The cytoplasmic expression of CD3 antigens in normal and malignant cells of the T lymphoid lineage

Anti-CD3 (T3) Ab reacting with different proportions of thymocytes (anti-CD3a: UCHT1, anti-CD3b: T10B9, and anti-CD3c: OKT3) were tested for cytoplasmic (cCD3) and membrane (mCD3) expression in the bone marrow, thymus, and blood in man and selected primates. The expression of cCD3a and cCD3c in the perinuclear and Golgi area of large, BrdU-incorporating, strongly TdT+ thymic blasts probably represents one of the earliest signs of T cell commitment, because these blast cells are CD1-, CD4-, CD8-, and mCD3-. The cCD3+, TdT+ cells are normally restricted to the thymus and are absent among the TdT+ cells of bone marrow. The anti-CD3b Ab used, T10B9, co-caps and co-modulates with the other anti-CD3 Ab and is a T cell-specific reagent at a membrane level but does not bind to perinuclear cCD3. Instead, this reagent cross-reacts with a filamentous cytoplasmic network in non-T cells in man and in primates S. oedipus and M. rhesus despite their T cell negativity for mCD3. The characteristics of all T-ALL cases studied: cCD3+, CD7+ along with nuclear TdT+ suggest lineage fidelity to early thymic blasts. As a marked contrast, cCD3 is absent in common ALL and in AML, including cases that concomitantly express CD7 and myeloid antigens. Thus, the cCD3, TdT combination provides a very sensitive assay for residual T-ALL blasts outside the normal thymus.     

Chimeric cells of maternal origin do not appear to be pathogenic in the juvenile idiopathic inflammatory myopathies or muscular dystrophy

IntroductionMicrochimeric cells have been studied for over a decade, with conflicting reports on their presence and role in autoimmune and other inflammatory diseases. To determine whether microchimeric cells were pathogenic or mediating tissue repair in inflammatory myopathies, we phenotyped and quantified microchimeric cells in juvenile idiopathic inflammatory myopathies (JIIM), muscular dystrophy (MD), and noninflammatory control muscle tissues.MethodFluorescence immunophenotyping for infiltrating cells with sequential fluorescence in situ hybridization was performed on muscle biopsies from ten patients with JIIM, nine with MD and ten controls.ResultsMicrochimeric cells were significantly increased in MD muscle (0.079 ± 0.024 microchimeric cells/mm² tissue) compared to controls (0.019 ± 0.007 cells/mm² tissue, p = 0.01), but not elevated in JIIM muscle (0.043 ± 0.015 cells/mm²). Significantly more CD4+ and CD8+ microchimeric cells were in the muscle of patients with MD compared with controls (mean 0.053 ± 0.020/mm² versus 0 ± 0/mm²p = 0.003 and 0.043 ± 0.023/mm² versus 0 ± 0/mm²p = 0.025, respectively). No differences in microchimeric cells between JIIM, MD, and noninflammatory controls were found for CD3+, Class II+, CD25+, CD45RA+, and CD123+ phenotypes, and no microchimeric cells were detected in CD20, CD83, or CD45RO populations. The locations of microchimeric cells were similar in all three conditions, with MD muscle having more microchimeric cells in perimysial regions than controls, and JIIM having fewer microchimeric muscle nuclei than MD. Microchimeric inflammatory cells were found, in most cases, at significantly lower proportions than autologous cells of the same phenotype.ConclusionsMicrochimeric cells are not specific to autoimmune disease, and may not be important in muscle inflammation or tissue repair in JIIM.     

