Immunopathology Atul K

Chapter  Immunopathology Atul K. Bhan, Robert B. Colvin, and Harold F. Dvorak

he application of immunology to clini- and when, beginning in the 1980s, the unit rap- Tcal medicine and the development of immu- idly implemented diagnostic immunohistochem- nopathology as a specialized fi eld stem largely from istry into the routine diagnostic service; and (3) the pioneering work of the great immunologist/ from 1991 to the present, when the unit contin- pathologist Paul Ehrlich, who received the 1908 ued to develop new immunological diagnostic Noble Prize for his work on immune responses in tests and conduct basic research. infection (1). Th e introduction of immunofl uorescence techniques by Dr. Albert Coons in the Early Years: Before  1940s set the stage for establishment of immu- Th e earliest manifestations of the then-nascent nohistochemical techniques and development of fi eld of immunopathology at MGH were the fl ow cytometric analysis of cells. Dr. Coons, who studies of Dr. Louis Dienes, a bacteriologist who had been trained in internal medicine at the Mas- was recruited to the Pathology department in 1930 sachusetts General Hospital (MGH), performed (see fi gure 21.3; chapters 5 and 21). He showed that most of his work at Harvard Medical School simple proteins (ovalbumin) could elicit a “tuber- (HMS), initially in the Department of Microbi- culin reaction” in the skin if the animal (guinea ology and Immunology and later in the Pathology pig) had previously been sensitized by inoculation Department. Over the years, immunopathology of the protein into a tuberculous lesion (2). At the has applied the knowledge obtained from basic time only products of organisms were thought to immunology research to achieve a better under- trigger this type of response, termed a delayed- standing of disease pathogenesis, develop sophis- type hypersensitivity reaction and later shown ticated tests for accurate clinical diagnosis, and to be a T cell mediated reaction. With Dr. Tracy implement new therapeutic strategies. Mallory (chapter 6) in 1932 he characterized the Th e history of immunopathology at MGH morphology of the lesions and contrasted them will be described for three periods: (1) before with antibody-mediated reactions (Arthus reac- 1975, when immunopathology concepts were tion). Tuberculin sensitivity could also be pro- being developed and when the Immunopathol- duced by sensitization with protein alone (no ogy Laboratory was established; (2) from 1975 tuberculous infection), although it was milder to 1991, when the Immunopathology Unit was (Jones-Mote reaction). Also with Dr. Mallory, in formed and fl ourished under the leadership of 1937, he showed that tuberculin hypersensitivity Dr. Robert T. McCluskey (chapter 14) to become developed simultaneously with the mononuclear the dominant research unit in MGH Pathology, infi ltrate surrounding tuberculous lesions.

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Starting in 1951, Dr. Byron Waksman (fi gure 23.1), a neurologist at MGH, showed that anal- ogous delayed immune reactions to autologous proteins (myelin) could produce a rapid-onset form of disseminated encephalomyelitis in rab- bits that had been immunized with rabbit spinal cord, characterized by a perivenous infi ltration of “round” cells and neuronal demyelination, which had pathologic similarities to multiple sclerosis (3). Dr. Waksman devoted most of his career to an understanding of the pathogenesis of delayed hypersensitivity as it occurred, not only in the brain, but also in joints (a model of rheumatoid arthritis) and skin of animals of several species. His laboratory was on Warren 3, above the Pathology department, and he attracted several outstanding pathologists into the fi eld of immunopathology (chapter 17). One of those attracted was an HMS student, Harold F. (“Hal”) Dvorak, who was impressed by Dr. Waksman’s lectures and delighted to accept his joint off er, with Dr. Benjamin Castleman Figure 23.1 Byron Waksman (chapter 8), to take a post-sophomore pathology fellowship in 1959–1960, splitting time between attracted to the group on Warren 3, in his case by diagnostic pathology and research. Dr. Dvorak, Dr. Arnason’s lectures, and he spent the summer who worked on cell transfer studies and devel- of 1966, after his second year at HMS, studying opment of in vitro immune assays, considered the antigens of the hippocampus in the labora- the fellowship one of the most