Reconstitution of the T-Cell Compartment After Bone Marrow Transplantation: Restoration of the Repertoire by Thymic Emigrants

We have studied the reconstitution of the T-cell compartment afterbone marrow transplantation (BMT) in five patients who received agraft-versus-host disease (GVHD) prophylaxis consisting ofmethotrexate, cyclosporin, and 10 daily injections (day 4 to day+5) of Campath-1G. This treatment eliminated virtually all T cells (7 ± 8 T cells/µL at day 14) which facilitated the analysis of the thymus-dependent and independent pathways of T-cell regeneration. During the first 6 months, the peripheral T-cell pool wasrepopulated exclusively through expansion of residual T cells with allCD4⁺ T cells expressing the CD45RO-memory marker. In twopatients, the expansion was extensive and within 2 months, the totalnumber of T cells (CD8>>CD4) exceeded 1,000/µL. In the otherthree patients, T cells remained low (87 ± 64 T cells/µL at 6 months) and remained below normal values during the 2 years of thestudy. In all patients, the firstCD4⁺CD45RA⁺RO T cellsappeared after 6 months and accumulated thereafter. In the youngestpatient (age 13), the increase was relatively fast and naiveCD4⁺ T cells reached normal levels (600 T cells/µL) 1 year later. In the four adult patients (age 25 ± 5), the levelsreached at that time-point were significantly lower (71 ± 50 Tcells/µL). In all patients, the T-cell repertoire that had been verylimited, diversified with the advent of theCD4⁺CD45RA⁺RO T cells. Cellsorting experiments showed that this could be attributed to thecomplexity of the T-cell repertoire of theCD4⁺CD45RA⁺RO T cells thatwas comparable to that of a normal individual and that, therefore, itis likely that these cells are thymic emigrants. We conclude that afterBMT, the thymus is essential for the restoration of the T-cellrepertoire. Because the thymic activity is restored with a lag time ofapproximately 6 months, this might explain why, in particular inrecipients of a T-cell-depleted graft, immune recovery is delayed.     

Intravascular Coagulation Activation in a Murine Model of Thrombomodulin Deficiency: Effects of Lesion Size, Age, and Hypoxia on Fibrin Deposition

We consecutively inactivated both alleles of the thrombomodulin (TM)gene in murine embryonic stem (ES) cells and generated TM-deficient(TM^/) chimeric mice. Quantitation of an ES-cellmarker and protein C cofactor activity indicates that up to 50% ofpulmonary endothelial cells are ES-cell derived and therefore TMdeficient. Infusions of ¹²⁵I-fibrinogen into mice show asignificant increase (fourfold, P < .005) in radiolabeledcross-linked fibrin in TM^/ chimeric mouse lung ascompared with wild-type mice. However, only chimeric mice that exhibitat least a 30% reduction in protein C cofactor activity and are atleast 15 months old display this phenotype. Immunocytochemicallocalization of TM in chimeras shows a mosaic pattern of expression inboth large and small blood vessels. Colocalization of cross-linkedfibrin and neo (used to replace TM) reveals that fibrin is deposited inTM^/ regions. However, the fibrin deposits werelargely restricted to pulmonary vessels with a lumenal area greaterthan 100 µm². The hypercoagulable phenotype can beinduced in younger chimeric mice by exposure to hypoxia, which causes afivefold increase in -fibrin levels in lung. Our findings show thatTM chimerism results in spontaneous, intravascular fibrin depositionthat is dependent on age and the magnitude of the TM deficiency.     

Variable protection of beta 3-integrin--deficient mice from thrombosis initiated by different mechanisms

Platelet integrin IIb3 (GPIIb/IIIa) plays a central role inthe initiation of arterial thrombosis, but its contribution todisseminated microvascular thrombosis is less well defined. Therefore,wild-type mice (3^+/+), 3-integrin-deficient mice(3^/), and wild-type mice treated with a hamstermonoclonal antibody (1B5) that blocks murine IIb3 function weretested in models of large-vessel and microvascular thrombosis. In thelarge-vessel model, ferric chloride was used to injure the carotidartery, and the time to thrombosis was measured. In 3^+/+mice, the median time to occlusion was 6.7 minutes, whereas occlusion did not occur in any of the 3^/ mice tested(P < .001). Fab and F(ab')₂ fragments of1B5 increased the median time to occlusion. To initiate systemicintravascular thrombosis, prothrombotic agents were administeredintravenously, and platelet thrombus formation was monitored by thedecrease in circulating platelet count. Three minutes after theinjection of adenosine diphosphate (ADP), collagen + epinephrine,or tissue factor, the platelet counts in 3^+/+ micedecreased by 289, 424, and 429 × 10³/µL, respectively.3^/ mice and wild-type mice pretreated with 1B5 Fab(1 mg/kg, IP) were nearly completely protected from the effects of ADP.In contrast, 3^/ mice were only partially protectedfrom the effects of collagen + epinephrine and minimally protectedfrom the effects of tissue factor. In all cases, less fibrin becamedeposited in the lungs of 3^/ mice than in wild-typemice. These results suggest that though IIb3 plays adominant role in large-vessel thrombosis, it plays a variable role insystemic intravascular thrombosis.     