Phenotypic heterogeneity of Gross virus-induced thymic lymphomas in the rat: cellular origins and migratory properties

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross leukemia virus (RAGV) were analyzed for a variety of differentiation markers. The neoplasms from individual rats all expressed the antigenic phenotype MP+, W3/13+, Thy-1+, RT-1+, RT-7+, W3/25-. However, approximately two-thirds of the neoplasms were positive for the OX 8 antigen, and one-third were negative. The OX 8- neoplasms only involved the thymus, whereas approximately 40% of the OX 8+ neoplasms involved the spleen as well as the thymus. Virtually all OX 8+ and OX 8- neoplastic cells contained terminal deoxynucleotidyl transferase (TdT), and both OX 8+ and OX 8- lymphomas expressed the lactate dehydrogenase (LDH)-5' isozyme and the primary, but not the secondary, ADA isozyme. This enzymatic phenotype is characteristic of thymocyte precursors, but not thymocytes. Our results therefore indicate that RAGV-induced lymphomas arise from transformed prethymic TdT+ cells which contain the LDH-5' and the primary ADA isozymes. These preleukemic cells presumably migrate to the thymus where they express the RT-7 pan-T-cell antigen and, in some instances, the OX 8 antigen during the development of overt leukemia. The OX 8+ neoplasms, being more differentiated than their OX 8- counterparts, then migrate to peripheral lymphoid tissues.     

The expression of T cell receptor-associated proteins during T cell ontogeny in man

The expression of TCR-associated molecules was examined in human fetal and postnatal tissues. From gestational wk 7 onward in the fetal liver, putative prothymocytes have been identified with cytoplasmic CD3 positivity (cCD3+). These immature cells are TdT- and do not express membrane CD3 (mCD3-) or TCR beta identified by beta F1, but show CD7 and CD45 positivity without CD1, CD2, CD5, CD4, CD8, CD10, and class II Ag. Their high proliferative activity is indicated by greater than 85% Ki67 positivity. After the 10th wk, beta F1+, mCD3+ cells also appear in the liver and these are mostly Ki67- but no TCR gamma delta-bearing cells can be identified at such an early stage of extrathymic development. In the mCD3- TdT-fetal thymus (10 1/2 to 18th wk) cCD3+, mCD3- CD1-blasts proliferate (Ki67+) and lack TCR-beta or TCR-gamma delta. The TdT-, CD1+ cortical thymocytes develop into TCR-beta + and WT31-positive (TCR-alpha beta +) cells. Subsequently TdT-positive thymocytes become detectable around 19 to 20 wk, and in such glands the peak of proliferative activity is seen among TdT+, cCD3+ cells which appear to acquire, in a regular sequence, cytoplasmic beta F1 (TCR-beta), mCD3, and TCR-alpha beta (WT31 positivity) together with the loss of TdT and Ki67 positivity. A newly described transitional population of cells is TdT-, beta F1+ but exhibits no detectable WT31 positivity. These cells correspond to the CD1+, mCD3+ thymocytes and are probably the targets of thymic selection. The cells of the TCR-gamma delta lineage, detected by mAb TCR-delta-1 and delta TCS1, are rare (0.02 to 0.5%) among thymocytes from gestational wk 10 1/2 onward through the whole span of thymic development, but these cells include a proportion (18 to 59%) of cells expressing CD1 Ag, suggesting that these TCR-gamma delta cells differentiate in the thymus. Among the CD1+, TCR-gamma delta + thymocytes, no TdT positivity can be detected.     

Murine anti-CD3 monoclonal antibody induces potent cytolytic activity in both T and NK cell populations

