Tumor Budding Correlates With the Protumor Immune Microenvironment and Is an Independent Prognostic Factor for Recurrence of Stage I Lung Adenocarcinoma.

BACKGROUND: Immune cell infiltration associated with tumor capsule disruption and tumor budding has been shown to reflect invasiveness, metastasis, and unfavorable prognosis in colorectal cancer. We investigated the influence of tumor budding on prognosis and its association with the immune microenvironment in lung adenocarcinoma. METHODS: Tumor slides from resected stage I lung adenocarcinomas were reviewed (n = 524 and n = 514, for training and validation cohorts, respectively) for assessment of tumor budding. CD3+ and forkhead box P3+ (FoxP3+) lymphocytes, CD68+ macrophages, IL-7 receptor, and IL-12 receptor ß2 were analyzed using tissue microarrays constructed from tumor and stroma. Probability of recurrence was calculated using the competing risks method. RESULTS: In the training cohort, risk of recurrence for high-grade tumor budding was higher than it was for low-grade tumor budding (32% vs 12%, P < .001), which was confirmed in the validation cohort (P = .005). Tumor budding stratified the risk of recurrence for acinar-predominant (22% vs 9%, P < .001), papillary-predominant (22% vs 13%, P = .045), and solid-predominant (39% vs 19%, P = .022) tumors. Tumor budding was associated with higher stromal FoxP3+ lymphocyte infiltration, higher stromal FoxP3/CD3 risk index, higher tumoral and stromal CD68+ macrophage infiltration, and IL-7 receptor overexpression (P < .001, all associations). Tumor budding remained independently associated with recurrence on multivariate analysis (hazard ratio, 1.61; P = .008). CONCLUSIONS: Tumor budding is an independent prognostic factor of stage I lung adenocarcinoma and correlates with the protumor immune microenvironment. Our findings advocate investigating tumor-immune cell interactions at the invading edge as a biologic driver of tumor aggressiveness.

Clonal evolution of CD8+ T cell responses against latent viruses: relationship among phenotype, localization, and function.

UNLABELLED: Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, interleukin-7 receptor α (IL-7Rα)-expressing cells contain the precursors for long-lived antigen-experienced CD8(+) T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here, we studied whether IL-7Rα-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool. Using flow cytometry and functional assays, we found that the IL-7Rα(+) hCMV-specific T cell population comprises cells that have a memory phenotype and lack effector features. We used next-generation sequencing of the T cell receptor to compare the clonal repertoires of IL-7Rα(+) and IL-7Rα(-) subsets. We observed limited overlap of clones between these subsets during acute infection and after 1 year. When we compared the hCMV-specific repertoire between PB and paired LNs, we found many identical clones but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only rarely identical to the unique LN clones. Thus, although PB IL-7Rα-expressing and LN hCMV-specific CD8(+) T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8(+) T cell pool during latency or upon antigen recall. IL-7Rα(+) PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8(+) effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naive CD8(+) T cell pool. IMPORTANCE: Insight into the self-renewal properties of long-lived memory CD8(+) T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is, however, not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: circulating acute-phase IL-7Rα-expressing hCMV-specific CD8(+) T cells and lymph node (LN)-residing hCMV-specific (central) memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation seldom originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues or are recruited from the naive CD8(+) T cell pool.

Clinical impact of immune microenvironment in stage I lung adenocarcinoma: tumor interleukin-12 receptor ß2 (IL-12Rß2), IL-7R, and stromal FoxP3/CD3 ratio are independent predictors of recurrence.

