Seasonal Variation and Sex Differences of Circulating Macrophages, Immunoglobulins and Lymphocytes in Healthy School Children

Subpopulations of T and B lymphocytes and levels of serum immunoglobulins G, A, M, E and subclasses G1, G2 and G3 were studied in 45 healthy school children aged 8–16 years during four seasons of the year. There were significant increases in CD4⁺ T helper cells, total T lymphocytes and CD4⁺/CD8⁺ (helper/cytotoxic) T-cell ratio during the spring season. While the levels of CD8⁺ T cells and total B lymphocytes remained statistically unchanged during all four seasons, the levels of natural (HNK-1) killer cells and macrophages increased significantly during the autumn and summer seasons respectively. The levels of immunoglobulins G, A, M and E remained statistically unchanged during all four seasons. Girls had higher levels of CD4⁺ T cells and a higher CD4⁺/CD8⁺ T-cell ratio than boys. Girls also had slightly higher levels of immunoglobulin G and M. These observations suggest that seasonal variations of some immunological parameters occur in healthy children. This may be an adaptive response to variable climatic and other environmental factors. These natural variations due to seasonal changes should be taken into account when immunological tests are used in clinical investigations.     

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.     

The role of B7 co-stimulation in activation and differentiation ofCD4+and CD8+T cells

Summary: The functional significance of B7 co-stimulation in T-cell activation was described first in the context of preventing the induction of anergy. The functions of this pathway are far more complex than initially appreciated in view of the existence of two B7 molecules which have specificities for both CD28 and CTLA-4, which serve to amplify and terminate T-cell responses respectively Mice lacking B7 co-stimulators and CD28 and CTLA-4 co-stimulatory receptors are helping to clarify the functions of this key immunoregulatory pathway. In this review we will focus on the role of B7 co-stimulation in the activation and differentiation of CD4⁺ helper cells and CD8⁺ cytotoxic cells. The contribution of B7 co-stimulation to CD⁺ responses depends upon the activation history of the T-cell and the strength of the T-cell antigen receptor signal. B7 co-stimulation contributes to in Cerleukin (IL)-2 production by both naive and previously activated CD4⁺ T cells. B7 co-stimulation is most critical for the differentiation of naive CD4+ T cells to IL-4 producers, but predominately influences IL-2 production by previously activated CD4+ cells. B7 co-stimulation is important in development of cytotoxic T cells through both effects on T-helper cells and by direct co-stimulation of CDS⁺ cells.     

T Helper-Independent Activation of Human CD8+ Cells: the Role of CD28 Costimulation

The concept that activation of MHC class I-restricted CD8⁺ cells entirely depends on help from MHC class II-restricted CD4⁺ T cells has recently been supplemented with an alternative model in which CD8⁺ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8⁺ T cells in two different in vitro models. Freshly isolated CD8⁺ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting FcγR⁺ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8⁺ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8⁺ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8⁺ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8⁺ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance.     

T-Lymphocyte Functions in Acute Leukaemia Patients with Severe Chemotherapy-Induced Cytopenia: Characterization of Clonogenic T-Cell Proliferation

Intensive chemotherapy for acute leukaemia is followed by a period of severe chemotherapy-induced leukopenia. We used a limiting dilution assay to investigate whether remaining CD4⁺ and CD8⁺ T lymphocytes derived from such leukopenic patients could be activated and undergo clonogenic proliferation. The activation signal in our model was accessory cells (irradiated normal peripheral blood mononuclear cells) + phytohaemagglutinin (PHA) + interleukin-2 (IL-2). During severe leukopenia a majority of circulating lymphocytes were CD4⁺ T cells. Clonogenic proliferating T lymphocytes were detected for all patients. Higher frequencies of clonogenic cells were detected in the CD8⁺ subset as compared to the CD4⁺ subset. However, for both subsets frequencies of proliferating cells were decreased compared with healthy individuals. The CD4⁺ and CD8⁺ lymphocytes were also capable of proliferation in response to alloactivation, and accessory cells mainly containing acute myelogenous leukaemia blast were efficient as accessory cells for activation. For the CD4⁺ cells, increased proliferation was detected in the presence of acute myelogenous leukaemia (AML) blasts compared with normal accessory cells. Based on our results we conclude that: (1) although acute leukaemia patients with therapy-induced leukopenia have both a quantitative and a qualitative T-cell defect, (2) the remaining T-cell population includes a subset capable of clonogenic proliferation. However, (3) proliferation of the clonogenic CD4⁺ cells can be modulated by AML blasts.     