The Fetal Origin of B-Precursor Leukemia in the E-ret Mouse

Before the clinical onset of B-precursor lymphoblastic leukemia,Eµ-ret mice have an expansion of late pro-B cells(CD45R⁺CD43⁺CD24⁺BP-1⁺)within the bone marrow. To characterize the early effects of thetransgene product on lymphopoiesis, we initially sequenced the Ig heavychain (IgH) rearrangements within the late pro-B cells in 24-day-oldEµ-ret and transgene negative mice. In both mouse populations, theIgH rearrangements were polyclonal, predominately nonproductive, andexhibited similar V, D, and J gene usage. However, the frequency of Nregions, a marker of postnatal lymphopoiesis, was notably different. Atthe VD junction, N regions were found in 25 of 25 (100.0%)rearrangements from transgene-negative mice compared with 12 of 36 (33.3%) rearrangements from Eµ-ret mice. At the DJ junction, Nregions were found in 21 of 25 (84.0%) rearrangements from transgenenegative mice compared with 4 of 36 (11.1%) rearrangements fromEµ-ret mice. Subsequently, we sequenced the clonal IgH rearrangements from 9 leukemias that developed in 10-to 38-week-old mice and foundthat 7 leukemias had a least 1 rearrangement that lacked N regions atthe DJ junction. In addition, V replacement events were observed in the1 leukemia studied in detail. Terminal deoxynucleotidyl transferase,the enzyme responsible for N region addition, was expressed at markedlylower levels in late pro-B cells from 7- to 10-day-old Eµ-ret micecompared with transgene-negative mice. Examination of fetallymphopoiesis in Eµ-ret mice identified a relative increase in early(CD45R⁺CD43⁺CD24⁺BP-1)and late pro-B cells and a decrease in more differentiatedCD43 B-lineage cells. Fetal early pro-B cells fromEµ-ret mice proliferated threefold to fivefold greater butdifferentiated to a lesser extent than those from transgene negativemice when cultured in vitro with interleukin-7. These data suggest thatthe B precursor leukemias in adult Eµ-ret mice arise from the progenyof pro-B cells generated in utero.     

Inhibition of the Differentiation of Dendritic Cells From CD34+ Progenitors by Tumor Cells: Role of Interleukin-6 and Macrophage Colony-Stimulating Factor

The escape of malignant cells from the immune response against thetumor may result from a defective differentiation or function ofprofessional antigen-presenting cells (APC), ie, dendritic cells (DC).To test this hypothesis, the effect of human renal cell carcinoma celllines (RCC) on the development of DC from CD34⁺progenitors was investigated in vitro. RCC cell lines were found torelease soluble factors that inhibit the differentiation of CD34⁺ cells into DC and trigger their commitment towardsmonocytic cells(CD14⁺CD64⁺CD1aCD86CD80HLA-DR^low)with a potent phagocytic capacity but lacking APC function. RCC CM werefound to act on the two distinct subpopulations emerging in the cultureat day 6 ([CD14⁺CD1a] and[CD14CD1a⁺]) by inhibiting thedifferentiation into DC of [CD14⁺CD1a]precursors and blocking the acquisition of APC function of the [CD14CD1a⁺] derived DC. Interleukin-6(IL-6) and macrophage colony-stimulating factor (M-CSF) were found tobe responsible for this phenomenon: antibodies against IL-6 and M-CSFabrogated the inhibitory effects of RCC CM; and recombinant IL-6and/or M-CSF inhibited the differentiation of DC similarly toRCC CM. The inhibition of DC differentiation by RCC CM was preceeded byan induction of M-CSF receptor (M-CSFR; CD115) and a loss ofgranulocyte-macrophage colony-stimulating factor receptor (GM-CSFR; CD116) expression at the surface of CD34⁺cells, two phenomenon reversed by anti-IL-6/IL-6R and anti-M-CSF antibodies, respectively. Finally, a panel of tumor cell lines producing IL-6 and M-CSF induced similar effects. Taken together, theresults suggest that the inhibition of DC development could represent afrequent mechanism by which tumor cells will escape immune recognition.     