PURPOSE: Mounting evidence suggests that tumor-infiltrating immune cells have prognostic value for patients with solid organ malignancies. Our aim was to investigate the prognostic significance of the immune microenvironment in patients with stage I lung adenocarcinoma (ADC). PATIENTS AND METHODS: Using tissue microarray and immunohistochemistry, we investigated eight types of tumor-infiltrating immune cells in the tumor nest and tumor-associated stroma as well as tumor expression of five cytokines in a uniform cohort of 956 patients with stage I lung ADC (478 each in training and validation cohorts). RESULTS: Although a high density of stromal forkhead box P3 (FoxP3) -positive cells was associated with shorter recurrence-free probability (RFP; P = .043), the relative proportion of stromal FoxP3 to CD3 was a stronger predictor of recurrence (5-year RFP, 85% for high v 77% for low ratio; P = .004). High expression of tumor interleukin-12 receptor ß2 (IL-12Rß2) was associated with better outcome (5-year RFP, 90% for high v 80% for low expression; P = .026), whereas high expression of tumor IL-7R was associated with worse outcome (5-year RFP, 76% for high v 86% for low expression; P = .001). In multivariate analysis, these immune markers were independently associated with recurrence. Although IL-7R remained significant for poor overall survival, all the markers remained prognostic for recurrence in patients with stages IA and IB disease as well as for patients with tumors ≤ 2 cm. CONCLUSION: Our investigation confirms the biologic and prognostic significance of the tumor immune microenvironment for patients with stage I lung ADC and provides support for its use to stratify clinical outcome and immunotherapeutic interventions.

Placental macrophage (Hofbauer cell) polarization is independent of maternal allergen-sensitization and presence of chorioamnionitis.

BACKGROUND: Macrophages can polarize in which M1/classically activated and M2/alternatively activated macrophages are considered to be the extremes. M1 macrophages are involved in inflammatory reactions, while M2 macrophages are suggested to be involved in homeostasis, parasite killing, tumor promotion, tissue remodeling and in allergic reactions. We hypothesized that polarization of placental macrophages (Hofbauer cells) is influenced by the allergen-sensitization status of the mother and/or the presence of chorioamnionitis, a placental inflammation. This Hofbauer cell polarization might be associated to the intrauterine environment and influence the risk of allergy development for the child. Therefore we aimed to determine the polarization status of Hofbauer cells in health and disease. METHODS: We determined the expression of CD68, CX3CR1, IL-7R, DC-SIGN/CD209 and CD163 in placentas of sensitized versus non-sensitized mothers (n = 17), and placentas with or without histological chorioamnionitis (n = 10) by means of immunohistochemical analysis and quantitative real-time PCR (qPCR). RESULTS: Protein expression of the M1 markers (CX3CR1, IL-7R and CCR7) could not be detected in any of the analyzed samples while the M2 markers (DC-SIGN, CD163 and mannose receptor/CD206) were readily detected. Significant differences between non-sensitized versus sensitized mothers and uncomplicated versus chorioamnionitis complicated pregnancies were not detected at protein or at mRNA expression level. CONCLUSIONS: These results suggest that Hofbauer cells have an M2 phenotype, and that their polarization is not affected by maternal allergen-sensitization or by presence of chorioamnionitis.

Bcl11b heterozygosity promotes clonal expansion and differentiation arrest of thymocytes in gamma-irradiated mice.

Bcl11b encodes a zinc-finger transcription factor and functions as a haploinsufficient tumor suppressor gene. Bcl11b(KO/KO) mice exhibit differentiation arrest of thymocytes during beta-selection as has been observed with other mouse models involving knockouts of genes in the Wnt/beta-catenin signaling pathway. Recurrent chromosomal rearrangement at the BCL11B locus occurs in human T-cell leukemias, but it is not clear how such rearrangement would contribute to lymphomagenesis. To address this issue, we studied clonal cell growth, cell number, and differentiation of thymocytes in Bcl11b(KO/+) mice at different time points following gamma-irradiation. Analysis of D-J rearrangement at the T cell receptor beta-chain (TCRbeta) locus and cell surface markers by flow cytometry revealed two distinct populations of clonally growing thymocytes. In one population, thymocytes share a common D-J rearrangement but retain the capacity to differentiate. In contrast, thymocytes in the second population have lost their ability to differentiate. Since the capacity to self renew and differentiate into multiple cell lineages are fundamental properties of adult stem cells, the differentiation competent population of thymocytes that we have isolated could potentially function as cancer stem cells. We also demonstrate increased expression of beta-catenin, a well-known oncogenic protein, in Bcl11b(KO/+) thymocytes. Collectively, the Bcl11b(KO/+) genotype contributes to clonal expansion and differentiation arrest in part through an increase in the level of beta-catenin.

