Immunity's Memories, Lost and Found

Courtesy of Mark Jenkins SLICED MICE: Single-cell-thick sections of adult mice expressing CD45.1 stained with nuclear dye (blue) and a monoclonal antibody specific for CD45.2 (red). The left panel shows a background level of CD45.2 staining. The center panel shows a mouse that received several million Salmonella peptide-specific CD4 T cells from a transgenic mouse expressing CD45.2. Transferred naïve T cells were found only in secondary lymphoid organs. At right, when also injected with Salmonella peptide three days prior to staining, CD4 T cells proliferated and migrated into nonlymphoid organs. Scientists have long believed that immunological memory--the record of infections past--is maintained by highly differentiated T cells and B cells that were generally thought to reside in the spleen and lymph nodes, the secondary lymphoid organs. Thus, the lymph nodes and blood (considered a surrogate for the spleen) are typically used to probe the long-term effects of immunological challenge. But as it turns out, these memory seekers may have been looking in all the wrong places. In this issue's Hot Papers, two research teams, using different but complementary techniques, discovered that nonlymphoid tissues such as the lungs and fat pads also harbor memory T cells. These cells seem to respond to antigenic rechallenge differently than their counterparts in the spleen and lymph nodes. Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. D. Masopust et al., "Preferential localization of effector memory cells in nonlymphoid tissue," Science, 291:2413-7, 2001. (Cited in 195 papers) R.L. Reinhardt et al., "Visualizing the generation of memory CD4 T cells in the whole body," Nature, 410:101-5, 2001. (Cited in 175 papers) "The magnitude of the populations of memory cells that you could find in these nonlymphoid tissues hadn't been appreciated," says Leo Lefrançois, of the University of Connecticut Health Center in Farmington and a principle investigator on one paper.1 "Our data showed that probably an equal number, if not a greater number, of memory cells were found in the nonlymphoid tissues at any time during the response to infection as there were in the lymphoid tissue." What Lefrançois' team found for CD8+ (cytotoxic) T cells, Marc Jenkins' University of Minnesota-Twin Cities team found for CD4+ (helper) T lymphocytes.2 The findings, says Jenkins, "really forced people to consider nonlymphoid tissue as a reservoir of memory cells." And that, says Susan Swain, director of the Trudeau Institute in Saranac Lake, NY, is one of the reasons that so many of her fellow immunologists have paid attention to the papers. "They didn't really think of [T] cells going to bizarre places like fat pads and other nonlymphoid sites," she says. It had been known for some time that activated cells, those that had recently encountered antigen, can migrate throughout the body. But these papers piqued the immunology community's interest for two reasons: First, not all the peripheral T cells Lefrançois and Jenkins looked at expressed the phenotypic markings of activation. Second, many had the characteristics of effector cells, which provide help to cytotoxic or phagocytic cells, or are themselves cytotoxic, much more so than even those seen in lymphoid tissues. VISUALLY STUNNING RESULTS In the years leading up to these publications, many labs had seen that the clonal expansion of T cells responding to an immunization peaked at about five to seven days. This was followed by a dramatic loss of antigen-specific cells from the lymphoid tissue, and finally stabilization in cell numbers. "We were wondering whether that loss phase could be explained partly, simply by exodus of cells from the lymphoid tissue into nonlymphoid tissue," Jenkins recalls. "We decided that to make the accounting exercise complete, we needed to sample the nonlymphoid body." There were, essentially, two ways to accomplish this goal. One was to "take out and grind up all the nonlymphoid organs, try to get the cells out, and see if we could find the antigen-specific cells there," Jenkins explains. The other was to "use a method that could sample everything at once," which is what his lab ended up doing. They refined a technique called whole-body thin sectioning to resolve single antigen-specific T cells. "[First author] Lee [Reinhardt] worked out a catalytic amplification system that gave him an intense signal from the primary antibody he was staining with," says Jenkins. By analyzing multiple sections through different animals, at different time points, he showed that "many of these cells appear in nonlymphoid tissue, even though none were there before immunization." The method is incredibly labor-intensive. Even after he had determined the ideal conditions for staining, Reinhardt still needed to take about 750 digital images for each section, and "jigsaw all those together in a precise way that created the total picture," Jenkins, his mentor, explains. "It took him four years to do that paper, basically." The results were "visually