Relationships among differentiated T-cell subpopulations. IV. Preferential induction of delayed footpad reaction to xenografts and its radioresistant nature.

Immune response in mice to xenogeneic cells was characterized by positive delayed footpad reaction and negative macrophage migration inhibition. Mice immunized with allogeneic cells exhibited negative delayed footpad reaction and positive macrophage migration inhibition. Cytotoxic T lymphocytes were detected only in mice immunized with allogeneic tumour cells. Delayed footpad reaction against xenogeneic cells was radioresistant and the immune lymphocytes responsible for such a reaction were presumed to have some relation to xenograft rejection.     

Relationships among differentiated T-cell subpopulations. III. Radioresistance of delayed hypersensitivity to heterologous erythrocytes.

Effects of whole body irradiation with 600 rad on delayed hypersensitivity, direct cytotoxicity and antibody production were examined in mice immunized with chicken erythrocytes (CRBC) or sheep erythrocytes (SRBC) in saline. Delayed footpad reactions to CRBC were not affected by the exposure to irradiation 3 h before or after antigenic stimulation. On the other hand, direct cytotoxic activities in spleen cells and antibody titres to CRBC were reduced by such exposures. Additionally, delayed footpad reactions to SRBC were not affected by the exposure to irradiation. Generation of effector cells responsible for delayed footpad reactions proved to be resistant to irradiation.     

Relationships among differentiated T-cell subpopulations: II. Cytotoxicity and other functions of T cells specific for nucleated chicken erythrocytes

Relationships among T-cell mediated cytotoxicity, tuberculin type hypersensitivity, Jones-Mote type hypersensitivity and activation of helper T cells were studied in AKR mice by means of target cell destruction (⁵¹Cr-release), footpad reaction, migration inhibition test and antibody production against the trinitrophenyl group. (1) Immunization with chicken red blood cells (CRBC) in saline, Freund's incomplete (FIA) or complete adjuvant (FCA) and fixed-CRBC (FRBC) in FIA or FCA induced delayed hypersensitivity as demonstrated by footpad swelling. (2) Migration inhibition was positive in the group immunized with CRBC in saline or FCA, or FRBC in FCA, but negative in those immunized with CRBC or FRBC in FIA. This may suggest that the former has to be assigned to tuberculin type and the latter to Jones-Mote type. (3) T-cell mediated cytotoxicity by immune spleen cells was detected only in mice immunized with CRBC in saline. (4) Pre-treatment with cyclophosphamide augmented delayed footpad reaction in mice immunized with CRBC in saline, but suppressed cytotoxic activity. (5) FRBC in saline scarcely induced delayed footpad reaction and cytotoxic activity, whereas they activated helper function efficiently. Thus, four types of immunological phenomena, attributable to the functions of T cells, may depend upon distinct subpopulations of differentiated T cells which are raised by different methods of immunization.     

Relationships among differentiated T-cell subpopulations. I. Dissociated development of tuberculin type hypersensitivity, Jones-Mote type hypersensitivity and activation of helper function.

Relationships among tuberculin type hypersensitivity, Jones-Mote type hypersensitivity and activation of helper T cells were studied in AKR mice by means of footpad reaction, migration inhibition test and antibody production against the trinitrophenyl group. (1) Immunization with SRBC in saline, Freund's incomplete adjuvant (FIA) or complete adjuvant (FCA) and fixed-SRBC (FRBC) in FIA- or FCA-induced delayed hypersensitivity as demonstrated by footpad swelling. (2) Migration inhibition was positive in the groups immunized with SRBC or FRBC in FCA, but negative in those immunized with SRBC in saline or FIA or FRBC in FIA. This may suggest that the former has to be assigned to tuberculin type and the latter to Jones-Mote type. (3) Both pre-treatment with BCG and with cyclophosphamide (CY) augmented delayed footpad reaction in the mice immunized with SRBC in saline. However, migration inhibition was positive only in the group pre-treated with BCG. BCG may convert the reaction from Jones-Mote type to tuberculin type, while CY may augment the reaction of Jones-Mote type. (4) FRBC in saline scarcely induced delayed footpad reaction, whereas they activated helper function efficiently. Thus, three types of immunological phenomena attributable to the functions of T cells may depend upon distinct subpopulations of differentiated T cells which are raised by different methods of immunization.     

Mechanisms of tissue injury in hyperacute xenograft rejection.

