J.W. Streilein

Evidence for multiple CD95-CD95 ligand interactions in anteriorchamber-associated immune deviation induced by soluble protein antigen.

We have investigated whether CD95–CD95 ligand interactions are important in anterior chamber‐associated immune deviation (ACAID) induced by soluble protein antigen, and if so, to identify the participating cells on which these molecules are expressed. Peritoneal exudate cells as antigen‐presenting cells (APC) obtained from B6.lpr/lpr, B6.gld/gld and C57BL/6 mice were cultured with ovalbumin (OVA) and transforming growth factor‐β2 (TGF‐β2) overnight, then injected intravenously into C57BL/6 or B6.lpr/lpr recipients. Some B6.lpr/lpr mice were reconstituted with naive T cells from wild‐type C57BL/6 donors. In other experiments, B6.lpr/lpr and B6.gld/gld mice received an anterior chamber injection of OVA followed 7 days later by subcutaneous immunization with OVA plus adjuvant. Delayed hypersensitivity (DH) was assessed with an ear swelling assay. T cells activated in vitro with OVA‐pulsed, TGF‐β‐treated APC were tested in vivo for their capacity to suppress DH expression in a local adoptive transfer assay. The results indicate that when ACAID was induced by in‐vitro generated ACAID‐inducing cells, the APC expressed CD95L, and recipient T cells expressed CD95. The capacity of in vitro generated regulatory T cells to suppress DH expression to OVA in vivo was not governed by CD95–CD95L interactions. When OVA was injected into the anterior chamber of naive mice, CD95 expression was required for ACAID induction, although ACAID was readily induced in CD95L‐deficient mice. We conclude that CD95–CD95L interactions are required in ACAID for the initial stage of APC presentation of eye‐derived antigens to T cells, and that CD95–CD95L interactions participate at one or more additional step in the process by which ACAID is induced by soluble protein antigens.

Studies on delayed systemic effects of ultraviolet B radiation on the induction of contact hypersensitivity, 3. Dendritic cells from secondary lymphoid organs are deficient in interleukin-12 production and capacity to promote activation and differentiation of T helper type 1 cells.

Ultraviolet‐B radiation (UVR) of mouse skin promotes both local and systemic immune aberrations that are thought to be important in the pathogenesis of cutaneous malignancies. Acute, low‐dose UVR regimens inhibit the induction of contact hypersensitivity (CH) in genetically susceptible mice by TNF‐α‐dependent mechanisms. In addition, these regimens also promote the development of tolerance when hapten is applied to the UVR‐exposed site at the completion of the radiation treatment protocol. A third immune abnormality is also observed in mice exposed to acute, low‐dose UVR. This abnormality, which develops within 48–72 hr of the completion of the UVR regimen, has been described among antigen‐presenting cells within secondary lymphoid organs, including lymph nodes that do not drain the site of irradiation. Dendritic cells (DCs) from lymph nodes and spleens of mice exposed to UVR lack the capacity to induce CH if they are derivatized with hapten and injected intracutaneously into naive mice. The DC defect is related to the production of and systemic dissemination of interleukin‐10 (IL‐10) by keratinocytes within the epidermis of the UVR‐exposed skin. We have now examined the nature of the functional aberration that exists among DCs within the secondary lymphoid organs of UVR‐exposed mice by examining the capacity of DCs to express co‐stimulatory molecules, and their ability to activate ovalbumin (OVA) ‐specific DO11.10 T‐cell receptor transgenic T cells in vitro. Our results indicate that DCs from UVR‐exposed mice produced insufficient amounts of IL‐12. When pulsed with OVA, these cells were capable of inducing proliferation among DO11.10 T cells in vitro, but the responding cells produced neither IFN‐γ nor IL‐10 and IL‐4. A similar antigen‐presenting cell defect was generated in mice treated with a subcutaneous injection of IL‐10. We conclude that acute, low‐dose UVR creates an IL‐10‐dependent functional deficit in DCs in secondary lymphoid organs, and that this defect robs UVR‐exposed mice of the capacity to develop CH when hapten is painted epicutaneously.

Studies of delayed systemic effects of ultraviolet B radiation (UVR) on the induction of contact hypersensitivity, 2. Evidence that interleukin-10 from UVR-treated epidermis is the critical mediator.

