Ikaros family transcription factors expression in rat thymus: detection of impaired development.

The expression of Ikaros family transcription factors and consequently their signalling pathway is limiting for hematopoietic and lymphocyte development in mice and human. Due to their importance, these transcription factors are highly homologous between species. As an initial approach to examining the possible involvement of Ikaros transcription factors in pathogenesis of rat lymphoid development, we analyzed the expression of all known Ikaros family members, Ikaros, Aiolos, Helios, Eos and Pegasus in the rat thymus. We established a semi-quantitative RT-PCR to detect mRNA of each transcription factor. For the first time we give evidence of the expression of Ikaros family transcription factors in the rat thymus. Further, we evaluated whether their mRNA expression was succumbed to changes when the rats were exposed to ethanol, as a known debilitating agent during development. Therefore we analyzed the thymus of adult rats whose mothers were forced to drink ethanol during gestation, to detect possible changes in thymus mRNA expression levels of Ikaros, Aiolos, Helios, Eos and Pegasus. We found that rats prenatally exposed to ethanol show a slightly higher expression of Ikaros family transcription factors in the adult thymus when compared to control rats, but these differences were not statistically significant. We further studied the distribution of the major lymphocyte subpopulations in the rat thymus according to CD3, CD4 and CD8 expression by four color flow cytometry. We found a higher incidence of CD3 positive cells in the double positive, CD4+CD8+ thymic subpopulation of rats prenatally exposed to ethanol when compared to non-exposed animals. Our findings indicate that ethanol exposure of pregnant rats might influence the development of CD3 positive cells in the thymus of the offspring but this result should be further tackled at the level of transcription factor expression.

Prolegomenon for chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a unique lymphoproliferative disorder that scarcely occurs under the age of 40; thereafter the incidence of CLL increases exponentially with age. CLL is characterized by progressive expansion of malignant CD5+ME+ B-cell clone accompanied by a myriad of cellular and humoral immune defects. Each of them might be linked to different clinically manifested complications such as increasing rate of infections, autoimmune disorders and disturbed immune surveillance against tumour cells. We assume that CLL occurs as a consequence of age-dependent, genetically related functional restrictions of the thymic microenvironment in supporting common lymphoid progenitor cells (CD5+ME+CD4-CD8-) to differentiate into mature T-cell and B-cell descendants. In conjunction with genetic abnormalities developing in B-cell progenitors, presumably expressing P glycoprotein (Pgp+), we postulate that developmentally altered T-cell descendants, along with quantitative imbalance among CD4+, their subsets and CD8+ lymphocytes in the peripheral blood, play an important additional role in facilitating the malignant B-cell clone emergence and in modulating the CLL clinical evolution. Namely, imbalance of any of T-cell-mediated cell interactive homeostatic mechanisms accompanied by imbalance in the production of various cytokines might in CLL influence leukaemic B-cell growth by deregulating inducer (c-myc and p53) and/or suppressor (bcl-2 and mutant p53) oncogenes responsible for the promotion or suppression of B-cell mitogenesis that may in turn further contribute to their impaired differentiation and/or differentiation arrest. In conclusion, CLL might be interpreted as a primary immunodeficiency syndrome developing in elderly population due to gradually evolving restriction of genetically controlled programs in the thymic microenvironment responsible for irregular maturation of common lymphoid progenitor cells that constitutively express CD5 antigen and ME receptor into T-cell and B-cell descendants.

Molecular insight into the diagnosis of lymphoma.

