Characterization of T-Cell Receptor Vβ Repertoire in Ovarian Tumour-Reacting CD3+ CD8+ CD4− CTL Lines

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T-cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3⁺ CD8⁺ CD4⁻ cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3⁺ CD8⁺ CD4⁻ lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8⁺ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour-reactive CD3⁺ CD8⁺ CD4⁻ CTL from different individuals of known HLA types.     

Mifepristone as an Anti-Implantation Contraceptive Drug: Roles in Regulation of Uterine Natural Killer Cells during Implantation Phase

Problem  To investigate the immunological mechanism of low-dose mifepristone acting as a contraceptive at the level of the endometrium.
Method of study  Endometrial explants were cultured in vitro with or without mifepristone treatment for 24 hr. Some tissues were fixed and immunostained for CD56, while other tissues were dissociated and cells analysed by three colour flow cytometry for CD3, CD56 and CD16.
Results and conclusion  Results showed a significant increase in the number of CD56⁺ natural killer (NK) cells and the percentages of CD3⁻ CD56⁺ CD16⁻ NK cell subset in the tissue treated with mifepristone, while the percentage of CD3⁻ CD56⁺ CD16⁺ NK cell subset remained unaffected. It shows that low-dose mifepristone increases the number of CD56⁺ NK cells and the percentage of CD3⁻ CD56⁺ CD16⁻ NK subset in receptive endometrium and provides new insights into the immunological mechanism of low-dose mifepristone as an anti-implantation contraceptive drug.     

CD3−4−8− Thymocyte Precursors with Interleukin-2 Receptors Differentiate Phenotypically in Coculture with Thymic Stromal Cells

CD3⁻4⁻8⁻ interleukin-2 receptor positive (IL-2R⁺) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL-2R⁺CD3⁻4⁻8⁻ thymocytes were obtained by positive panning of IL-2R⁺ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2½ days significant numbers of CD4⁺8⁺ and CD3⁺4⁺8⁻ cell populations developed, the latter expressing the αβ T-cell receptor (αβ-TCR). Thus heterogeneous stromal cell cultures support the development of IL-2R⁺ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal-dependent step.     

Peripheral T-cell Lymphomas Immunologic and Enzyme Histochemical Analysis of 15 Cases

Ffteen cases of peripheral T cell lymphoma were studied to evaluate the respective properties of various histologic types using enzyme histochemical and ultrastructural examinations in addition to immunological methods. Eleven cases in an ATLA negative group manifested various histologic patterns such as IBL like, pleomorphic and Lennert's lymphomas in comparison with the relatively monomorphic proliferation of neoplastic lymphoid cells in the 4 ATLA positive cases. The presence of neoplastic clear cells is characteristic of peripheral T-cell malignancies, and is likely to be found in CD4 lymphomas. There is an occasional reaction of epithelioid histiocytes and plasma cells with eosinophils, the former being designated Lennert's lymphoma and the latter IBL like T-cell lymphoma. Immunological examination revealed four immunophenotypic patterns: (1) CD2⁺3⁺4⁺8⁺, (2) CD2⁺ 3⁻4⁺8⁻, (3) CD2⁺3⁺4⁻8⁺, and (4) CD2⁺3⁺4⁺8⁺, but did not provide information concerning the intimate relationship between histologic types and immuno phenotyes. β-Glucuronidase reactivity, however, contributed to the distinction between helper and suppressor T cell malignancies, suggesting its usefulness for distinguishing these two cell types and their malignant counterparts.     