Potential role for IL-7 in Fas-mediated T cell apoptosis during HIV infection.

IL-7 promotes survival of resting T lymphocytes and induces T cell proliferation in lymphopenic conditions. As elevated IL-7 levels occur in HIV-infected individuals in addition to high Fas expression on T cells and increased sensitivity to Fas-induced apoptosis, we analyzed whether IL-7 has a regulatory role in Fas-mediated T cell apoptosis. We show that IL-7 up-regulates Fas expression on naive and memory T cells through a mechanism that involves translocation of Fas molecules from intracellular compartments to the cell membrane. IL-7 induced the association of Fas with the cytoskeletal component ezrin and a polarized Fas expression on the cell surface. The potential role of IL-7 in Fas up-regulation in vivo was verified in IL-7-treated macaques and in HIV-infected or chemotherapy treated patients by the correlation between serum IL-7 levels and Fas expression on T cells. IL-7 treatment primed T cells for Fas-induced apoptosis in vitro and serum IL-7 levels correlated with the sensitivity of T cells to Fas-induced apoptosis in HIV-infected individuals. Our data suggest an important role for IL-7 in Fas-mediated regulation of T cell homeostasis. Elevated IL-7 levels associated with lymphopenic conditions, including HIV-infection, might participate in the increased sensitivity of T cells for activation-induced apoptosis.

Four functionally distinct populations of human effector-memory CD8+ T lymphocytes.

In humans, the pathways of memory and effector T cell differentiation remain poorly defined. We have dissected the functional properties of ex vivo effector-memory (EM) CD45RA-CCR7- T lymphocytes present within the circulating CD8+ T cell pool of healthy individuals. Our studies show that EM T cells are heterogeneous and are subdivided based on differential CD27 and CD28 expression into four subsets. EM(1) (CD27+CD28+) and EM(4) (CD27-CD28+) T cells express low levels of effector mediators such as granzyme B and perforin and high levels of CD127/IL-7Ralpha. EM(1) cells also have a relatively short replicative history and display strong ex vivo telomerase activity. Therefore, these cells are closely related to central-memory (CD45RA-CCR7+) cells. In contrast, EM(2) (CD27+CD28-) and EM(3) (CD27-CD28-) cells express mediators characteristic of effector cells, whereby EM(3) cells display stronger ex vivo cytolytic activity and have experienced larger numbers of cell divisions, thus resembling differentiated effector (CD45RA+CCR7-) cells. These data indicate that progressive up-regulation of cytolytic activity and stepwise loss of CCR7, CD28, and CD27 both characterize CD8+ T cell differentiation. Finally, memory CD8+ T cells not only include central-memory cells but also EM(1) cells, which differ in CCR7 expression and may therefore confer memory functions in lymphoid and peripheral tissues, respectively.

The blood contains multiple distinct progenitor populations with clonogenic B and T lineage potential.

The thymus is seeded by bone marrow-derived progenitors that circulate in the blood. Multiple cell types can be found in the thymus early after i.v. administration or in steady state, but most fail to satisfy the known characteristics of true T progenitors. Cells that do conform to classical definitions retain multilineage potential, but surprisingly, cannot make B cells. Because acquisition of the T lineage fate among noncommitted progenitors is a lengthy process, the absence of B cell potential in early thymocytes suggests that B and T lineages diverge prethymically. To test this suggestion, we screened numerous presumptive progenitor populations for T cell growth and differentiation potential, as well as for clonogenic T or B cell development. We find that blood and marrow each contain multiple distinct subsets that display growth and differentiation potential consistent with being canonical T progenitors. Assessment of clonogenic potential further shows that although all blood and marrow populations have high T cell cloning potential, no T/non-B cells are apparent. These data suggest that either true thymic reconstitution potential derives from a small T/non-B cell subset of one of these populations, or that most of the cells defined as canonical progenitors within the thymus do not, in fact, reside in the mainstream of T progenitor differentiation.