Malignant lymphoma may be very difficult to diagnose with routine histopathological methods because they may recapitulate benign architecture or contain benign infiltrates. The best method of diagnosis is to establish the clonal profile of the lymphocyte population, since a monoclonal proliferation is highly suggestive of neoplasia. By means of a PCR (polymerase chain reaction) method it is possible to detect the immunoglobulin heavy chain (IgH) gene rearrangement and therefore establish the lymphocyte clonality. PCR with primers complementary to relatively conserved regions called frameworks (FR1-FR3) laying among hyper variable regions (CDR1-CDR3) of IgH gene unable us to detect monoclonal versus polyclonal B-cell population. The length of the PCR product with these primers is unique if we deal with a monoclonal population. On the contrary, a polyclonal population gives PCR products of a different size. In this retrospective study we used a semi-nested PCR to analyse 37 paraffin-embedded specimens. All of them had been evaluated previously by pathohistological and immunophenotypic criteria. A number of polyclonal (PBL and tonsils from healthy donors) and monoclonal cells (PBL from CLL patients, Raji cell line) were analyzed as controls. Clonality was successfully determined in all specimens. Our results support the concept that molecular techniques such as PCR provide a helpful approach for detection of monoclonal immunoglobulin rearrangements in malignant lymphoma. This is especially true for border cases, but always in the combination with other pathohistological methods.

Monoclonality in Helicobacter pylori-positive gastric biopsies: an early detection of mucosa-associated lymphoid tissue lymphoma.

Mucosa-associated lymphoid tissue (MALT) is not present in healthy gastric mucosa, but it can develop in sites of long-persisting inflammation and is connected with the development of MALT lymphoma. A monoclonal lymphocyte population is one of the characteristics of such lymphomas. In this study we analyzed gastric biopsies (formalin fixed and paraffin embedded or frozen) in 93 patients with dyspepsia accompanied by Helicobacter pylori infection. We applied PCR and single-cell immunocytochemistry to detect the clonality of the gastric B-cell population. Immunocytochemistry performed on 33 frozen biopsies showed two samples with monoclonal pattern. PCR analysis of immunoglobulin heavy-chain (IgH) gene rearrangements revealed two monoclonal populations out of 161 biopsies from 60 patients. We conclude that PCR analysis was the most sensitive method, which gave us insight into the nature of the earliest stage of MALT lymphoma in gastric biopsies.

Development of T lymphocytes at extrathymic sites.

T lymphocytes expressing both CD4 and CD8 are the predominant cell type in the thymic cortex but are extremely rare outside the thymus of normal mice. In this article, we show that if precursor thymocytes (CD4-CD8-) from fetal or adult donors are injected i.v. into irradiated recipients, some of these cells will lodge in lymph nodes and develop into both CD4+CD8+ (double-positive) and CD4+ or CD8+ (single-positive) cells. This phenomenon also occurred in thymectomized recipients, strongly suggesting it is genuine extrathymic development. Prethymic precursors (e.g., fetal liver), were unable to use the lymph node for T cell development, without thymic processing. The data suggest that given unusual circumstances (irradiation or thymectomy and availability of appropriate precursors), the lymph nodes can support T cell development.

Stem cell antigen 2 expression in adult and developing mice.

Stem cell antigen 2 (Sca-2) expression can distinguish the most immature T-lymphocyte precursors in the thymus from the hemopoietic stem cells. Sequence analysis of the Sca-2 protein showed that Sca-2 is a glycosylphosphatidylinositol (GPI) anchored molecule that shares some characteristics with the members of the Ly-6 multigene family, and that it is the same as the thymic shared antigen-1 (TSA-1). Here we extend these studies and critically reassess the expression of the Sca-2/TSA-1 antigen in hematopoietic tissues of adult and developing mice. With more sensitive methods we show that the distribution of Sca-2/TSA-1 differs from existing reports. We find especially high expression of Sca-2/TSA1 at day 14 of fetal development.

CD4 and CD8 expression and T cell antigen receptor gene rearrangement in early intrathymic precursor cells.