stunning," says Graham Le Gros, director of research for New Zealand's Malaghan Institute. Lefrançois' group took the other route to their findings, grinding samples from different tissues. They isolated lymphocytes from both lymphoid and nonlymphoid tissues of mice that had been previously infected with virus or bacteria, and used flow cytometry, staining for antigen-specific T-cell receptors and other markers, to determine the percentages of memory cells in different organs. Even 296 days after infection, virus-specific CD8+ T cells were seen in nonlymphoid tissues in significant numbers. Says Lefrançois: "We performed a major cataloging of the immune response to infection throughout the entire body, which hadn't been done before." CENTRAL AND EFFECTOR MEMORY To the surprise of Lefrançois and his fellow immunologists, the cells found in the periphery "had immediate effector function," he says. This helped to prove a hypothesis put forward a year and a half earlier by Antonio Lanzavecchia and his colleagues at the Basel Institute for Immunology in Switzerland. The hypothesis states that there are at least two functional phenotypes of memory cells.3 In a recent review, Lefrançois explains: "Central memory T cells are located in the secondary lymphoid tissues, have little cytolytic activity, and are proposed to have limited migratory potential. Effector memory T cells reside in nonlymphoid tissues, are cytolytic, and have promiscuous circulatory potential."4 Meanwhile, Jenkins says he was surprised to find that peripheral CD4+ cells made interferon-gamma (IFN-*), indicating that they, too, were effector cells. His group had spent considerable time analyzing lymphoid tissue, but previously could not find many IFN-*-producing cells. These cells should be plentiful, given that the mice were making IgG2a antibodies, an indication of IFN-* production. "Both papers inform us very clearly that we must look at the behavior of lymphocytes in their true, most relevant, context: peripheral tissues," says Le Gros. All the paradigms have been built up based on lymph node T cells, but behavior and properties of lymphocytes in peripheral tissues cannot be ignored. They are "very disease-relevant, and very relevant to the design of vaccines," Le Gros adds. When physicians or vaccine designers such as the National Institutes of Health's Robert Sedar want an indication of the human response to immunization, they typically look for effector cytokines such as IFN-* or interleukin-4 (IL-4), but only in the circulating lymphocytes. "So we're massively underestimating, probably, the effector response by limiting ourselves in humans just to blood," Sedar says. Lefrançois' and Jenkins' work "opened our eyes." Le Gros says that in his studies of asthma, much drug discovery is aimed at controlling the activation and proliferation of T cells in a lymph-node context, but this may be wrong. He and his colleagues have recently reported that activated T cells in the lymph nodes can migrate to the lung and airway. Once there, while able to produce considerable amounts of the asthmatic cytokine IL-4 upon antigenic challenge, they are completely unable to divide or migrate back to the lymph nodes.5 Swain's group has found that "there is continuum of effector heterogeneity, and only the most differentiated effectors can migrate into an inflammatory" or other nonlymphoid site.6 They suggest that these "heterogeneous effector populations may give rise to corresponding memory populations."7 The raison d'etre for extralymphatic effector lymphocytes is easy to guess. It just makes sense for antigen-specific effector cells to be where an infection is likely to recur. Many researchers now are teasing out additional differences between these and the cells in the lymphatic organs. Josh P. Roberts (tcwriter@msn.com) is a freelance writer in Minneapolis Minn. (Note: Marc Jenkins was a member of Roberts' PhD committee at the University of Minnesota.) References 1. D. Masopust et al., "Preferential localization of effector memory cells in nonlymphoid tissue," Science, 291:2413-7, 2001. (Cited in 195 papers) 2. R.L. Reinhardt et al., "Visualizing the generation of memory CD4 T cells in the whole body," Nature, 410:101-5, 2001. (Cited in 175 papers) 3. F. Sallusto et al., "Two subsets of memory T lymphocytes with distinct homing potentials and effector functions," Nature, 401:708-12, 1999. 4. K.S. Schluns, L. Lefrançois, "Cytokine control of memory T-cell development and survival," Nat Rev, 3:269-79, April 2003. 5. N.L. Harris et al., "Differential T cell function and fate in lymph node and nonlymphoid tissues," J Exp Med, 195:317-26, 2002. 6. S.L. Swain et al., "Regulation of memory CD4 T cells: Generation, localization and persistence," Adv Exp Med Biol, 512:113-20, 2002. 7. E. Roman et al., "CD4 effector T cell subsets in the response to influenza: heterogeneity, migration, and function," J Exp Med, 196:957-68, 2002. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Immunity's Memories, Lost and Found

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