CD127+CCR5+CD38+++ CD4+ Th1 effector cells are an early component of the primary immune response to vaccinia virus and precede development of interleukin-2+ memory CD4+ T cells.

The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-gamma)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-gamma and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.

Infection of CD127+ (interleukin-7 receptor+) CD4+ cells and overexpression of CTLA-4 are linked to loss of antigen-specific CD4 T cells during primary human immunodeficiency virus type 1 infection.

We recently found that human immunodeficiency virus (HIV)-specific CD4+ T cells express coreceptor CCR5 and activation antigen CD38 during early primary HIV-1 infection (PHI) but then rapidly disappear from the circulation. This cell loss may be due to susceptibility to infection with HIV-1 but could also be due to inappropriate apoptosis, an expansion of T regulatory cells, trafficking out of the circulation, or dysfunction. We purified CD38+++CD4+ T cells from peripheral blood mononuclear cells, measured their level of HIV-1 DNA by PCR, and found that about 10% of this population was infected. However, a small subset of HIV-specific CD4+) T cells also expressed CD127, a marker of long-term memory cells. Purified CD127+CD4+ lymphocytes contained fivefold more copies of HIV-1 DNA per cell than did CD127-negative CD4+ cells, suggesting preferential infection of long-term memory cells. We observed no apoptosis of antigen-specific CD4+ T cells in vitro and only a small increase in CD45RO+CD25+CD127dimCD4+ T regulatory cells during PHI. However, 40% of CCR5+CD38+++ CD4+ T cells expressed gut-homing integrins, suggesting trafficking through gut-associated lymphoid tissue (GALT). Furthermore, 80% of HIV-specific CD4+ T cells expressed high levels of the negative regulator CTLA-4 in response to antigen stimulation in vitro, which was probably contributing to their inability to produce interleukin-2 and proliferate. Taken together, the loss of HIV-specific CD4+ T cells is associated with a combination of an infection of CCR5+ CD127+ memory CD4+ T cells, possibly in GALT, and a high expression of the inhibitory receptor CTLA-4.

Memory lineage relationships in HTLV-1-specific CD8+ cytotoxic T cells.

Cytotoxic memory T cells play a critical role in combating viral infections; however, in some diseases they may contribute to tissue damage. In HAM/TSP, HTLV-1 Tax 11-19+ cells proliferate spontaneously in vitro and can be tracked using the Tax 11-19 MHC Class I tetramer. Immediately ex vivo, these cells were a mix of CD45RA-/CCR7- TEM and CD45RA+/CCR7- TDiff memory CTL. The subsequent proliferating Tax 11-19 tetramer+ population expressed low levels of IL-7Ralpha, failed to respond to IL-7 and IL-15, and did not develop a TCM phenotype. Thus, chronic exposure to viral antigen may result in a sustained pool of TEM cells that home to the CNS and mediate the spinal cord pathology seen in this disease.

Optimization of culture and storage conditions for an in vitro system to evaluate thymocyte phenotype and function.

Studies on thymopoiesis are critical to the understanding of T-cell homeostasis as well as the host response to T-cell depletion. Various in vitro culture systems have been used in the study of thymocyte development; however it is unclear if current co-culture methods have been fully optimized. In this study in vitro suspension cultures have been re-evaluated and the optimal storage conditions for thymocytes have been established by evaluating various methods of storing/isolating thymic tissue and isolated thymocytes as well as the source of thymic epithelial cells (TEC). It was determined that thymocytes must be freshly isolated from whole thymic tissue and ideally stored at 4 degrees C prior to co-culture. Co-culture with either autologous or allogeneic TEC results in similar thymocyte subset distribution as well as interleukin-7 receptor-alpha (CD127) expression on these subsets. To evaluate the influence of the source of TEC on one aspect of thymocyte function the effect of IL-7 stimulation on the expression of CD127 was evaluated. IL-7 stimulation resulted in a downregulation of the expression of CD127 on all thymic subsets similar to that observed in circulating CD8+ T-cells. The effect of this was the same whether TEC were autologous or allogeneic. Optimizing culture techniques and facilitating the study of individual thymocyte subsets will lead to a better understanding of thymic function and development. It could also lead to therapeutic approaches that enhance immune recovery after T-cell depletion in HIV infection, bone marrow transplantation or following chemotherapy.