Population Dynamics of CD4+ T Cells Lacking Thy-1 in Murine Retrovirus-Induced Immunodeficiency Syndrome (MAIDS)

Increased numbers of CD4⁺ Thy-1 cells have been described in the spleen (SP) of mice with retrovirusinduced immunodeliciency (MAIDS). Since this phenotypic abnormality might have considerable functional importance, the expansion of the CD4⁺ Thy-1 subset in MAIDS was characterized further. CD4⁺ Thy-1⁻ and Thy-1⁺ T-cell is from infected mice expressed similar densities of CD3 and TCR γ/β. In contrast, the Thy-I⁻ subset was uniformly CD44^hi, even early in the disease when part of Thy-I⁺ cells were still CD44¹⁰. The emergence of CD4⁺ Thy-1⁻cells occurred first in SP and lymph nodes and was observed later in thymus. The important fraction ofCD4⁺ cells lacking Thy-1 normally present in Peyer's patches was only weakly modified. Despite the major expansion of the CD4⁺ Thy-1⁻ phenotype. the proliferating fraction was not higher in this subset than in CD4⁺ Thy-1⁺ cells from infeeted miee. Persistence after hydroxyurea administration was identical in both subsets, indicating similar mean cell lifespans. Taken together, these results show that the major expansion of CD4⁺ Thy-I⁻ T-cells in MAIDS is not ascribable solely to increased proliferation within this subset. Phenotypic analysis suggests that CD4⁺ Thy-I⁻ cells result from the differentiation of Thy-I⁺ cells induced by activation signals related to retroviral infection.     

GD2 Ganglioside-Specific Monoclonal Antibody Reacts with Murine Cytotoxic T Lymphocytes Reactive with FBL-3N Erythroleukaemia

Ganglioside expression on cytotoxic T lymphocytes induced against the mouse erythroleukaemia line FBL-3N, was analysed, compared with that of naive T lymphocytes in the thymus, lymph nodes and spleen. Although normal uncultured and cultured T lymphocytes expressed no GD2, GD3 or GM2 gangliosides, cytotoxic T cells with CD4⁺ CD8⁻, CD4⁻CD8⁺, and CD4⁻CD8⁻ phenotypes reacted with anti-GD2 monoclonal antibody with various intensities. The reactivity with anti-GD2 was associated with the intensity of cytotoxic activity as analysed using cytotoxic T lymphocyte (CTL) clones established from the bulk CTLs of each phenotype. An increase of the mRNA expression of GM2/GD2 synthase gene was demonstrated by Northern blot hybridization using RNA extracted from thymocytes, spleen cells, Con A-treated spleen cells and various types of CTL cells. These results indicated that GD2 ganglioside expression might be associated with the functional differentiation of murine T lymphocytes.     

Human NK Cells Expressing α4β1/β7 Adhere to VCAM-1 Without Preactivation

The authors demonstrate that resting CD56⁺/CD3⁻ NK cell adhesion to the endothelial VCAM-1 is over three-fold higher than CD56⁻/CD3 ⁺ T-cell adhesion. T-cell, but not NK-cell adhesion, to VCAM-1 is enhanced significantly by stimulation. The expression of VCAM-I receptor subunits α4 and β1 on both effector cells remains unchanged upon stimulation. A subpopulation of NK cells, as well as of T cells, was found to express β7, whose expression was not altered upon stimulation. The authors conclude that the adhesive properties of the same receptor structures on these distinct cell populations are regulated in a different manner, according to the specific functions of the effector cells of the immune system.     

Expression of CD28 on CD8+ and CD4+ Lymphocytes During HIV Infection

CD28 interaction with B7 molecules, expressed on the membranes of antigen-presenting cells, costimulates cytokine production, T-cell proliferation and generation of cytotoxic lymphocytes. The expression of CD28 markers on CD4⁺ and CD8⁺ lymphocytes was studied in a group of subjects at various stages of HIV infection. A reduction in the percentage of CD28-bearing CD4⁺ and CD8⁺ cell subsets was observed during the asymptomatic stage of the disease. This reduction was more pronounced in AIDS than in non-AIDS patients. At the same time, an increase in the absolute CD8⁺CD28⁻ cell number (greater in stage A than in stage B and C subjects) was observed in HIV-infected patients. The finding of an altered pattern of CD28 expression on T cells might per se explain certain early defects in the cytokine pattern and in the immune response peculiar to HIV-infected patients.     