Minimal Residual Disease Status Before Allogeneic Bone Marrow Transplantation Is an Important Determinant of Successful Outcome for Children and Adolescents With Acute Lymphoblastic Leukemia

The efficacy of allografting in acute lymphoblastic leukemia (ALL)is heavily influenced by remission status at the time of transplant.Using polymerase chain reaction (PCR)-based minimal residual disease(MRD) analysis, we have investigated retrospectively the impact ofsubmicroscopic leukemia on outcome in 64 patients receiving allogeneicbone marrow transplantation (BMT) for childhood ALL. Remission BMspecimens were taken 6 to 81 days (median, 23) before transplant. Allpatients received similar conditioning therapy; 50 received grafts fromunrelated donors and 14 from related donors. Nineteen patients weretransplanted in first complete remission (CR1) and 45 in second orsubsequent CR. MRD was analyzed by PCR of Ig or T-cell receptor  or rearrangements, electrophoresis, and allele-specific oligoprobing.Samples were rated high-level positive (clonal band evident afterelectrophoresis; sensitivity 10² to 10³),low-level positive (MRD detected only after oligoprobing; sensitivity 10³ to 10⁵), or negative. Excluding 8 patients transplanted in CR2 for isolated extramedullary relapse (allMRD), MRD was detected at high level in 12 patients, lowlevel in 11, and was undetectable in 33. Two-year event-free survivalfor these groups was 0%, 36%, and 73%, respectively (P < .001). Follow-up in patients remaining in continuing remission is 20 to96 months (median, 35). These results suggest that MRD analysis couldbe used routinely in this setting. This would allow identification ofpatients with resistant leukemia (who may benefit from innovative BMTprotocols) and of those with more responsive disease (who may becandidates for randomized trials of BMT versus modern intensive relapsechemotherapy).     

Influence of Cytokines on the Growth Kinetics and Immunophenotype of Daughter Cells Resulting From the First Division of Single CD34+Thy-1+lin- Cells

There is a need to determine whether culture conditions may existfor ex vivo expansion of hematopoeitic stem cells (HSC), which favorsolely proliferative self-renewal of HSC as opposed to proliferationwith differentiation. Using single cells, we studied the effects ofindividual and combinations of cytokines in serum-free medium on thekinetics of the first cell doubling and the resulting phenotype of eachof individual daughter cell. CD34⁺Thy-1⁺lincells were plated 1 cell per well in Terasaki plates inserum-free medium containing cytokines. Each well containing a singlecell was monitored daily over 7 days for maintenance, division, or death. When division occurred in an individual well, the phenotype ofthe daughter cells was determined by staining with anti-CD34 fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated lineage specific antibodies. The cumulative percent of wells with anundivided single cell, wells in which the cell had divided, and wellsin which the cell had died were scored. The number of doublets withconserved phenotype (CD34⁺lin) wascompared to those wells with one or more differentiated daughter cells(CD34⁺lin⁺). Over 7 days, cells cultured insingle factors showed that between 13% (interleukin-6 [IL-6]) and29% (thrombopoietin [TPO]) of the cells were undivided, between 13%(IL-1) and 35% (TPO) of the cells doubled, and between 35% (TPO) andgreater than 60% (IL-11, IL-1, or hepatocyte growth factor[HGF]) died. When combinations of cytokines were usedover 7 days, between 5% (FLT-3 ligand [FLT-3L], stemcell factor [SCF], IL-3, IL-6, granulocytecolony-stimulating factor [G-CSF],  nerve growth factor[NGF]) and 22% (FLT-3L + HGF) of the cellsremained undivided, between 15% (HGF, IL-1, IL-11, G-CSF) and 68%(SCF + TPO) of the cells had doubled and between 27% (FLT-3L + TPO) and 70% (HGF, IL-1, IL-11, G-CSF) died. The combination of FLT-3L + TPO induced the highest total percent (64.6%) of cells withconserved phenotype (percent conserved doublets + percent with 1 cellconserved), followed by SCF + TPO, (50%) and the combination ofFLT-3L, SCF, IL-3, IL-6, G-CSF, NGF (53%). These combinations alsoproduced the highest yield of cells with conserved phenotype after onedivision (FLT-3L + TPO  81 cells/100 initial cells, SCF + TPO  68 cells/100 initial cells) (P = .01). Observation of thetime of the initial cell division and phenotype of the daughter cellsallowed us to identify candidate combinations of cytokines that promotemaintenance of lin cells (TPO), or recruit the primitivecells to divide and undergo phenotypic self-renewal (FLT-3L + TPO,SCF + TPO).     