Interleukin-2 and Serum Thymic Factor Enable Autologous Rosette-forming T Lymphocytes to Generate Helper and Cytotoxic Functions

The autologous rosette-forming T cells (Tar cells) isolated by means of their ability to form rosettes with autologous erythrocytes were characterized by the use of OKT monoclonal anti-human T-cell subset antibodies and a monoclonal anti-HLA-DR antibody. We found that the phenotype of Tar cells was OKT 3⁺4⁺8⁺Dr⁻ as determined by both indirect imnrunofluorescence microscopy and complement-mediated killing of ⁵¹Cr-labelled Tar cells. In addition, we found that Tar lymphocytes were able to develop cytotoxicity against allogeneic and trinitrophenol (TNP)-conjugated autologous target cells in the presence of interleukin-2 (IL-2) or serum thymic factor. However, these cells showed little or no cytotoxicity in the absence of interleukin-2 or serum thymic factor. Tar lymphocytes generated helper function for B lymphocytes in the presence of interleukin-2 in both pokeweed mitogen (PWM)- and purified protein derivative (PPD)-stimulated cultures. Nevertheless, non-IL-2-treated Tar cells did not exhibit any helper activity on B cells. Finally, pretreatment of Tar cells with 1000–1500 rad of X ray made these cells unable to develop helper function for B lymphocytes. It is concluded that: (1) OKT 3⁺4⁺8⁺Dr⁻ Tar cells are able to generate cytotoxicity against alloantigens and TNP-labelled self structures provided they are stimulated by IL-2 or serum thymic factor; (2) these cells need both to proliferate and to receive help from IL-2 to develop helper cells capable of assisting B-lymphocyte differentiation into plasma cells in both PWM- and PPD-stimulated cultures.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

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.     

Activation Signal Induces the Expression of B Cell-Specific CD45R Epitope (6B2) on Murine T Cells

Tlymphocytes express multiple forms of the leukocyte-common antigen CD45, transcribed by alternative usage of leukocyte-common antigen exon 4 6. The various isoforms of CD45R expressed differentially on T cells are involved in different stages of development and activation. The monoclonal antibody (MoAb) RA3-6B2 is established as a B cell-type isoform (B220)-specific marker. However, it reacts with certain activated T cells although the relationship between 6B2 expression and T-cell activation is unclear. We have examined the 6B2 expression on activated T cells and found that concanavalin A, anti-CD3 antibody and staphylococcal enterotoxin B (SEB) induced 6B2 expression on T cells. The expression was found on both CD4⁺ and CD8⁺ T cells and also was induced by SEB in vivo predominantly on CD8⁺ T cells. The 6B2⁺ T cells are IL-2R⁺ and blasted cells according to flow cytometry analysis. Therefore, the 6B2⁺ T cells are supposed to be in an activated stage. Enzymatic analysis demonstrated that trypsin treatment decreased the 6B2 expression, whereas neuraminidase increased the intensity on activated T cells. Neither endo-D or endo-H have any effect on the expression and there are no differences, in the results of immunoprecipitation and RT-PCR analysis, between control T cells and activated T celts. Taken together, the 6B2 epitope is presumed to be the product of CD45R modification and is expressed on activated T cells. These results illustrate a novel classification of a T-cell subpopuiation bearing a 6B2 epitope.     

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.     

Restricted and Individual Usage of T-cell Receptor β-Gene Variables in Nickel-Induced CD4+ and CD8+ Cells

Nickel allergy is manifested as contact allergic eczema elicited by delayed-type hypersensitivity, the reaction being mediated by T lymphocytes. We examined the T-cell receptor (TCR) β-chain variable gene segment (Vβ) use of nickel-induced CD4⁺ and CD8⁺ T cells in the peripheral blood of nickel-sensitive and non-sensitized subjects. The results show that each patient had an individual Vβ repertoire overexpressed, these being in CD4⁺ cells Vβ10 and Vβ13 (in subject A); Vβ1, Vβ2, Vβ13 and Vβ21 (subject B); Vβ1 and Vβ10 (subject C); Vβ9 and Vβ19 (subject D). Thus, no single Vβ gene dominated in a majority of the CD4⁺ samples. The Vβ genes overexpressed in patient CD8⁺ nickel-induced T cells were Vβ1 (in subject A), Vβ1 (subject B), Vβ1 and Vβ2 (subject C) and Vβ7 (subject D), domination of Vβ1 being seen in most of the CD8⁺ samples (75%). No specific overexpression of any Vβ genes in the nickel-allergic subjects was found in comparison with the non-sensitized subjects. In conclusion, an individual pattern of restricted Vβ genes was induced with nickel in CD4⁺ and CD8⁺ T cells in each nickel allergy patient.     

