Peptide immunotherapy with CD4 T cell epitopes has been used extensively in mouse models to prevent and ameliorate antigen-specific inflammatory responses and is the subject of current clinical trials in allergy and autoimmunity (Larche and Wraith, 2005
). Improved understanding of mechanisms is likely to significantly improve efficacy. In our clinical studies, Fel d 1 peptides were delivered i.d., at low dose, to redirect allergen-specific responses, decrease symptoms, and reduce inflammation. Systemic doses of peptide as low as 5 µg reduced skin allergen sensitivity and proinflammatory PBMC responses to allergen (Oldfield et al., 2001
; Oldfield et al., 2002
; Alexander et al., 2005a
; Alexander et al., 2005b
). Reduced proliferative responses to allergen were associated with increased IL-10 production from PBMC and induction of functional CD4+
regulatory T cells (Verhoef et al., 2005
To define the immunological mechanisms underlying the effects of peptide immunotherapy, we performed a detailed analysis of peptide-specific responses in allergic asthmatic subjects treated with a therapeutic peptide vaccine or with placebo. Peptide treatment reduced proliferative and cytokine responses to both treatment and nontreatment peptides, which is indicative of linked epitope suppression, a process through which cells rendered tolerant to one epitope suppress the function of cells specific for other epitopes within the same molecule (Holan and Mitchison, 1983
). To determine the functional effects of peptide immunotherapy on T cells from the lung parenchyma and airways, which are more relevant to clinical asthma but cannot be studied in human volunteers, we generated a novel mouse model of peptide immunotherapy. The model was specifically designed to closely mimic our human experience. Mice were treated with a single Fel d 1 peptide, which forms part of the therapeutic clinical vaccine used in human studies. Presentation of the peptide to T cells was restricted by the only MHC class II molecule expressed by the mice, the product of a human HLA-DR1 transgene. Using this novel model we were able for the first time to directly track T cell responses to the treatment peptide using HLA-DR1 tetramer reagents. Tetramer analysis revealed reduced antigen-specific proliferative responses of both DR1Feld1(29–45)
cells and tetramerneg
cells supporting the observation of linked epitope suppression in the clinical study. Given the marked reduction in lung inflammation and decreases in Th2 responses after peptide treatment in the mouse model, it is clear that the peptide-specific T cell population was capable of down-regulating an established inflammatory response driven by multiple T cell epitopes. Indeed, in this model, targeting of a relatively rare population of peptide-specific T cells was associated with the production of IL-10 by a much larger proportion of the T cell pool. Similar induction of IL-10–producing “bystander” T cells was recently reported in a related model after transfer of antigen-specific CD4+
regulatory T cells (Kearley et al., 2005
). “Infectious” expansion of IL-10–producing T cell populations has been described previously in other disease scenarios (Qin et al., 1993
Peptide therapy markedly increased BAL levels of IL-10 and numbers of IL-10+ T cells in lung tissue, the latter increasing threefold. Lung digest T cells cultured for 7 d with recombinant allergen showed lower levels of proliferation after Fel d 1 peptide treatment compared with control peptide. This may have been caused by suppressive effects of increased numbers of IL-10+ T cells, and/or caused by a reduction in the numbers of allergen-specific T cells through clonal deletion.
PBMCs from allergen peptide-treated subjects, and heat shock protein peptide-treated subjects with type I diabetes and rheumatoid arthritis, demonstrated enhanced levels of antigen-stimulated IL-10 production in vitro (Akdis and Blaser, 1999
; Raz et al., 2001
; Oldfield et al., 2002
; Prakken et al., 2004
cells have been found to increase in number in blood and tissues taken in clinical studies of grass pollen and insect venom immunotherapy (Bellinghausen et al., 1997
; Nasser et al., 2001
; Nouri-Aria et al., 2004
) and a higher frequency of IL-10–secreting T cells is found in peripheral blood of nonatopic versus atopic individuals (Akdis et al., 2004
). Adoptive transfer of IL-10–secreting cells has been shown to ameliorate allergic airway inflammation. We have shown that transfer of CD4+
regulatory T cells suppresses allergic lung inflammation by an IL-10–dependent mechanism (Kearley et al., 2005
). Furthermore, IL-10–transduced dendritic cells down-regulate allergic airway inflammation in mice by induction of IL-10–expressing T cells (Henry et al., 2008
), and OVA-specific T cells engineered to express IL-10 also inhibit Th2-induced AHR and inflammation (Oh et al., 2002
). In this study, neutralization of IL-10 activity via blockade of IL-10R reversed peptide-induced tolerance, as shown by lung function analysis, elevation of lung and systemic Th2 responses, and reversal of peptide treatment effects on proliferation of Feld1(29–45)
-specific T cells. These results specifically demonstrate the IL-10 dependence of peptide therapy in this model, which does not rely on the transfer of manipulated cells.