Preapoptotic phenotype of viral epitope-specific CD8 T cells precludes memory development and is an intrinsic property of the epitope.

Virus-specific CD8 T cells after clearance of infection reduce their number in lymphoid organs by apoptotic death and by migration into peripheral tissues. During and after infection, many lymphocytic choriomeningitis virus (LCMV)-specific CD8 T cells in lymphoid but not peripheral tissues are in a preapoptotic state, as detected by the early apoptosis marker annexin V. In this report, we investigated the significance of this preapoptotic state and how it may be influenced by viral epitope specificity. Stimulation with anti-CD3 or IL-2 in vitro postponed DNA fragmentation in annexin V+ cells, but adoptive transfer studies in vivo showed that this preapoptotic phenotype precluded the development of functional memory. CD8 T cells specific to LCMV epitopes NP396 and gp33 differed in their preapoptotic state, with NP396-specific T cells binding more annexin V than gp33-specific T cells. These epitope- and tissue-dependent differences were seen in primary, memory, and secondary responses and in mice receiving different displays of Ag by infection with LCMV strains of different tropisms or by infection with vaccinia virus recombinants expressing LCMV proteins. Thus, the epitope-dependent differences in apoptosis were independent of virus tropisms, duration of Ag exposure, and competition within APCs, and were an intrinsic property of the epitope. The tissue-dependent and epitope-dependent preapoptotic state correlated with reduced expression of IL-7Ralpha.

IL-7-dependent homeostatic proliferation in the presence of a large number of T cells in gld mice.

In gld mice, CD4 and 8-double-negative (DN) T cells as well as naive and memory-phenotype T cells accumulate in the peripheral lymphoid organs. Although Fas ligand (L) defect accounts for the progressive accumulation of abnormal DN T cells, the existence of other mechanisms which may be involved in the defective homeostasis in gld mice has been unclear. In this study, we analyze T-cell homeostasis in gld mice using adoptive transfer systems. It was shown that a gld, but not C57BL/6 (B6), environment led to augmented proliferation of B6 T cells transferred without up-regulation of CD69. Thus, the augmented T-cell proliferation seemed to result from mal-homeostatic proliferation even in the presence of a large number of recipient T cells. T cells from lpr mice showed no significant proliferation in the B6 environment, suggesting that the absence of Fas-Fas L interaction was not responsible for the mal-homeostatic proliferation. Although similar levels of IL-7 mRNA were detected in gld and B6 spleens, the intensity of CD127 and the proportion of CD127+ cells in the T cells were significantly lower in gld mice than in B6 mice, suggesting that IL-7 excess in a gld environment is responsible for the abnormal proliferation of transferred T cells. The administration of anti-CD127 antibody inhibited the proliferation of transferred lymphocytes. Thus, IL-7-dependent proliferation seems to be involved in the abnormal proliferation of lymphocytes in gld recipients.

Selective expression of IL-7 receptor on memory T cells identifies early CD40L-dependent generation of distinct CD8+ memory T cell subsets.

Several recent studies have demonstrated that T-helper cell-dependent events during the initial priming period are required for the generation of CD8(+) T cell-mediated protective immunity. The underlying mechanisms of this phenomenon have not yet been determined, mostly because of difficulties in studying memory T cells or their precursor populations at early stages during immune responses. We identified IL-7 receptor (CD127) surface expression as a marker for long-living memory T cells, most importantly allowing the distinction between memory and effector T cells early after in vivo priming. The combination of surface staining for CD127 and CD62L further separates between two functionally distinct memory cell subsets, which are similar (if not identical) to cell subsets recently described as central memory T cells (CD127(high) and CD62L(high)) and peripheral effector memory T cells (CD127(high) and CD62L(low)). Using this new tool of memory T cell analysis, we demonstrate that CD8(+) T cell priming in the absence of T cell help or CD40L specifically alters the generation of the effector memory T cell subset, which appears to be crucial for immediate memory responses and long-term maintenance of effective protective immunity. Our data reveal a unique strategy to obtain information about the quality of long-term protective immunity early during an immune response, a finding that may be applied in a variety of clinical settings, including the rapid monitoring of vaccination success.