The earliest T precursor population in the adult mouse thymus, considered to have the surface phenotype CD4lo8-3-44+25-Thy-1lo c-kit+ (termed the low CD4 precursor), has been shown to have the capacity to produce B cells and dendritic cells, as well as T cells, and to have the T cell antigen receptor (TCR) C beta gene region in germ-line configuration. Because of evidence that this precursor population may have low levels of CD8 as well as CD4 on the cell surface, it was isolated, stained for surface CD4 and CD8 and assayed for the expression of messenger RNA (mRNA) for CD4 and CD8 by the reverse transcriptase polymerase chain reaction (RT-PCR). The low CD4 precursors gave definite, moderate levels of staining for both CD8 and CD4, in contrast to downstream precursors which showed only marginal staining and so could be considered as genuine CD4-8-3- triple negatives. The low CD4 precursor expressed a significant level of mRNA for CD4, indicating that the surface CD4 was produced by these cells. However, the low CD4 precursor did not express a detectable level of mRNA for CD8, suggesting that the surface CD8 was acquired from other cells. Since the low CD4 precursor population was found already to express mRNA for enzymes involved in TCR gene rearrangement, including in this study terminal deoxynucleotidyl transferase (TdT), a PCR procedure was used to assay early precursors for D-J rearrangements at the TCR beta gene locus. However, the low CD4 precursor had the TCR beta D-J genes in germ-line configuration, D-J gene rearrangements being first detected several stages downstream in the CD3-4-8-44-25-+ precursor population. We conclude that a transient synthesis of CD4, but not of CD8, characterize these early thymus precursors. Although they have initiated synthesis of some recombination-associated enzymes, full commitment to the T lineage and TCR gene rearrangement is a later event.

Purified murine long-term in vivo hematopoietic repopulating cells are not prothymocytes.

Analysis of kinetics of thymic repopulation by Rh123low, Lin-, Ly6A/E+, c-kit+ (Rh123low) cells, highly enriched for long-term in vivo hematopoietic repopulating cells, reveals that this population is deficient in thymic repopulation at week 3 after intravenous transplantation when compared to normal bone marrow cells. This suggests that the marrow prothymocytes have been depleted from this population, and analysis of thymic repopulation at week 3 can therefore be used to differentiate prothymocytes and their precursors. Using this short-term assay, the Rh123high, Lin-, Ly6A/E+, c-kit+ (Rh123high) population has been found to be relatively more efficient at early thymic repopulation, suggesting that this population contains the prothymocytes. In addition, the differentiation potential and reconstitution behavior of the Rh123high population observed after intravenous and intrathymic transfer strongly indicates that this population is at the transitional stage between the marrow primitive pluripotential and thymic more mature lymphoid restricted stem cells. We propose that the thymic repopulating ability of the Rh123low population is through generation of the more mature Rh123high progeny, presumably in the marrow.

Thymic stem cells in mouse bone marrow.

There is still controversy concerning the nature of the stem cells from bone marrow that colonize the thymus during embryogenesis and continually throughout life. To identify the bone marrow stem cells that home to and populate the thymus, we screened murine bone marrow cells for the presence of a population of surface phenotype similar to the earliest known intrathymic precursor. We have identified a population characterized by expression of an intermediate level of heat stable antigen, a very low level of Thy-1, and high levels of CD44 and class I major histocompatibility complex antigens. It is negative for B-cell, granulocyte, macrophage, and erythrocyte markers (B220, Gr-1, Mac-1, and TER 119). All these markers are common to the intrathymic precursors and bone marrow stem cells. However, this new bone marrow population is Sca-2+, similar to the intrathymic precursor, which makes a clear distinction from the Sca-2- bone marrow hematopoietic stem cells previously characterized. The frequency of the new population in the normal mouse bone marrow is about 0.25%. When transferred intrathymically or intravenously to lethally irradiated mice, it has a higher expansion potential (2 x 10(5)) than has been found for the intrathymic precursors (10(3)), but less than was found for the Sca-2- multipotent stem cell (10(7)). These transfer studies also showed that it was pluripotent, in that its precursor activity was not restricted to the production of T or B lymphocytes. However, it gave a reduced spleen colony number and smaller colonies (day-12 colony-forming unit spleen) when compared with multipotent stem cells. Thus, the cell we have identified appears to be the latest pluripotent cells so far identified in bone marrow and is therefore a good candidate for a bone marrow prothymocyte, but it appears not to be T-cell-committed.

