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Appreciation An appreciation of Ralph Marvin Steinman (1943–2011) Carol L. Moberg Ralph Steinman, an editor at the Journal of Experimental Medicine since 1978, shared the 2011 Nobel Prize in Physiology or Medicine for his discovery of dendritic cells (DCs) and their role in immunity. Ralph never knew. He died of pancreatic cancer on September 30, 3 days before the Nobel announcement. Unaware of his death at the time of their announcement, the Nobel Committee made the e n unprecedented decision that his award i c would stand. Ralph was the consum- i d mate physician-scientist to the end. e After his diagnosis, he actively partici- M pated in his 4.5 years of treatments, l creating experimental therapies using a t his own DCs in conjunction with the now known that this cell-mediated University as a postdoctoral fellow in n e therapies devised by his physicians, immunity involves T lymphocytes in the laboratory of Zanvil (Zan) Cohn, m all the while traveling, lecturing, and many different helper, cytotoxic, and the founder of modern macrophage ri most of all pursuing new investiga- suppressor forms, and that they are all biology. This was an ideal place to di- pe tions in his laboratory. For 38 years— controlled by DCs. Ralph’s discovery rectly test whether macrophages would x from his discovery of DCs to his Nobel thus provided the missing link between trap intact antigens and present them to E Prize—Ralph pioneered the criteria and innate and adaptive immunity. DCs are lymphocytes. The laboratory was founded of methods used to identify, isolate, grow, essential for understanding how the im- by the premier microbiologist René mune system works during health and Dubos, who recognized the need to study and study DCs. He and his colleagues l a how diseases develop. They are un- the host during infection. He had been demonstrated that DCs are initiators n avoidable targets for identifying new the ideal mentor for James Hirsch and r of immunity and regulators of toler- u preventions and therapies. Zan, two physicians devoted to infec- o ance. In his most recent studies, Ralph tious disease, who pursued elegant careers J was harnessing the specialized fea- e tures of DCs to design improved vac- with phagocytes. Dubos, Hirsch, and h Discovery Cohn were all editors of this journal. T cines. The following synopsis describes During his medical training, Ralph was Ralph’s initial research focused on some of his seminal discoveries. challenged to learn how an antigen endocytosis of proteins in macrophages. provokes an immune response. He rec- But when he was unable to find reser- Until Ralph Steinman discovered DCs, ognized that MacFarlane Burnet’s clonal voirs of intact antigen on peritoneal the innate and adaptive systems had been selection theory could not account for macrophages, he turned to the spleen to considered separate entities for nearly a how the body responds to substances— see if this organ might harbor cells re- century. Innate immunity involved Elie both foreign and self—by generating a sponsible for generating immunity Metchnikoff’s phagocytes, particularly diverse repertoire of immune cells, each (Steinman and Cohn, 1972). macrophages that internalize and kill with a single, distinct antibody as its Ralph and Zan discovered DCs in microbes. Paul Ehrlich’s adaptive immu- receptor (Burnet, 1957). Because clonal 1973. Looking through a phase contrast nity involved lymphocytes that produce selection could not be initiated by add- microscope, they encountered a popula- the antibodies that are so often used ing foreign proteins to lymphocytes, tion of cells from the spleen that had for clinical benefit. A new dimension to it was thought that mysterious “acces- Ehrlich’s work was first described in the sory” cells were also required to induce © 2011 Moberg This article is distributed under the terms of an 1940s by Merrill Chase, a researcher at immunity. One idea was that accessory Attribution–Noncommercial–Share Alike–No Mirror Sites license the Rockefeller Institute who showed cells were macrophages. for the first six months after the publication date (see http://www .rupress.org/terms). After six months it is available under a Creative that lymphocytes, rather than antibodies, The story of DCs began in 1970, Commons License (Attribution–Noncommercial–Share Alike 3.0 Un- ported license, as described at http://creativecommons.org/licenses/ bring about adaptive immunity. It is when Ralph joined the Rockefeller by-nc-sa/3.0/). The Rockefeller University Press J. Exp. Med. Vol. 208 No. 12 2337-2342 2337 www.jem.org/cgi/doi/10.1084/jem.20112294 never been seen before and did not Maggi Witmer-Pack, he evaluated their between DCs and all other immune cell look like macrophages. The cells were capacity to stimulate the mixed leuko- types. Michel