Reply

I thank Nina Bhardwaj and Marie Larsson for their comments indicating where I might have erred and offering me the opportunity to clarify my thoughts. In my Opinion article1, I do contend that peptide–heat-shock protein (HSP) complexes in apoptotic cells might be an important and physiologically relevant source of antigen for the immune system. In fact, some of these ideas were conceived while I worked in Bhardwaj's laboratory through a collaboration with Pramod Srivastava.

The question of cell death and its role in immunity has been of much interest and some dispute. My Opinion article was intended to emphasize the importance of incorporating molecular and biochemical data into the definitions of cell death that are used by immunologists. I agree that freeze–thawed vaccinia-virus-infected monocytes provide a source of antigen for the immune system and that this lysate might contain peptide–HSP complexes, as well as whole proteins that are a source of antigen for cross-presentation. However, I think the definitions of death that are used in the study of Bhardwaj, Larsson and colleagues2 require more rigour. I stand corrected that the authors did not report on HSP upregulation in the vaccinia-virus-infected monocyte model2. But, I argue that infection as a source of stress would result in marked upregulation of HSPs3,4, so 'necrotic death', in their model, should be considered to be secondary necrosis (that is, secondary to early apoptotic cell death). Their stated conclusion, “regardless of the source of antigens ... a common pathway is used to present cell-derived antigens”, might be appropriate for understanding vaccine strategies but does not necessarily offer insight into the physiology of the situation. Our current research efforts are aimed at understanding physiological and pathological situations, and I believe it remains to be shown how dying cells influence immune responses in vivo.

Regarding protein aggregates versus pre-processed antigen as a source of MHC epitopes for cross-presentation, I am at a disadvantage as our work has yet to be published. I look forward to opportunities in the near future to discuss this issue further. Until then, I hope that readers will approach the study of Shen and Rock5 with some healthy skepticism. I argue that using nitrogen cavitation to disrupt cells might also disrupt peptide–HSP complexes. Shen and Rock5 do not show that the HSPs prepared from their lysates actually remain bound to their peptides. Furthermore, it is not clear to me that purified HSP complexes are as efficiently cross-presented as membrane-bound complexes (as would be the case when dying cells are the source of the peptide–HSP complex). Recent studies by Norbury et al.6 and Wolkers et al.7 might also be included in this discussion. I would like to note that I do not exclude the importance of whole protein or protein fragments as a source of antigen for cross-presentation. That said, my Opinion article focused on outlining the active role that apoptotic cells might have in an immune response. Although some model systems show the efficiency of protein processing by a phagosome-to-cytosol pathway, there might be important differences depending on the epitope studied or the context of cell death and antigen capture.

I commend the editors of Nature Reviews Immunology for including a Perspectives section in their journal, and I hope that readers will be stimulated by the ideas I have put forth. Already, I have received generous and challenging responses. Through careful experimentation and the use of well-defined terminology, I hope that we might come closer to understanding the importance of cell death in directing the immunological outcome of antigen processing and presentation.