Acute, low‐dose ultraviolet B radiation (UVR) alters cutaneous immunity at the local site as well as systemically. Within 2–3 days of UVR exposure, recipient mice lose their capacity to develop contact hypersensitivity (CH) when hapten is painted on unexposed skin. This loss correlates temporally with a functional deficit among dendritic antigen‐presenting cells within non‐draining lymph nodes and spleen. In the experiments described, the delayed systemic immune deficiency following acute, low‐dose UVR exposure was found to be eliminated with neutralizing anti‐interleukin‐10 (IL‐10) antibody. Intracutaneous injection of IL‐10 generated a deficiency of systemic immunity as well as a functional deficit among lymph node dendritic cells that was similar to that induced by UVR. The skin itself was found to be the source of the IL‐10 responsible for these defects, and epidermis (presumably keratinocytes) rather than mast cells was found to be the source of IL‐10 within UVR‐exposed skin. The potential relationships are discussed between the delayed systemic immune deficit created by acute, low‐dose UVR, and the systemic immune deficits caused by chronic, high‐dose UVR and by a single, high‐dose UVR exposure.

Anterior chamber‐associated immune deviation‐inducing cells activate T cells, and rescue them from antigen‐induced apoptosis

Immune responses to antigens injected into the anterior chamber of the eye are devoid of T helper 1 (Th1)‐type responses of the delayed hypersensitivity type, which has been termed anterior chamber‐associated immune deviation (ACAID). Recently, it has been found that peritoneal exudate cells (PEC) from normal mice can be made to acquire the capacity to induce ACAID in vivo when the cells are pulsed with antigen in vitro in the presence of transforming growth factor‐β2 (TGF‐β2), a major cytokine in the ocular microenvironment. We now report that when ovalbumin (OVA)‐specific T cells from DO11.10 transgenic mice, or from OVA‐primed normal mice, were activated in vitro by normal (untreated) PEC pulsed with OVA, the responding T cells were induced to undergo apoptosis. However, when PEC were first treated with TGF‐β2 and then used to stimulate DO11.10 T cells in the presence of OVA, T‐cell proliferation occurred without evidence of increased apoptosis. The ability of TGF‐β2 to rescue responding T cells from apoptosis rested with the capacity of this cytokine to inhibit interleukin‐12 (IL‐12) production by PEC. Untreated PEC produced large amounts of IL‐12 upon interaction with responding T cells. Under these conditions, tumour necrosis factor‐α (TNF‐α) production was up‐regulated, and this cytokine, in turn, triggered apoptosis among T cells stimulated with OVA‐pulsed PEC. From these results, we conclude that TGF‐β2‐treated APC promote ACAID by rescuing antigen‐activated T cells from apoptosis, and by conferring upon these cells the capacity to down‐regulate delayed hypersensitivity.

MHC class II tolerant T cells undergo apoptosis upon re-exposure to tolerogen in vivo

Tolerance of MHC class II alloantigens can be achieved by intravenous injection of semiallogeneic hematopoietic cells into neonatal mice. Lymphoid cells of tolerant mice fail to proliferate or secrete interleukins IL-2 or IL-4 when stimulated in vitro with tolerogen. Since the lymphoid organs of B10.T(6R) tolerant mice contain normal levels of I-E reactive (V beta 11+) CD4+ T cells, deletion of alloreactive T cells does not appear to be the mechanism involved in the tolerance induction. To test whether T cells from tolerant animals can become activated under conditions that do not involve alloantigen stimulation, we stimulated these cells with immobilized anti-V beta 11 antibodies. Spleen cells from grafted tolerant and rejector mice proliferated in response to anti-V beta 11+ antibodies, suggesting they were not inert. We then tested whether V beta 11+ T cells from grafted mice can be induced to proliferate following stimulation with alloantigen in vivo. We adoptively transferred T cells from grafted tolerant and rejector mice into irradiated (B10.AQR x B10.T(6R))F1 mice and harvested the lymphoid organs after 65 h. Cells from both grafted tolerant and rejector mice underwent blast transformation, but only cells from rejector mice proliferated when exposed to immobilized anti-V beta 11 antibodies. The failure of V beta 11+ cells from tolerant mice to proliferate after in vivo stimulation may be because they are apoptotic. To test this hypothesis, spleen cells from naive or neonatally tolerized (with (B10.AQR x B10.T(6R))F1 cells) B10.T(6R) mice were adoptively transferred into irradiated (B10.AQR x B10.T(6R))F1 mice and bcl-2 expression was analysed in harvested V beta 11+ cells. Large cells recovered from recipients of naive 6R cells expressed bcl-2 mRNA. By contrast, large cells harvested from recipients of tolerized 6R cells did not express bcl-2 mRNA, suggesting bcl-2 mRNA expression was downregulated in these mice. Moreover, in another experiment, large V beta 11+ cells from grafted tolerant animals recovered after transfer into irradiated (B10.AQR x B10.T(6R))F1 mice did not express the bcl-2 protein as determined by flow cytometry, and contained fragmented DNA as assessed by the TUNEL method. Taken together, these data suggest that MHC class II tolerant T cells undergo apoptosis upon re-exposure to tolerogen in vivo.