IL-7 enhances peripheral T cell reconstitution after allogeneic hematopoietic stem cell transplantation.

We used clinically relevant murine allogeneic bone marrow transplantation (BMT) models to study the mechanisms by which IL-7 administration can improve posttransplant peripheral T cell reconstitution. After transplant we could distinguish two populations of mature donor T cells: (a) alloreactive T cells with decreased expression of CD127 (IL-7 receptor alpha chain) and (b) nonalloreactive T cells, which express CD127 and undergo homeostatic proliferation. IL-7 administration increased the homeostatic proliferation of nonalloreactive T cells, but had no effect on alloreactive T cells and the development of graft-versus-host disease. Allogeneic transplant of purified hematopoietic stem cells and adoptive transfer of thymocytes into lethally irradiated hosts suggested that recent thymic emigrants can undergo homeostatic proliferation and acquire a memory-like phenotype. We found by BrdU pulse-chase, cell cycle, and annexin V analyses that IL-7 administration has significant proliferative and antiapoptotic effects on posttransplant peripheral T cells. We conclude that homeostatic expansion is important for T cell reconstitution after allogeneic BMT and involves both transferred mature T cells and recent thymic emigrants. Apart from its thymopoietic effects, IL-7 promotes peripheral T cell reconstitution through its selective proliferative and antiapoptotic effects on nonalloreactive and de novo-generated T cells, but has no effect on alloreactive T cells.

Characteristics of t(8;21) acute myeloid leukemia (AML) with additional chromosomal abnormality: concomitant trisomy 4 may constitute a distinctive subtype of t(8;21) AML.

t(8;21)(q22;q22) is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), especially in FAB M2. Clinically, this type of AML often shows eosinophilia and has a high complete remission rate with conventional chemotherapy. t(8;21) AML is also frequently associated with additional karyotypic aberrations, such as a loss of the sex chromosome; however, it is unclear whether these aberrations change the biological and clinical characteristics of t(8;21) AML. To investigate this issue, 94 patients with t(8;21) AML were categorized according to their additional karyotypic aberrations, which were detected in more than three cases, and then morphologic features, phenotypes, expression of cytokine receptors, and clinical features were compared to t(8;21) AML without other additional aberrant karyotypes. t(8;21) AML with loss of the sex chromosome and abnormality of chromosome 9 were found in 27 cases (29.3%) and 10 cases (10.6%), respectively; however, no differences were observed from the t(8;21) AML without other additional karyotypes in terms of morphological and phenotypic features. There was also no significant difference in the clinical outcome among these three groups. On the other hand, trisomy 4 was found in three cases (3.2%) and these cells showed low expressions of CD19 (P=0.06) and IL-7 receptor (P=0.05), and high expressions of CD33 (P=0.13), CD18 (P=0.03), and CD56 (P=0.03) when compared to t(8;21) AML without additional karyotypes. Moreover, all three t(8;21) AML cases with trisomy 4 did not show eosinophilia in their bone marrow and died within 2.4 years. These observations suggest that additional karyotypic aberration, t(8;21) with trisomy 4 is rare, but it may constitute a distinctive subtype of t(8;21) AML.

In vitro identification of human pro-B cells that give rise to macrophages, natural killer cells, and T cells.

In this study we report the molecular and functional characterization of very early interleukin 7 receptor alpha (IL-7Ralpha)+-CD79a+CD19- B-cell progenitors, produced by human CD34+CD19-CD10- cord blood cells grown in the presence of stromal cells and cytokines. Purified IL-7Ralpha+CD79a+CD19- cells transcribed the B-lymphoid specific genes E2A, EBF, TdT, Rag-1, had initiated DJH rearrangements, but almost lacked Pax-5 mRNA. When exposed to appropriate environmental conditions, these cells repressed B-cell genes and completely differentiated into CD14+ macrophages, CD56+ natural killer cells, and CD4high T cells. Retention of the DJH rearranged genes in both CD14+ and CD56+ cells unambiguously demonstrates that early B-cell genes, expressed prior to Pax-5, can be activated in a multipotent human progenitor cell whose final fate, including in non-B lineages, is determined by external signals.

