γδ T-Cell Precursor-Derived CD4− CD8−αβ T Cells Retain γδ Cell Function

We have previously shown that some of the DNαβ⁺ T cells arising in TcRα-chain transgenic mice are of γδ T cell origin, based on phenotypic data and on their status of TcR gene rearrangements. In the present report we investigated the impact of αβ TcR expression on the functional programme of the mature γδ precursor-derived DNαβ⁺ T cells. Our results demonstrate that both their proliferative capacity and their cytokine production profile are similar to that of γδ T cells. Furthermore, both transgenic DNαβ⁺ T cells and DNγδ⁺ T cells up-regulate CD8α expression after activation, but, in contrast to CD4⁺αβ T cells, are unable to induce proliferation of naive B cells. Thus, our results suggest that the effector functions of mature T cells are determined independently of the TcR isotype, probably at an early stage of differentiation, and thereby have important implications for current models of T-cell lineage commitment.     

Adhesion of Activated T Lymphocytes to Vascular Smooth Muscle Cells and Dermal Fibroblasts is Mediated by β1- and β2-Integrins

Several studies during recent years have demonstrated the potential for vascular smooth muscle cells (SMC) and dermal fibroblasts to participate in immune interactions such as antigen presentation and alloreactivity. The molecular interactions mediating lymphocyte adhesion to these mesenchymal cells have, however, not previously been characterized in detail. In the present study we demonstrate ICAM-1 (CD54) expression by cultured human SMC and its up-regulation by IL-1, IFN-gamma, and bacterial lipopolysaccharide. Monoclonal antibodies were used to define the molecular interactions in the adhesion of 51Cr-labelled T lymphoblasts to adherent SMC and fibroblasts. ICAM-1 appeared to mediate adhesion of T lymphocytes by binding to the beta 2-integrin CD11a/CD18 (LFA-1) expressed by the lymphoblasts. We present evidence for the involvement of at least three different mechanisms in the adhesion of activated T lymphocytes to cultured fibroblasts. It was found that beta 2-integrin-mediated interaction could only account for less than half of the binding activity. The remaining adhesion was partly mediated by beta 1-integrins, presumably via VLA-5 since an anti-VLA-5 antibody and an RGD-containing peptide blocked adhesion to the same degree. However, antibodies to beta 1-, beta 2-, and beta 3-integrin subunits added together only inhibited adhesion by approximately 50%. The residual adhesion could be blocked by inhibition of cell metabolism and was increased by stimulation of the lymphocytes with phorbol ester, suggesting involvement of other, as yet undefined, adhesion molecules. The molecular interactions between lymphocytes and mesenchymal cells demonstrated in this study may have implications in several inflammatory conditions such as vasculitis, atherosclerosis, and connective tissue diseases.     

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.     

Analysis and Characterization of the Interleukin 2 Receptor α and β Chain Expression inCD4−CD8− Cells

The differential effects that the binding of interleukin 2 (IL-2) to its beta or alpha beta receptors might induce in two different CD4-CD8- T-cell lines were analysed. While LD1.T3b, a double-negative T cell derived from MRL/lpr mice, constitutively expressed high levels of the IL-2R beta chain, YAC-1, a Moloney sarcoma virus-transformed CD4-CD8- T cell, expressed (as an activated T cell) the beta and alpha chains. The presence of IL-2 in the culture medium was lethal for LD1.T3b cells, while it had no effect on the growth of YAC-1 cells. IL-2 increased the expression of the beta chain and, to a lesser extent, of the alpha chain in YAC-1 cells. In addition, other markers such as CD4 and CD5 were induced by IL-2 in this cell line.     

Activated CD3−CD16+ Natural Killer Cells Express a Subset of the Lymphokine Genes Induced in Activated αβ+ and γω+ T cells

In this study we analysed the potential of highly purified polyclonal TcR alpha beta+, TcR gamma delta + and CD3- NK cells, to produce lymphokines in response to mitogenic stimulation. RNA hybridizations were performed to detect with high sensitivity the induction of multiple lymphokine genes. Upon stimulation with lectin and phorbol ester TcR gamma delta + lymphocytes expressed the same set of lymphokine genes as the TcR alpha beta + lymphocytes expressed the same set of lymphokine genes as the TcR alpha beta + lymphocytes, which included IL-2, -3, -4, -5, GM-CSF, TNF alpha and beta, IFN gamma. In contrast, a more limited set of lymphokine genes (GM-CSF, TNF alpha and beta, IFN gamma) was induced in activated CD3- NK cells, thus indicating that this subpopulation of cells may display different regulatory functions, with respect to CD3+ T lymphocytes.     