Intrathymic lymphoid precursor cells during fetal thymus development.

Our previous studies have demonstrated the presence, in the adult mouse thymus, of a population of early precursor cells able to give rise to T and B lymphocytes but not myeloid cells. This population of cells expresses low levels of CD4 and has been termed the "low CD4 precursors." All these precursors were found to be c-kit positive, and they precede the better known CD4-CD8- precursor stage. In this study, embryonic and neonatal thymuses were examined to see whether a similar low CD4 precursor was part of the pathway of T cell development during ontogeny. A population with the phenotypic characteristics of the adult low CD4 precursor was found from day 15 of embryonic development, although the expression of low levels of CD4 was apparent only from embryonic day 17. Functional tests of these putative precursors showed they had no thymus-reconstituting ability when isolated from thymuses at any time during embryonic life, and very low reconstituting ability even 24 days after birth. These results raise questions about the adult low CD4 precursor as an obligatory stage in the development of T cells in the thymus.

Functional differences of T cells in B-chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (B-CLL) is a disease characterized by an accumulation of monoclonal lymphocytes of B cell origin. Although the neoplastic process involves the B lymphocyte compartment, phenotypic and functional defects within the T lymphocyte population implicate their possible role in the pathogenesis of the disease. We analyzed the functional and morphological integrity of T lymphocytes from the peripheral blood of 64 patients with B-CLL. The activation of B-CLL T cells after PHA stimulation was determined by measuring [3H]-thymidine incorporation, assessing cell numbers in parallel cultures, and by monitoring the lymphocyte subsets during 9 days of cultivation. Our results indicate the presence of three functionally different populations of T cells in the peripheral blood of B-CLL patients. We present evidence for an increased proliferative potential of T lymphocytes from a group of patients with B-CLL.

In vivo bidirectional regulation of mouse natural killer (NK) cell cytotoxic activities by Leu-enkephalin: reversibility by naloxone.

Intraperitoneal injection of Leu-enkephalin (LENK, 10 or 7.5 mg/kg) induced bidirectional modulation of natural cytotoxic activities in spleens of CBA mice (suppression followed by enhancement). NK-cytotoxic activity was more affected than the ADCC. Early suppression of NK activity could be reversed by 4 x M excess of naloxone injected 20 min before LENK, suggesting that the suppression was mediated by opioid receptors. Subsequent increase of NK activity could not be abrogated by naloxone, at least not completely. Naloxone itself decreased NK activity 12 hours after treatment, but enhanced ADCC at 24 and 48 hours. This increase was abrogated by LENK. In addition to functional alterations, LENK also induced phenotypic changes of spleen cells, i.e. a decrease in the percentage of asialo-GM-1+ cells 24 hours posttreatment. There was no correlation between LENK-induced alterations of cytotoxic function and the percentage of cells with NK phenotype (GM-1+). Thus, LENK modulates cytolytic functions and the phenotype of NK cells in vivo in a complex way, which besides opioid mechanisms may also include non-opioid ones.

Developmental potential of the earliest precursor cells from the adult mouse thymus.