We did not observe an increase in the number of DR1Feld1(29–45)tetramer+ nondividing cells which would have been expected had they been anergized. Thus, we further conclude that peptide therapy does not result in the induction of anergy in the target T cell population, a possibility we have been thus far unable to exclude in clinical studies.
Resolution of allergic airway disease in our mouse model was achieved with administration of only a single peptide. Chai et al. (2004)
prevented graft rejection by prophylactic administration of 9 µg (3 administrations) of peptide intranasally (i.n.). Apostolou and Von Boehmer (2004)
induced de novo conversion of naive T cells to CD4+
antigen-specific regulatory cells through chronic exposure (10 µg/d for 14 d) to peptide. By directly targeting dendritic cells with an influenza peptide integrated into an antibody to DEC-205, Kretschmer et al. generated antigen-specific regulatory T cells with the equivalent of 500 pg of epitope (Kretschmer et al., 2005
). Here we report the induction of antigen-specific tolerance and resolution of inflammation after the single administration of 1 µg of peptide without adjuvant or cell-targeting strategy. Thus, ultra low-dose delivery of CD4 T cell epitopes can induce T cells with regulatory function that are capable of reversing existing pathology.
Using lavage and whole lung tissue, we demonstrated a marked reduction in airway and tissue eosinophilia and reduced in situ Th2 inflammation. BAL and lung tissue IL-4, IL-5, and IL-13 cytokine production was reduced. Local IFN-γ production was not elevated after Feld1(29–45) treatment, suggesting that peptide therapy did not result in deviation from a Th2 to a Th1 response. Levels of the Th2-associated chemokines CCL11, CCL17, and CCL22 were also significantly decreased. Possibly as a direct result of this, we observed fewer CD4+ T cells expressing IL-4 and IL-5 and reduced recruitment of Th2 cells to lung tissue and BAL after peptide treatment.
TGF-β has been implicated in T cell regulation of immune responses through conversion of naive CD25−
T cells to regulatory CD25+
cells through induction of Foxp3 expression and reduction of T cell proliferation (Chen et al., 2003
; Apostolou and Von Boehmer, 2004
). However, in this study, we found no change in levels of active TGF-β1 in BAL or lung tissue homogenates after peptide challenge. This implies that TGF-β does not have a significant role in suppression of pulmonary pathophysiology in this model. We have described similar findings in another lung model (Kearley et al., 2005
). Because Foxp3 expression is thought to be a marker for CD4+
regulatory T cells (Hori et al., 2003
) we measured intracellular Foxp3 expression in CD4+
T cells isolated from BAL, lung tissue, and peribronchial lymph nodes. Peptide treatment did not result in increased numbers of Foxp3+
cells in any of these tissues. These data may discount a role for CD4+
regulatory T cells in peptide-directed resolution of pathophysiology, in agreement with our published clinical findings (Smith et al., 2004
), but importantly they highlight a role for IL-10–secreting regulatory cells.
In summary, our data indicate that peptide immunotherapy ameliorates allergic inflammation in a mouse model via an IL-10–dependent mechanism and substantially reduces clinical surrogate markers of allergy in human cat allergic asthmatics through a process involving linked epitope suppression associated with induction of IL-10. No evidence for the induction of clonal T cell anergy was obtained. Treatment of mice was associated with reduced eosinophilia and mucus production, improved lung function, reduced Th2 cytokine and chemokine levels, lower total and specific IgE, and reduced numbers of Th2 cells infiltrating the lung tissue and BAL. Moreover, we demonstrate, for the first time, the direct effect of peptide administration on functional responses of lung parenchymal T cells specific for the treatment peptide using MHC class II tetramers. The percentage of IL-10–producing T cells was markedly enhanced in the lung after peptide therapy and was substantially greater than the percentage of cells specific for the treatment peptide, an observation that is reminiscent of infectious tolerance. Finally, tolerance induction in the mouse model appeared to be independent of TGF-β and Foxp3 expression. These studies further our understanding of mechanisms of peptide-induced tolerance in allergic asthma. These results will inform the design and evaluation of peptide interventions to ameliorate chronic allergic and autoimmune diseases.