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Background: It is well recognized that CD8 T cells inhibit IgE responses. In this study, we investigated the mechanism of CD8 T cell-mediated IgE suppression by comparing the capacity of T cytotoxic 1 (Tc1) and T cytotoxic 2 (Tc2) CD8 T cells to inhibit IgE responses to ovalbumin (OVA)., Methods: Tc1 and Tc2 CD8 T cells were generated from OVA(257-264)-specific Vbeta5.2 T cell receptor (TcR) transgenic mice by stimulation with anti-CD3 and anti-CD28 under Tc1 and Tc2 polarizing conditions. Tc1 and Tc2 Vbeta5.2 TcR CD8 T cells (10(6)) were adoptively transferred to syngeneic mice, and following immunization with 100 micro of OVA/alum, serum IgE antibodies were measured by passive cutaneous anaphylaxis and expressed as the highest dilution that gave a detectable skin response., Results: Both Tc1 and Tc2 CD8 T cells from OT-I mice inhibited IgE., Conclusion: Both Tc1 and Tc2 CD8 T cells promote Th1 immunity and inhibit IgE responses. This process appears to be independent of CD8 T cell-derived IFN-gamma, as both Tc2 (IFN-gamma-) and Tc1 (IFN-gamma+) CD8 T cells inhibited IgE., (Copyright 2001 S. Karger AG, Basel)

Previous studies in both rats and humans have shown that interleukin (IL) 4 can suppress the generation of IL-2-producing CD8 T cells. In an attempt to elucidate the mechanism by which this suppression is brought about, we set out to investigate whether the IL-4 signal interferes with the IL-2 receptor system. It has already been reported that IL-2 can affect the expression of its own receptor and thus provide a means of controlling its own activities. In this study, we demonstrate that the IL-2 Ralpha- and the IL-2 Rgamma-chains are dramatically upregulated following stimulation of CD8 T cells, whereas lower levels of beta-chain are observed. IL-4 did not affect the expression of the alpha- or beta-chains, but was found to inhibit the generation of common gamma-chain-expressing cells. Moreover, CD4 T cells were found to express much lower levels of this subunit and appeared less sensitive to the effects of IL-4. We postulate that the differential expression of the gamma-chain subunit, in the presence and absence of IL-4, may provide a tool for identifying functionally distinct subpopulations of CD8 T cells.

The aim of this study was to compare the effects of IL-4, IL-12 and IFN-gamma on the production of T helper-1 (Th1) and T helper-2 (Th2)-type cytokines from human peripheral blood 'naive' CD45RA and 'memory' CD45RO CD4 T cells. CD45RA or CD45RO CD4 T cells were cultured for 4 days with phorbol myristate acetate (PMA) and ionomycin and either IL-4, IFN-gamma or IL-12 and their ability to proliferate and secrete IFN-gamma and IL-4 determined. Purified CD45RO CD4 T cells stimulated with PMA and ionomycin secreted higher levels of IL-4 and IFN-gamma, as measured by ELISA, than CD45RA CD4 T cells which secreted little IL-4 or IFN-gamma. However, CD45RA and CD45RO CD4 T cells proliferated to the same extent and IL-4, IFN-gamma and IL-12 had no effect on this. IL-12 and IFN-gamma had no effect on the amount of IL-4 secreted by PMA and ionomycin-stimulated CD45RO CD4 T cells, but culture with IL-4 enhanced IL-4 production 7-fold. IL-12 increased the amount of IFN-gamma produced by CD45RO CD4 T cells 2- to 3-fold. Small amounts of IFN-gamma production were induced in CD4 CD45RA T cells by IL-12 and IFN-gamma. These results indicate: (1) that CD45RA cells cannot make significant amounts of IL-4 under the conditions used, (2) that CD45RO cells can produce both Th1 and Th2 cytokines immediately upon restimulation, (3) that IL-12 favours Th1 cytokine production in both CD45RA and CD45RO CD4 T cells, and (4) that IFN-gamma favours IFN-gamma production in CD45RA but not CD45RO cells.