A new, numerically minute population of cells representing the earliest T precursor cells in the adult mouse thymus has recently been isolated. This population has been shown to be similar to bone marrow hemopoietic stem cells in surface antigenic phenotype and to express moderate levels of CD4. We now show, by fluorescence-activated cell sorting and intrathymic transfer to irradiated mice, that this apparently homogeneous population differs from multipotent stem cells in expressing the surface stem cell antigen 2 (Sca-2), that it differs from most early B lineage cells in lacking B220 and class II major histocompatibility complex expression, and that it binds rhodamine 123 like an activated rather than a quiescent cell. Irradiated recipient mice differing at the Ly 5 locus were used to compare the developmental potential of these early intrathymic precursors with bone marrow stem cells. Only T lineage product cells were detected when the intrathymic precursor population was transferred back into an irradiated thymus. However, when the intrathymic precursor population was transferred intravenously, it displayed the capacity to develop into both B and T lymphoid cells in recipient bone marrow, spleen, and lymph nodes, but no donor-derived myeloid cells were detected. The absence of myeloid and erythroid precursor activity was confirmed by showing that the intrathymic precursor population was unable to develop into myeloid or erythroid spleen colonies on intravenous transfer or to form colonies in an agar culture. These findings indicate that this earliest intrathymic precursor population has become restricted (or strongly biased) to lymphoid lineage development, but not exclusively to T lymphocytes.

Insulin and insulin-like growth factor I (IGF I) in early mouse embryogenesis.

Growth factors have an important role in the regulation of cell growth, division and differentiation. They are also involved in the regulation of embryonic growth and differentiation. Insulin and insulin-like growth factor I (IGF I) play an important part in these events in the later stages of embryogenesis, when organogenesis is completed. In this study, we are presenting evidence that insulin and IGF I are also secreted by embryonic tissues during the prepancreatic stage of mouse development. We found measurable amounts of insulin and IGF I in 8- to 12-day-old mouse embryos. We also showed that embryonic cells derived from 8-, 9- and 10-day-old mouse embryos secrete insulin, IGF I and/or related molecules. Furthermore, the same growth factors, when added to the culture of 9-day-old mouse embryonic cells, stimulate their proliferation. These results lead to the conclusion that insulin can stimulate the growth of embryonic cells during the period when pancreas is not yet formed, which is indirect evidence for a paracrine (or autocrine) type of action.

Antilymphocytic antibodies and marrow transplantation. IX. T-cell depletion in marrow donors with C1q high and low affinity antibodies for suppression of GVHD in fully mismatched mice.

Rat monoclonal antibodies (mAbs) of the same specificity (anti-Thy-1) but different immunoglobulin (Ig) subclass were investigated for their effect on suppression of graft-versus-host disease (GVHD) by depleting the marrow donors of T cells in vivo. Transplantation to homozygous, fully mismatched mice of spleen and bone marrow cells from unthymectomized mice injected with the mAbs revealed that two rat anti-Thy-1 mAbs with high affinity for C1q (IgG2b) suppressed and prevented acute and chronic mortality of GVHD. In contrast, rat mAbs with low affinity for C1q (IgM, IgG2c) barely delayed acute mortality. This correlated with findings on the degree of splenic T-cell depletion in donor mice with the IgG2b mAb, able to deplete 97%, and the IgG2c and IgM mAbs, only 83% and 75% of T cells, respectively. An effect akin to the one achieved with IgG2b was seen, however, when donor mice were thymectomized and then treated with three injections of IgG2c isotype. The rat IgM mAb was not immunosuppressive even under such conditions. Immunocytochemical and immunohistochemical examination of the donor lymph nodes after single injection of either mAb showed that only 84% of T cells were eliminated, and in contrast to the spleen, none of the tested antibodies could deplete T cells further. The thymus did not appear depleted at all, although the cortical thymocytes were coated with either of the injected mAbs.

Distribution of injected anti-Thy-1 monoclonal antibodies in mouse lymphatic organs: evidence for penetration of the cortical blood-thymus barrier, and for intravascular antibody-binding onto lymphocytes.

