PERSPECTIVE
Immunotherapy with Antigens and Epitopes
Pro or Con

Lanny J. Rosenwasser and Erwin W. Gelfand

Departments of Medicine and Pediatrics, National Jewish Medical and Research Center, Denver, Colorado

Allergen injection immunotherapy is generally considered to produce a state of tolerance in both animal models and in human allergic and asthmatic disease (1). The potential mechanisms that underlie this tolerance induction is thought to focus on two separate circumstances, both of which utilize the T cell as an important target (2). The first involves anergy, clonal silence, or actual clonal deletion with elimination of allergen-reactive T cells. The second relates to the role of immunoregulation-induced immune deviation. In this latter circumstance, while specificity of reactions are maintained, the quality of the reaction is mediated by differing outputs of cytokines in the milieu of the responding tissue. These two alternatives (anergy versus immune deviation) are not necessarily mutually exclusive. In fact, clonal anergy and/or clonal deletion has not been distinguished in many of the models of injection immunotherapy. The possibility of immune deviation, although suggested by a number of studies, also has not been proven outright to underlie allergen-induced tolerance. The relevance of the mechanisms involved is not just theoretical. The actual mechanism for tolerance induced by allergen injection immunotherapy may provide clues to utilize pharmacotherapy in a more focused and direct way to alter allergic diseases and asthma. However, the injection of protein antigens and, in particular, allergens to moderate tissue target responder organs in the therapy of allergic diseases and airway hyperresponsiveness still remains a mystery in terms of the mechanism of action. The specificity of the reaction at the level of a single T cell clearly involves recognition of short linear sequence structures identified as T-cell epitopes within the larger structure of the protein antigen. The role of specific epitopes in this process has only recently been studied. Since T lymphocyte responses play a key role in the pathogenesis of airway hyperresponsiveness, asthma, and atopic disease, the mechanism by which these T cells play a role in these responses has become critical to understand (6, 7). The differentiation between epitope specific responses using an immunodominant T-cell epitope and overall responses based on a complete protein antigen therefore takes on significant implications (8, 9). Table 1 summarizes the immune changes involved in immunotherapy.

Original ideas concerning immunotherapy suggested that humoral immune function and the development of antigen-specific immunoglobulin (Ig)G, including IgG4, could be associated with specific responses in ameliorating airways and allergic responses. However, since very often changes in humoral immune function during traditional immunotherapy are not associated temporally with improvement in clinical responses, the actual role of such potential "blocking" antigen-specific IgG molecules remains under skepticism. The role of T lymphocytes in these responses in both animal models and in humans over the past 10 years has been clearly elucidated. T helper 2 (Th2) T cells are increased at tissue sites in allergic diseases and asthma, accumulate at sites of allergen challenge, and correlate with disease symptoms. Cytokines made by these Th2 cells, including interleukin (IL)-4 and IL-5, are relevant to promote class switching to IgE and can initiate and stimulate the differentiation, survival, and chemotaxis of eosinophils and other immune cells that release mediators in the target tissues (10). Thus, the ability to downmodulate this Th2-type T-cell response has been a major target for understanding the potential mechanisms of immunotherapy. Modulation of production or downregulation of cytokines such as IL-4, IL-5, and IL-13, and upregulation of  interferon and IL-12 and IL-18 are associated with the presumed induction of an ameliorating Th1 response at a prior site of a Th2 response (13). The mechanisms for these changes are not entirely clear, but it represents immune deviation in the response to antigen. How this immune deviation is accomplished could be achieved by changes in local signaling patterns generated by alterations in the cytokine milieu at the sites of inflammation. It also could represent predetermined function identified by differentiated cells of Th1 or Th2 lineage in the tissues. Regardless of the mechanism, such findings demonstrating immune deviation have been well established in the literature (13). The advantage of using epitopes even in traditional immunotherapy is supported by the fact that whole allergens that may be recognized by IgE would have a similar degree of therapeutic efficacy if relevant T-cell epitopes are used. This approach would eliminate any of the dangers generated by excess whole antigen, such as IgE-mediated anaphylaxis and actual exacerbation of the disease via IgE-dependent mechanisms. Another advantage of potential epitope or peptide immunotherapy is that they may be administered at a large excess, which may further enhance the possibilities of T-cell downregulation and epitope-specific anergy or tolerance. The exact mechanisms underlying these responses are not delineated. (A summary of potential mechanisms in immunotherapy is presented in Table 2.)