formative parts tory directly above Dr. Castleman’s offi ce. of his life. Dr. Waksman had a coterie of inter- Recognizing the increasing importance of national postdoctoral fellows in his lab at that immunology in the understanding and treat- time, including Branislov Jankovich (Yugo- ment of many diseases, Dr. Castleman decided to slavia), Barry Arnason (Canada), and Timo form an Immunopathology Laboratory in 1959 Kosunen (Finland), all of whom were working under the direction of Dr. Martin H. Flax (fi gure on aspects of cellular immunity. Dr. Kosunen 23.2), a protégé of Dr. Waksman. Th e concept of made use of the recently available isotope triti- immune mediated injury was extended to other ated thymidine to perform autoradiography and organs by Drs. Flax and Waksman in 1963 stud- demonstrated for the fi rst time that a substantial ies on adjuvant disease (an autoimmune arthri- fraction of cells accumulating in tuberculin-type tis) and in 1963–1966 studies on experimental reactions were actively dividing (4). Dr. Arnason allergic thyroiditis (5) with Drs. Stuart Sell and was one of the fi rst to demonstrate that neonatal James Billote; on allergic contact dermatitis with thymectomy severely compromised the immune Dr. James Caulfi eld; and on beryllium granu- system, so that rats were able to “tolerate” and lomatous hypersensitivity with Dr. Sidney Les- not reject grafts of foreign tissues. Dr. Robert B. kowitz in Medicine (6). As the early focus of the Colvin (chapter 25) was another HMS student laboratory was diagnostic renal biopsies, a close

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relationship developed between the Immunopa- basophil content, the reaction was called cuta- thology Laboratory and nephrologists as well as neous basophil hypersensitivity (CBH). Simi- the Renal Transplantation Unit. lar, basophil-rich reactions were found in other Dr. Vivian Pinn started working with Dr. Flax organs and tissues in response to an immune in 1967, when she joined MGH as a resident in challenge: for example, the eye in ocular hyper- Pathology (see fi gure 7.26). Dr. Pinn moved with sensitivity and human kidneys undergoing acute Dr. Flax to Tufts University Medical School in allograft rejection (8, 9). Dr. Dvorak remembers 1970, when Dr. Flax was made the Chairman of showing one-micron Epon slides to Dr. Castle- the Department of Pathology there. She remained man; he was impressed but asked for a hematoxy- involved in immunopathology of the kidney and lin and eosin (H&E) section of the same lesions subsequently became Professor and Chairman of and had no trouble detecting basophils! Pathology at Howard University College of Med- Th ese studies were extended to human cellular icine, the fi rst African American woman to chair immunity when Dr. Dvorak developed poison an academic pathology department. ivy on Martha’s Vineyard. He biopsied his rash Dr. Colvin, who at that time was a resident and found it was swarming with basophils. He in Pathology, worked on renal and transplant secured the help of Dr. Colvin and Dr. Martin pathology with Drs. Pinn and Flax and became C. Mihm Jr. of Dermatopathology (chapter 18) the resident expert in this area when they left to extend these studies to other contact allergens, for Tufts. Dr. Colvin was able to resuscitate an employing volunteers who were biopsied, includ- ancient Reichert fl uorescent microscope for ing the investigators. Dr. Ann Dvorak (Hal’s immunofl uorescence, which had to be done at night, since the room on Warren 1 had no shades on the windows. Dr. Dvorak had come back to MGH for an internship and residency in Pathology in 1963 and then, working with Dr. Flax, made important observations regarding immunological unre- sponsiveness induced by intravenous tolerizing antigen. After two years of research at NIH, Dr. Dvorak returned to the MGH in 1967 and began developing and directing immunopathology research. Dr. Dvorak used elegant one-micron Giemsa-stained Epon sections, prepared by his research assistant, Eleanor Manseau, to study the cellular details of delayed-hypersensitivity reactions by light microscopy (7). He was, therefore, for the fi rst time able to appreciate infi ltrating basophil leukocytes in tissues and their participation in a type of delayed-hypersensitivity reaction (Jones-Mote reaction) induced by immunization with protein antigens in incomplete Freund’s adjuvant (which were probably what Drs. Dienes and Mallory had created by injecting ovalbumin without tubercle bacillus). Because of its high Figure 23.2 Martin Flax