and Ralph used this elongated with unusual stellate, or tree- cyte reaction (MLR). This T cell reac- monoclonal antibody to selectively de- like, processes that were constantly tion had been used for 15 years to detect plete DCs, but not monocytes, from forming and retracting. Ralph named differences in MHC antigens between spleen suspensions to show that the DC them dendritic cells (from the Greek potential donors and recipients of organ was the primary stimulator of the MLR word dendreon for tree; Steinman and transplants. At the time, it was termed in bulk spleen (Steinman et al., 1983). Cohn, 1973, 1974). mixed lymphocyte reaction because it Another student, Wes van Voorhis, was thought that B cells from the po- identified DCs in human blood tential transplant donor triggered the (Van Voorhis et al., 1982) and devel- Purification division of T cells of the potential trans- oped a monoclonal antibody that killed It took nearly 5 years to get pure popu- plant recipient. Ralph showed that the monocytes, but not DCs. These experi- lations of DCs and to compare their newly identified DCs were 100 times ments were important because they pro- functions with those of other cell types. more potent than bulk spleen cells at vided evidence that DCs also existed in DCs were rare (<1% of mouse spleen inciting the MLR, and that antigen- humans and set the stage for later immu- cells), and their only unique markers bearing B cells and macrophages con- nization experiments (Inaba et al., 1983). were their unusual shape and move- tributed little to initiating the reaction. Ralph and Kayo Inaba demon- ment. Fortunately, George Palade, Phil The DCs were converting transplanta- strated a role for DCs in stimulating Siekevitz, David Sabatini, Günter tion antigens into “immunogens” capa- naive B cells to produce antibodies ca- Blobel, and Christian de Duve—who ble of eliciting cell and organ rejection. pable of binding sheep red blood cells were in the process of inventing In 1978, Ralph predicted that DCs and protein antigens. They showed that modern cell biology—were neighbors would “prove to be a critical accessory once helper T cells had been activated of the Cohn laboratory in Rockefeller’s cell required in the generation of many by DCs, the T cells could interact effi- Bronk Building. The de Duve labora- immune responses” (Steinman and ciently with antigen-presenting B cells tory’s expertise in density gradient cen- Witmer, 1978). However, this inter- to drive B cell clonal expansion and anti- trifugation for subcellular fractionation pretation was not accepted by most body production (Inaba and Steinman, was key to Ralph’s purification meth- immunologists because of the unusual 1984, 1985; Inaba et al., 1984). They ods. The elegant and ingenious steps features of the MLR in comparison to noted that antigen-reactive T cells clus- Ralph worked out started with centri- other immune responses. For example, tered on the surfaces of the very small fuging spleen cells on a bovine serum the antigens responsible for initiating number of DCs, typically 1% or less of albumin gradient, on which semipuri- the MLR were not well defined, and the total cells, in their experimental sys- fied DCs rose to the top. He then placed vigorous in vitro responses apparently tems. Analyzing these T cell–DC clus- this fraction on glass for one hour, deli- required no priming. ters, they showed that DCs provide the cately washed away all but the DCs and “microenvironment” for T cell activa- some macrophages, and cultured these tion and generation of cellular immu- cells overnight to let the DCs detach Antigen presentation nity (Inaba et al., 1987). from the culture dish and float into sus- The next few years were very produc- In all of these systems, small num- pension. The lingering few macrophages tive, and DCs were quickly shown to bers of DCs would elicit a T cell re- were removed by adding antibody- effectively present antigens to T cells. sponse, whereas much larger numbers coated sheep red blood cells and centri- Michel Nussenzweig, the first student of other cell types failed to do so. These fuging them out (Steinman et al., 1979). to work on DCs, showed that DCs cap- experiments led to the realization that This procedure was laborious, and the ture antigens and present them to cyto- there are two stages to every immune DC yield was poor. As a result, few lytic T lymphocytes. Michel modified response. The first is an afferent stage laboratories attempted to reproduce it syngeneic thymocytes with trinitrophe- during which DCs stimulate the growth or to work with DCs for many years. nyl and showed that DCs could cap- and differentiation of rare quiescent After an antibody was developed to ture, process, and present the antigen precursors of antigen-specific helper CD11c, an antigen expressed by DCs, to