In the paper by Janssen and colleagues, immunotherapy with ovalbumin and the immunodominant T-cell epitope associated with ovalbumin is examined in a murine model of allergic inflammation, airways hyperresponsiveness, and presumed allergic asthma (14). Repeated ovalbumin challenge increased serum levels of ovalbumin-specific IgG1, IgE, airway eosinophilia, and hyperresponsiveness compared with control animals. IL-4 and IL-5 production also was increased in this model when lymph node T cells were examined in vitro. Pretreatment with ovalbumin- injection therapy given subcutaneously significantly reduced ovalbumin-specific IL-4 and IL-5 production. In contrast, immunotherapy with the immunodominant OVA peptide 323-339 aggravated airway eosinophilia and bronchial hyperresponsiveness, whereas levels of immunoglobulins, including IgG1, IgG2a, and IgE specific for ovalbumin, and in vitro IL-4 and IL-5 production were not affected. Hence, the whole allergen was capable of moderating the complete response in a standard manner as seen with immunotherapy, whereas the epitope actually stimulated many of the responses (15, 16). The explanation for this variability and the opposite result that one would predict for the immunodominant epitope may relate to the kinetics of the response, the role of accessory molecules or organ-specific antigen-presenting cells in presenting the epitope as opposed to the whole allergen. Many other potential mechanisms could alter the balance between Th2-inducing cytokines such as IL-4, IL-5, IL-13, and Th1- inducing cytokines such as IL-12 and IL-18. While there are some aspects of this response that have been recapitulated in peptide immunotherapy trials for cat allergens, by and large the results with the ovalbumin (OVA)-peptide were unexpected (8, 9, 14, 17).

The mechanism by which the peptide stimulates (as opposed to inducing tolerance) needs to be examined, especially since other reports in the literature suggest that immunization with epitopes is actually more efficient than injection of whole allergen in the induction of peripheral T-cell tolerance. The possibility that IgG molecules, including IgG4, directed to alternate portions of the overall OVA antigen may stimulate and tolerize effector cells, such as mast cells and basophils, through stimulation of Fc receptors on the surface of mast cells and basophils needs to be considered. Other potential mechanisms may be involved in the immunomodulation associated with immunotherapy. For example, in addition to effects on antigen presentation, T-cell responses, and humoral effects on effector cells like mast cells and basophils after immunotherapy, there may be direct effects on B cells that can be associated with potential improvement. For example, engagement of the B-cell antigen receptor for whole allergen may negatively regulate the capability of that B cell to switch for IgE isotype production, or it may be directly inhibitory to the capability of the IgE B cell already switched and committed to actually secrete and produce IgE, hence producing an ameliorative effect (15, 16, 18). Other possibilities include the potential role of antigen-induced immunotherapy in generating an antibody response whose complexes are altered and handled by antigen-presenting cells expressing high and low affinity IgE Fc receptors to produce and modulate IgE-dependent responses. Antigen-specific IgE and IgG complexed with allergen may alter the actual epitopes that may be potentially recognized by T cells, thus possibly altering T-cell reactivity in this indirect fashion.

One is still left with the quandary of how immunotherapy works. Further insight into the potential mechanisms of immunotherapy may be obtained by examining data generated in relation to the use of either vaccines for protein antigens or allergens encoded by cDNA, and the propensity for the induction of Th1-like responses under these circumstances. The identification of immunostimulatory DNA sequences that may activate a specific pathway involved in selection of this Th1 predilection during priming and sensitization will also be informative. Some of these mechanisms may also be related to the route of administration. As a result, injection versus inhalation may produce different responses with antigens, epitopes, and DNA vaccines. More importantly, these DNA vaccines may provide a means to elucidate the inhibitory arm of the overall immunomodulatory effects seen with immunotherapy. It is possible that immunostimulatory sequences within DNA vaccines, in conjugate between nucleotides and protein antigens, and allergens encoded by cDNA, can have an indirect effect on immune deviation by the induction of IL-12 and IL-18 during the immunologic recognition of these vaccine-related materials. This cytokine milieu would naturally favor the production of Th1-type T cells, consonant with stimulation of previously uncommitted cells that have the correct T-cell receptor to see the epitopes encoded for, either by the complementary DNA or expressed in the nucleotide protein conjugants. Defining the mechanism and identifying the precise role of T cells, T-cell receptors, cellular interactions, and downstream mediators, which regulate the response to immunotherapy, is critical for determining the most effective strategies for immunologic intervention in antigen-specific and antigen-mediated atopic diseases such as asthma, rhinitis, and eczema.

	Footnotes

Address correspondence to: Erwin W. Gelfand, M.D., Chairman, Department of Pediatrics, Professor of Pediatrics, and Microbiology/Immunology, University of Colorado Health Sciences Center, 1400 Jackson Street, Denver, CO 80206. E-mail: gelfande{at}njc.org

(Received in original form May 19, 1999).

	References

1. Durham, S. R., and S. J. Till. 1998. Immunologic changes associated with allergen immunotherapy. J. Allergy Clin. Immunol 102: 157-164 [Medline].

2. Creticos, P. S., N. F. Adkinson Jr., A. Kagey-Sobotka, D. Proud, H. L. Meier, R. M. Naclerio, L. M. Lichtenstein, and P. S. Norman. 1985. Nasal challenge with ragweed pollen in hay fever patients: effect of immunotherapy. J. Clin. Invest. 76: 2247-2253 .

3. Varney, V. A., Q. A. Hamid, M. Gaga, S. Ying, M. Jacobson, A. J. Frew, A. B. Kay, and S. R. Durham. 1993. Influence of grass pollen immunotherapy on cellular infiltration and cytokine mRNA expression during allergen-induced late-phase cutaneous responses. J. Clin. Invest 92: 644-651 .