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wife), who had done a postdoctoral fellowship in research fellowship at Children’s Hospital, moved electron microscopy with Dr. Morris Karnovsky to MGH to pursue immunopathology research. at HMS and had helped demonstrate basophils as Th e Immunopathology Laboratory at that time an important feature of other cellular immunity was located on the fi rst fl oor of the Warren Build- in guinea pigs, also participated in the studies. ing. Dr. Elizabeth Hammond, who graduated Dr. Ann Dvorak showed through ultrastructural from the University of Utah School of Medicine studies the activation of microvascular endothe- in 1967, joined the lab fi rst as a research fellow lial cells and pericytes in the venules from which in 1972 and subsequently as a staff member in lymphocytes, basophils, and other infl ammatory 1974. Dr. Stephen J. Galli, who graduated from cells were extravasating, and the degranulation of HMS in 1973, joined the lab as a research fel- both basophils and mast cells as features of cellu- low in 1977. Both Dr. Hammond and Dr. Galli lar immunity. She found that basophils and mast had been residents in Pathology at MGH. At that cells in cellular immune reactions underwent time as well, Dr. Ann Dvorak was recruited from not the explosive degranulation characteristic of Tufts to establish a clinical and research electron anaphylaxis, but, rather, a partial type of piece- microscopy laboratory. meal degranulation in which granule contents In the 1970s MGH administration committed were released gradually over time by a vesicular new resources to the Pathology department, add- transport mechanism. Her concept of piecemeal ing much needed space for research on the fi fth degranulation is now widely accepted not only in and eighth fl oors of the recently built Cox Build- basophils but also in degranulating eosinophils, ing. A committee composed of Drs. Hal and Ann which she subsequently studied after moving Dvorak, Bhan, Hammond, and Mihm designed to Beth Israel Hospital in 1979. One important the Cox space for research as well as for diagnos- observation made in these studies was that fi brin tic immunopathology and electron microscopy. deposited outside the vessels was related to the Th e highly functional open design has been induration, a characteristic feature of tubercu- copied by the other major research laboratories lin hypersensitivity (10). Proof that fi brin was at MGH (e.g., the Wellman/Th ier, Martin, and responsible for causing induration came from Simches buildings). Th e fi fth-fl oor Immunopa- subsequent studies by Dr. Colvin in two patients thology Unit was completed by January 1976 and with congenital afi brinogenemia; both developed the eighth-fl oor animal facilities in April 1977. erythematous delayed reactions with a typical Th e only glitch was that the electron microscopes cellular infi ltrate but lacked fi brin deposition and were installed over the massive ventilation fans induration. bolted to the ceiling of the fourth fl oor; the solu- tion was to suspend the microscopes on bungee –: The McCluskey Era cords attached to a 3-inch-thick iron plate. As Dr. Dvorak’s studies were progressing, Dr. Th e new laboratories in the Cox Building Robert McCluskey succeeded Dr. Castleman provided the infrastructure for the development as Chief of Pathology (chapter 13). During Dr. of the nationally recognized Immunopathology McCluskey’s tenure at Children’s Hospital in Bos- Unit. Th e unit, directed by Hal Dvorak, was ton (1971–1974), he established immunopathol- staff ed initially by Drs. Bhan, Hammond, John ogy research and developed collaborations that Long, Galli, Neil Orenstein, and Colvin, who continued after his move to MGH. In 1975 Dr. had returned to MGH after working as an experi- Atul K. Bhan, who had been a postdoctoral fel- mental pathologist at Walter Reed Army Institute low with Dr. McCluskey following completion of of Pathology from 1972 to 1975. Dr. Hammond Chief Residency in Pathology and a postdoctoral studied macrophage migration inhibitory factor,