CBFbeta -SMMHC, Expressed in M4eo Acute Myeloid Leukemia, Reduces p53 Induction and Slows Apoptosis in Hematopoietic Cells Exposed to DNA-Damaging Agents

CBF-SMMHC is expressed in M4Eo acute myeloid leukemia (AML) as aresult of inv(16), but how it contributes to leukemogenesis isunknown. p53 mutations are rare in de novo AML, but theyare common in many malignancies. Expression of CBF-SMMHC in Ba/F3 cells reduced p53 induction in response to ionizing radiation oretoposide 3- to 4-fold. However, p53 induction was normal in Ba/F3cells expressing a CBF-SMMHC variant that does not interfere withDNA binding by CBF, indicating that a CBF genetic target regulates p53induction. The p53 gene may be regulated by CBF, because p53 mRNAlevels were reduced by CBF-SMMHC. Reduced p53 induction was notcaused by slowed cell proliferation, a consequence of CBF-SMMHCexpression, because p53 was induced similarly in control cultures andin cultures propagated in 10-fold less interleukin-3 (IL-3).CBF-SMMHC did not slow apoptosis resulting from IL-3 withdrawal,where p53 induction is minimal, but slowed apoptosis in Ba/F3 cellsexposed to 10 Gy of ionizing radiation or 3 to 8 µg/mL etoposide,providing 2-fold protection at 6 or 18 hours. Inhibition of apoptosiswas temporary, because all the cells exposed to these doses ultimatelydied, and clonal survival assays performed using 0.04 µg/mL etoposidedid not show protection by CBF-SMMHC. p21 levels were increased incells subjected to DNA damage, regardless of CBF-SMMHC expressionand attenuated p53 induction. Bcl-2, bcl-x_L,bcl-x_S, and bax levels were unaffected by CBF-SMMHC. Attenuated p53 induction may contribute to leukemogenesis byCBF-SMMHC by slowing apoptosis via a p21-independent mechanism.     

Most Acute Myeloid Leukemia Progenitor Cells With Long-Term Proliferative Ability In Vitro and In Vivo Have the Phenotype CD34+/CD71-/HLA-DR-

Acute myeloid leukemia (AML) occurs as the result of malignanttransformation in a hematopoietic progenitor cell, which proliferates to form an accumulation of AML blasts. Only a minority of these AMLcells are capable of proliferation in vitro, suggesting that AML cellsmay be organized in a hierarchy, with only the most primitive of thesecells capable of maintaining the leukemic clone. To further investigatethis hypothesis, we have evaluated a strategy for purifying theseprimitive cells based on surface antigen expression. As an in vitroendpoint, we have determined the phenotype of AML progenitor cellswhich are capable of producing AML colony-forming cells (CFU) for up to8 weeks in suspension culture (SC) and compared the phenotype with thatof cells which reproduce AML in nonobese diabetic/severe combinedimmunodeficiency (NOD/SCID) mice. AML cells were fluorescence-activatedcell sorted (FACS) for coexpression of CD34 and CD71, CD38,and/or HLA-DR and the subfractions were assayed in vitro and invivo at various cell doses to estimate purification. While the majorityof primary AML CFU lacked expression of CD34, most cells capable ofproducing CFU after 2 to 8 weeks in SC wereCD34⁺/CD71. HLA-DR expression washeterogeneous on cells producing CFU after 2 to 4 weeks.However, after 6 to 8 weeks in SC, the majority of CFU were derivedfrom CD34⁺/HLA-DR cells. Similarly, themajority of cells capable of long-term CFU production from SC wereCD34⁺/CD38. Most cells that were capableof engrafting NOD/SCID mice were alsoCD34⁺/CD71 andCD34⁺/HLA-DR. Engraftment was not achievedwith CD34⁺/CD71⁺ or HLA-DR⁺subfractions, however, in two patients, both the CD34⁺and CD34 subfractions were capable of engrafting theNOD/SCID mice. A three-color sorting strategy combining these antigensallowed approximately a 2-log purification of these NOD/SCID leukemia initiating cells, with engraftment achieved using as few as 400 cellsin one experiment. Phenotyping studies suggest even higher purificationcould be achieved by combining lack of CD38 expression with theCD34⁺/CD71 or CD34⁺/HLADR phenotype. These results suggest that most AML cellscapable of long-term proliferation in vitro and in vivo share theCD34⁺/CD71/HLA-DR phenotypewith normal stem cells. Our data suggests that in this group ofpatients the leukemic transformation has occurred in a primitiveprogenitor, as defined by phenotype, with some degree of subsequentdifferentiation as defined by functional assays.     