Vascularized organs transplanted between phylogenetically distant species are subject to hyperacute rejection. The pathologic features of hyperacute rejection appear to be caused by profound dysfunction of small blood vessels in the graft. The author describes a model for the initiation and pathogenesis of hyperacute rejection. The model focuses on the endothelial cell as a target of the recipient immune reaction. Rejection is initiated by the binding to donor endothelial cells of recipient xenoreactive natural antibodies, which, in turn, triggers the complement cascade. These events lead to activation of graft endothelial cells, which promotes the thrombus formation and loss of vascular integrity characteristic of hyperacute rejection.     

Herpes simplex virus infection of human T-cell subpopulations.

The ability of herpes simplex virus type 1 to productively infect human T-cell subpopulations was examined. Unstimulated helper/inducer (T4+) and cytotoxic/suppressor (T8+) lymphocytes limited herpes simplex virus replication as effectively as unseparated peripheral blood T cells (T3+). Phytohemagglutinin stimulation before infection resulted in equivalently productive herpes simplex virus infections in the three cell fractions.     

Inhibition of skin xenograft rejection by depleting T-cell receptor alpha beta-bearing cells without T-cell receptor gamma delta-bearing cells or natural killer cells by monoclonal antibody.

We compared the effects of in vivo administration of the anti-T-cell receptor (TCR) alpha beta monoclonal antibody (mAb) (H57-597) to those of the anti-CD3 mAb (145-2C11), with or without anti-NK1.1 mAb (PK136), on xenogeneic skin graft survival in mice. In anti-TCR alpha beta mAb-treated B6 mice, F344 rat skin grafts survived for about 54 days, whereas in anti-CD3 mAb-treated B6 mice with or without anti-NK1.1 mAb treatment grafts survived about 25 days. In anti-TCR alpha beta mAb-treated B6 mice, TCR alpha beta-bearing T-lymphocyte function was completely abrogated, although TCR gamma delta-bearing T-lymphocyte function was still intact on day 9. In the anti-CD3 mAb-treated mice, the functions of both types of T lymphocytes were completely abrogated. On day 32, when most of the skin xenografts had been rejected in the anti-CD3 mAb-treated mice, the functions of both T lymphocytes had recovered considerably, and could actually respond to F344 antigens. In contrast, the function of TCR alpha beta-bearing cells had only partially recovered in the anti-TCR alpha beta mAb-treated mice. Finally, natural killer (NK) activity in the anti-TCR alpha beta mAb-treated mice was intact on day 32, when rat skin grafts still survived. In contrast, NK activity in the anti-CD3 mAb plus anti-NK1.1 mAb-treated mice did not recover on day 32, when skin xenografts had already been rejected. These results suggest that TCR gamma delta-bearing T cells and NK cells by themselves, at least in the absence of TCR alpha beta-bearing T cells, do not mediate xenogeneic skin graft rejection in mouse/rat combinations.     

Serotonin regulation of T-cell subpopulations and of macrophage accessory function.

The role of serotonin as an immune modulator was investigated by measuring the functional competence of T cells from control mice versus from mice whose intracellular stores of serotonin had been depleted by pretreatment with p-chlorophenylalanine (PCPA). While the proportions of splenic CD4+ and CD8+ T cells isolated from control and PCPA-treated mice were similar, the level of expression of the alpha-chain interleukin-2 receptor (IL-2R) was reduced on splenic CD4+ cells but not on CD8+ cells. Culture with the T-cell mitogen concanavalin A (Con A) failed to induce expression of the IL-2R on either CD4+ or CD8+ cells of PCPA-treated mice, although IL-2R was induced on control cells. The proliferative response to Con A by these spleen cells from PCPA-treated mice was also reduced compared to that by control spleen cells. Both expression of IL-2R and proliferation in response to Con A by spleen cells from serotonin-depleted mice were increased or completely restored by supplementation of the cultures with serotonin. Studies to identify the mechanisms for the reduction in T-cell activation when serotonin levels were reduced implicated a defect in the capacity of macrophages from PCPA-treated mice to provide accessory help for T-cell activation. Splenic macrophages from control mice were able to restore the blastogenic capability of lymphocytes from PCPA-treated mice, although macrophages from PCPA-treated mice were unable to support normal lymphocyte blastogenesis unless the cultures were supplemented with serotonin. These results show the requirement of autologous serotonin for optimal T-cell activation and suggest the importance of serotonin in macrophage accessory function for T-cell activation.     

Prevention of pancreatic islet xenograft rejection by dietary vitamin E.

