Development of rat CD45+ 13-day-old fetal liver cells in SCID mouse fetal thymic organ cultures.

A phenotypic analysis of the lympho-hemopoietic cells which occur in the liver of 13-day-old fetal rats was achieved by flow cytometry in an attempt to further characterize the rat lymphoid progenitor cells. A small fraction of rat 13-day-old fetal liver (r13FL) cells, which weakly expressed the leukocyte common antigen CD45, constituted a homogeneous Thy-1(hi), CD71(-), CD44(+), MHC class I+, CD43(+) cell subpopulation negative for CD45RC, CD3, TCRalphabeta, TCRgammadelta, CD2, CD5, CD4, CD8, CD25, CD28, NKR-P1a and sIg. On the contrary, the CD45(-) cells were a heterogeneous cell subset which expressed Thy-1, CD71 and CD44 at distinct levels. After MACS separation, the CD45(+) r13FL cells, but not the CD45(-) cell subset, in vitro repopulated 14-day-old SCID mouse fetal thymic lobes providing rat T cells, both TCRalphabeta and TCRgammadelta, NK cells, and thymic dendritic cells but not B lymphocytes. Interestingly, NKR-P1a(lo) TCRalphabeta+ or TCRgammadelta+ cells developed in the xenogeneic cultures, and a rare CD4(+)CD8(+) double-positive subpopulation among the TCRgammadelta-expressing cells accumulated in the oldest cultures. These results are discussed from the double perspective of the nature of the precursor cells which colonize the fetal thymus and the relevance of the xenogeneic SCID mouse fetal thymic microenvironment for supporting rat lymphopoiesis.     

Characterization of human lymphocytes separated from fetal liver and spleen at different stages of ontogeny

Membrane markers on human lymphocytes separated from fetal liver and spleen were studied. Depending on the period of intrauterine development, a growing percentage of T- and B-lymphocytes (up to 16% and 45%, respectively) among spleen cells was seen, but in liver the number was low independent of the gestational age (T cells less than 10% and B cells less than 15%). The majority of early CD3+ spleen cells (21st-28th week) expressed TCR alpha beta but not TCR gamma delta, although a significant proportion of these cells was still lacking CD4, CD8, and CD5 differentiation antigens, suggesting their immaturity. Later spleen T cells (29th-36th week) expressed the phenotype as mature adult-type T cells (CD3+TCR alpha beta +CD4/8+CD5+). During ontogeny in fetal spleen, a growing number of B cells could be estimated without any changes in the proportion of subsets, expressing the different light and heavy chains. However, the proportion of CD5+ B cells decreased with gestational age. The results suggest that the functional immaturity of antenatal splenocytes could not be caused by dramatic phenotypical differences in comparison with adult-type splenic lymphocytes.     

CD8 lymphocytosis in primary cytomegalovirus (CMV) infection of allograft recipients: expansion of an uncommon CD8+ CD57- subset and its progressive replacement by CD8+ CD57+ T cells.

Allograft recipients undergoing cytomegalovirus infection present increased proportions of circulating CD8+ lymphocytes. A longitudinal study of 11 kidney and five liver allograft recipients with primary CMV infection but no other etiological factor of graft dysfunction revealed selective imbalances of peripheral blood CD8+ T cell subsets. Initially, CMV viraemia is associated with elevated CD8+bright T cell numbers and T cell activation. Activation markers fall to normal when viral cultures become negative (before the end of the first month). During the second to sixth month, most (12/16) patients keep up high CD8+ T cell counts (1050-2900 CD8+ cells/mm3), comprising an uncommon CD8+ T cell subset, as 45-73% of CD8+bright lymphocytes were CD3+ and TCR alpha beta+, but were not stained by anti-CD28, CD11b, CD16, CD56, and CD57 antibody. Unexpectedly, CD8+CD57+ T cells, a hallmark of CMV infection, do not appear until the second to sixth month of primary CMV infection, and their numbers increase progressively thereafter. They become the predominant CD8+ T cell subset after 6 months of infection and their persistence for several (up to 4) years is strongly correlated (r = 0.87) with expansion of CD8+ cells. By analysis with MoAbs, there was no bias towards the use of particular TCR-V beta gene families at any time of primary CMV infection. Persistence of CD8 lymphocytosis is thus directly related to the rate of expansion of an uncommon CD8+CD57- subset and its progressive replacement by CD8+CD57+ T cells that are chronically elicited by CMV.     