4. Durham, S. R., S. Ying, V. A. Varney, M. R. Jacobson, R. M. Sudderick, I. S. Mackay, A. B. Kay, and Q. A. Hamid. 1996. Grass pollen immunotherapy inhibits allergen-induced infiltration of CD4+ T lymphocytes and eosinophils in the nasal mucosa and increases the number of cells expressing messenger RNA for interferon-. J. Allergy Clin. Immunol 97: 1356-1365 [Medline].

5. Gleich, G. J., E. M. Zimmermann, L. L. Henderson, and J. W. Yunginger. 1982. Effect of immunotherapy on immunoglobulin E and immunoglobulin G antibodies to ragweed antigens: a six-year prospective study. J. Allergy Clin. Immunol 70: 261-271 [Medline].

6. Bond, J. F., A. W. Brauer, D. B. Segal, A. K. Nault, B. L. Rogers, and M. C. Juo. 1993. Native and recombinant Fel d I as probes into the relationship of allergen structure to human IgE immunoreactivity. Mol. Immunol 30: 1529-1541 [Medline].

7. Briner, T. J., M. C. Kuo, K. M. Keating, B. L. Rogers, and J. L. Greenstein. 1993. Peripheral T-cell tolerance induced in naive and primed mice by subcutaneous injection of peptides from the major cat allergen Fel d I.  Proc. Natl. Acad. Sci. USA 90: 7608-7612 [Abstract/Free Full Text].

8. Péne, J., A. Desroches, L. Paradis, B. Lebel, M. Farce, C. F. Nicodemus, H. Yssel, and J. Bousquet. 1998. Immunotherapy with Fel d 1 peptides decreases IL-4 release by peripheral blood T cells of patients allergic to cats. J. Allergy Clin. Immunol 102: 571-578 [Medline].

9. Marcotte, G. V., C. M. Braun, P. S. Norman, C. F. Nicodemus, A. Kagey-Sobotka, L. M. Lichtenstein, and D. M. Essayan. 1998. Effects of peptide therapy on ex vivo T-cell responses. J. Allergy Clin. Immunol 101: 506-513 [Medline].

10. Robinson, D., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A. M. Bentley, C. Corrigan, S. R. Durham, and A. B. Kay. 1992. Predominant Th2-like bronchoalveolar T-lymphoctye population in atopic asthma. N. Engl. J. Med 326: 298-304 [Abstract].

11. Robinson, D., Q. Hamid, A. Bently, S. Ying, A. B. Kay, and S. R. Durham. 1993. Activation of CD4+ T cells, increased Th2-type cytokine mRNA expression, and eosinophil recruitment in bronchoalveolar lavage after allergen challenge in patients with atopic asthma. J. Allergy Clin. Immunol 92: 313-324 [Medline].

12. Robinson, D. S., S. Ying, A. M. Bently, Q. Meng, J. North, S. R. Durham, A. B. Kay, and Q. Hamid. 1993. Relationships among numbers of bronchoalveolar lavage cells expressing messenger ribonucleic acid for cytokines, asthma symptoms, and airway methacholine responsiveness in atopic asthma. J. Allergy Clin. Immunol 92: 397-403 [Medline].

13. Hamid, Q. A., E. Schotman, M. R. Jacobson, S. M. Walker, and S. R. Durham. 1997. Increases in IL-12 messenger RNA+ cells accompany inhibition of allergen-induced late skin responses after successful grass pollen immunotherapy. J. Allergy Clin. Immunol 99: 254-260 [Medline].

14. Janssen, E. M., M. H. M. Wauben, E. H. Jonker, G. Hofman, W. Van Eden, F. P. Nijkamp, and A. J. M. Van Oosterhout. Opposite effects of immunotherapy with ovalbumin and the immunodominant T cell epitope on airway eosinophilia and hyperresponsiveness in a murine model of allergic asthma. 21:21-29.

15. Oshiba, A., E. Hamelmann, K. L. Bradley, J. E. Loader, H. Renz, G. L. Larsen, and E. W. Gelfand. 1996. Pretreatment with allergen prevents immediate hypersensitivity and airway hyperresponsiveness. Am. J. Respir. Crit. Care Med 153: 102-109 [Abstract].

16. Oshiba, A., E. Hamelmann, A. Haczku, K. Takeda, D. H. Conrad, H. Kikutani, and E. W. Gelfand. 1997. Modulation of antigen-induced B and T cell responses by antigen-specific IgE antibodies. J. Immunol 159: 4056-4063 [Abstract].

17. Norman, P. S., J. L. Ohman Jr., A. A. Long, P. Creticos, M. A. Gefter, Z. Shaked, R. A. Wood, P. A. Eggleston, K. B. Hafner, P. Rao, L. M. Lichtenstein, N. H. Jones, and C. F. Nicodemus. 1996. Treatment of cat allergy with T-cell reactive peptides. Am. J. Respir. Crit. Care Med 154: 1623-1628 [Abstract].

18. Oshiba, A., and E. W. Gelfand. 1999. Engagement of the B-cell antigen receptor by antigen negatively regulates IgE production by antigen-specific B cells. J. Allergy Clin. Immunol 103: 341-348 [Medline].