A different ontogenesis for chronic lymphocytic leukemia cases carrying stereotyped antigen receptors: molecular and computational evidence.

Chronic lymphocytic leukemia (CLL) is uniquely characterized by the existence of subsets of cases with quasi-identical, 'stereotyped' B-cell receptors (BCRs). Herein we investigate this stereotypy in 2662 patients with CLL, the largest series yet, using purpose-built bioinformatics methods based on sequence pattern discovery. Besides improving the identification of 'stereotyped' cases, we demonstrate that CLL actually consists of two different categories, based on the BCR repertoire, with important biological and ontogenetic differences. The first ( approximately 30% of cases) shows a very restricted repertoire and is characterized by BCR stereotypy (clustered cases), whereas the second includes cases with heterogeneous BCRs (nonclustered cases). Eleven major CLL clusters were identified with antigen-binding sites defined by just a few critically positioned residues, regardless of the actual immunoglobulin (IG) variable gene used. This situation is closely reminiscent of the receptors expressed by cells participating in innate immune responses. On these grounds, we argue that whereas CLL cases with heterogeneous BCRs likely derive from the conventional B-cell pool, cases with stereotyped BCRs could derive from progenitor cells evolutionarily adapted to particular antigenic challenges, perhaps intermediate between a true innate immune system and the conventional adaptive B-cell immune system, functionally similar to what has been suggested previously for mouse B1 cells.

Limited impact of the thymus on immunological recovery during and after chemotherapy in patients with diffuse large B-cell lymphoma.

To investigate the impact of thymus on immunological recovery after dose-dense chemotherapy a prospective study of 17 patients diagnosed with diffuse large B-cell lymphoma (DLBCL) was conducted. Patients were monitored before, during and until 3 months after chemotherapy. The thymus was visualized using computer tomographic scans. Patients were divided into two groups according to thymic size, one group comprising of patients without detectable thymus and one group of patients with detectable thymus. Naïve CD4 and CD8 counts were measured by flow cytometry, and to measure thymic output determination of CD4+ cells containing T-cell receptor excision circles (TREC) was done. During chemotherapy, the naïve CD4 count decreased significantly as did the CD4-TREC%. Significant difference in recovery of naïve CD4 counts between patients with detectable and undetectable thymic tissue during treatment with chemotherapy was not found. CD4-TREC% was associated with lower age. It was not possible to demonstrate an association between thymic size and recovery of the naïve CD4+ cells. The study terminated 3 months after the last cycle of chemotherapy, and at that time point the naïve CD4 counts and the CD4-TREC% had not returned to pretreatment levels. However, patients with detectable thymic tissue had higher naïve CD4 counts after the first cycles of chemotherapy, suggesting that these patients may be less susceptible to infectious complications related to chemotherapy.

Soluble CD40 ligand induces selective proliferation of lymphoma cells in primary mantle cell lymphoma cell cultures.

Interaction between CD40 and the CD40 ligand (CD40L) is critical for the survival and proliferation of B cells during immunopoiesis. However, the role of CD40L in the pathogenesis of malignant lymphomas is ambiguous. Primary mantle cell lymphoma (MCL) cells were cultured in the presence of recombinant human CD40L trimer (huCD40LT), and a significant time- and dose-dependent induction of DNA synthesis was observed in thymidine incorporation assays (n = 7, P <.04). The maximal rate of DNA synthesis was reached at huCD40LT doses of 100 ng/mL and above after 4 days of culture, but a significant increase of DNA synthesis was detected already at doses of 1 ng/mL (P =.03). HuCD40LT never inhibited the basal level of DNA synthesis. These findings established 400 ng/mL of huCD40LT for 4 days as standard conditions in the system. Under these conditions, huCD40LT significantly increased the proportion of cells in the S/G(2)/M phases of the cell cycle in 4 of 7 studied cases, while the fraction of apoptotic cells remained unchanged (n = 7). HuCD40LT also induced expression of CD80/B7-1, CD86/B7-2, and CD95/Fas and up-regulated the expression of HLA-DR (n = 6). With the use of bromodeoxyuridine incorporation in triple-color flow cytometric analysis, it was found that huCD40LT induced cell-cycle progression in light chain-restricted cells only, of which a median of 14% (range, 0.5% to 29%; n = 4) returned to G(0/1) phase DNA content after bromodeoxyuridine incorporation, demonstrating completion of at least one cell cycle in the presence of huCD40LT. Thus, primary clonal MCL cells are activated and can proliferate in the presence of huCD40LT as a single agent.