Highly Efficient Transduction of the Green Fluorescent Protein Gene in Human Umbilical Cord Blood Stem Cells Capable of Cobblestone Formation in Long-Term Cultures and Multilineage Engraftment of Immunodeficient Mice

Purified CD34⁺ andCD34⁺CD38 human umbilical cord blood (UCB)cells were transduced with the recombinant variant of Moloney murineleukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFPexpression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producercell line (GP+envAm12). SF-EGFP was produced in the PG13 cellline pseudotyped for the gibbon ape leukemia virus (GaLV) envelopeproteins. Using a 2-day growth factor prestimulation, followed by a2-day, fibronectin fragment CH-296-supported transduction, CD34⁺ and CD34⁺CD38 UCBsubsets were efficiently transduced using either vector. The use of theSF-EGFP/PG13 retroviral packaging cell combination consistentlyresulted in twofold higher levels of EGFP-expressing cells than theMFG-EGFP/Am12 combination. Transplantation of 10⁵ inputequivalent transduced CD34⁺ or 5 × 10³input equivalent CD34⁺CD38 UCB cells innonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentagesof 8% and 5%, respectively, which showed that the in vivorepopulating ability of the cells had been retained. In addition, miceengrafted after transplantation of transduced CD34⁺ cellsusing the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFPwith median values of 2% and 23% of human CD45⁺ cells,respectively, which showed that the NOD/SCID repopulating cells weresuccessfully transduced. EGFP⁺ cells were found in allhuman hematopoietic lineages produced in NOD/SCID mice including humanprogenitors with in vitro clonogenic ability. EGFP-expressing cellswere also detected in the human cobblestone area-forming cell (CAFC)assay at 2 to 6 weeks of culture on the murine stromal cell lineFBMD-1. During the transduction procedure the absolute numbers of CAFCweek 6 increased 5- to 10-fold. The transduction efficiency of thisprogenitor cell subset was similar to the fraction ofEGFP⁺ human cells in the bone marrow of the NOD/SCID micetransplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transducedCD34⁺ cells, ie, 6% and 27%, respectively. The studythus shows that purified CD34⁺ and highly purifiedCD34⁺CD38 UCB cells can be transducedefficiently with preservation of repopulating ability. The SF-EGFP/PG13vector/packaging cell combination was much more effective intransducing repopulating cells than the MFG-EGFP/Am12 combination.     

The NF-kappaB subunit Rel A is associated with in vitro survival and clinical disease progression in chronic lymphocytic leukemia and represents a promising therapeutic target

In this study, we characterized nuclear factor B (NF-B) subunit DNA binding in chronic lymphocytic leukemia (CLL) samples and demonstrated heterogeneity in basal and inducible NF-B. However, all cases showed higher basal NF-B than normal B cells. Subunit analysis revealed DNA binding of p50, Rel A, and c-Rel in primary CLL cells, and Rel A DNA binding was associated with in vitro survival (P = .01) with high white cell count (P = .01) and shorter lymphocyte doubling time (P = .01). NF-B induction after in vitro stimulation with anti-IgM was associated with increased in vitro survival (P < .001) and expression of the signaling molecule ZAP-70 (P = .003). Prompted by these data, we evaluated the novel parthenolide analog, LC-1, in 54 CLL patient samples. LC-1 induced apoptosis in all the samples tested with a mean LD₅₀ of 2.8 µM after 24 hours; normal B and T cells were significantly more resistant to its apoptotic effects (P < .001). Apoptosis was preceded by a marked loss of NF-B DNA binding and sensitivity to LC-1 correlated with basal Rel A DNA binding (P = .03, r² = 0.15). Furthermore, Rel A DNA binding was inversely correlated with sensitivity to fludarabine (P = .001, r² = 0.3), implicating Rel A in fludarabine resistance. Taken together, these data indicate that Rel A represents an excellent therapeutic target for this incurable disease.     