A2B5 Cells from Human Glioblastoma have Cancer Stem Cell Properties

Glioblastomas, like other cancers, harbor small cell populations with the capability of sustaining tumor formation. These cells are referred to as cancer stem cells. We isolated cells expressing the surface marker A2B5 from three human glioblastomas (GBM) and showed that after grafting into nude mice, they generated dense and highly infiltrative tumors. Then, we extensively studied A2B5⁺ cells isolated from 11 human GBM. These cells display neurosphere-like, self-renewal, asymmetrical cell division properties and have multipotency capability. Stereotactic xenografts of dissociated A2B5⁺-derived secondary spheres revealed that as few as 1000 cells produced a tumor. Moreover, flow cytometry characterization of A2B5⁺-derived spheres revealed three distinct populations of cells: A2B5⁺/CD133⁺, A2B5⁺/CD133^- and A2B5^-/CD133^-, with striking proportion differences among GBM. Both A2B5⁺/CD133⁺ and A2B5⁺/CD133^- cell fractions displayed a high proliferative index, the potential to generate spheres and produced tumors in nude mice. Finally, we generated two green fluorescent protein-cell lines that display—after serum induction—distinct proliferative and migratory properties, and differ in their CD133 level of expression. Taken together, our results suggest that transformed A2B5⁺ cells are crucial for the initiation and maintenance of GBM, although CD133 expression is more involved in determining the tumor's behavior.     

3, 3', 4, 4'-Tetrachlorobiphenyl Inhibits Proliferation of Immature Thymocytes in Fetal Thymus Organ Culture

The environmental pollutant 3, 3', 4, 4'-tetrachlorobiphenyl (TCB) leads to thymic atrophy and immuno-suppression, the former possibly causing the latter. TCB binds lo the cytosolic aryl-hydrocarbon receptor (AhR) and transforms it into a DNA-binding state. The development of fetal thymocyles is severely affected by TCB and other AhR-binding xenobiotics, leading to a skewed pattern of thymocyte maturation stages. Murine thymocyte proliferation after exposure to TCB was studied in fetal thymus organ culture (FTOC). C57BL/6 fetus thymic lobes from day 15 of gestation were explanted and grown for 2, 4, 6. and 8 days in organ culture in the presence or absence of 3.3 μM TCB. Subsets of thymocytes were defined by CD4 and CD8 surface markers, and their cell cycle was analysed by DNA staining with 7-amino-actinomycin D (7-AAD). Exposure of fetal thymi in vitro to 3.3 μM TCB significantly reduced the total number of thymocytes. and fewer thymocytes were in S/G₂M phase. The inhibition of cell proliferation induced by TCB treatment affected mainly the CD4⁻ CD8⁻ (double-negative, DN) and CD4⁻ CD8⁺ (single-positive, SP) subsets, and these inhibition appeared mainly in more immature thymocytes, i. e. DNCD3⁻ and CD8⁺CD3⁻ subpopulations, whereas no effect of TCB on CD4⁺CD8⁺ (double-positive, DP) cell proliferative activity was observed. Analysis of the relation of cell proliferation and development of subsets in differentiating fetal ihymocytes suggests that TCB enhanced thymocyte differentiation into mature CD8⁺ cells.     

Natural CD4+ CD25+ regulatory T cells. Their role in the control of superantigen responses

Summary: CD4 regulatory T cells have a major role in controlling the immune response to self and foreign antigens. Natural CD4⁺ CD25⁺ T cells are a major component of the regulatory subset. Their absence is associated with the development of autoimmune and inflammatory diseases and with abnormal peripheral T-cell homeostasis. Two main characteristics discriminate natural CD4⁺ CD25⁺ T cells from their CD4⁺ CD25⁻ counterparts, namely their cytokine production profile and their behavior during tolerance induction. Natural CD4⁺ CD25⁺ T cells produce interleukin (IL)-10, a cytokine that contributes to their regulatory role. They do not produce IL-2 and are dependent on exogenous IL-2 for proliferation in vitro and in vivo . Studies of their response to superantigen administration in vivo show that they are resistant to clonal deletion but can be tolerized by anergy. Their resistance to apoptosis may contribute to their continuous regulatory function, as it allows them to maintain permanent control over effector T cells.     