It has been demonstrated previously that repeated exposure of mice to chemical allergens of different types results in the development of qualitatively divergent immune responses characterized by the production by draining lymph node cells (LNC) of distinct cytokine patterns. Chronic exposure of mice to contact allergens, such as 2,4-dinitrochlorobenzene (DNCB), resulted in the secretion by LNC of low or undetectable levels of interleukins 4 and 10 (IL-4 and IL-10), but comparatively high levels of interferon gamma (IFN-gamma); the latter cytokine being produced by both CD4+ and CD8+ cells. In contrast, chronic exposure of mice to trimellitic anhydride (TMA), a respiratory allergen associated in humans with occupational asthma, induced instead the production by LNC of relatively high concentrations of IL-4 and IL-10, but little IFN-gamma. The low levels of IFN-gamma secretion which were provoked by treatment with TMA were shown to derive from CD8+ cells exclusively. In the present investigations we have sought to determine whether the polarized responses observed following repeated exposure to these chemical allergens are reflected by cytokine secretion patterns provoked by primary exposure. To this end, mice of BALB/c strain were exposed epicutaneously daily for 3 consecutive days to concentrations of DNCB and TMA (1 and 10%, respectively), or to oxazolone, another contact allergen (0.25%), that resulted in substantial proliferative activity in draining lymph nodes. The production by draining LNC of IFN-gamma and of mitogen-inducible IL-4 was measured by enzyme-linked immunosorbent assay and the relative contribution of CD4+ and CD8+ cells to the patterns of cytokine secretion observed was analyzed using both positive and negative selection methods. It was found that primary exposure to DNCB, oxazolone and TMA each resulted in the production by LNC of both IFN-gamma and IL-4. Selective depletion of, or enrichment for, CD4+ and CD8+ cells revealed that only CD4+ cells elaborated mitogen-inducible IL-4. Depletion of neither CD4+ nor CD8+ cells compromised the production by TMA- or DNCB-activated LNC of IFN-gamma, although positively selected CD8+ cells were considerably less able than CD4+ cells to elaborate this cytokine, presumably secondary to a lack of appropriate accessory cells. Taken together the results demonstrate that early during immune responses to DNCB or oxazolone and TMA there is no evidence for the selectivity of cytokine secretion patterns that characterizes responses following more chronic exposure. Moreover, it is clear that exposure to TMA initially induces the production of IFN-gamma by both CD4+ and CD8+ cells, whereas after more chronic treatment the secretion of this cytokine is a function of CD8+ cells exclusively. Collectively, these results indicate that the polarized responses that develop in mice following chronic exposure to different classes of chemical allergen are not reflected by the characteristics of primary immune responses. As such the development of qualitatively divergent immune responses to chemical allergens provides a paradigm for the evolution of differentiated T cell function with time and/or with antigen exposure.

The role of CD8+ T cells in the regulation of IgE was investigated by in vivo CD8+ T-cell depletion. Following intraperitoneal immunization with ovalbumin (OVA), OVA-specific T cell responses were first detected in the draining lymph nodes (LN) and subsequently in the spleen. In vitro depletion of LN CD8+ cells reduced the OVA-specific LN CD4+ T cell response; while depletion of splenic CD8+ cells enhanced the OVA-specific splenic CD4+ T cell response, suggesting an early helper and later suppressor function. CD8+ cell depletion in vivo up to 7 days after immunization failed to enhance IgE production, but depletion of CD8+ T cells between day 12 and day 18 increased IgE levels to over 10 microg/ml. Adoptive transfer of 1 x 10(7) purified CD8+ T cells (> 95% CD8+ > 98% CD3+ < 1% NK, < 1% CD4+) from parallel immunized rats or of 1 x 106 cells of an OVA-specific, MHC class I restricted CD8+ T cell line suppressed the IgE, but not IgG, response by > 95%. These results provide further evidence that CD8+ T cells inhibit IgE production, possibly by preventing the generation of adequate amounts of appropriate CD4+ T cell help.