Multicentric Castleman disease is associated with polyfunctional effector memory HHV-8-specific CD8+ T cells

Multicentric Castleman disease (MCD) is a devastating human herpesvirus 8 (HHV-8)–related lymphoproliferative disorder that occurs in immunocompromised persons. To determine the role of immune responses in MCD, we studied the frequency, antigenic repertoire, differentiation, and functional profile of HHV-8–specific CD8⁺ T cells in MCD patients and in human immunodeficiency virus–coinfected asymptomatic HHV-8 carriers (AC). Screening CD8⁺ T-cell responses with ELISpot interferon- (IFN-) assays using 56 peptides on 6 latent and lytic HHV-8 proteins showed that MCD and AC patients had responses of similar magnitude and antigenic repertoire and identified a new 10-mer human leukocyte antigen B7 CD8 epitope in K15. Intracellular IFN- staining showed significantly more CD45RA^–CCR7^–CD27^– CD8⁺IFN-⁺ cells (late phenotype) and significantly fewer CCR7^–CD27⁺CD45RA^– cells (early and intermediate phenotype) in MCD than in AC patients. This phenotypic shift was not found for Epstein-Barr virus–specific CD8⁺ T cells tested as controls. HHV-8 viral loads were negatively correlated with early and intermediate effector memory cells. HHV-8–specific T cells were polyfunctional (secretion of IFN-, tumor necrosis factor-, macrophage inflammatory protein-1β, and/or CD107a) in both MCD and AC patients. In conclusion, MCD is not associated with a lack of HHV-8–specific CD8⁺ T cells or limitation of their functional profile. Their differentiation increases with HHV-8 viral load. These results offer new insight into the pathophysiology of MCD.     

Cytokine Gene Polymorphisms Associating With Severe Acute Graft-Versus-Host Disease in HLA-Identical Sibling Transplants

It is now well known that the initial phase of graft-versus-hostdisease (GVHD) involves cytokine release during preconditioning of therecipient of an allogeneic bone marrow transplant (BMT). Tumor necrosisfactor (TNF), in particular, has been implicated in pathologicaldamage and is released pretransplant due to irradiation and cytotoxicpreconditioning regimens. Interleukin-10 (IL-10), a naturalimmunosuppressant of TNF , may be involved in downregulation ofthese responses, which may be an individual patient-specific effect. Inthis study, we determined the genotype for polymorphisms associatedwith TNF and IL-10 in 80 potential allo-BMT recipients andcorrelated the genotype with the severity of GVHD in 49 patients forwhom clinical data relating to GVHD was available. The widely studiedTNF 308 polymorphism does not show any significantassociations, but the d3 homozygous allele of the TNFd microsatelliteis preferentially associated with grade III/IV GVHD (7 of 11 patients)compared with its occurrence in 8 of 38 patients with grade 0/II GVHD(P = .006). Alleles of the IL-10 1064 promoterregion microsatellite polymorphism that possess greater numbers ofdinucleotide (CA) repeats also significantly associate with more severeGVHD. This region has been demonstrated to be important in theregulation of the IL-10 promoter. Eighteen of 38 patients with grade0-II GVHD possessed alleles with greater numbers (12 or more) ofdinucleotide repeats, compared with 9 of 11 cases with grade III-IVGVHD (P < .02). Of the 38 patients with grade 0-II GVHD, 3 of38 had a both TNFd3/d3 and IL-10 (12-15) genotype,compared with 6 of 11 patients with grade III-IV GVHD (P < .001). There was no association of either the TNFd or IL-10 microsatellite polymorphisms with mortality (P = .43 and .51, respectively). Our results suggest that patient cytokine gene polymorphism genotypes may influence GVHD outcome by affecting cytokineactivation during the pretransplant conditioning regimens, and theseresults are the first to suggest a genetic predisposition to thisimportant transplant-related complication.     

Human Granulocyte Colony-Stimulating Factor (G-CSF) Stimulates the In Vitro and In Vivo Development But Not Commitment of Primitive Multipotential Progenitors From Transgenic Mice Expressing the Human G-CSF Receptor

