M de Boer

Prevention of Experimental Colitis in SCID Mice Reconstituted with CD45RBhigh CD4+ T Cells by Blocking the CD40-CD154 Interactions

Increased expression of CD40 and CD40 ligand (CD40L or CD154) has been found in inflamed mucosa of human inflammatory bowel disease (IBD), and interactions between these molecules seem to be involved in local cytokine production by macrophages. However, the precise role of CD40 signaling in the pathogenesis of IBD is still poorly understood. The aim of the present study was to investigate the in vivo relevance of CD40 signaling in experimental colitis in SCID mice reconstituted with syngeneic CD45RBhighCD4+ T cells. The results demonstrated that CD40+ and CD40L+ cells as well as their mRNA levels were significantly increased in inflamed mucosa. Administration of anti-CD40L neutralizing mAb over an 8-wk period starting immediately after CD45RBhighCD4+ T cell reconstitution completely prevented symptoms of wasting disease. Intestinal mucosal inflammation was effectively prevented, as revealed by abrogated leukocyte infiltration and decreased CD54 expression and strongly diminished mRNA levels of the proinflammatory cytokines IFN-γ, TNF, and IL-12. When colitic SCID mice were treated with anti-CD40L starting at 5 wk after T cell transfer up to 8 wk, this delayed treatment still led to significant clinical and histological improvement and down-regulated proinflammatory cytokine secretion. These data suggest that the CD40-CD40L interactions are essential for the Th1 inflammatory responses in the bowel in this experimental model of colitis. Blockade of CD40 signaling may be beneficial to human IBD.

Increased cytolytic T lymphocyte activity and decreased B7 responsiveness are associated with CD28 down-regulation on CD8+ T cells from HIV-infected subjects.

The CD28 receptor on CD4+ and CD8+ T cells interacts with B7 molecules on antigen‐presenting cells (APC) to generate essential costimulatory signals. The cytolytic potential of CD8+ T cells could be linked to CD28 expression. Since HIV induces dysfunction of both CD4+ and CD8+ T cells, we evaluated CD28 expression and function in both subsets during HIV infection. CD28 expression on CD8+ T cells from HIV+ subjects was strongly reduced in a disease stage‐related fashion. CD28‐CD8+ T cells preferentially expressed CD57 and CD11b, but lacked CD26 and IL‐2Rα. The CD8+ T cells from the patients showed a significantly reduced proliferative response to co‐stimulation with cell‐bound anti‐CD3 and B7. Nevertheless, when stimulated with plate‐fixed anti‐CD3, CD8+ T cells from HIV‐infected subjects proliferated normally, and normal levels of IL‐2Rα nod transferrin‐receptor could be induced on CD28‐CD8+ T cells from the patients. In addition, stimulation with plate‐fixed anti‐CD3 induced proliferative responses in highly purified CD28‐CD8+ T cells from both HIV‐ and HIV+ persons. Furthermore, the increased cytotoxic activity of peripheral blood mononuclear cells (PBMC) from HIV+ subjects, measured in an anti‐CD3 redirected assay, was predominantly exerted by CD28‐CD57+ T cells. CD4+ T cells from the patients showed a slight but significant CD28 down‐regulation and were slightly hyporesponsive to B7 co‐stimulation. Decrease of CD28 on CD8+ T cells from HIV+ subjects is associated with an impaired response to co‐stimulation via B7. CD28‐CD8+ T cells from seropositives, however, are not completely inert, since they contain in vivo activated CTL and they can be additionally activated through a B7‐independent stimulation.

Cytokine production by phytohemagglutinin-stimulated human blood cells: Effects of corticosteroids, T cell immunosuppressants and phosphodiesterase IV inhibitors

The ability of dexamethasone and prednisolone (corticosteroids), FK506 and cyclosporin A (T cell immunosuppressants), and of nitraquazone and rolipram (phosphodiesterase IV inhibitors) to inhibit cytokine production by stimulated human blood was investigated. Heparinized human blood obtained from normal healthy volunteers was stimulated with phytohemagglutinin (PHA) in the presence or absence of drug. After different incubation times, supernatant levels of interleukin (IL)-2, IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF) and interferonγ (IFN-γ) were quantified by ELISA. Dexamethasone strongly inhibited the production of IL-5 (IC50 = 0.004 µM), was less potent against IL-2 and IFN-γ (IC50 = 0.02–0.05 µM) and showed a relatively weak effect against GM-CSF (IC50 = 0.6 µM). Similarly prednisolone potently suppressed IL-5 generation (IC50=0.05 µM), displayed a more modest activity on IL-2 and IFN-γ (IC50 = 0.2–0.3 µM) and exerted only partial effects (43% inhibition at 1 µM) on GM-CSF. FK506 strongly suppressed the production of IL-2 (IC50 = 0.01 µM) and GM-CSF (IC50 = 0.03 µM), but was inactive (<30% inhibition at 1 µM) against IL-5 and IFN-γ. Similarly, cyclosporin A reduced the generation of IL-2 (IC50 = 0.4 µM) and GM-CSF (IC50 = 0.6 µM) while barely affecting the other two cytokines. Nitraquazone and rolipram were most active in reducing the production of IL-5 (IC50 = 0.8 and 1.3 µM, respectively), while their potency against IL-2, GM-CSF and IFN-γ was 3–6 times lower, with IC50s between 2.4 and 8.0 µM. These data indicate that corticosteroids, T cell immunosuppressants and phosphodiesterase IV inhibitors affect cytokine production by PHA-stimulated human blood cells in a differential and “pharmacotypical” manner.

Prolonged skin graft survival by administration of anti-CD80 monoclonal antibody with cyclosporin A

Costimulation via the B7/CD28 pathway is an important signal for the activation of T cells. Maximal inhibition of T-cell activation and the induction of alloantigen-specific nonresponsiveness in vitro was achieved using anti-CD80 monoclonal antibody (mAb) in combination with cyclosporin A (CsA). Based on this knowledge, the efficacy of the prophylactic treatment of anti-CD80 mAb and CsA on allogeneic skin graft survival was tested in a preclinical rhesus monkey model. No side effects have been observed. Administration of anti-CD80 mAb resulted in high mAb serum levels that decreased to undetectable values around day 7. At the same time, the anti-mouse antibody response started to develop. The anti-CD80 mAb bound to peripheral blood mononuclear cells and was detectable in lymph node and grafted skin during the treatment period. The skin graft survival time of untreated or suboptimally CsA-treated rhesus monkeys was 10 days. Treatment with CsA (blood levels of 100-160 ng/ml) in combination with anti-CD80 mAb (0.5 mg/kg) resulted in a significantly increased skin graft survival time to 14 days. Eventually, skin grafts in all rhesus monkeys were rejected, which coincided with an increase in helper and cytotoxic T-cell frequency and induction of an antibody response directed against the donor antigens. Therefore, treatment of anti-CD80 mAb in combination with CsA has significant immunosuppressive potency, but was unable to induce donor-specific nonresponsiveness in skin graft recipients.

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.