Tumour necrosis factor-alpha induced CD70 and interleukin-7R mRNA expression in BEAS-2B cells.

Over the past few years, evidence has emerged for the potential role of the human bronchial epithelial cell in the initiation and progress of inflammation of the airway. Thus, the aim of this study was to investigate the expression pattern of cytokines and immunomodulatory factors in the human bronchial epithelial cell. To elucidate this highly complex expression and regulation pattern, the simian virus-40 transformed human bronchial-epithelial cell line BEAS-2B was stimulated with human recombinant tumour necrosis factor (hrTNF)-alpha (10 ng x mL(-1) (specific activity, 2.86 x 10(7) U x mg(-1))) and messenger ribonucleic acid (mRNA) expression pattern was analysed by complementary deoxyribonucleic acid (cDNA) array analysis. Among 375 arrayed cDNA clones, 173 (46%) were detected in BEAS-2B cells. The levels of expression of 17 genes, including those of monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, growth-related oncogene (GRO) alpha, beta, gamma, interleukin (IL)-7 receptor, CD70, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and regulated in activation, normal T-cell expressed and secreted (RANTES) were elevated after TNF-alpha stimulation. The differential character of 12 clones was further characterised and verified by real time polymerase chain reaction (PCR) analysis of total ribonucleic acid (RNA) isolated from BEAS-2B cells after 4 or 16 h incubation with increasing TNF-alpha concentrations (1 pg-10 ng x mL(-1)). The authors semiquantified concentration-dependent mRNA upregulation of cytokines and immunology factors identified in the array and could determine threshold values of mRNA increases at 10 pg x mL(-1)-1 ng x mL(-1) TNF-alpha by real-time PCR. For CD70 (CD27 ligand) and interleukin-7 receptor, which to the best of the author's knowledge have not yet been described in the human bronchial epithelial cell, a rapid and continuous messenger ribonucleic acid increase induced by 100 pg x mL(-1) tumour necrosis factor-alpha after only 60-90 min was shown. A potential role for these genes in the inflammatory process in the human bronchial epithelial cell is proposed.

Growth of human T cell acute lymphoblastic leukemia lymphoblasts in NOD/SCID mouse fetal thymus organ culture.

The in vitro proliferation of T cell acute lymphoblastic leukemia (T-ALL) cells in its entirety has not been well delineated because of a lack of an appropriate culture system that mimics the growth pattern in a living body. We applied a NOD/SCID mouse fetal thymus organ culture (FTOC) for leukemic cells from fresh (one case) and frozen (seven cases) bone marrow (BM) samples of children with T-ALL. Cell growth was observed in all seven samples in the culture, reaching a proliferational peak at 4 weeks, and it was calculated that the proliferation potential was 212-to 319-fold. The FTOC-derived T-ALL cells showed similarity to the original cells morphologically and immunophenotypically, still possessed clonalities and were able to regenerate overt leukemia in NOD/SCID mice. These FTOC-derived T-ALL cells differed from ordinary cell lines because they always need FTOC support. Thus, we established a new in vitro culture for T-ALL cells. A comparison of the original and FTOC-derived T-ALL cells revealed that the proportion of cells expressing IL-7R increased in all seven cases. Sorting and re-seeding of FTOC-derived IL-7R+ and IL-7R- cells into secondary FTOC resulted in a predominant generation of IL-7R+ cells from both fractions, while IL-7R- cells proliferated more potently than did IL-7R+ cells, suggesting that a pathway for the conversion of IL-7R- to IL-7R+ exists during the proliferation of T-ALL lymphoblasts. Addition of exogenous IL-7 or neutralization with anti-IL-7 antibody did not influence the growth pattern of T-ALL cells in FTOC. The current study provides a unique assay system for the exploration of the hierarchy within human T-lymphoid leukemic cells, and should facilitate the establishment of novel therapeutic modalities.