In pancreatic islet transplantation, the adhesion of activated leukocytes to endothelial cells and the loss of microvascular integrity represent the critical microcirculatory events, which promote loss of graft function due to rejection. With the view that oxygen radicals may contribute to graft rejection, we studied the effect of the antioxidant vitamin E on microvascular rejection of islet grafts. Islets were transplanted syngeneically and xenogeneically (rat) into dorsal skin-fold chambers of hamsters, which received a non-vitamin-E-supplemented laboratory chow. Treated animals with xenografts were fed with a diet supplemented with vitamin E in a low (150 mg/kg) and high (8000 mg/kg) concentration. Intravital fluorescence microscopy demonstrated complete vascularization of syngeneic grafts at day 10 after transplantation, intact islet microcirculation at day 20 with a functional capillary density of 653 +/- 6 cm-1, and only few leukocytes adherent to the endothelial lining of the islets' microvasculature (88 +/- 23 mm-2). Xenogeneic islets showed initial signs of rejection at day 6, including adhesion of leukocytes to the microvascular endothelium (610 +/- 110 mm-2) and loss of endothelial integrity. After 20 days, functional capillary density was significantly lower (173 +/- 68 cm-1) when compared with syngeneic grafts, indicating failure of graft acceptance. Supplementation of the diet with low and high concentrations of vitamin E resulted in a significant (P < 0.05) reduction of xenograft leukocyte-endothelium interaction (146 +/- 29 mm-2 and 109 +/- 42 mm-2) at day 6 after transplantation and and adequate development of functional capillary density at day 20 (478 +/- 36 cm-1 and 539 +/- 86 cm-1; P < 0.05), indicating prevention of microvascular rejection. We conclude that dietary supplementation of the lipophilic antioxidant vitamin E attenuates leukocyte-endothelial cell interactions, preserves microvascular integrity, and thus inhibits microvascular rejection in a dose-dependent fashion. Our study underscores the pivotal mediator role of reactive oxygen species in islet xenograft rejection and, furthermore, suggests that dietary vitamin E may act as an adjunct anti-rejection treatment in clinical islet transplantation.     

Processed MHC class I alloantigen as the stimulus for CD4+ T-cell dependent antibody-mediated graft rejection.

The traditional view of graft rejection is one of direct recognition of allogeneic MHC molecules by effector T cells, the phenotype of which may be predicted by the nature of the MHC disparity. In this article, Andrew Bradley and colleagues discuss recent evidence that suggests this view may be an oversimplification, and argue that additional effector mechanisms, such as alloantibody, need to be reconsidered.     

T-cell colony formation from murine thymocyte subpopulations and pre-T cells.

Murine thymocytes are able to proliferate on clonal basis as colonies in the semisolid medium methylcellulose. It has previously been shown that the colony-forming cells are predominantly rather mature thymocytes and presumably exclusively single positive CD8+ cells. The results of the present study point to that also immature CD4-CD8- (DN = double negative) thymocytes and pre-T cells from nude mice are able to form colonies in methylcellulose culture. The numbers of colonies are increased by the presence of phorbolester, and it is shown by the addition of various interleukins to the culture that IL-2 is the essential interleukin in colony formation. It is further shown that all colony cells derived from DN thymocytes are of the CD4-CD8+ phenotype, while nude mouse precursors give rise to either CD8+ or DN colony cells. Thus, no CD4+ T cells were able to proliferate as colonies by the present culture method, even after exposure to a number of different interleukins or phorbolesters. This makes the method a reliable tool for clonal growth of both immature and mature CD8+ thymocytes and T cells.     

T-cell subpopulations in tuberculosis and the effects of rifampicin

T-cell subpopulations were evaluated by several recent methods in 38 tuberculosis patients (24 active and 10 quiescent cases of pulmonary tuberculosis; two of miliary and two of active extra-pulmonary tuberculosis) before and during rifampicin (RMP) treatment. There was a significant reduction in the total number of T cells (E-RFC and OKT3+ cells) and of helper T cells (OKT4+) coinciding with an increase in the number of suppressor T cells when the 38 tuberculosis patients were compared with 21 healthy control subjects. When the changes of T-cell subpopulations in groups of subjects and patients with different clinical forms of the disease were analysed, these changes could be clearly shown with both sets of assays (receptor assays and monoclonal antibody assays) among those with the active pulmonary form of tuberculosis while similar changes could be demonstrated only by one or the other assay among those with the other forms of the disease. The effects of one month of RMP treatment on these parameters were much more obvious among the clinically active patients than the quiescent patients, i.e. a recovery of total T cells from a low pre-treatment to a near normal level accompanied by a significant reduction in the number of suppressor cells (OKT8+). In fact, among quiescent patients the number of suppressor cells (as TG) appeared to rise further with RMP treatment.     

T-cell receptor expression in intestinal intra-epithelial lymphocyte subpopulations of normal and athymic mice.