Regulatory T cells in spontaneous autoimmune encephalomyelitis

Summary: Spontaneous experimental autoimmune encephalomyelitis (EAE) develops in 100% of mice harboring a monoclonal myelin basic protein (MBP)-specific CD4⁺αβ T-cell repertoire. Monoclonality of the αβ T-cell repertoire can be achieved by crossing MBP-specific T-cell receptor (TCR) transgenic mice with either RAG^−/− mice or TCR α^−/−/TCR β^−/− double knockout mice. Spontaneous EAE can be prevented by a single administration of purified CD4⁺ splenocytes or thymocytes obtained from wild-type syngeneic mice. The regulatory T cells (T-reg) that protect from spontaneous EAE need not express the CD25 marker, as effective protection can be attained with populations depleted of CD25⁺ T cells. Although the specificity of the regulatory T cells is important for their generation or regulatory function, T cells that protect from spontaneous EAE can have a diverse TCR α and β chain composition. T-reg cells expand poorly in vivo , and appear to be long lived. Finally, precursors for T-reg are present in fetal liver as well as in the bone marrow of aging mice. We propose that protection of healthy individuals from autoimmune diseases involves several layers of regulation, which consist of CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁻ T-reg cells, and anti-TCR T cells, with each layer potentially operating at different stages of T-helper cell-mediated immune responses.     

Variation in Gut-Homing CD27-Negative Lymphocytes in Inflammatory Colon Disease

Prolonged antigenic stimulation results in lymphocyte shedding of CD27, a member of the tumour necrosis factor receptor (TNFR) family, and transformation to a stable phenotype capable of synthesizing interleukin-4 (IL-4). Co-expression of α4β7 identifies those cells with gut-homing potential. We have investigated these cell populations in patients with inflammatory colonic disease. Circulating and lamina propria mononuclear cells were isolated from patients with Crohn's disease (CD), ulcerative colitis (UC), non-inflammatory bowel disease (non-IBD) colonic inflammation and healthy controls. Double and triple colour flow cytometry for CD3, CD4, CD27, α4β7 and intracellular cytokines was performed. Circulating CD4+CD27– populations were increased in patients with CD (8.8 ± 0.8%, P  < 0.001), UC (12.2 ± 1.9%, P  < 0.001) and non-IBD colitis (10.5 ± 1.3%, P  < 0.01) as compared with controls (6.1 ± 0.5%). CD4⁺CD27⁻α4β7⁺ cells were increased in CD ( P  < 0.01). Lamina propria CD4⁺CD27⁻ populations were depressed significantly in CD ( P  < 0.05), UC ( P  < 0.02) and non-IBD colitis ( P  < 0.03). Mucosal CD4⁺CD27⁻ cells synthesized IL-4 in preference to interferon-γ. Thus, colonic inflammation is associated with alterations in gut-tropic circulating and mucosal populations of differentiated memory T cells with the phenotype of predominantly IL-4-synthesizing cells.     

