In vitro analysis of CD40-CD154 and CD28-CD80/86 interactions in the primary T-cell response to allogeneic "nonprofessional" antigen presenting cells.

BACKGROUND: Recently, several ligand interactions have been examined in detail as potential mediators of costimulatory signaling. The CD154/CD40 and CD28/B7 interactions have been highlighted as being among the more-significant contributors to proper activation of unprimed T lymphocytes. Human keratinocytes (HK) and human dermal fibroblasts (HF) are capable of expressing Class II HLA and CD40 antigens after interferon-gamma exposure, yet neither express significant levels of B7. HK and HF have been characterized as "nonprofessional" antigen presenting cells (APC) and their poor APC function has been partially attributed to deficient costimulatory activity. METHODS: In this study, we examined whether substituting for costimulatory signaling events through the addition of cross-linked monoclonal antibodies against the T-cell ligand/s (CD28 and/or CD154) could restore allostimulation. Mixed lymphocyte reactions were performed combining enriched human peripheral blood T cells and allogeneic HK or HF with or without stimulatory anti-CD28 and/or anti-CD154 antibodies. RESULTS: The results show that the addition of anti-CD28 alone permitted HF but not HK to present alloantigen effectively. In contrast, addition of both anti-CD154 and anti-CD28 was required to generate even a moderate proliferative response to allogeneic HK. Further, adding a monomorphic anti-HLA-DR antibody substantially inhibited these responses. Additional experiments suggest that signaling through CD40/CD154 directs HK to produce TGF-beta, which would adversely affect T-cell activation. CONCLUSIONS: The data presented highlight significant differences in signaling capacities for HK versus HF and provide evidence for a partial mechanism by which allogeneic human skin equivalents might be immunologically null upon engraftment.

Effects of immunoregulatory cytokines on the immunogenic potential of the cellular components of a bilayered living skin equivalent.

The purpose of this study was to determine if the immunocompatibility of an allogeneic living skin equivalent (LSE) would be affected by cytokines that would be potentially present at the wound site. Specifically, the ability of interleukin-1alpha (IL-1a), interleukin-6 (IL-6), or interleukin-12 (IL-12) to induce an allogeneic T cell response to "nonprofessional" antigen presenting cells (APC) was investigated in this series of experiments. Since cytokine concentrations at the wound site can vary greatly, recombinant IL-1a, IL-6, and IL-12 were used over a wide range of concentrations. These cytokines were either added directly to a mixed lymphocyte reaction (MLR) culture system or used to pretreat APC prior to use in the MLR culture. The addition of IL-12, IL-1alpha, or IL-6 into an MLR was examined as a possible means of providing the necessary costimulatory signal for functionally deficient APC, such as human keratinocytes (HK) and dermal fibroblasts (HF). While the results show that IL-1a and IL-12 can significantly augment a primary allogeneic response against appropriately equipped antigen presenting cells, the same was not true for HK or HF. Further experiments showed that pretreatment of HK, HF, or human umbilical vein endothelial cells (HUVEC) with Interferon-gamma (IFNgamma) and either IL-12, IL1alpha, or IL-6 had no significant affect on their ability to present alloantigen to immune-reactive T lymphocytes over IFNgamma-treatment alone. The data suggest that exposure of HK or HF to IL-1alpha, IL-6, or IL-12 in combination with IFNgamma does not provide the additional signal(s) required by these cells to effectively present alloantigen to unprimed T cells. The data suggests that exposure to these immunoregulatory cytokines in the wound bed would be unlikely to affect the immunocompatibility of the LSE.

Improving coronary angiograms.

Coronary angiography is an important procedure, and the best quality radiographs possible are essential to accurate diagnosis. Aside from the technical factors necessary to produce good images, the ability of the patient to breathe properly greatly influences the radiographic outcome. The breathing technique described here has been found effective, and teaching patients how to do it is an integral part of the technologist's role in the examination.