The localization of monoclonal anti-Thy-1 binding in mouse thymus, spleen, and lymph nodes was studied at early intervals after intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.) injection of a single dose of the monoclonal antibody (MAb). Five minutes after i.v. injection, anti-Thy-1 was bound to cortical thymocytes surrounding capillaries in the thymic cortex, to thymic cells beneath the thymic capsule and to medullary thymocytes around venules of the thymus medulla. When anti-Thy-1 was injected i.p. or s.c. the MAb was first deposited in capillary walls in the thymus cortex and did not appear on thymocytes outside of capillaries until 60 min after injection. These findings suggest that thymic cortical capillaries are permeable for anti-Thy-1 MAb contrary to the generally accepted principle of a blood thymus barrier to antigens in thymic cortex. Some cortex capillaries also became permeable for peroxidase when injected 15 min after anti-Thy-1 MAb. Anti-Thy-1 MAb penetration into spleen white pulp and lymph node paracortex occurs along the circulatory pathway of the vascular system in the spleen and of lymphatics in lymph nodes. But those lymphocytes with a strong anti-Thy-1 MAb loading always appeared along the pathways of lymphocyte circulation indicating that the most intense contact between anti-Thy-1 MAb and T-lymphocytes occurs not in the lymphatic organs but during the intravascular period of recirculation of lymphocytes.

Simultaneous demonstration of two antigens on single T cells using antibodies with contrasting labels.

We describe a double labeling method for the discrimination of 2 antigens on single cells. It consists of a combination of 3H-immunoautoradiography and immunocytochemistry applied to cells previously fixed on poly-L-lysine (PLL)-coated multispot slides. The method has been applied to various mouse cells contemporaneously labeled with 2 different monoclonal antibodies. In order to distinguish the attached antibodies unambiguously, they were labeled with contrasting markers. One of the antibodies was marked with tritium blackening the photographic film that covers the slide. The other was detected with the peroxidase-anti-peroxidase (PAP) method forming a reddish precipitate. The contrast between the reddish reaction product of the PAP-labeled antibody and the black silver grains allows cells, specifically labeled with both antibodies, to be distinguished from cells labeled with only one or neither of the antibodies. Tritium-labeled antibodies were introduced because of their advantage over antibodies labeled with iodine in the closer localization of the silver grains to the bound antibody and their much longer halflife (60 days versus 12 years). In this study we applied a tritium-labeled anti-Thy-1.1 together with anti-Lyt-1 monoclonal antibody for studying the distribution of the corresponding antigens on lymphocytes in the mouse thymus and lymph node cells.

The relationship between membrane characteristics, functional reactivity of T-lymphocytes, and the progression rate of B-cell chronic lymphocytic leukemia.

Study performed on 192 patients has demonstrated that the progression rate of chronic lymphocytic leukemia (CLL) is associated with the existence of T-cell defect(s). A dynamic classification system based on evaluation of tumor mass growth rate, response to therapy, and myelopoietic failure (MF) has been devised for evaluation of progression rate of CLL. Besides four basic groups (Group 1: CLL without therapy; Group 2: CLL on therapy, without remission; Group 3: CLL on therapy, partial remission; Group 4: CLL on therapy, complete remission) patients were further classified into phase (type) A (stable, indolent CLL) or phase (type) B (active, progressive CLL). The major criteria for phase A were: total tumor mass (TTM) doubling time (DT) longer than 12 months, no MF, and/or good response to therapy. The major criteria for phase B were: TTM DT less than 12 months and/or accompanying MF and/or no response to therapy. The following major findings have been demonstrated: (1) altered quantitative relationship between active and nonactive parts of T-cell compartment (E/A ratio) in the progressive phase of CLL; (2) altered B/T gamma ratio in the progressive phase of CLL; (3) more than 50% increased percentage of T gamma cells in the stable phase of CLL; (4) very low stable and absent seeding efficiency of T-cells in the progressive phase of CLL; (5) altered (delayed) DNA synthesis pattern in the progressive phase of CLL; and (6) negative local xenogeneic graft versus host reaction in the progressive phase of CLL. Based on reported results, a hypothesis regarding the possible role of T-cells in the pathogenesis of CLL was suggested.