Expression of high molecular weight isoforms of CD45 by mouse thymic progenitor cells

We have studied the expression of isoforms of CD45 (leukocyte common antigen, LCA) among T cell precursors using the organ culture system of Jenkinson et al. (Eur. J. Immunol. 1982. 12: 583). These experiments show that cells capable of recolonizing alymphoid embryonic thymic lobes in vitro can be detected in the thymus of fetal and adult mice and are enriched when thymocytes are depleted of cells bearing CD4 or CD8. These data are consistent with results from in vivo experiments of Fowlkes et al. (J. Exp. Med. 1985. 162: 802) indicating that T cell precursors lie within the double-negative (CD4-CD8-) compartment. No precursors were detected among the reciprocal populations of cells bearing CD4 and/or CD8 (single and double positives). Double-negative cell fractions were then divided on the basis of reactivity with monoclonal antibodies RA3-2C2 and RA3-3A1. These antibodies recognize the high molecular weight species of the LCA or, more accurately, a product defined by exon A of the CD45 gene. Recolonizing cells were found predominantly in the CD45RA+ (RA3-2C2 and RA3-3A1 reactive) fraction of double-negative thymocytes; CD45RA- enriched populations had increased efficiency of recolonization and CD45RA- depleted populations had decreased ability to recolonize as compared with the whole CD4-CD8- fraction. To clarify whether progenitors enriched in the CD45RA+ fraction were capable of giving rise to mature CD4+, CD8+ and CD4+ CD8+ cells, we analyzed the progeny of lobes seeded with CD4-CD8-CD45RA+ fractions. After 7-9 days in organ culture the proportion of CD4+, CD8+ or CD4+ CD8+ cells had increased to 35.2%, 18.6% and 23.7%, respectively (mean of five experiments), indicating that progenitors among the CD45RA+ population were indeed multipotent. These results suggest that the majority of T stem cells in the thymus are among thymocytes that express the CD45RA molecule, an hypothesis supported by our finding that removal of CD45RA-expressing cells (using complement and antibody) eliminated recolonizing capacity of thymic cell fractions.     

Establishment of efficient reaggregation culture system for gene transfection into immature T cells by retroviral vectors

To overcome low efficiency of retroviral infection into immature T cells, we modified reaggregation fetal thymus organ culture by closely packed co-culture with virus-producing cells (VPC). The viral vector was constructed in chimeric vector, pMX, with IRES and tailless-rat CD2 as a surface marker of infected cells. A rearranged TCR beta gene (Vbeta8.2) was further inserted into the construct for investigating effect of the introduced gene in T cell development. Using this system, we succeeded to transfer the viral vector into immature thymocytes at a remarkably higher efficiency compared to conventional methods using medium containing retrovirus. Moreover, the introduced TCR beta gene was expressed on thymocytes of RAG2-deficient mice to induce in the transition of CD4-CD8- double-negative (DN) into CD4+CD8+ double-positive (DP) cells by transducing beta-selection signaling. Thus, our modified reaggregation culture system is useful for studying the molecular mechanism of T cell development due to a highly efficient gene transfer into immature T cells.     

T cell receptor-γδ-expressing fetal mouse thymocytes are generated without T cell receptor Vβ selection

We investigated whether fetal mouse T cell receptor (TCR) γδ cells have been subjected to so-called TCRβ selection at the CD25 stage of thymus development. To this end, we carried out a comparative three-color flow microfluorimetric analysis of TCRβδ cells developing in the fetal, neonatal and adult thymus using monoclonal antibodies to CD2, CD8, CD24, CD25 and CD44. Day-15 fetal TCRγδ cells were CD2⁺, suggesting an origin at a post-CD25 stage. Molecular analysis of TCRβ rearrangements were also carried out. Thus, by semi-quantitative polymerase chain reaction (PCR) amplification of Vβ6 and Vβ8 to Jβ2 rearrangements day-15 fetal TCRγδ showed extensive TCRβ rearrangements, a finding confirmed by PCR amplification from single micromanipulated cells. Finally, sequencing analysis of 104 PCR-amplified TCR VDJβ2 fragments showed that the majority (58%) were rearranged out of frame. Taken together, these phenotypic and molecular analyses suggest that fetal TCRγδ cells have not been subject to TCRβ selection.     