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Brigham and Women’s Hospital (9). Dr. Dvorak found that supernatants from nearly all human and animal tumors generated intense blue spots in the Evans blue dye permeability assay, whereas those from several normal cells did not (fi gure 23.4). He called this tumor supernatant permeabi- lizing activity vascular permeability factor, or VPF (11). Subsequently, VPF was shown to be a weak endothelial cell mitogen, was renamed vascular endothelial growth factor (VEGF), or, more specifi cally, VEGF-A, and became a therapeutic target in cancer and macular degeneration. Subse- quent studies revealed that VPF/VEGF was over- expressed by most malignant tumors and at lower levels in a number of normal adult tissues. After moving to Beth Israel Hospital in 1979 (taking with him his wife, Ann, and Stephen Galli), Dr. Dvorak continued his work and found that VPF/ VEGF had a central role in wound healing and chronic infl ammatory diseases such as psoriasis, rheumatoid arthritis, and induced lymphangio- genesis as well as angiogenesis; others discovered Figure 23.3 Harold Dvorak working in the that it was essential for the development of the Immunopathology Laboratory, Cox 5, late 1970s normal vasculature. Th us, Dr. Dvorak’s discovery of VPF resulted not only in the fi nding of and she and Dr. Colvin got their fi rst R01 grant a new molecule but also in the elucidation of a together on the clotting system in delayed-type process—one that, although not yet completely hypersensitivity. She soon left to accompany her understood, is responsible for initiating stroma husband to Salt Lake City, however, where she formation and tissue repair following injury. became Professor of Pathology at the University Unfortunately, this same process also facilitates of Utah and Chair of the Department of Pathol- the growth and spread of malignant tumors, as ogy at the LDS Hospital. Dr. Galli and his wife, well as having central roles in diabetic retinopa- Anne, continued the work on basophils with Hal thy and several types of infl ammatory diseases. Dvorak, demonstrating that purifi ed guinea pig Because of Dr. McCluskey’s research interest in basophils were able to synthesize histamine. Dr. renal diseases, the immunopathogenesis of kid- Galli subsequently focused his research on mast ney diseases became a major research and diag- cells, and he became one of the world’s leading nostic focus of the Immunopathology Unit (fi g- authorities on mast cell biology; he moved from ure 23.5). He built a strong research team, includ- Beth Israel to become Chair of the Department ing Dr. Colvin, Dr. Bhan, and Bernard Collins of Pathology at Stanford University School of to study mechanisms involved in renal diseases. Medicine in 1999. Dr. Eveline E. Schneeberger, who had worked Dr. Hal Dvorak (fi gure 23.3) extended his previously with Dr. McCluskey at Children’s studies of the immune response to tumors and Hospital, joined the team in 1979. Drs. Bhan collaborated with Dr. W. Hallowell Churchill at and McCluskey explored novel mechanisms of

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developed many tests that were off ered as part of the departmental reference laboratory in renal pathology, including the Western blot assay for the detection of antibodies to glomerular basement membrane (GBM), which is the defi nitive diagnostic test for anti-GBM antibodies. Immu- nofl uorescence studies were also applied for the diagnosis of skin disorders, including indirect immunofl uorescence tests for the detection of circulating autoantibodies in patients with bul- lous lesions of the skin. Dr. John Niles, a nephrologist, started postdoctoral research training with Dr. McCluskey in 1985. Together they investigated the role of anti- neutrophil cytoplasmic autoantibodies (ANCA) in the pathogenesis of Wegener’s granulomatosis. Th ese studies led to identifi cation of one of Figure 23.4 Harold and Ann Dvorak in front of the the ANCA autoantigens, proteinase-3, and the research poster describing tumor secreted mediators, development of quantitative diagnostic assays. Cox 5, late 1970s Dr. Niles has achieved international recognition as an expert in ANCA-related diseases and glomerulonephritis. By adoptive transfer experi- directs the MGH Pathology reference laboratory ments, they conclusively showed that T cells for ANCA testing (14, 15). could induce glomerular injury against a planted Robert Colvin became the Director of the glomerular antigen, challenging the dogma that Immunopathology Unit in 1979. In Dr. Colvin, T cells had no role in glomerulonephritis (12). Dr. McCluskey found a colleague with a com- Dr. McCluskey recruited Dr. Lynn Baird to mon interest in renal pathogenesis and develop- identify