Allergic disease presents an increasing health problem in industrialised countries. Despite improved diagnosis and drug therapy, the number of fatalities from asthma and anaphylaxis continues to rise. A key element in the aetiology of the allergic response to inert protein antigens is an inherent imbalance in the atopic allergen specific CD4+ T cells, resulting in a dominance of the pathological, IL-4 producing, T helper 2 phenotype which favours B cell IgE production. Previous work from our laboratory, and elsewhere, has established that CD8+ T cells play an important role in regulating the magnitude and duration of IgE responses and may be vital for the restoration of IgE immunologic homoeostasis in 'normal' non-atopic humans and low/medium responder rodents. We have found that CD8+ T cell depletion during a critical period after immunization produces a high and persistent IgE response in otherwise IgE hyporesponsive animals. We and others have demonstrated that small, but important, quantities of soluble antigen can enter the MHC class I pathway and, using ovalbumin (OVA)-transfected antigen-presenting cells, have succeeded in cloning OVA-specific, MHC class I-restricted rat alpha/beta T cell receptor (TcR) positive CD8+ T cells. These cells universally secrete IFN-gamma (5-69 ng/ml) and IL-2 (7.6-37.0 U/ml) and occasionally IL-4 (16.0-8/1.0 IU). When adoptively transferred, 1 x 10(6) of these cloned cells inhibit IgE production in vivo by as much as 50-fold and this effect can be titrated down to 1 x 10(3) cells. The IgE regulatory activity of the clones appears unrelated to their cytolytic potential or to their capacity to make IFN-gamma.

The effect of ricin treatment on IgE levels and airway inflammation has been examined in ovalbumin (OA)-sensitized rats. Ricin augmented the total IgE titre and caused about a 10-fold increase in the antigen-specific IgE titre. Repeat antigen challenges (every second day) with an OA aerosol increased the number of eosinophils and neutrophils in bronchoalveolar lavage fluid and produced airway hyperresponsiveness (AHR) 24 h after the third challenge. Ricin pretreatment caused a significantly larger influx of eosinophils, neutrophils and lymphocytes into lavage fluid, but there was no further increase in AHR. The present study demonstrates that ricin augments IgE production and the inflammatory response, but that AHR is unrelated to the number of infiltrating inflammatory cells.

We have systematically investigated the capacity of highly purified human peripheral CD8+ T cells to produce interleukin (IL)-4 and interferon (IFN)-gamma when triggered by different stimuli. CD8+ T cells were isolated from peripheral blood by positive selection to > 99% purity and stimulated with one of three different stimuli: phytohaemagglutinin (PHA) and IL-2, phorbol myristate acetate (PMA) and ionomycin, and plate-bound anti CD3 and PMA. On their own, ionomycin and IL-2 failed to stimulate significant CD8+ T cell proliferation while PHA, plate-bound anti-CD3 and PMA induced weak proliferation. A combination of PHA and IL-2, PMA and ionomycin, or plate-bound anti-CD3 and PMA all induced vigorous CD8+ T cell proliferation. IFN-gamma was produced following all three stimuli, but was greatest from cells cultured with PMA and ionomycin. However, IL-4 secretion was only detected in cell cultures stimulated with PMA and ionomycin. These results indicate that, with sufficient stimulation, human CD8+ T cells have the potential to produce Th2 as well as Th1 cytokines.

The ability of CD8+ T cells to synthesise and secrete cytokines before and after culture with interleukin (IL)-4 and anti-interferon (IFN)-gamma was investigated. Purified rat splenic CD8+ and CD4+ T cells and CD4-CD8- cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for 6 h and expression of mRNA for IL-2, IL-4, IL-5, IL-6, IL-10 and IFN-gamma determined using a quantitative PCR technique. Secreted IFN-gamma and IL-2 were measured by ELISA and bioassay, respectively, 24 h after restimulation. The highest levels of IL-4, IL-5 and IFN-gamma mRNA were expressed by CD8+ T cells, the highest levels of message for IL-2 were expressed by CD4+ T cells and the highest levels of message for IL-6 and IL-10 by CD4-CD8- splenocytes. Purified rat splenic CD8+ T cells were cultured for 6 days with PMA, ionomycin and IL-2 with or without IL-4 or anti-IFN-gamma and restimulated with PMA and ionomycin. Those CD8+ T cells cultured with IL-4 expressed increased levels of IL-4, IL-5 and reduced levels of IL-2 and IL-6 mRNA, while CD8+ T cells cultured with IL-4 and anti-IFN-gamma expressed increased levels of mRNA for IL-10 and IFN-gamma. Comparable effects were seen for secreted IFN-gamma and IL-2. These results indicate that CD8+ T cells have the potential to produce large amounts of Th2 cytokines and are able to differentiate into different subpopulations with distinct cytokine profiles under the control of IL-4 and IFN-gamma.