Generation of alphabeta T-cell receptor+ CD4- CD8+ cells in major histocompatibility complex class I-deficient mice upon activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Gene-targeted mice lacking the beta2 microglobulin gene (beta2m-/- mice), and hence functional major histocompatibility complex (MHC) class I molecules, do not develop CD4- CD8+ cells. We show here that both in vitro and in vivo treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a trans-activating ligand of the endogenous aryl hydrocarbon receptor (Ah-R), bypasses the need for MHC class I molecules for selection into the CD4- CD8+ cell pool. When beta2m-/- dams were given a single dose of 50 microg of TCDD, approximately 13% of CD4- CD8+ thymocytes could be detected in their newborn pups. In TCDD-exposed fetal thymus organ cultures of beta2m-/- mice, approximately 35% CD4- CD8+ thymocytes were detectable. About 16% of these CD4- CD8+ cells bore the alpha beta T-cell receptor (TCR) and approximately 33% bore CD3. Only a minority of the CD8+ cells were heat-shock antigen positive. The cells possessed killing activity as shown using the 51Cr-release assay comprising gamma delta TCR- CD4- CD8+ thymocytes from 3 to 4-day-old b2m-/- mice. Thus, TCDD leads to a significant increase of mature CD4- CD8+ thymocytes in relative and absolute numbers. High numbers of CD4- CD8+ thymocytes developed also in organ cultures from thymi, lacking both MHC class I and class II molecules, exposed to TCDD. A 10-fold transient increase of Notch1 mRNA in thymocytes from fetal thymus organ culture, exposed for 4 days to TCDD, was detected in CD4+ CD8+ cells compared with controls. We suggest that TCDD affects thymic selection and directs the lineage commitment of CD4+ CD8+ thymocytes towards CD4- CD8+ cells, possibly via up-regulation of the Notch1 gene.     

Interleukin-7 treatment promotes the differentiation pathway of T-cell-receptor-alpha beta cells selectively to the CD8+ cell lineage.

In this report we have studied the influence of interleukin-7 (IL-7) on thymocyte differentiation by evaluating the effects of IL-7 on the generation of T-cell receptor-alpha beta (TCR-alpha beta) and TCR-gamma delta thymocyte subpopulations in rat fetal thymus organ culture. IL-7 enhanced the differentiation pathway of TCR alpha beta thymocytes, first increasing the numbers of immature CD8+ cells, and later those of both CD4+ CD8+ and mature thymocytes. The kinetics of thymocyte migration out of thymic lobes was also accelerated, and the average number of mature TCR-alpha beta phi emigrants per day was increased in the presence of IL-7. Moreover, mature CD4- CD8+ thymocytes were preferentially generated after IL-7 administration. This TCR-alpha beta hi cell population was not actively dividing, indicating that IL-7-promoted thymocyte differentiation was selective to the CD8 cell lineage. Distribution of some TCR-V alpha and TCR-V beta segments among mature thymocytes was also modified in IL-7-treated thymic lobes. On the contrary, the maturation of TCR-gamma delta was not affected by IL-7 addition during the first days of culture, but their numbers sharply increased by day 6 of culture. These results were confirmed with IL-7-treated cultures for 24 hr, showing that IL-7 responsiveness was acquired by TCR-gamma delta cells late in thymus ontogeny. The present results thus indicate a key role for IL-7 in the maturation of TCR-alpha beta thymocytes and the expansion of thymic TCR-gamma delta cells.     