produce MHC-restricted cytolytic and cytolytic T cells. The second stage activated monocytes, and NK cells, T lymphocytes (Nussenzweig et al., is an efferent limb during which T cells purification became routine (Metlay 1980). Michel also developed 33D1, the that have been activated by DCs divide et al., 1990). first DC-specific monoclonal antibody in response to encounter with antigens (Nussenzweig et al., 1982). Although that have been processed and presented Mixed leukocyte reaction 20 years passed before the antigen rec- by other cells, including B cells and Functional studies on DCs began once ognized by 33D1 was characterized at macrophages. This second stage yields Ralph had pure DC populations. He the molecular level (Dudziak et al., large numbers of effector T cells that noted that DCs expressed high levels of 2007), 33D1 was important because it participate in many immunological re- MHC proteins. Therefore, along with revealed the first molecular distinction actions (Steinman and Nussenzweig, 2338 An appreciation of Ralph Marvin Steinman | Moberg Appreciation 1980; Steinman, 1991). Together, these had discovered that Flt-3L, a hemato- of developing monocyte-derived DCs afferent and efferent limbs solve the poietin that causes a dramatic 10–15-fold in vitro (Trombetta et al., 2003). They conundrum of how rare antigen- expansion of DCs in mice and in proposed that this adaptation allows specific lymphocytes are selected and humans, can be used to grow authentic DCs to efficiently capture, process, and mobilized to bring about vigorous spe- DCs in vitro. Once methods to gener- present antigens by preventing the cific responses. ate large numbers of DCs became avail- complete catabolism of protein antigens able, scientists could more easily study (Inaba et al., 2000). By allowing pep- their immunizing properties. This de- tide–MHC II complexes to form inside Maturation velopment triggered a major expansion the endocytic system, DCs optimized To gain information about DCs in non- in research and greatly accelerated the their subsequent display at the cell lymphoid tissues, Ralph turned to the pace of discovery. surface (Turley et al., 2000). The regu- epidermal Langerhans cells, which were Pursuing the concept that DCs are lation and composition of the endo- also known to express high levels of “nature’s adjuvant,” Ralph’s group cytic system points to a hallmark MHC class II proteins. With Gerold incubated maturing DCs with anti- difference between DCs and macro- Schuler, Niki Romani, and Kayo, gens in vitro and injected these cells phages. Whereas macrophages are spe- Ralph found that Langerhans cells into mice and, eventually, humans. The cialized for continual antigen scavenging needed to differentiate, or mature, to DCs stimulated T cell become typical immune stimulatory growth, and the ex- cells (Schuler and Steinman, 1985; panded T cells recognized Inaba et al., 1986; Romani et al., 1989). the specific antigens pre- Although Langerhans cells were later sented on the MHC shown to have a separate cellular origin products of the injected from that of DCs (Merad et al., 2002), DCs (Inaba et al., 1990). they served as a model to understand These included micro- DC function. Ralph and his colleagues bial antigens in mice discovered that maturing cells exhibited (Inaba et al., 1993b) and different functional attributes during model antigens in humans different periods of time. The first func- (Dhodapkar et al., 1999). tion, antigen capture and presentation, Prior to these discover- is exhibited by immature DCs. The ies, research on antigen second function, accessory or co- presentation had empha- stimulatory activity, is mediated by ma- sized tissue culture sys- ture DCs. Although all DCs process tems and used previously and present antigens, the maturation of activated lymphocytes or DCs determines the type and quality of cloned lymphocyte lines. the immune response that will result With DCs, Ralph took from recognition of DC-presented the problem into immu- antigens by T cells. There was also evi- nologically intact mice dence that the environment in which Ralph Steinman. Photo by Robert Reichert, The Rockefeller and humans. By showing University, 1992. DCs mature exerts a major influence on that DCs initiated im- the form of adaptive immunity they pro- munity specific for the mote. Thus, maturation of DCs serves as antigens they had captured, he justified and destruction, DCs process antigen in the critical link between innate and adap- using these cells to develop immuno- a manner that generates the peptides tive T cell–dependent immunity. therapeutic strategies. that bind to different types of antigen- presenting molecules. Growing DCs Endocytosis Ralph’s studies with Michel on Early research on DCs was limited by Ralph had assumed that splenic and endocytic receptors