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Immunological techniques Subtract, add, mix and match: techniques for exploring the immune system Editorial overview Nilabh Shastri

Addresses Division of Immunology, LSA 421, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA; e-mail: nshastri@socrates.berkeley.edu

Current Opinion in Immunology 1998, 10:123-124

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© Current Biology Ltd ISSN 0952-7915

Why develop methods? Especially when most reviewers of grant applications respond to such proposals with the platitude "...technologically driven. Little biological relevance..." Yet, a small number of researchers continue to (often surreptitiously) devote their time, effort and resources into these undertakings. Some are driven to push the limits of established methods, some by the desire to see if things can be done differently, and some, curiously, by 'laziness'. Paradoxically they often end up spending a year's hard work to enable them, and eventually others in the field to do something in a day which could otherwise be done in a week! Regardless of the motivation for their development, robust assays eventually provide relief from drudgery at the bench, and new paths for exploring the unknown.

All approaches to study of modern immunology use protocols that, in the final analysis, aim to subtract, add, mix and match. This is certainly true for the reductionist goal of stripping biological phenomenon of all their natural complexity to enable their study in isolation. Other components are later mixed in to match the natural context. But even at the level of the whole animal, as previous years' contributions show, valuable insights are emerging from the study of knockouts (subtractions), transgenics and knockins (additions and/or subtractions). This year's annual issue covers molecular and subcellular techniques which have been applied to different areas of immunology with significant success.

Differential expression of a common set of inherited genes is the key to development of specialized functions in multicellular systems. Subtractive hybridization was the original method of choice for identification of such genes because prior knowledge of their function was not required. Here Hess, Laumen and Wirth (pp 125-130) describe several new variations and refinements of the polymerase chain reaction (PCR) and sequence-based current versions of this technique and their applications to analysis of differential gene expression in tumors and during lymphoid development. With the development

of microchip arrays of DNA sequences, changes in expression of thousands of transcripts can now be detected simultaneously. The ability to examine the expression of virtually all genes was recently accomplished in the yeast and should be possible for mammalian cells in the foreseeable future. The ability to simultaneously subtract and add all expressed genes in lymphocytes promises a new perspective for understanding their development and responses.

Apoptosis, or programmed cell death, occurs at discrete checkpoints during development in species ranging from worms to humans. In the immune system, the birth and death of lymphocytes and their antigenic targets is a fact of everyday life. Wallach and colleagues (pp 131-136) review the triumphs and limitations of the original yeast two-hybrid screening technique in the discovery of mediators that activate or inhibit apoptosis. Given the concerted nature of the steps leading to cell death, the method has been rather successful in revealing the identity of the proximal interacting proteins as well as their modes of interaction via death domains. The authors also discuss the gaps in knowledge of more distal, downstream mediators of apoptosis and possible refinement of the methods that may provide the way to their discovery.

Immune surveillance by CD8 ÷ T cells depends upon the mechanism of antigen processing that results in the display of processed peptide/MHC class I complexes on the cell surface. Recent years have defined an increasing number of proteins involved in the transport and assembly of the peptide/MHC complexes in the endoplasmic reticulum. My colleagues and I (pp 137-144) review the methods for analysis of the antigen-processing pathway from the perspective of the antigen. The recent discoveries of novel peptide/MHC complexes-- the output of the antigen pro- cessing pathway--are yielding not only potential targets for immune intervention in cancer and transplantation, but also insights into the source of naturally processed peptides and their proteolytic intermediates.

Finally, Pierre and Mellman (pp 145-153) remind the re- ductionists that a cell is far more than a membrane bound collection of nucleic acids and their translation products. They critically examine the available methods for analysis of subcellular compartments where peptide/MHC class II complexes are assembled. The inherent challenges in the analysis of ephemeral endosomes that ferry the MHC molecules before and after peptide loading, are

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overcome by a combination of conventional and elec- trophoretic techniques. Most impressively, the application of these techniques has revealed that expression of these endosomal compartments is developmentally regulated in specialized dendritic cells.

Together the contributions in this section testify that the simplest arithmetic operations when applied to biology can provide the keys to understanding some of the most complex immunological processes.