Antibodies specific for the CD3 complex have the capacity to both stimulate and inhibit a variety of T cell functions. We show here that a monoclonal antibody to the epsilon chain of CD3 can induce efficient non-MHC-restricted cytolytic activity in murine lymphocytes with peak activity occurring after 48 hr of incubation. In a panel of targets, the anti-CD3-activated effectors lysed tumor cells but not normal lymphoblasts. Cytolysis was not dependent on the presence of the antibody in the cytolytic assay. Moderate to high cytolytic activity was elicited from lymph nodes, spleen, and thymus by anti-CD3 treatment in vitro, whereas only low activity was apparent in bone marrow. The precursors of anti-CD3-activated cells consisted largely of mature T cells, although a smaller component of immature T cells was also involved. Thus, separation of thymocytes based on adhesion to peanut agglutinin revealed that both positive (immature) and negative (mature) fractions could be activated, while cytotoxic pretreatment of spleen cells with an antibody (J11d) to immature T cells before anti-CD3 activation significantly decreased the resulting cytotoxicity. The majority of precursors in spleen were Thy 1+ and CD8+ and/or AGM1+. Antibody depletion studies showed that the effector cells have both a T and a NK component consisting of Thy 1+, CD5+, CD8+, CD4-, and AGM1- cells and Thy 1-, CD5-, CD8-, CD4-, and AGM1+ cells, respectively. The production of significant amounts of IL-2 and TNF in culture following anti-CD3 treatment, along with the synergistic effect of exogenously added IL-2, suggests that one or both of the effector cell types could be induced by lymphokines. The intraperitoneal administration of the anti-CD3 antibody induces cytolytic activity in vivo. Therefore, the direct activation of cytolysis by anti-CD3 antibody and the additional effects, both direct and synergistic, of lymphokines produced by the activated lymphocytes could conceivably provide a potent anti-tumor therapy.     

The development of helper T cell precursors in mouse thymus

We have examined the appearance in mouse ontogeny of thymocyte precursors for Ag-specific, MHC-restricted Th. These cells are first detectable at day 18 of fetal life, about 1 day after alpha/beta, TCR-positive cells begin to appear. These early Th precursors are not dependent on the thymus for priming with Ag and MHC, and are L3T4+, Lyt-2-. Thus, these cells already have the phenotype of mature Th. In neonatal F1 animals expressing both IAk and IAb, the appearance of Th precursors restricted by either IAk or IAb is specifically inhibited by treatment of the mice with anti-IAk or anti-IAb antibodies, respectively. These results indicate that cells of mature T cell phenotype and function can arise fairly rapidly from immature, receptor-bearing precursors, once these appear. Moreover the results are in line with those previously obtained in chimeric animal experiments which suggested that specific interaction of TCR on thymocytes with class II alleles in the thymus is required for the subsequent appearance of T cells restricted by those class II alleles.     

Phenotypic features and proliferative activity of B cell progenitors in X-linked agammaglobulinemia

In this study, we applied mAb and heterologous antisera in double marker combinations to investigate the phenotype and the proliferative activity of immature B lineage cells in XLA. Bone marrow (BM) samples from eight male adult patients with no circulating B lymphocytes were studied. The proportions and the phenotype of the earliest identifiable B cell progenitors, expressing nuclear terminal deoxynucleotidyl transferase (TdT), cytoplasmic CD22, and membrane CD19 and CD10 were identical to those observed in normal BM. In XLA these cells represented 1.2% to 22% of BM mononuclear cells; 5% to 42% and 1% to 45% of such cells weakly expressed CD20 and CD37, respectively, and invariably lacked CD13 and CD33. Cytoplasmic mu+ sIg- pre-B cells were seen in low numbers (0.1% to 0.3%) in four samples and were undetectable in the remaining four. Consequently, the ratio TdT+/c mu+ was greater than 100 in five out of eight samples studied in contrast to the less than 10 values seen in normal individuals. The proliferative activity of B lineage progenitor cells was studied by using Ki67 and anti-bromodeoxyuridine mAb. Although the proliferation of TdT+ cells in XLA was comparable with that seen in normal BM samples (24% to 59% of TdT+ were Ki67+ and 11% to 27% incorporated bromodeoxyuridine), this was dramatically reduced in the c mu+ cells (no c mu+, Ki67+ seen in three samples where pre-B cells were observed). Thus, the abnormalities of B cell differentiation in XLA are first seen at the c mu+ pre-B stage and suggest a maturation block in the transition between TdT+, c mu- pre-pre-B cells and c mu+ pre-B cells. The severity of this block may be variable, allowing the generation of a near normal number of pre-B cells in some patients, which nevertheless have a defective proliferative activity. Finally, our study further supports the concept that the effects of the "XLA gene" are confined within the B lineage by demonstrating that the proportions of T cells bearing TCR-alpha beta and TCR-gamma delta in XLA are similar to those seen in normal individuals.     