Vaccination with β2-Microglobulin-Deficient Dendritic Cells Protects Against Growth of β2-Microglobulin-Deficient Tumours

Defects in cell surface expression of major histocompatibility complex class I antigen molecules are common in tumour cells. We have previously described the generation of adaptive immunity to tumour cells deficient in the transporter associated with antigen processing molecule. In this study, we demonstrate enhanced in vivo protection against growth of β₂-microglobulin-deficient tumour cells in syngeneic C57Bl/6 mice, following vaccination with β₂-microglobulin-deficient dendritic cells. In vitro analysis suggested that vaccinated mice produced CD3⁺ cells, which could induce apoptosis in syngeneic β₂-microglobulin-deficient tumour and non-malignant cells. Further investigation of target cell recognition suggested that also tumour cells lacking expression of classical major histocompatibility complex class I heavy chains and functional transporter associated with antigen processing molecules were recognized by CD3⁺ effector cells from vaccinated mice. Histopathological examination of organs from vaccinated mice showed no significant vaccination-induced pathology. The present findings point to a new possible strategy to counteract the growth of major histocompatibility complex class I-deficient tumour cells.     

Resistance and Susceptibility to Cyclosporin A as CD3− 4− 8− Human Thymocytes Differentiate In Vitro

Human T-cell development appears to be relatively resistant to cyclosporin A (CsA). Children exposed to CsA mw/eroaspart of kidney transplant maintenance have few abnormalities. The objective of the study described here was to analyse the effects of CsA on the development in vitro of human multinegative (MN) (CD3-4-8-) thymocytes as a model system for thymic progenitor development in vitro. MN thymocytes, prepared by depletion methods, differentiated in vitro to acquire CD3 and undergo transitions in CD45 isoform expression analogous to those postulated to occur in vivo. In this work MN thymocytes were cultured with IL-2 and on thymic epithelial cells (TEC) with or without lL-2, either in ihe presence or absence of CsA. For many thymocyte preparations, differentiation in the presence of CsA resulted in almost complete inhibition of the acquisition of CD3and ofthe low Mr isofomi CD45R0. Expression of CD45RA and of total CD45 were reduced but not eliminated and the density of CD29 was unaffected. For others, neither CD3 nor CD45 expression was affected., but selective inhibition of TCR5 expression occurred. At all doses of CsA (0.1–100 μg/ml), MN thymocytes continued to cycle indicating a CsA-resistant generative compartment. Treatment of peripheral blood T cells with CsA had no effect on surface expression of CD3 or CD45 isoforms but did reduce the amount of de novo-synthesized CD45R0 mRNA. Culture of MN thymocytes on TEC rendered them virtually resistant to the negative effects of CsA. CD3 acquisition was unhindered and total CD45 remained high, but the transition from CD45RA to CD45R0 appeared to be delayed. In the absence of TEC, expression of both TCRδ and of TCRδ was inhibited, but on TEC, TCRδ was actually up-regulated in some conditions. The effects of CsA on human thymocyte development appeared to be modulated by the physiological state of the donor and the growth conditions to which the ceils were subjected. Conditions which most closely approximated those manifest in vivo rendered thymocytes most resistant to the negative effects of CsA. The amount of CsA required to affect differentiation in vitro was significantly higher than could be attained in vivo suggesting that the immunomodulatory effects of CsA in the maintenance of organ transplants may from an as yet uncharacterized mechanism     

Serial Measurements of Pregnancy-specific β1-glycoprotein (β1SP1) in Patients with Trophoblastic Disease

A radioimmunoassay for pregnancy-specific-β₁-glycoprotein (Bohn. 1971) has been established with a limit of detection of 2 μMg/litre in serum. The assay has been used to measure serial levels of pregnancy-specific-β₁-glycoprotein (β₁SP₁) in the serum of patients with choriocarcinoma and teratoma for comparison with measurements of the β-subunit of human chorionic gonadotrophin. The value of the assay for β₁SP₁, in the management of these patients is discussed.     

Increased expression of collagen receptors: α1β1 and α2β1 integrins on blood eosinophils in bronchial asthma

BACKGROUND: Eosinophils are one of the major effector cells in bronchial asthma. Their infiltration of airways correlates with the asthma severity. Recruitment and activation of eosinophils are partially mediated by integrins alpha4beta1 and alpha4beta7. Collagens type I and IV constitute important components of extracellular matrix and vascular basement membrane, respectively. Therefore, collagen-binding integrins (alpha1beta1 and alpha2beta1) may also play a role in eosinophil lung infiltration. OBJECTIVE: To evaluate the possible presence of alpha1beta1 and alpha2beta1 integrins on peripheral blood eosinophils from asthmatic subjects. METHODS: Collagen receptors were studied on eosinophils separated by immunomagnetic CD16-negative method from healthy donors (n=13) and patients with moderate persistent atopic bronchial asthma (n=15). Surface receptor identification was performed by flow cytometry and cell adhesion assay. RESULTS: Eosinophils isolated from the patients showed increased expression of both alpha1beta1 and alpha2beta1 integrins as compared with healthy controls. Moreover, adhesive function of eosinophils to collagen type IV was inhibited by snake venom disintegrins: viperistatin and obtustatin. These disintegrins contain KTS active motif and are specific inhibitors of alpha1beta1 integrin. CONCLUSION: We demonstrated for the first time that collagen receptors: alpha1beta1 and alpha2beta1 integrins are overexpressed on the surface of peripheral blood eosinophils of asthmatic subjects. Further studies may reveal potential application of KTS-disintegrins or their structural analogs for therapy of bronchial asthma.     