Granulocyte colony-stimulating factor (G-CSF) stimulates theproliferation and restricted differentiation of hematopoietic progenitors into neutrophils. To clarify the effects of G-CSF onhematopoietic progenitors, we generated transgenic (Tg) mice that hadubiquitous expression of the human G-CSF receptor (hG-CSFR). In clonalcultures of bone marrow and spleen cells obtained from these mice,hG-CSF supported the growth of myelocytic as well as megakaryocytic,mast cell, mixed, and blast cell colonies. Single-cell cultures oflineage-negative(Lin)c-Kit⁺Sca-1⁺ orSca-1 cells obtained from the Tg mice confirmed thedirect effects of hG-CSF on the proliferation and differentiation ofvarious progenitors. hG-CSF also had stimulatory effects on theformation of blast cell colonies in cultures using5-fluorouracil-resistant hematopoietic progenitors and clone-sortedLinc-Kit⁺Sca-1⁺ primitivehematopoietic cells. These colonies contained different progenitors inproportions similar to those obtained when mouse interleukin-3 was usedin place of hG-CSF. Administration of hG-CSF to Tg mice led tosignificant increases in spleen colony-forming and mixed/blast cellcolony-forming cells in bone marrow and spleen, but did not alter theproportion of myeloid progenitors in total clonogenic cells. Theseresults show that, when functional G-CSFR is present on the cellsurface, hG-CSF stimulates the development of primitive multipotentialprogenitors both in vitro and in vivo, but does not induce exclusivecommitment to the myeloid lineage.     

Pharmacokinetics and Pharmacodynamics of Oral Methotrexate and Mercaptopurine in Children With Lower Risk Acute Lymphoblastic Leukemia: A Joint Children's Cancer Group and Pediatric Oncology Branch Study

We prospectively assessed the pharmacokinetics of methotrexate,mercaptopurine, and erythrocyte thioguanine nucleotide levels in ahomogenous population of children with lower risk acute lymphoblastic leukemia and correlated pharmacokinetic parameters with disease outcome. The maintenance therapy regimen included daily oralmercaptopurine (75 mg/m²) and weekly oral methotrexate (20 mg/m²). One hundred ninety-one methotrexate doses and 190 mercaptopurine doses were monitored in 89 patients. Plasma drugconcentrations of both agents were highly variable. The area under theplasma concentration-time curve (AUC) of methotrexate ranged from 0.63 to 12 µmolh/L, and the AUC of mercaptopurine ranged from 0.11 to8 µmolh/L. Drug dose, patient age, and duration of therapy didnot account for the variability. Methotrexate AUC was significantly higher in girls than boys (P = .007). There was considerableintrapatient variability for both agents. Erythrocyte thioguaninenucleotide levels were also highly variable (range, 0 to 10 pmol/g Hgb)and did not correlate with mercaptopurine dose or AUC. A Cox regression analysis showed that mercaptopurine AUC was a marginally significant (P = .043) predictor of outcome, but a direct comparison ofmercaptopurine AUC in the remission and relapsed patient groups failedto show a significant difference. Methotrexate and mercaptopurineplasma concentrations and erythrocyte thioguanine nucleotide levelswere highly variable, but measurement of these pharmacokineticparameters at the start of maintenance will not distinguish patientswho are more likely to relapse.     

Targeting NF-kappaB in Waldenstrom macroglobulinemia

The nuclear factor-B (NF-B) path-way has been implicated in tumor B-cell survival, growth, and resistance to therapy. Because tumor cells overcome single-agent antitumor activity, we hypothesized that combination of agents that target differentially NF-B pathway will induce significant cytotoxicity. Therapeutic agents that target proteasome and Akt pathways should induce significant activity in B-cell malignancies as both pathways impact NF-B activity. We demonstrated that perifosine and bortezomib both targeted NF-B through its recruitment to the promoter of its target gene IB using chromatin immunoprecipitation assay. This combination led to synergistic cytotoxicity in Waldenstrom macroglobulinemia (WM) cells that was mediated through a combined reduction of the PI3K/Akt and ERK signaling pathways, found to be critical for survival of WM cells. Moreover, a combination of these drugs with the CD20 monoclonal antibody rituximab further increased their cytotoxic activity. Thus, effective WM therapy may require combination regimens targeting the NF-B pathway.      

The Src family kinase Hck regulates mast cell activation by suppressing an inhibitory Src family kinase Lyn

IgE/antigen-dependent mast cell activation plays a central role in immediate hypersensitivity and other allergic reactions. The Src family tyrosine kinase (SFK) Lyn is activated by the cross-linking of high-affinity IgE receptors (FcRI). Activated Lyn phosphorylates the FcRI subunits, ß and , leading to subsequent activation of various signaling pathways. Lyn also plays a negative regulatory function by activating negative regulatory molecules. Another SFK, Fyn, also contributes to mast cell degranulation by inducing Gab2-dependent microtubule formation. Here we show that a third SFK, Hck, plays a critical role in mast cell activation. Degranulation and cytokine production are reduced in FcRI-stimulated hck^–/– mast cells. The reduced degranulation can be accounted for by defects in Gab2 phosphorylation and microtubule formation. Importantly, Lyn activity is elevated in hck^–/– cells, leading to increased phosphorylation of several negative regulators. However, positive regulatory events, such as activation of Syk, Btk, JNK, p38, Akt, and NF-B, are substantially reduced in hck^–/– mast cells. Analysis of lyn^–/–hck^–/–, lyn^–/–FcRIß^–/–, and hck^–/–FcRIß^–/– cells shows that Hck exerts these functions via both Lyn-dependent and Lyn-independent mechanisms. Thus, this study has revealed a hierarchical regulation among SFK members to fine-tune mast cell activation.      