the antigen of membranous glomeru- ing a strong diagnostic renal pathology program. lonephritis, a goal he had long pursued. Th ese While a house offi cer in Pathology at MGH, Dr. studies and a subsequent collaboration with Dr. Colvin had become interested in renal and trans- John Smith led to the identifi cation and clon- plant pathology. Dr. Paul Russell, a distinguished ing of an autoantigen located in rat renal tubules surgeon who established the MGH Transplant and podocytes (megalin), which was the target in Service, became a mentor and longtime friend Heyman nephritis (13), a rat model of membra- in this fi eld. From 1971 to 1972, Dr. Colvin was nous glomerulonephritis (MGN); however, this an NIH Research Fellow under the mentorship antigen did not prove to be the human autoan- of Dr. Harold Dvorak, working on the role of tigen they were seeking. Dr. McCluskey contin- the clotting system in T cell mediated immune ued to work on this theme after his retirement as reactions. Dr. Colvin extended the studies to the chief in 1991 and even secured an NIH grant to role of fi bronectin in infl ammatory reactions and support his studies. Dr. Collins and Colvin con- wound healing with a fellow, Dr. Richard Clark; tinue working on the target antigens of MGN in together they defi ned the concept of a “provi- 2011. Collins also took a major initiative in devel- sional matrix” (16). Dr. Colvin worked on the oping and expanding the application of immu- pathogenesis of autoimmune tubulointerstitial nofl uorescence in human renal biopsies and diseases, continuing the work on anti-tubular

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had close associations with members of the MGH Pathology staff (chapter 7). Both Benja- min Castleman and Walter Putschar had visited AIIMS to help the development of the Pathology department. Dr. Bhan’s research experience while at Children’s Hospital from 1972 to 1975 and collaborative studies with Dr. Stuart Schlossman at Dana-Farber Cancer Institute set the foundation for his future work on lymphoid cells in normal and diseased states. One of the important fi ndings of these studies was the demonstration that T cells, but not B cells, migrate to rejecting allografts (18). Th e development of hybridoma-derived monoclonal antibodies in 1975 had a profound infl u- ence on all aspects of biological sciences, including immunopathology. One of the immediate eff ects was on the characterization of normal and neoplastic hematopoietic cell lineages. Dr. Bhan Figure 23.5 Robert McCluskey (left) with Richard performed important early work to identify and Dickersin (looking through electron microscope) and Ann Brescia (electron microscopy technician), characterize lymphoid cell diff erentiation and Cox 5, late 1970s dendritic cells in the lymphoid tissues and at the infl ammatory sites (19, 20). Dr. Sibrand Poppema, basement membrane disease and in transplan- a visiting fellow from the University of Groningen, tation. His close interactions with Dr. Benedict participated in the initial studies to identify stages Cosimi and other members of the renal trans- of T cell and B cell location and diff erentiation in plant group resulted in seminal observations the lymphoid tissues (19); Dr. Poppema, interna- and publications and recognition of MGH as a tionally recognized for his research on Hodgkin’s major center of renal transplantation. Dr. Colvin disease, became Professor of Pathology at the Uni- was a key member of the team that tried the fi rst versity of Groningen in 1995. Dr. George Murphy therapeutic monoclonal antibody, OKT3, in a (chapter 18), a resident in Pathology at MGH and patient who developed acute renal allograft rejec- a trainee of Dr. Mihm’s, carried out studies on the tion on October 20, 1980 (17). Th e team gath- analysis of Langerhans cells and histiocytosis X ered around the prototype fl ow cytometer oper- with monoclonal antibodies in collaboration with ated by Dr. Colvin the evening after the fi rst dose Drs. Bhan and Terence Harrist (21). and observed that all the T cells had disappeared In 1981 the Diagnostic Immunoperoxidase from the circulation within an hour of treatment. Laboratory was established by Dr. Bhan with Someone in the fl ow cytometry room quoted Dr. the help of Bruce Kaynor, a talented and dedi- John Collins Warren (chapter 1) and said, “Gen- cated technician. Dr. Nancy L. Harris (see fi g- tlemen, this is no humbug!” ure 24.9), who had joined the Immunopathol- Atul Bhan had been trained in anatomic and ogy Unit in 1978 as a research fellow, published experimental pathology at the All India Insti- several important papers with Dr. Bhan on tute of Medical Sciences (AIIMS), New Delhi, immunohistochemical characterization of lym- under Dr. Vulimiri Ramalingaswami, who had phoid cells for the diagnosis and classifi cation of