The differential regulation of Lck kinase phosphorylation sites by CD45 is critical for T cell receptor signaling responses

The molecular mechanisms whereby the CD45 tyrosine phosphatase (PTPase) regulates T cell receptor (TCR) signaling responses remain to be elucidated. To investigate this question, we have reconstituted CD45 (encoded by Ptprc)-deficient mice, which display severe defects in thymic development, with five different expression levels of transgenic CD45RO, or with mutant PTPase null or PTPase-low CD45R0. Whereas CD45 PTPase activity was absolutely required for the reconstitution of thymic development, only 3% of wild-type CD45 activity restored T cell numbers and normal cytotoxic T cell responses. Lowering the CD45 expression increased CD4 lineage commitment. Peripheral T cells with very low activity of CD45 phosphatase displayed reduced TCR signaling, whereas intermediate activity caused hyperactivation of CD4+ and CD8+ T cells. These results are explained by a rheostat mechanism whereby CD45 differentially regulates the negatively acting pTyr-505 and positively acting pTyr-394 p56(lck) tyrosine kinase phosphorylation sites. We propose that high wild-type CD45 expression is necessary to dephosphorylate p56(lck) pTyr-394, suppressing CD4 T+ cell lineage commitment and hyperactivity.     

细胞毒颗粒相关蛋白TIA-1在鼻NK/T细胞淋巴瘤及淋巴增生组织中的表达

目的 观察２７例鼻ＮＫ／Ｔ细胞淋巴瘤之瘤细胞表达细胞毒颗粒相关蛋白ＴＩＡ－１的情况及其与该肿瘤的免疫表型,基因型及ＥＢ病毒感染的关系。方法 采用ＳＰ法免疫组织化学染色,选用的抗体有：ＴＬＡ－１,ＣＤ５６,ＣＤ３ε,ＣＤ４５ＲＯ,ＣＤ８和ＣＤ２０等;聚合酶链反应,作ＴＣＲγ链及免疫球蛋白ＪＨ链基因重排,ＥＢＥＲ１／２原位杂交及与ＴＬＡ－１和ＣＤ８等的双标记染色,还与１０例鼻咽淋巴增生病例进行了比较。     

Presence of murine fetal liver cells capable of being induced to differentiate in vitro into T cell receptor-positive cells.

Mouse fetal liver cells were analyzed for the surface expression of T cell markers. Fetal liver cells prepared from mouse embryos at 14.5 days of gestation contained a small number of CD4+ cells (1.4%), but virtually no cells positive for any other T cell markers such as CD8, CD3 and T cell receptor (TcR). When a fetal liver cell suspension prepared from BALB/c(male) x AKR(female) F1 embryos at 14.5 days of gestation was cultured in medium supplemented with culture supernatants of both WEHI-3 and concanavalin A-stimulated rat spleen cells, TcR alpha beta+ and CD4+ cells were generated, whereas CD8+ and TcR gamma delta+ cells were hardly detectable. Most of TcR alpha beta+ and CD4+ cells were H-2d+, thus clearly showing their fetal origin. Treatment with anti-CD4, anti-CD3 or anti-TcR alpha beta antibodies plus complement or electronic sorting to remove cells expressing these markers failed to inhibit the generation of T cell marker-positive cells following culture in vitro. On the other hand, depletion of Thy-1.2+ cells reduced their generation. These findings indicate the presence of some progenitor T cells in fetal liver with the Thy-1+, CD3-, CD4-, CD8-, TcR- phenotype, which can be induced to differentiate into TcR alpha beta+ cells in the presence of specific humoral supplements without the influence of the thymus.     