The Interaction of Hemoglobin E With beta Thalassemia: A Study of Hemoglobin Synthesis in a Family of Mixed Burmese and Iranian Origin

A 5-yr-old girl with hemoglobin E- thalassemia was discovered in a family of mixedorigin. The father is Iranian (-thalassemiatrait) and the mother is Burmese (hemoglobin-E trait). Hemoglobin synthesis wasstudied in vitro in the blood of theproposita and family members. In the subjects with hemoglobin E trait the ratio ofthe quantity of hemoglobin A to hemoglobin E was 3:1. However. the ^A/^Esynthesis ratio in reticulocytes was in therange of 1.5-2.18, and the specific activityof ^E was 31%-49% greater than ^A, suggesting instability of hemoglobin E withpreferential destruction of abnormal hemoglobin. The blood of the proposita exhibitedonly hemoglobin F and hemoglobin E andreticulocytes and bone marrow showed no^A synthesis. This Iranian -thalassemiagene is therefore of the ° type. The ^E/synthesis ratio (approximately 0.74) inblood of the proposita was similar to the^A/ ratio in mildly affected relatives withthalassemia trait. These results suggestthat the severity of the hemoglobin E-thalassemia syndrome is attributable toboth instability and defective synthesis ofhemoglobin E in association with absent^A synthesis due to a ° thalassemia gene.

Submitted on March 19, 1973 Revised on May 4, 1973 Accepted on May 8, 1973     

Perforin and interferon-gamma activities independently control tumor initiation, growth, and metastasis

Perforin (pfp) and interferon- (IFN-) together inC57BL/6 (B6) and BALB/c mouse strains provided optimal protection in 3 separate tumor models controlled by innate immunity. Using experimental (B6, RM-1 prostate carcinoma) and spontaneous (BALB/c, DA3 mammary carcinoma) models of metastatic cancer, mice deficient in both pfp andIFN- were significantly less proficient than pfp- or IFN--deficient mice in preventing metastasis of tumor cells to thelung. Pfp and IFN--deficient mice were as susceptible as micedepleted of natural killer (NK) cells in both tumor metastasis models,and IFN- appeared to play an early role in protection frommetastasis. Previous experiments in a model of fibrosarcoma induced bythe chemical carcinogen methylcholanthrene indicated an important rolefor NK1.1⁺ T cells. Herein, both pfp and IFN- playedcritical and independent roles in providing the host with protectionequivalent to that mediated by NK1.1⁺ T cells. Furtheranalysis demonstrated that IFN-, but not pfp, controlled the growthrate of sarcomas arising in these mice. Thus, this is the first studyto demonstrate that host IFN- and direct cytotoxicity mediated bycytotoxic lymphocytes expressing pfp independently contribute antitumoreffector functions that together control the initiation, growth, andspread of tumors in mice.     

Adult murine hematopoiesis can proceed without beta1 and beta7 integrins

The function of 41 and 47 integrins in hematopoiesis is controversial. While some experimental evidence suggests a crucial role for these integrins in retention and expansion of progenitor cells and lymphopoiesis, others report a less important role in hematopoiesis. Using mice with a deletion of the 1 and the 7 integrin genes restricted to the hematopoietic system we show here that 41 and 47 integrins are not essential for differentiation of lymphocytes or myelocytes. However, 17 mutant mice displayed a transient increase of colony-forming unit (CFU-C) progenitors in the bone marrow and, after phenylhydrazine-induced anemia, a decreased number of splenic erythroid colony-forming units in culture (CFUe's). Array gene expression analysis of CD4⁺CD8⁺ double-positive (DP) and CD4^–CD8^– double-negative (DN) thymocytes and CD19⁺ and CD4⁺ splenocytes did not provide any evidence for a compensatory mechanism explaining the mild phenotype. These data show that 41 and 47 are not required for blood cell differentiation, although in their absence alterations in numbers and distribution of progenitor cells were observed.