Leukaemic T Cells from Patients with Chronic Lymphocytic Leukaemia of T-Cell Origin Respond to Staphylococcus aureus Enterotoxin Superantigens

We investigated the in vitro responsiveness of peripheral blood lymphocytes from two patients with T-cell chronic lymphocytic leukaemia (T-CLL) to Staphylococcus aureus enterotoxin (SE) superantigens. T-cell receptor (TcR) αβ(Vβ 7.1)-expressing CD4⁺ leukaemic T cells from patient HE (white blood cell count 480,000/μl) proliferated in response to SEA and, only at 1000-fold higher concentrations, to SEB, SED, and SEE. CD4⁺ CD8⁺ TcRαβ (Vβ 12.1)-expressing leukaemic T cells from patient KO (white blood cell count 120,000/μl) were activated by SEB but not by the other tested SEs. In both instances, the activation of leukaemic T cells by SE was dependent on the presence of HLA-DR⁺ cells. Southern blot analysis of TcRβ gene rearrangement confirmed that the proliferating cells were derived from the leukaemic T-cell clone and not from contaminating normal T cells. These data indicate that leukaemic T cells from patients with T-CLL exert a clonally variable responsiveness to SE superantigens. We conclude that recognition of specific antigen and subsequent signal transduction can be initiated via the TcR of leukaemic T-CLL cells.     

Defective Calcium-Dependent Signal Transduction in T Lymphocytes of Ataxia-Telangiectasia

T-cell functions of two patients with ataxia-telangiectasia were investigated. Patients with ataxia-telangiectasia had reduced percentages of circulating CD3⁺ cells and CD4⁺ cells, although neither patient had a reduced percentage of circulating CD8⁺ cells. The proliferative responses and interleukin-2 production of peripheral blood mononuclear cells to T-cell mitogens were reduced in the patients. The intracellular calcium concentration in T cells or CD4⁺ cells from both patients was only slightly increased after phytohaemagglutinin stimulation. Moreover, the concentration after OKT3 stimulation was not or only slightly increased in T cells or CD4⁺ cells from both patients. Our results suggest that the functional defect of T cells is caused by defective Ca²⁺-dependent signal transduction through the CD3 complex of the surface in T cells of ataxia-telangiectasia.     

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.     

Immunophenotypic and Functional Characteristics of Haemopoietic Cells from Human Cord Blood

Cord blood (CB) as a new source for bone marrow transplantation represents advantageous features concerning stem cell and leucocyte compartments and function. We attempted to get more information about the phenotypes and function of CB cells by investigating their cell surface markers and also the production of IL-2, IFN-γ and IL-6 by mitogen and alloantigen stimulation. The CB cells were characterized by a low proportion of CD3⁺ T cells, CD4⁺ T subpopulation, activated T cells and CD3⁺ CD16/CD56⁺ cytotoxic cells, suggesting reduced graft versus host potential. The significant increase of CD19/CD3 double positive cells and decrease of CD19/HLA-DR double positive mature B cells reflect that immature B cells exist in CB. In the functional studies, a 27- and 5-fold reduction was observed in the production of IFN-γ by CB cells stimulated with PHA and allogeneic cells, respectively. The production of IL-2 in PHA-stimulated CB cells also showed a 50% determination. Decrease in the production of these cytokines by CB cells is supported by the decline of the proportion of CD3⁺ T cells. However, an increase was observed in the production of IL-6 by CB cells stimulated with allogeneic cells as compared with the controls. These results suggest a difference in the functional activity of the T helper cell subsets between the CB and peripheral blood and/or differences in the functional maturity of T helper cell subsets and B cells in these compartments.     

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.     

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.     

CD4+CD25+ regulatory cells in acquired MHC tolerance

Summary: Tolerance to self-antigens is an ongoing process that begins centrally during T-cell maturation in the thymus and continues throughout the cell's life in the periphery by a network of regulated restraints. Remaining self-reactive T-cells that escape intrathymic deletion may be silenced within the peripheral immune system by specialized regulatory CD4⁺ cells. By analogy, regulatory CD4⁺ cells that control immunity to "acquired self" should arise in circumstances where the immune system acquires tolerance to foreign MHC, such as the tolerance that develops following the exposure to foreign MHC antigens during the neonatal period. We have used this classic model of neonatal tolerance to examine the role of regulatory CD4⁺ cells in acquired tolerance to disparate class I and class II MHC. Adoptive transfer of unfractionated but not CD4⁺-depleted spleen cells from neonatal tolerant mice into SCID recipients inhibited skin graft rejection by immunocompetent CD8⁺ T cells. Using 5-bromo-2'-deoxyuridine incorporation, standard cytotoxic T-lymphocyte assays, short-term interferon-γ ELISPOT, and intracellular FACS analysis to study CD8⁺ T-cell effector function, we demonstrated that neonatal tolerant mice contain CD4⁺CD25⁺ cells that suppress the development of anti-donor CD8⁺ T-cell responses in vitro . We conclude that regulatory CD4⁺CD25⁺ cells initiate and/or maintain tolerance by preventing the development of CD8⁺ T-cell alloreactivity.