IFN-γ Rα Is a Key Determinant of CD8+ T Cell-Mediated Tumor Elimination or Tumor Escape and Relapse in FVB Mouse

During the past decade, the dual function of the immune system in tumor inhibition and tumor progression has become appreciated. We have previously reported that neu-specific T cells can induce rejection of neu positive mouse mammary carcinoma (MMC) and also facilitate tumor relapse by inducing neu antigen loss and epithelial to mesenchymal transition (EMT). Here, we sought to determine the mechanism by which CD8+ T cells either eliminate the tumor, or maintain tumor cells in a dormant state and eventually facilitate tumor relapse. We show that tumor cells that express high levels of IFN-γ Rα are eliminated by CD8+ T cells. In contrast, tumor cells that express low levels of IFN-γ Rα do not die but remain dormant and quiescent in the presence of IFN-γ producing CD8+ T cells until they hide themselves from the adaptive immune system by losing the tumor antigen, neu. Relapsed tumor cells show CD44+CD24- phenotype with higher rates of tumorigenesis, in vivo. Acquisition of CD44+CD24- phenotype in relapsed tumors was not solely due to Darwinian selection. Our data suggest that tumor cells control the outcome of tumor immune surveillance through modulation of the expression of    IFN-γ Rα.     

Homeostatic Properties and Phenotypic Maturation of Murine CD4+ Pre-Thymic Emigrants in the Thymus

After a tightly regulated developmental program in the thymus, “mature” single positive (SP) thymocytes leave the thymus and enter the periphery. These newly arrived recent thymic emigrants (RTEs) are phenotypically and functionally immature, and will complete a dynamic maturation in the peripheral lymphoid organs before being licensed to be resident naïve T cells. To study the early events occurring in the RTE maturation process, we identified the phenotype of CD4⁺ pre-RTEs, a population of CD4⁺ SP thymocytes that have acquired the thymus egress capability. Compared to peripheral naïve T cells, CD4⁺ pre-RTEs displayed superior survival capability in lymphoreplete mice and faster proliferation under lymphopenic condition. The differences in Bcl2/Bim expression and/or heightened IL-7 signaling pathway may account for the pre-RTEs’ better responsiveness to homeostatic signals. Qa2, the expression of which indicates the phenotypic maturation of SPs and RTEs, was found to be upregulated in CD4⁺ pre-RTEs in thymic perivascular space. Migratory dendritic cells that surround this region contribute to Qa2 expression in pre-RTEs. The dendritic cell-driven Qa2 induction of CD4⁺ pre-RTEs is independent of MHC class II and Aire molecules.     

The Involvement of Immune Semaphorins in the Pathogenesis of Inflammatory Bowel Diseases (IBDs)

ResultsThe percentage (%) of T regulatory cells (Tregs) expressing sema3A in patients with active CD (64.5% ±14.49%) and active UC (49.8% ±16.45%) was significantly lower when compared to that of healthy controls (88.7% ±3.6%, p< 0.001 and p< 0.0001, respectively). This expression was seen to be in negative correlation with CD activity. Serum levels of Sema4A were significantly lower in patients with CD and UC when compared to that of controls (5.69±1.48ng\ml for CD, 5.26±1.23 ng/ml for UC patients vs 9.74±2.73ng/ml for normal controls, P<0.001). Sema4A was highly expressed in lymphocytes of the lamina propria of CD and UC patients but absent in patients with diverticulitis or in normal individuals.ConclusionsAltered % of Tregs expressing sema3A in patients with inflammatory bowel diseases (IBD) is partially responsible for their failure in preventing CD4+ effector T cell induced inflammation in IBD in peripheral blood. The increased expression of sema4A in bowel biopsies from CD and UC patients is suggestive of its central role in regulating local tissue inflammation in the bowel.     