Oncostatin M and interleukin 6 inhibit cell cycle progression by prevention of p27kip1 degradation in HepG2 cells.

We analysed the regulation of G1-phase progression in relation to cytokine receptor signalling in HepG2 hepatoma cells, stably transduced with the IL-10 receptor after stimulation with Oncostatin M (OSM), IL-6, Leukaemia Inhibitory Factor (LIF) and IL-10. All cytokines induced STAT3 phosphorylation to approximately the same level, but only OSM, and to a lesser extent IL-6, induced STAT5 phosphorylation. The cytokines also stimulated phosphorylation of ERK in the order of decreasing effectiveness: OSM > IL-6 > LIF > IL-10. The same order of activity of the cytokines was observed on inhibition of DNA synthesis and accumulation of cells in the G1-phase of the cell cycle. These processes were accompanied by a decrease in cyclin A expression and CDK2 activity, and enhanced accumulation of p27kip1. The level of p27kip1 mRNA expression was unaffected by the cytokines, and maintenance of the elevated level of p27kip1 occurred independently of de novo protein synthesis. Furthermore, inhibition of proteasomal activity increased the level of p27kip1 in the unstimulated cells to the same level as in OSM-treated cells. Inhibition of MEK activation completely abrogated OSM and IL-6 induced p27kip1 accumulation, while expression of dominant negative STAT5 decreased the OSM and IL-6 mediated inhibition of DNA-synthesis and partially inhibited p27kip1 accumulation.

The cellular biology of B-cell chronic lymphocytic leukemia.

In conclusion, B-CLL cells through their immunophenotype have the functional potential required to interact with cells in what has been called the immunological synapse, i.e. the cognate interactions between T-cells, antigen-presenting cells and B-cells during immunopoiesis. The data reviewed herein provides substantial evidence to suggest that B-CLL cells in fact can interact, not only with T-cells but also with endothelial cells and stromal cells in the bone marrow. These interactions, in particular signaling through CD40, contribute to extended survival and proliferation of B-CLL cells and, thereby, the risk of complete malignant transformation of the clone. Therefore, this review would suggest that the answers to how B-CLL is initiated may be found in molecules responsible for the normal regulation of immunopoiesis. Transformation to malignancy, by contrast, is likely to be caused by loss of control over the G1 restriction in the cell cycle in B-CLL cells.

The prognostic significance of chromosomal analysis and immunophenotyping in 117 patients with de novo acute myeloid leukemia.

Chromosomal abnormalities is one of the most important prognostic factors in acute myeloid leukemia (AML). Other parameters which may influence the prognosis include age, French-American-British-type, clinical variables and possibly the expression of certain immunophenotypic surface makers. However, only rarely has the expression of these markers been analyzed in multivariate models including the information from cytogenetics and clinical variables. We conducted a retrospective study of 117 consecutive adult patients with de novo AML diagnosed and treated in our institution during a 6-year period. Following standard induction chemotherapy with daunomycin and cytosine arabinoside 75 patients (64%) achieved complete remission (CR). The overall 5 year survival rate was 23% and, for patients achieving CR, 30%. When all patients were analyzed age, chromosomal aberration and lack of CD33 expression were of independent prognostic value. The overall 5 year survival rate was 28% for patients aged 55 years or younger, 25% for patients aged 56-65 years and 4% for those > 65 years, P = 0.041. Patients with good-risk chromosomal abnormalities presented an overall 5 year survival of 36%, compared to 25% in patients with normal karyotype, 22% in patients with intermediate risk abnormalities and 5% in patients with poor-risk abnormalities, P = 0.004. Patients with CD33+ myeloblasts had an overall survival of 25% at 5 years compared to 0% in the CD33- patients, P = 0.021. Analysis of the expression of CD7, CD34 and terminal deoxynucleotidyl transferase on myeloblasts had no impact on overall survival in a multivariate analysis. Thus, this study confirmed the prognostic value of age and cytogenetic risk group and defined CD33 as a novel factor of independent prognostic importance in adult de novo AML.