Rat bone marrow cells undergo thymopoiesis in mouse fetal thymic organ culture

We have developed an in vitro differentiation assay allowing the study of thymopoiesis from rat bone marrow cells. In this assay, Wistar rat bone marrow cells repopulated fetal Swiss mouse thymic lobes depleted in endogeneous lymphoid cells by deoxyguanosine treatment. Due to the xenogeneic situation, repopulating rat cells from any hemopoietic lineage could be easily recognized by anti-rat monoclonal antibodies such as anti-Thy-1.1 that did not react with Swiss mouse thymocytes. After 15 days in vitro, 80% of the developing rat cells were Thy-1.1+ lymphoid cells and about 70% of the Thy-1.1+ cells expressed CD5, CD2 and leukosialin. The percentages of cells expressing pre-B cell, B cell and myeloid determinants were less than 20%. The developing thymocytes comprised CD4-CD8- T cell receptor (TcR) alpha/beta-, CD4-CD8+TcR alpha/beta low and CD4+CD8+TcR alpha/beta low cells, indicating that the early stages of rat thymopoiesis occurred within mouse thymic lobes. Limiting dilution assays showed that 50% of positive assays were obtained with 3000 nucleated bone marrow cells, which is in good agreement with recent estimates derived from in vivo reconstitution after intrathymical transfer. Moreover the limiting dilution assays proved to be sensitive enough to evidence a tenfold enrichment of pre-T cell activity in the low-density fraction of rat bone marrow. This xenogeneic system might greatly facilitate studies on prethymic and intrathymic stages of rat T cell development and permit new in vitro approaches of the colonizing bone marrow T cell precursor properties.     

Role of IL-2 in rat fetal thymocyte development

Early during rat thymus ontogeny, an important proportion of thymocytes expresses IL-2R and contains IL-2 mRNA. To investigate the role of the IL-2-IL-2R complex in rat T cell maturation, we supplied either recombinant rat IL-2 or blocking anti-CD25 mAb to rat fetal thymus organ cultures (FTOC) under several experimental conditions. The IL-2 treatment initially stimulated the growth of thymocytes and, as a result, induced T cell differentiation, but the continuous addition of IL-2 to rat FTOC, as well as the anti-CD25 administration, resulted in cell number decrease and inhibition of thymocyte maturation. These results indicate that immature rat thymocytes bear functional high- affinity IL-2R and that IL-2 promotes T cell differentiation as a consequence of its capacity to stimulate cell proliferation. Modifications in TCR alpha beta repertoire and increased numbers of NKR- P1+ cells, largely NK cells, were also observed in IL-2-treated FTOC. Furthermore, IL-2-responsiveness of different thymocyte subsets changed throughout thymic ontogeny. Immature CD4-CD8-cells responded to IL-2 in two stages, early in thymus development and around birth, in correlation with the maturation of two distinct waves of thymic cell progenitors. Mature CD8+ thymocytes maximally responded to IL-2 around birth, supporting a role for IL-2 in the increased proliferation of mature thymocytes observed in vivo in the perinatal period. Taken together, these findings support a role for IL-2 in rat T cell development.      

Microanatomy of the liver immune system

The critical metabolic functions of the liver often eclipse any perception of its role as an immune organ. However, the liver as a mediator of systemic and local innate immunity and an important site of immune regulation is now an accepted concept. Complex repertoires of lymphoid and non-lymphoid cells are key to hepatic defense and immunoregulation. Hepatic cells of myeloid lineage include Kupffer cells and dendritic cells. Intrahepatic lymphocytes are distinct both in phenotype and function from their counterparts in any other organ and include both conventional (CD4+ and CD8+ αβ T cell receptor (TCR)+ T cells, B cells, natural killer (NK) cells) and nonconventional lymphoid cells (natural killer T (NKT) cells, γδTCR+ T cells, CD4? CD8? T cells). Many hepatic T cells express the TCR at an intermediate level and the great majority of them either coexpress NK cell markers (NKT cells) or they are apoptosing peripheral T cells. The percentage of activated (CD69+) and memory (CD45RBlow+) lymphocytes is much higher while naïve (CD62Lhigh) and resting T cells as well as B lymphocytes are underrepresented in the liver. The discovery of major populations of lymphoid cells in the liver that differ phenotypically, functionally and even perhaps developmentally from populations in other regions has been key to the evolving perception of the liver as a regulatory lymphoid organ. This chapter will focus on these populations and how they contribute to immune surveillance against malignant, infectious and autoimmune disease of the liver.     