CD4+CD25+ Regulatory T cell ontogeny and preferential migration to the cecal tonsils in chickens

Thymic CD4(+)CD25(+) cells have regulatory-T-cell-like properties in chickens. This study examined the ontogeny of CD4(+)CD25(+) cells in the thymus and in peripheral compartments in chickens. CD4(+)CD25(+) cells started to appear in the thymus at day 15 of incubation (E15), although at low percentages. Expressed as a percentage of CD4(+) cells, CD4(+)CD25(+) cells increased (P<0.01) from 1.7% at E20 to 7.3% at 0 d post-hatch (D0). CD4(+)CD25(+) cells did not appear in the spleen or cecal tonsils of embryos. Expressed as a percentage of CD4(+) cells, CD4(+)CD25(+) cells increased (P<0.01) from 0% at D0 to 27% at D1 in cecal tonsils and from 0% at D0 to 11% at D1 in the spleen. Expressed as a percentage of all mononuclear cells, cecal tonsils at D1 had approximately 3.5-fold higher percentage of CD4(+)CD25(+) cells than the spleen at D1. CD4(+)CD25(+) cells from cecal tonsils of chicks at D1 were suppressive. CD4(+)CD25(+) cells from D0 thymus, when injected back into MHC-compatible chicks, migrated to cecal tonsils and lungs and were detected until 10 d post-injection. CD4(+)CD25(+) cells from cecal tonsils had a higher (P = 0.01) relative amount of CCR9 mRNA than CD4(+)CD25(+) cells from the thymus. It could be concluded that in chickens CD4(+)CD25(+) cells migrate from the thymus immediately post-hatch and preferentially colonize the gut associated lymphoid tissues. CD4(+)CD25(+) cells' preferential migration to cecal tonsils is likely directed through the CCR9 pathway in chickens.     

CD49f Is an Efficient Marker of Monolayer- and Spheroid Colony-Forming Cells of the Benign and Malignant Human Prostate

Stem cells may play a role in the development and maintenance of proliferative diseases of the prostate such as prostate cancer and benign prostatic hyperplasia. Cell membrane protein markers, CD49f, CD133 and CD44, have been shown to identify putative prostate stem cells, but a lack of consensus exists with regards to the most efficient marker(s) for stem-like cell identification. This study aimed to determine whether previously reported markers had equal capacity to select monolayer and spheroid colony-forming cells (CFCs), which were used as surrogate readouts of stem-like cells, and to characterize the expression of CD49f, CD44 and CD133 by flow cytometry and immunohistochemistry.In benign prostate cells, CD49f+, CD44+, and CD133+ cells represented 5.6±3.1%, 28.2±4.1% and 0.10±0.06% of total cells. Both monolayer- and spheroid-CFCs existed at a frequency of approximately 0.5% of total cells. CD49f+, CD44+, and CD133+ subpopulations differed significantly in their ability to select benign CFCs. The highest recovery of CFCs was achieved by CD49f+ selection (98%), whereas CD44+ or CD133+ selection led to poor CFC-recovery (17% and 3%, respectively). For the first time, we show highly efficient recovery of CFCs from advanced prostate cancer by CD49f+, but not by CD44+ or CD133+ selection. Furthermore, CD133 expression (AC133 clone) could not be detected in benign prostate cells by either immunohistochemistry or flow cytometry. We conclude that CD49f, but not previously described stem cell markers CD133 and CD44, to be optimal for selection of monolayer- and spheroid-CFCs in the benign and malignant prostate.     

Dendritic cells generated either from CD34+ progenitor cells or from monocytes differ in their ability to activate antigen-specific CD8+ T cells.