ASRI2005-88 Comparable levels of CD4+CD25+ CTLA4+ Treg cells in peripheral blood of pre-eclampsia and normal pregnant patients

The acceptance of the semiallogeneic fetus within the maternal environment requires tolerance mechanisms not fully characterized yet. Normal pregnancy is known to be associated with a Th2 profile. Furthermore, T-regulatory cells were proposed to regulate the Th2/Th1 balance at early stages of pregnancy. Treg may avoid the shift to a Th1 profile preventing miscarriage. Accordingly, spontaneous abortion is characterized by a Th1 dominance and diminished levels of Tregulatory cells (Treg). The major aim of the present work was to investigate if pre-eclampsia, a late immunological complication of pregnancy, is characterized by similar hallmarks. Therefore, we measured the surface antigens CD4, CD25, CD8, CTLA4 (as well as the secretion of IL-10) in peripheral blood from patients suffering from pre-eclampsia (n = 8) and age-matched patients undergoing normal pregnancies (n = 9) by 4-colour flow-cytometry. We were not able to find any significant differences in the levels of CD4⁺, CD25⁺, CD8⁺, CTLA4, CD4⁺/CD25⁺, CD4⁺/CD25^bright, CD4⁺/CTLA4, CD25⁺/CTLA4, CD4⁺/CD25⁺/CTLA4, CD8⁺/CD25⁺, CD8⁺/CTLA4 or CD8⁺/CD25⁺/CTLA4 cell subsets. Our data suggest that Treg may not participate in the onset of pre-eclampsia and suggest other regulatory mechanisms during late pregnancy.     

Effect of 12 Neutralizing Anti-Cytokine Antibodies on In Vitro Activation of B-Cells. Interleukin-12 is Required by B1a but not B2 Cells

Normal human peripheral blood mononuclear cells, depleted of most monocytes and virtually all CD8-positive cells, were stimulated in vitro with pokeweed mitogen plus Staphylococcus aureus Cowan I in the presence or absence of variousneutralizing anti-cytokine antibodies. Numbers of CD5⁺ and CD5⁻ immunoglobulin-secreting cells were determined using the protein A haemolytic plaque assay after labelling B1a cells with anti-CD5-coated beads. Antibodiesagainst IL-2, IL-5 and IL-10 had little or no effect on plaque-forming cell (PFC) induction; anti-IL-6, -TNFα and -TGFβ enhanced PFC induction; anti-IL-1α, -IL-1β, -IL-4, -IFNγ and -IL-13 suppressed PFC induction. B1a and B2 cells were equally affected by cytokine deprivation using these 11 neutralizing antibodies. In contrast, neutralizing anti-IL-12 suppressed induction of CD5⁺ but not CD5⁻ PFC. Furthermore, recombinant IL-12, if added during thefirst 48 h of culture, enhanced CD5⁺ PFC induction while marginally suppressing (IgG-) or not affecting (IgA-, IgM-) induction of CD5⁻ PFC. IL-12 did not preferentially increase survival in culture of B1a cells norinduce expression of CD5 on B2-cells. Further studies are required to determine whether manipulation of B1a and B2 subsets in vivo using IL-12 could be achieved in clinical situations where imbalances in the two populations have beenobserved.     

Peripheral Lymphoid Hyperplasia and Central Lymphoid Depletion in Mice Treated with a Bacterial B-Cell Mitogen (F3'EP-Si/p90)

In order to further understand the mechanism mediating the mitogenic and immunosuppressor effects of p90, a protein produced by Streptococcus intermedius , flow cytometric studies were performed on peripheral and central lymphoid organs of mice treated with this protein. p90 induced a strong blastogenic B-cell response in the spleen and lymph nodes, followed by a slight but significant polyclonal T-cell activation. B-cell repertoire analysis indicated that polyclonal B-cell responses affected similarly both CD5⁺ and conventional (CD5⁻) B cells in the spleen. Repertoire analysis of T cells failed to reveal any preferential stimulation of the Vβ T-cell receptor (Vβ-TcR) families studied. Peripheral lymphoid hyperplasia was observed concomitantly with central lymphoid depiction. In the bone marrow, pre-B and B cells were profoundly depleted, with a more pronounced effect on small pre-B cells. In the thymus, double-positive (CD4⁺ CD8⁺) thymocytes were preferentially eliminated, with a relative enrichment of single positive (either CD4⁺ or CD8⁺) and double-negative (CD4⁻CD8⁻) thymocytes.     