Characterization of interleukin-10 receptor expression on B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (B-CLL) cells accumulate in vivo in the G0/G1 phase of the cell cycle, suggesting that their malignant expansion is due, at least in part, to a delay in cell death. However, the cellular or molecular factors responsible for a delay in B-CLL cell death are unknown. B-CLL cells do express receptors for interferon-alpha (IFN-alpha) and IFN-gamma, and activation of both has been shown to promote B-CLL survival in vitro by preventing apoptosis. The interleukin-10 (IL-10) receptor is another member of the IFN receptor family, but its ligand, IL-10, has been reported to induce apoptosis in B-CLL cells. In the current study, we undertook a biochemical analysis of IL-10 receptor expression on freshly isolated B-CLL cells and characterized the functional responsiveness of IL-10 binding to its constitutively expressed receptor. We show that B-CLL cells bind IL-10 with significant specificity and express between 47 and 127 IL-10 receptor sites per cell, with a dissociation constant in the range of 168 to 426 x 10(-12) mol/L. Ligand binding and activation of the IL-10 receptor expressed on B-CLL cells results in the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3 proteins. This pattern of STAT protein phosphorylation is identical to IL-10 receptor activation on normal cells and similar to IFN-alpha (STAT1 and STAT3) and IFN-gamma (STAT1) receptor activation in CLL. Further, in consecutive samples of fresh blood obtained from patients with B-CLL cells, the addition of IL-10 inhibited B-CLL proliferation, enhanced B-CLL differentiation, but did not induce apoptosis. Indeed, IL-10, like IFN-gamma, was able to significantly reduce the amount of B-CLL cell death caused by hydrocortisone-induced apoptosis. We conclude that cytokines, which signal through the interferon family of receptors, have comparable functional effects on B-CLL cells.

Minimal region of loss at 13q14 in B-cell chronic lymphocytic leukemia.

Allelic loss at nonrandom chromosomal sites is thought to mark the position of tumor suppressor genes involved in the pathogenesis and progression of human malignancies. Solid tumors in particular have been found to harbor multiple genetic changes resulting in loss of function mutations. Tumor suppressor genes have also been found to be involved in the progression of lymphoid tumors. Previous reports have suggested the involvement of a tumor suppressor gene located on the long arm of chromosome 13, between the retinoblastoma (RB) and D13S25 loci, in the pathogenesis and or progression of more than 40% of B-cell chronic lymphocytic leukemia (B-CLL), a common lymphoid malignancy whose molecular etiology remains largely unknown. In the present study, we report the construction and characterization of a YAC contig spanning a region of approximately 3 cM between the RB gene and the D13S31 locus. We also screened 60 paired normal/tumor B-CLL samples for allelic loss on chromosome 13 with nine microsatellite markers located between RB and D13S25. This analysis has allowed us to narrow the smallest region of loss to a segment of 550 kb located between the 206XF12 and D13S25 markers.

Persistence of the AML1/ETO fusion transcript in patients treated with allogeneic bone marrow transplantation for t(8;21) leukemia.