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lymphoid malignancies (22). She continued to allograft rejection and tumor infi ltrating lym- apply immunohistochemistry to hematological phocytes. Many of Dr. Bhan’s studies were sup- malignancies and achieved national and interna- ported by an NIH grant to study immunocom- tional recognition (chapter 16). Subsequently, the petent cells infi ltrating human breast cancer. application of immunohistochemical techniques Dr. James T. Kurnick joined the staff of the extended to all specialties of surgical pathology. Immunopathology Unit in 1980, following Dr. Salim Kabawat, who joined the Immunopa- pathology training at the University of Colo- thology Unit as a research fellow in Dr. Colvin’s rado and postdoctoral work in immunology in laboratory in 1983, described the reactivity of a Denver and Sweden. His work cloning human T monoclonal antibody, OC125, produced against lymphocytes, including the fi rst propagation of an ovarian tumor by Dr. Robert Bast at Dana- helper T cells in any species, brought him to the Farber Cancer Institute (23). (As a medical stu- MGH; here he extended his work with normal dent, Dr. Bast had spent time in MGH Pathol- T cell responses to soluble and cellular antigens, ogy; chapter 7.) Th e antigen CA125 recognized by including work propagating activated T lympho- the antibody has become an established marker cytes from sites of infl ammation in transplants, of ovarian tumors, and the serum levels of this myocarditis, rheumatoid arthritis, pyelonephri- antigen are used for diagnosis and monitoring of tis, and cancers. Th e work led to functional and patients with some forms of ovarian cancer. genetic studies on cells isolated from infl amed Th e refi nement of immunohistochemical tissues, including the demonstration of clonal techniques and introduction of antigen retrieval dominance of T cell infi ltrates in several condi- methods in the 1980s made it possible to iden- tions, such as rheumatoid arthritis and tumors tify an increasing number of tumor markers with (24, 25). Dr. Ivan Stamenkovic (see below) par- antibodies that were preferentially selected for ticipated in the studies of rheumatoid arthritis. their ability to recognize antigens in routinely Dr. Richard Kradin, who had a joint clinical fi xed specimens in surgical pathology. As Direc- appointment in both Pathology and Medicine tor of the Immunohistochemistry (Immunoper- (Pulmonary Unit), joined Dr. Kurnick’s research oxidase) Laboratory, Dr. Bhan took a leading role and developed a protocol in which patients with in integrating immunohistochemistry into surgi- lung cancer were treated with in vitro expanded cal pathology and selecting panels of antibodies tumor infi ltrating lymphocytes (fi gure 23.6). Th is that allowed characterization of tumor cells, not led to the treatment of advanced melanomas only by their morphologic features, but also by and non–small cell lung cancers with infusion their molecular profi le. His interactions with the of autologous tumor infi ltrating lymphocytes Surgical Pathology staff led to the development (26). Dr. Kurnik’s work on the immune response of strategies for the characterization of a wide to cancers has continued in recent years with spectrum of tumors, including hematopoietic, the realization that tumors can escape immune soft tissue, endocrine, and metastatic epithelial detection and destruction by the reversible loss of tumors (unknown primary); perhaps the most targeted diff erentiation antigens (27). Following signifi cant eff ect was in the diagnosis of poorly a decade in the Immunopathogy Unit on Cox diff erentiated malignant tumors. 5, Dr. Kurnick led a departmental expansion to Th e availability of monoclonal antibodies the new research facilities (Pathology Research at to functionally distinct lymphoid cells had an MGH East) in Charlestown. immediate infl uence on the immunohistochemi- Dr. Kradin continued his work in pulmonary cal characterization of a wide variety of infl am- immunology and interstitial lung diseases (28, matory conditions. Th e analysis also included 29), addressing the T cell, dendritic cell, and