Intra-epithelial lymphocytes (IEL) in murine small intestine were analysed for the presence of cell-surface antigens and T-cell receptor allotype in normal and athymic BALB/c mice by immunoperoxidase histochemistry on frozen sections and immunofluorescence on isolated IEL. In frozen sections, IEL of normal mice were 97.7% CD45+, 93.5% CD3+, 46.2% Thy-1+, 91.1% CD8+, 10.7% CD4+ and 21.1% KJ16+ (V beta 8.1 and 8.2). FACS analysis of isolated IEL confirmed the level of KJ16 expression and also demonstrated that 25% of IEL were F23.1+ (V beta 8.1-8.3). Immunofluorescent double-staining revealed a skewed distribution of T-cell receptor (TcR) expression on Thy-1+ and Thy-1- IEL. KJ16 and F23.1 were expressed on 25.9% and 32.7% of Thy-1+ IEL, respectively; however, the frequency of V beta 8 expression was diminished on Thy-1- IEL (4.1% KJ16+ and 12.1% F23.1+). IEL are present in athymic mice, but at reduced levels. In frozen sections these cells were 91.9% CD45+, 69.5% CD3+, less than 1% Thy-1+, 83.6% CD8+, less than 1% CD4+ and less than 1% KJ16+. Thus it appears that in normal mice there may be two distinct lineages of IEL, a thymus-dependent Thy-1+ population which utilizes the alpha beta T-cell receptor and a thymus-independent Thy-1- population (represented in athymic mice), which may possibly utilize the alternative gamma delta TcR.     

T-cell subpopulations in the development of atopic and contact allergy

Remarkable progress has been made in our understanding of the pathogenesis of skin diseases mediated by T cells. T-cell subsets responsible for the expression and regulation of allergic contact dermatitis to small chemicals or 'haptens' have been defined further, and the dynamics of T cells involved in the pathogenesis of atopic dermatitis have been clarified. In addition, studies are beginning to reveal the important contribution of skin resident cells to atopic dermatitis and the underlying molecular mechanisms.     

Dynamic changes in circulating and antigen-responsive T-cell subpopulations post-Mycobacterium bovis infection in cattle.

Bovine tuberculosis is a threat to animal and human health in several countries. Greater understanding of the immunology of the disease is required to develop improved tests and vaccines. This study has used a model of bovine tuberculosis, established in the natural host, to investigate the dynamic changes that occur in the circulating T-cell subpopulations after infection. When the phenotypic composition of the peripheral blood lymphocytes was determined pre- and post-experimental infection, the response to disease comprised three phases. Firstly, the WC1/gamma delta T cells decreased and then increased, suggesting localization to developing lesions and clonal expansion. Secondly, the CD4:CD8 ratio increased. Thirdly, the CD4:CD8 ratio decreased to less than pre-infection measurements. The latter changes suggested sequential involvement of CD4 and then CD8 T cells. The proportion of cells expressing interleukin-2 receptor (IL-2R) also increased. Panels of T-cell clones were established at various stages post-infection and all clones that exhibited antigen responsiveness were phenotyped. T-cell clones from early infection were WC1/gamma delta and CD4 in phenotype, while CD8 clones appeared later in infection, eventually becoming dominant. Therefore, from in vivo and in vitro evidence, it was suggested that there is a dynamic progression in the T-cell subpopulations involved dominantly in responses to mycobacteria.     

Additive effects of leflunomide and tacrolimus in prevention of islet xenograft rejection

Leflunomide is a low molecular weight immunosuppressive drug which inhibits the enzymes dehydroorotate dehydrogenase and protein tyrosine kinase, both of which are important components in the immune response. As the mechanisms of action of leflunomide and tacrolimus are different, we postulated an additive or synergistic effect of the two drugs and investigated the effects of leflunomide alone, or in combination with a suboptimal dose of tacrolimus, on xenogeneic islet transplantation in a rat-to-mouse model. A total of 1200-1500 rat islets were transplanted under the left kidney capsule of streptozotocin-induced diabetic BALB/c mice. The median survival time (MST) of the untreated group was 6 days. Leflunomide at 5, 10 and 20 mg/kg/d administrated for 10 days significantly prolonged MST to 10, 16 and 20 days. A dose of tacrolimus (2 mg/kg/d) was associated with a graft survival of 9 (range 6-12) days; most grafts rejected during ongoing therapy. When tacrolimus (2 mg/kg/d) was combined with leflunomide (10 mg/kg/d), the survival time of the islet xenografts was increased further to 22 days, significantly longer than with leflunomide or tacrolimus alone. In summary, our findings demonstrate that leflunomide prolonged xenogeneic islet graft survival, and that its immunosuppressive effect was improved when combined with tacrolimus.