Targeting CD45RB alters T cell migration and delays viral clearance

CD45 is a receptor tyrosine phosphatase essential for TCR signaling. One isoform, CD45RB, is down-regulated in memory cells and targeting CD45RB with a specific antibody has been shown to inhibit graft rejection. Its role in immunity to infection, however, has not been tested. Here, we report the effect of anti-CD45RB antibody treatment on the induction of anti-influenza CD8+ T cells and viral clearance. Anti-CD45RB-treated mice had delayed pulmonary viral clearance compared with untreated mice whose infection was completely cleared by day 8 post-infection. In anti-CD45RB-treated mice, the total CD4+ and CD8+ T cell numbers in both the lungs and mediastinal nodes were substantially reduced at days 5 and 8; this effect was less marked for the spleen. CD8+ T cells specific for influenza virus were also reduced compared with the control group in all three organs at day 8. By day 11, when both treated and control groups showed no virus remaining in the lungs, specific CD8+ T cell numbers were at similar low levels. Homing to lymph nodes and lung of dye-labeled T cells was greatly inhibited (by >80%) by anti-CD45RB treatment. This reduced homing corresponded with reduced CD62L and beta1-integrin expression in both uninfected and infected mice. Since CD62L plays a critical role in homing lymphocytes to lymph nodes, and high levels of CD62L and alpha4beta1-integrin are expressed by lymphocytes that home to bronchus-associated lymphoid tissue, we suggest that reduced expression of these molecules is a key explanation for the delay in immune responses.     

Human intrathymic T cell differentiation

The human thymus develops early on in fetal gestation with morphologic maturity reached by the beginning of the second trimester. Endodermal epithelial tissue from the third pharyngeal pouch gives rise to TE3+ cortical thymic epithelium while ectodermal epithelial tissue from the third pharyngeal cleft invaginates and splits during development to give rise to A2B5/TE4+ medullary and subcapsular cortical thymic epithelium. Fetal liver CD7+ T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression on thymocytes of other T lineage surface molecules. Human thymic epithelial cells grown in vitro bind to mature and immature thymocytes via CD2 and CD11a/CD18 (LFA-1) molecules on thymocytes and by CD58 (LFA-3) and CD54 (ICAM-1) molecules on thymic epithelial cells. Thymic epithelial cells produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF--molecules that likely are important in various stages of thymocyte activation and differentiation. Thymocytes can be activated via several cell surface molecules including CD2, CD3/TCR, and CD28 molecules. Finally, CD7+ CD4-CD8- CD3- thymocytes give rise to T cells of both the TCRab+ and TCR gd+ lineages.     

Functional maturation of recent thymic emigrants in the periphery: development of alloreactivity correlates with the cyclic expression of CD45RC isoforms.

The transition from fully developed CD4+CD8- single-positive (SP) thymocytes into fully mature recirculating peripheral T cells is both poorly understood with regard to the expression of restricted isoforms (CD45R) of the leukocyte common antigen and in terms of T cell function. The present investigation monitored the extrathymic development of CD4+CD8- SP thymocytes in euthymic recipients using allotype-marked donor cells and monoclonal antibody OX22 which recognizes an epitope on the C exon of rat CD45R. We established that donor-derived cells in the blood 1 day later bore the phenotype of the injected SP thymocytes (CD4+ Thy-1+ CD45RC-). T cells with the identical phenotype were also present in the thoracic duct lymph of uninjected rats, suggesting that the Thy-1+ CD45RC- T cells represent recent thymic emigrants (RTE) which have migrated to the periphery of their own accord. During extrathymic maturation donor-derived peripheral RTE lost Thy-1 within 3 days and expressed the CD45RC+ high molecular weight isoform by day 7; between days 8 and 14 a proportion (25%-30%) of the donor cells once again lost the high molecular weight isoform (CD45RC-). The transition of SP (CD45RC-) thymocytes to fully mature CD45RC+ CD4 T cells via intermediate peripheral RTE was accompanied at each stage by an increased ability of the maturing T cells to induce skin allograft rejection. Unexpectedly, the subsequent loss of the high molecular weight isoform, following presumed antigen encounter, was associated with a significant reduction in the ability of this Thy-1-CD45RC- subpopulation to effect graft rejection. The cyclic expression of CD45RC isoforms on both immature and mature CD4 T cells and the fact that the low molecular weight isoform was found in the periphery on both RTE (unquestionably naive) and antigen-experienced CD4 T cells, makes it unlikely that this isoform uniquely identifies memory T cells, at least in the rat.