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.     

CD4+, CD8+, and CD4− CD8− T Cells in CSF and Blood of Patients with Multiple Sclerosis and Tension Headache

Two-colour flow cytometric analysis was performed on paired samples of peripheral blood (PB) and cerebrospinal fluid (CSF) of patients with untreated multiple sclerosis (MS) and, for reference, subjects with muscular tension headache (TH) using anti-CD3, anti-CD4, anti-CD8, and anti-HLA-DR monoclonal antibodies in different combinations. CD4+/CD8+ T-cell ratio was increased in CSF compared to PB in both MS patients and TH subjects to a similar extent. This was mainly due to higher CD4+ T-cell levels in the CSF compartment. The proportion of HLA-DR+ T cells was higher in CSF than PB in both MS and TH; this increase of DR+ T cells in CSF was more prominent in MS. The level of CD4+ CD8+ T cells, which represent a subset of activated T cells, was not different between CSF and PB, either in MS or in TH. The proportion of CD4- CD8- T cells, which were found generally not to be blast cells, was lower in CSF compared to PB in both patient groups. However, their CSF level was higher and their PB level lower in MS compared to TH. Results point to an accumulation of activated T-helper cells in the CSF of both MS patients and healthy subjects. Fetal-type CD4- CD8- T cells bearing the unusual T-cell receptor gamma/delta seem to be selectively recruited to the CSF of MS patients.     

Pregnancy Specific β1-glycoprotein in Threatened Abortion

The aim of this study was to elucidate the prognostic value of the pregnancy specific β₁-glycoprotein (SP-I == PS/βG) of maternal serum in threatened abortion.
Determinations were made by rocket-immunoelectrophoresis with a detection limit of I mg/1.
59 patients with vaginal bleeding and positive pregnancy tests entered the final material. 33 gave birth after the 28th week, 26 had a spontaneous abortion.
Serum samples were taken until birth or spontaneous abortion.
After the 9th gestational week a separation of the serum concentrations was observed between the patients who had a spontaneous abortion and those who gave birth. It is concluded that the SP-1 determination might be of great prognostic value in threatened abortion.     

Auto- and Polyreactivity of IgM from CD5+ and CD5− Cord Blood B Cells

The presence of the CD5 (67 kDa) molecule on the surface of B cells has been considered a marker for cells producing auto- and polyreactive antibodies. Cord blood B lymphocytes (rich in CD5+ B cells) have been sorted into CD5 positive and negative populations by flow cytometry using monoclonal antibodies to CD20 and CD5. Clones of these populations were obtained by immortalization with Epstein-Barr virus. Clones derived from both CD5+ and CD5- B cells produced IgM which was auto- and polyreactive with a higher frequency of these specificities in the CD5+ population. These data indicate that expression of surface CD5 on cord blood B cells is not a definitive marker of an auto/polyreactive population.     

CD4− CD8− C.B-17 SCID Thymocytes Enter the CD4+ CD8+ Stage in the Presence of Neonatally Grafted T Cells

The aim of this work was to study the selection of donor T cells and their influence on thymic development in C.B-17 scid/scid (severe combined immunodeficient; SCID) mice during chronic graft-versus-host disease (GVHD). Recipient SCID mice (H-2^d), neonatally grafted with allogeneic peripheral T cells from CBA/J strain (H-2^k) of mice, only developed a mild acute GVHD, and were, at the chronic stage, devoid of pathological symptoms. Thymic cell numbers of injected mice differed from 10⁵ to 1.2 × 10⁷ at 2–3 weeks post-injection (p.i.), and from 4 × 10⁵ to 8.5 × 10⁷ at 2 months p.i. In these mice, the thymus size was correlated to the CD4^? CD8^? (double negative; DN) to CD4⁺ CD8⁺ (double positive; DP) cell ratio, where at 2 months p.i, 8 out of 16 treated SCID mice contained 5 × 10⁶ cells or more and also possessed the highest frequencies of endogenous DP cells (25–95%). In contrast to previous findings, peripheral donor T cells from allogeneic and syngeneic mice, infiltrating the host thymus, had a positive effect on the development of endogenous DP thymocytes. Furthermore, these thymocytes were developmentally blocked at the DP stage, occasionally in combination with the expression of CD25, CD44 and CD117 but in the absence of T-cell receptor (TCR) expression. Also, at this time-point, the CBA/J donor TCR Vβ repertoire was equal to that of normal CBA/J mice, but purified responding donor cells were proliferatively inhibited against H-2^d stimulators in ex vivo mixed lymphocyte cultures. In contrast, the same responders showed a pronounced proliferation against syngeneic H-2K^k stimulators, suggesting either a reversion from anergy of autoreactive CBA/J T cells or a vast expansion of multiple self-reactive T-cell clones, when parked in a milieu with a lower concentration of self-antigens.