granulocytic series in the bone marrow (BM), we have studied granulocytopoiesis in 32 adults with CIN by evaluating the number and survival characteristics of cells in several stages of granulocyte differentiation using flow cytometry and BM culture assays. We found that patients with CIN displayed a low percentage of CD34(+)/CD33(+) cells, defective granulocyte colony-forming unit (CFU-G) growth potential of BM mononuclear or purified CD34(+) cells, and low CFU-G recovery in long-term BM cultures (LTBMCs), compared with controls (n = 46). A low percentage of CD34(+)/CD33(+) cells in patients was associated with accelerated apoptosis and Fas overexpression within this cell compartment compared with controls. No significant difference was documented in the percentage of apoptotic cells or the Fas(+) cells within the fractionated CD34(+)/CD33(-), CD34(-)/CD33(+), and CD34(-)/CD33(-)/CD15(+) BM subpopulations or the peripheral blood neutrophils, suggesting that the underlying cellular defect in CIN probably concerns the committed granulocyte progenitors. LTBMC stromal layers from patients produced abnormally high amounts of tumor necrosis factor alpha and cytokine levels in culture supernatants inversely correlated with the number of myeloid progenitor cells and positively with the proportion of apoptotic CD34(+) cells. Patient LTBMC stromal layers displayed pathologic interferon gamma and Fas-ligand mRNA expression and failed to support normal myelopoiesis. These data suggest that impaired granulocytopoiesis in CIN is probably due to overproduction of inflammatory cytokines by immune cells within the BM microenvironment that may exert an inhibitory effect on myelopoiesis by inducing Fas-mediated apoptosis in the granulocyte progenitors.

Abstract 4556 Members of the tumor necrosis factor / tumor necrosis factor receptor (TNF/TNFR) superfamily are involved in bone marrow (BM) homeostasis by regulating the survival, apoptosis, proliferation, and differentiation of hematopoietic progenitor and precursor cells. The CD40 Ligand (CD40L)/CD40 molecules belong to the TNF/TNFR superfamily; we have recently showed that the CD40/CD40L interaction on BM CD34+ cells accelerates the apoptotic cell death. The aim of the present study is to investigate the distribution and function of the CD40/CD40L molecules in the BM granulocytic progenitor and precursor cell populations as well as the effect of their interactions on the stromal release of cytokines related to granulopoiesis. BM samples were obtained from posterior iliac crest aspirates from 19 hematologically healthy subjects after informed consent. We evaluated: (a) the surface expression of CD40 and CD40L on immunomagnetically sorted CD34+, CD34-/CD33+, CD34-/CD33-/CD15+ cells representing sequential stages of the granulocytic development, using flow cytometry in steady state conditions and following 20-hour incubation with 25ng/ml recombinant-human (rh) TNFα; (b) the proportion of apoptotic cells and the level of caspase-3 activation following CD40 engagement in the above cell populations using flow cytometry and a chromo-enzymatic method, respectively; (c) the production of granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) by long-term BM culture (LTBMC) stromal cells upon induction with rhCD40L (1μg/ml), using an enzyme-linked immunoabsorbent assay (ELISA) in culture supernatants. We found that the basal expression of CD40 and CD40L on the CD34+, CD34-/CD33+, CD34-/CD33-/CD15+ cells was 3.34%±3.90% and 1.8%±1.03%, 16.74%±14.69% and 6.96%±4.68%, 3.22%±3.36% and 2.26%±2.71%, respectively. Following incubation with rhTNFα a substantial increase was obtained in the expression of CD40 in all the above cell populations compared to baseline (37.18%±12.51%, 29.25%±14.81%, 15.84%±6.28%, respectively) (p Disclosures: No relevant conflicts of interest to declare.