The AML1/ETO fusion transcript is expressed in virtually all patients with t(8;21) (q22;q22) acute myeloid leukemia (AML). The fusion transcript can be detected by reverse transcription-polymerase chain reaction (RT-PCR) in most of these patients in long-term complete remission (CR) following conventional chemotherapy or autologous bone marrow transplantation (BMT). However, AML1/ETO expression has not been analyzed in a series of patients following allogeneic BMT. We examined CR bone marrow (BM) samples and, in some cases, blood samples from 10 patients with t(8;21) leukemia who underwent allogeneic BMT in either first or second remission or first or second relapse. A variety of myeloablative regimens were used. Eight patients received non-T-cell depleted BM from matched sibling donors, one patient received a T-cell depleted haploidentical BM, and one patient received a non-T-cell depleted BM from a matched unrelated donor (MUD). Five patients developed acute and/ or chronic graft versus host disease (GVHD). The furthest time points analyzed for the AML1/ETO transcript in the 10 patients in CR following allogeneic BMT ranged from 7.5 to 83.0 months. Sufficient RNA was extracted from the most recent BM or BM and blood samples from nine patients to assay for presence or absence of the AML1/ETO fusion transcript by RT-PCR. The fusion transcript was detected by RT-PCR in all nine of these patient samples; eight were positive in BM and one was negative in BM, but positive in blood. The fusion transcript could not be detected in a BM sample from the tenth patient obtained 7.5 months after BMT, but the amount of RNA available was suboptimal. Hematopoietic chimerism could be demonstrated in sorted CD34+ BM cells from two of four patient CR BM samples with RT-PCR evidence of the fusion transcript. Additionally, in one of the two cases with chimerism, we demonstrated an abnormal clonal population of recipient cells in the CR BM sample by fluorescence in situ hybridization. One patient died of complications from GVHD, while the other nine patients remain alive without evidence of relapse, with a median follow-up time of 27 (range, 7.5 to 87) months post-BMT. These data suggest that allogeneic BMT, like conventional chemotherapy and autologous BMT, is not sufficient to eradicate cells expressing AML1/ETO, and that a positive RT-PCR for the fusion transcript post allogeneic BMT is compatible with continued CR.

Receptors for interleukin (IL)-10 and IL-6-type cytokines use similar signaling mechanisms for inducing transcription through IL-6 response elements.

The cytoplasmic domain of the receptor for interleukin 10 (IL-10R) contains two box 3 sequence motifs that have been identified in the signal-transducing receptor subunits for IL-6-type cytokines and noted to be required for activating STAT3 and inducing transcription through IL-6-responsive elements. To determine whether the IL-10R has signaling functions similar to IL-6R in cells normally expressing these receptors, leukocytes of the B-, T-, and NK-cell lineages were treated with either cytokine. Both cytokines activated factors that bound to the sis-inducible element and included STAT1 and STAT3. The cell response to IL-10 characteristically differed from that to IL-2/IL-15, IL-4, and interferon gamma. The signaling capabilities of the IL-10R for activating specific STAT proteins and inducing gene transcription were defined by reconstitution of receptor functions in transfected tissue culture cells. COS-1 cells, co-expressing the human IL-10R and individual STAT proteins, confirmed a preference of the IL-10R for STAT3 and STAT1. Unlike many hematopoietin receptors, the IL-10R did not detectably activate STAT5. The IL-10R, together with reporter gene constructs containing different IL-6-responsive gene elements, reconstituted in hepatoma cells an induction of transcription by IL-10 that was comparable to that by IL-6. This regulation could not be appreciably modified by enhanced expression of STAT proteins. The similar actions of IL-10R and IL-6R on the induction of endogenous IL-6-responsive genes were demonstrated in hepatoma cells stably expressing the IL-10R. These receptor functions required the presence of the box 3 motifs, as shown by the analysis of the mouse IL-10R constructs containing progressively truncated cytoplasmic domains. The data demonstrate that the IL-10R, unlike other members of the interferon receptor family, is highly effective in recruiting the signaling pathways of IL-6-type cytokine receptors.

Improved vaccination response during ranitidine treatment, and increased plasma histamine concentrations, in patients with B cell chronic lymphocytic leukemia.