expressed by DCs the need to isolate these scarce cells skin DCs were fully ready to initiate introduced yet another field of study: from primary tissues. During the early immunity because of their high content how to increase the immunogenicity 1990s, Ralph and colleagues identified of MHC II molecules. But when for- of proteins by exploiting specific path- methods for stimulating bone marrow mer Cohn laboratory member Ira Mell- ways for antigen uptake. The research cells to grow and differentiate in vitro man and colleagues at Yale brought began with biomedical student William into cells that displayed many of the their expertise in cellular molecular bi- Swiggard, who isolated DEC-205 (also phenotypic characteristics of DCs ology to the DC field, they found that called CD205; Swiggard et al., 1995). (Inaba et al., 1992a,b, 1993a; Romani the MHC II proteins were mostly se- Wanping Jiang, a postdoctoral fellow in et al., 1994). By 2000, other investigators questered within the endocytic system Michel’s laboratory, cloned the gene JEM Vol. 208, No. 12 2339 and showed that it encoded an endo- describes a way to use DCs to generate RNA (poly ICLC) stimulated a broad cytic receptor, the first such receptor antigen-specific T reg cells capable of pre- innate immune response mimicking that identified on DCs in situ (Jiang et al., venting graft versus host disease in mice. induced by a live viral vaccine. 1995). By incorporating antigens into Whereas inducing tolerance in fully Ralph was that rare scientist who monoclonal antibodies specific for DEC- developed lymphocytes formerly re- lived through a period in which his en- 205 and administering these antibodies quired high doses of nontargeted anti- deavors came to fruition. He experi- to living animals, Daniel Hawiger, a stu- gens and met with limited success, the enced a revolution in immunology and dent in Michel’s laboratory, first showed targeted delivery of antigen to DCs both performed and witnessed work that that targeting antigens to DCs increased more efficiently and specifically silenced explained how DCs provide the physio- the efficiency of presentation by 100- the immune system. logical means to initiate clonal expan- fold (Hawiger et al., 2001). Although sion, bring about tolerance, differentiate the potency of DCs after antigen cap- lymphocytes into disease-fighting cells, ture had been emphasized in the past, DC-based vaccines and develop memory, each in an anti- Ralph and Michel’s work on antigen From the beginning, Ralph planned to gen-specific manner. Ralph was excited handling revealed that this function of take DCs into medicine. His deep in- when he found novel cells in 1973, and DCs can be dramatically improved in sights into the biology of DCs and his he died convinced that these cells had intact animals. interest in the control of immunity led become a novel force in medicine. him to explore two novel approaches to Immune tolerance stimulate innate immunity. Carol L. Moberg ([email protected]) is a Beginning in 2000, in close collabora- One strategy was an active immuni- senior research associate in the Steinman laboratory who has written extensively about tion with Michel and Hawiger, Ralph zation against advanced cancer. Madhav biomedical science. Her biography of René Dubos uncovered roles for DCs in controlling Dhodapkar, Ralph, and others devel- was published by ASM Press in 2006 and her immune tolerance (Hawiger et al., 2001; oped ways to remove DCs from a pa- history of the pioneering cell biology laboratories Steinman and Nussenzweig, 2002). To tient, charge them with specific antigens, at the Rockefeller University will be published by analyze tolerance, Ralph took an ap- and deliver them back into the patient the Rockefeller University Press in 2012. proach first suggested by their discovery (Chang et al., 2005). This approach, re- Michel Nussenzweig, Svetlana Mojsov, and that immature DCs induce antigen- sulting in expanded and sustained T cell Maggi Witmer-Pack made valuable contri- specific tolerance in humans (Dhodapkar responses, recently approved by the US butions to this manuscript. et al., 2001). Ralph studied two forms Food and Drug Administration, is now of antigen. One consisted of peptides being used by many other laboratories. and proteins incorporated into antibod- The other vaccine strategy harnesses REFERENCES ies that recognize DEC-205, which is the major features of DCs—location, Boscardin, S.B., J.C. Hafalla, R.F. expressed on DCs in lymphoid tissues presentation, maturation, and forma- Masilamani, A.O. Kamphorst, H.A. (Hawiger et al., 2001). The other con- tion of subsets—in situ. It involves en- Zebroski, U. Rai, A. Morrot, F. Zavala, sisted of antigen within dying cells gineering disease-relevant antigens into R.M. Steinman, R.S. 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