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Medicine in Ottawa; Robert Burton, collabora- tor in the early OKT3 studies, who went from being a fellow at MGH to Professor of Surgery at Newcastle, Australia; and Anthony Warrens, now a Professor of Medicine at Hammersmith Hos- pital in London. Dr. Frederic I. Preff er, who had research training at Roswell Park Memorial Insti- tute at Buff alo, joined the Immunopathology Unit as Dr. Bhan’s postdoctoral fellow in 1982. He made fl ow cytometry his fi eld and found the technology fascinating (30); he later described his experience as “love at fi rst sight.” He was chosen to direct the Clinical Flow Cytometry Lab, which was established 1985, and he directs it to this day. Figure 23.6 Richard Kradin (left) and James Kurnick, He also directs the Research Flow Cytometry Immunopathology Laboratory, Cox 5, 1980s Core, which has numerous state-of-the-art fl ow sorters and analyzers, and a premier research/ macrophage responses to inhaled antigens and cell sorting lab in the MGH research campus in migratory properties of dendritic cells, especially Charlestown as well as a facility in the Simches to local lymph nodes, and edited a textbook, Building on the main campus (31, 32). Immunopathology of Lung Disease. Five of Dr. Dr. Nadine Cerf-Bensussan, a postdoctoral Kradin’s laboratory research fellows, including research fellow from France, joined Dr. Bhan’s Drs. Patricia Finn and Steven Dubinett, subse- laboratory in 1981 to pursue research in mucosal quently pursued successful research careers and immunology. She identifi ed a molecule expressed are currently department chairmen in the United exclusively by intraepithelial lymphocytes (IEL) States and internationally. in rats (33) and subsequently in human IEL Th e Flow Cytometry Laboratory was started (αΕβ7, CD103), an integrin that is involved in in 1978 by Dr. Colvin with a gift of a FACS II cell the interaction with intestinal epithelial cells sorter from the Arthur D. Little Company. Ini- by binding to E-cadherin. Dr. Cerf-Bensussan tially it was housed in an unused animal room on is now an internationally recognized mucosal Cox 8 before it moved to new facilities on War- immunologist. Dr. Gary Russell, a pediatric gas- ren 5. Flow cytometry was used initially in the troenterologist working with Dr. Bhan, extended evaluation of tumor ploidy as well as for detec- these studies by making several similar antibodies tion of lymphocyte surface antigens with mono- against mucosal lymphocytes propagated from clonal antibodies. In the early 1980s, through col- intestinal biopsies from patients with celiac dis- laboration with Ortho Diagnostics, a prototype ease (34). Monoclonal antibodies specifi c to rat automated fl ow analyzer was installed and used Kupff er cells made in Dr. Bhan’s laboratory with for pioneering work to characterize circulating T the help of Drs. Richard Moscicki (who had been cell subsets with the new monoclonal antibod- a research fellow in the unit) and Kurt Bloch, ies. Among the research fellows working with Dr. Director of Clinical Immunology, confi rmed the Colvin whose careers were infl uenced by their hypothesis that cells express unique functionally fl ow cytometry studies were Dr. Andrew Lazarov- important molecules depending on their loca- its, who created the monoclonal antibody Act-I tion and environment. Dr. Masafumi Maruiwa, to α4β7 integrin and later became a Professor of a postdoctoral fellow from Japan in Dr. Bhan’s

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laboratory, demonstrated in collaboration with selected primarily from the MGH Pathology Dr. Amin Arnaout of the MGH Renal Unit that Training Program and clinical services, but there one of these antibodies recognized a unique com- were also individuals trained at other institutions. plement receptor on Kupff er cells (35). Early trainees included Drs. Galli, Harris, James Th e infl uence of immunopathology on diag- Faix, Raymond Sobel, Dobri Kiprov, Th eodore nostic pathology grew in the 1970s and 1980s. Mayer, Kradin, Andrew Rosenberg, Moscicki, Th e U.S. and Canadian Academy of Pathology Preff er, Niles, and Johnson Wong. Dr. Sobel (USCAP) invited Drs. McCluskey and Colvin to (chapter 17) joined the Immunopathology Unit organize a special course entitled “Immunopatho- in 1981 as a research fellow with Dr. Colvin, after logic Techniques in