Patients with B cell chronic lymphocytic leukemia (B-CLL) have decreased capacity to mount relevant antibody responses upon immunization, and development of hypogammaglobulinemia is part of the natural history of the disease. We investigated the influence of histamine type-2 (H2) receptor blockade by ranitidine on the in vivo antibody production in B-CLL patients following vaccination. Anti-polysaccharide antibodies in B-CLL patients, vaccinated with a tetanus-toxoid conjugated vaccine against Haemophilus influenzae type-B (Hib), reached long-term protective levels in more than 90% of B-CLL patients randomized to ranitidine treatment, as compared to 43% of the untreated patients (P = 0.024). No difference in the response to vaccination against influenza virus types A and B protein could be detected between the two groups. Plasma histamine levels were 2-fold to 20-fold higher in 23 out of 31 B-CLL patients, compared to normal controls, and these levels showed a significant positive correlation to disease duration. These findings indicate the possibility of improving in vivo antibody production against a highly relevant pathogen in B-CLL patients by histamine type-2 receptor blockade, and the combined finding of an immune-stimulatory effect of ranitidine and increased plasma histamine levels, strongly suggests the involvement of histamine in the pathogenesis of B-CLL immunodeficiency.

Treatment of hypogammaglobulinaemia in chronic lymphocytic leukaemia by low-dose intravenous gammaglobulin.

Intravenous immunoglobulin replacement therapy reduces the number of bacterial infections in B-cell chronic lymphocytic leukaemia (B-CLL) patients. However, due to the complexity of immunodeficiency in B-CLL and the cost-effectiveness of replacement therapy, it is important to identify patients who are likely to benefit from the treatment and to investigate which dose should be used. 15 patients with hypogammaglobulinaemia and a history of recurrent infections received a fixed dose of 10 grams of gammaglobulin intravenously every 3 weeks. Serum IgG levels were significantly higher after three doses (p = 0.0002), and stabilized just above lower reference value after 11 doses. The total number of infection-related events during 168 months before therapy was compared to the total number of infection-related events in 169 months during therapy. The number of antibiotic prescriptions was reduced from 78 to 54 (N.S.), the number of admissions to hospital due to infections was reduced from 16 to 5 (p = 0.047) and the number of febrile episodes was reduced from 63 to 31 (p = 0.004). We conclude that a fixed low dose of gammaglobulin intravenously can restore normal serum IgG levels in hypogammaglobulinaemic B-CLL patients, and leads to a decreased number of febrile episodes and admissions to hospital due infections.

No reinnervation of hepatic sympathetic nerves after liver transplantation in human subjects.

Transplantation of the liver results in surgical denervation of the organ. However, it is not known whether and to what extent sympathetic reinnervation occurs postoperatively in the transplanted human liver. Thirty-two liver biopsies (right lobe) were obtained from 13 liver-transplanted patients 1, 3, 6, 12 or 30 months after transplantation and 11 biopsies were obtained from 11 non-transplanted subjects with normal liver tests. The concentrations of the sympathetic neurotransmitter norepinephrine and of epinephrine were determined in liver tissue homogenates. The concentration of norepinephrine was 0.019 +/- 0.05 nmol. g wet liver tissue-1 (mean and SE, n = 32) in the transplanted patients, which was only 1% of the concentration in biopsies from control subjects (2.180 +/- 0.420 nmol.g wet liver tissue-1). The hepatic norepinephrine concentration did not increase significantly over time in liver-transplanted patients during the observation period (0.015 +/- 0.008 nmol.g wet wt-1 (1 month post) (n = 8) vs. 0.024 +/- 0.018 nmol.g wet wt-1 (12 months post) (n = 6) and 0.012 +/- 0.006 nmol.g wet wt-1 (30 months post) (n = 5)) (p < 0.05). The liver tissue concentration of epinephrine was markedly lower in liver-transplanted subjects (0.01 +/- 0.003 nmol.g wet tissue-1) than in control subjects (0.04 +/- 0.007 nmol.g-1) (p < 0.01). This study indicates that within the first years after transplantation, there is no evidence of sympathetic liver nerve reinnervation in liver-transplanted patients.