II. The Effects of Thalidomide Treatment on Autoimmune-Prone NZB and MRL Mice are Consistent with Stimulation of the Central Immune System

We describe here some immunomodulatory effects of thalidomide on autoimmune-prone mice. The highly increased synthesis of splenic IgM in NZB mice, of splenic and lymph node IgG of different subclasses in MRL/n mice, and of splenic and lymph node IgGl in MRL/lpr mice was markedly inhibited by thalidomide treatment. After a single treatment with 3mg of thalidomide, the following changes were observed in NZB mice: (i) an initial decrease in the numbers of large CD5⁺μ^high, and in the numbers of total CD5⁺μ⁻, CD5⁻μ^high, CD5⁺μ^high lymphocyte populations of the pleural cavity followed by a late increase in the numbers of large cells of the three cell populations; (ii) a consistent increase in the numbers of a CD5^lowμ^low pleural lymphoid population; (iii) a consistent reduction in the numbers of splenic large CD5⁺ B cells and an oscillatory increase in the number of cells with CD5⁻ phenotype; (iv) a late reduction in the numbers of splenic total CD5⁺ B cells. These results are consistent with the notion that thalidomide controls a disease-associated expansion of B cells in autoimmune prone mouse strains through a stimulatory effect of the drug on the immune system.     

Strict Adherence to a Common Rank Order of T-Cell Receptor Vβ Usage in Human Leucocyte Antigen Disparate Individuals

In order to investigate the T-cell receptor (TCR) Vβ usage in different T-cell subsets, the authors performed flow cytometric analyses using a large panel of TCR Vβ-specific monoclonal antibodies on CD4⁺, CD8⁺ CD28⁺ and CD8⁺ CD28⁻ T cells from 15 random blood donors, six umbilical cords and seven human leucocyte antigen (HLA) identical non-twin sibling pairs. The authors found that the proportion of T cells expressing each Vβ gene product was similar within CD4⁺ and CD8⁺ CD28⁺ T cells from all samples studied. For these T-cell subsets a rank order of Vβ usage could be identified which was adhered to by all donors. In contrast, within CD8⁺ CD28⁻ T cells a wide variation of Vβ usage was found between individuals, and no rank order correlation could be detected. Members of HLA identical sibling pairs were found to be no more similar in their usage of Vβ gene products than pairs of HLA disparate random blood donors. Groups of individuals sharing HLA antigens were no different from the groups not sharing such antigens in their usage of Vβ segments. The results suggest that HLA polymorphisms play no more than a minor part in determining TCR Vβ usage.     

CD 127- and FoxP3+ expression on CD25+CD4+ T regulatory cells upon specific diabetogeneic stimulation in high-risk relatives of type 1 diabetes mellitus patients

Abnormalities in CD4⁺CD25⁺ regulatory T cells (Treg) may contribute to type 1 diabetes (T1D) development. First-degree relatives of T1D patients are at increased risk especially when they carry certain HLA II haplotypes. Using two novel markers of CD4⁺CD25⁺ Treg (CD127⁻ and FoxP3⁺ respectively), we evaluated number and function of Treg after specific stimulation with diabetogeneic autoantigens in 11 high-risk (according to HLA-linked risk) relatives of T1D patients and 14 age-matched healthy controls using a cytokine secretion assay based on interferon- γ (IFN- γ ) production. High-risk relatives of T1D patients had significantly lower pre- and post-stimulatory number of CD127⁻ Treg than that of healthy controls ( P  < 0.05). Labelling Treg with FoxP3⁺ demonstrated similar trend but did not reach statistical significance. Although the stimulation with diabetogenic autoantigens did not lead to a significant change in number of Treg in both groups, the defective function of Treg was performed by significantly higher activation of diabetogeneic T cells in high-risk relatives of T1D patients compared to healthy controls ( P  ≤ 0.02). Individuals at increased HLA-associated genetic risk for T1D showed defects in Treg.