Dendritic cells (DC) can be generated in vitro from monocytes (M-DC) or from CD34+ hemopoietic progenitor cells (CD34-DC) but their precursors are not equivalent cells, prompting a comparison of the functional capacities of these APC. Both types of DCs established from the same individuals using the same cytokines displayed a comparable phenotype of mature DC (CD1a+, CD83+, CD86+, CD4+, HLA-DR++, CD14-, CD15- ) and were equally potent stimulators of allogeneic T cell proliferation, being both more powerful than immature M-DCs. An autologous panel of APCs produced in HLA-A2+ individuals, including CD34-DC, M-DC, monocytes, and EBV-lymphoid cell line was comparatively evaluated for presentation of the Erb-B2 peptide E75 to a CTL line. After short exposures (5 h) to E75-loaded APCs, similar levels of intracellular IFN-gamma were induced in Ag-specific CD8+ T cells regardless of APC type. In sustained cultures (4-14 days), more Ag-specific T cells were obtained when peptide was presented on CD34-DC (p < 0.05) rather than on M-DC, EBV-lymphoid cell lines, or monocytes, and these effects were dose-dependent. Activated T cells expressed 4-1BB, and the presence of 4-1BB-Ig fusion protein partially blocked Ag-specific CD8+ cell activation after CD34-DC or M-DC presentation. Our results show that 34-DC have a preferential capacity to activate CD8+ T cells and that this property is not strictly correlated to their ability to induce allogeneic T cell proliferation but due to mechanisms that remain to be defined.     

DNA polymerases of human tonsil and chicken bursa: absence of a distinct B cell specific terminal deoxynucleotidyl transferase.

The extracts obtained from chicken bursas and human tonsils, both of which are known to contain predominantly B lymphocyte precursor cells, were examined for the presence of putative B cell specific terminal deoxynucleotidyl transferase (B-TdT). Neither of the two types of tissue extracts revealed the presence of specific TdT, although the presence of DNA polymerase α, β, and γ could be easily demonstrated.     

Stage-specific induction of terminal deoxynucleotidyl transferase in a T-lymphoid line upon coculture with a thymic stromal line

We previously reported an in vitro T-cell differentiation system in which the L4 lymphoid clone was cocultured with the St3 stromal line derived from the same murine thymic tumor, 15#4T. L4 cells in L4—St3 cocultures sequentially express Thy-1 and CD4 in a manner typical of normal thymocytes. In contrast, L4 cells grown in medium alone retain their Thy-1⁻CD4⁻ phenotype. We also isolated L4 subclones from the coculture with increasingly differentiated phenotypes with respect to Thy-1 and CD4. We now report induction of an additional thymocyte differentiation marker, terminal deoxynucleotidyl transferase (TdT) in 15#4T cells (and to a lesser extent subcloned L4 cells) upon coculture with St3 stroma. Coculture of 15#4T cells with St3 stroma resulted in expression of TdT as measured by ribonuclease protection for TdT RNA and Western immunoblotting for TdT protein. Cocultured L4 cells were induced for TdT expression to a lesser degree and for a shorter period of time. The magnitude of TdT RNA induction was maximal for cell lines with the least mature differentiation phenotype (15#4T and L4: Thy-1⁻CD4⁻) and decreased proportionally for subclones with increasingly mature phenotype, e.g., L4E cells (Thy-1⁺CD4⁺). TdT protein was undetectable by Western immunoblotting and immunofluorescent staining of the L4E subclone on or off stroma. Recombination-activating gene-1 (RAG-1), which is expressed in immature thymocytes during T-cell receptor rearrangement, but suppressed in mature thymocytes, was also examined using the ribonuclease protection assay. In contrast to TdT, RAG-1 expression was suppressed by coculture with St3 cells for 15#4T and also more mature subclones, indicating regulation by a mechanism independent from TdT. The ordered induction of TdT, Thy-1, and CD4, as well as regulation of RAG-1 in the 15#4T-St3 system, supports the conclusion that this in vitro system is a valuable model for characterizing regulation of these markers in normal thymocytes.