Diagnostic Pathology.” Th e completing neuropathology training at Stanford. course was fi rst given by the members of the unit Th e focus of his research was immune responses at the annual meeting of the society in Boston in in the brain, including experimental allergic 1976 and subsequently every two years from 1977 encephalomyelitis (EAE), which extended Dr. to 1987. Th e success of the course led to the pub- Waksman’s work (37). Dr. Sobel returned to lication in 1988 of a textbook entitled Diagnostic Stanford in 1992. Immunopathology, edited by Drs. Colvin, Bhan, and McCluskey. A second edition of the book,  to Present containing a more comprehensive review of immu- In 1991 Dr. Colvin succeeded Dr. McCluskey nopathologic mechanisms and diseases, transplan- as Chief of the Pathology Service at MGH (fi g- tation, immunohistochemistry of tumors, and ure 23.7). Dr. Colvin appointed Dr. Bhan the techniques, was published in 1995 (36). Director of the Immunopathology Unit, which Immunopathology was approved by the at this time was composed of research laborato- American Board of Medical Specialties in 1983. ries staff ed by Drs. Bhan (mucosal immunology), Dr. McCluskey was among the fi rst to be certi- Kradin (pulmonary immunology and infl amma- fi ed and become a member of the test committee. tion), Niles (ANCA), Schneeberger (permeability Subsequently, both Drs. Colvin and Bhan were properties of the air/blood barrier in the lung), certifi ed and served on the test committee. By Preff er (fl ow cytometry), Wong (immunother- 1999 the American Board of Pathology stopped apy of HIV infection and transplant rejection), issuing Special Certifi cation in Immunopathol- and Colvin (transplantation immunology). Dr. ogy; since every trainee in pathology needed to G. Richard Dickersin, who had succeeded Ann know immunological mechanisms and immu- Dvorak as Director of the Diagnostic Electron nology-based laboratory tests, the Board decided Microscopy Laboratory (chapter 16), shared the to include immunopathology as a part of Ana- electron microscopic area of the Cox 5 laboratory tomic and Clinical Pathology certifi cation. space with Dr. Schneeberger. Th e NIH general postdoctoral research train- Dr. Colvin continued to be involved in trans- ing program that started in the Pathology depart- plant research; among his notable research con- ment in 1957 served as a forum for enlisting train- tributions were the identifi cation of the diagnos- ees in immunopathology as well as other areas. tic criteria for acute antibody-mediated rejection Support for research training in immunopathol- and the discovery of chronic antibody-mediated ogy beginning in 1980 was largely through an rejection, using peritubular capillary deposi- NIH institutional training grant titled “Immu- tion of the complement fragment, C4d, as a key nology and Tumor Biology,” submitted originally marker (38, 39, 40, 41). His criteria have been by Harold Dvorak; Dr. Colvin was the fi rst pro- accepted by the Banff schema and have become gram director. Th e fi ve concurrent trainees were incorporated into standard clinical practice. Dr.

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Figure 23.7 Farewell to Robert Colvin as Director of Immunopathology Unit upon becoming Chief of Pathology, Cox 5 staff , 1991. Front row, seated, left to right: Atul Bhan, Robert Colvin, Richard Dickersin, Eveline Schneeberger. First row standing: Bruce Kaynor, Chen-Kwen Hsuing, Guoli Pan, Wei Lin. Second row standing: Luba Zugachin (behind Atul Bhan), Ruth Manozzi, Wei Jia Xia, Colleen Hagan (partially hidden), Marcia Levy, unidentifi ed, Chris Howard. Th ird row standing: John Niles, Masafumi Mauiwa, Gary Russell (partially hidden behind Wei Jia Xia), Richard Kradin, Bernard Collins, Volker Nickelit (behind Marcia Levy), Malgorzata Stronska, unidentifi ed, Gertrude Fondren (behind Chris Howard). Back row: Fred Preff er (behind Gary Russell), Ray Sobel (behind and right of M. Stronska). Colvin was one of the initial authors on the Banff Th e establishment of liver transplantation pro- classifi cation in 1993, and he continues as a leader gram in 1983 extended Dr. Bhan’s collaboration of this biannual international meeting that pro- with Dr. Jules Dienstag (MGH Gastrointestinal motes scientifi c progress and standardization of Unit), with whom he had investigated the immu- practice in transplantation. In studies with Dr. nopathogenesis of primary biliary cirrhosis and Paul Russell, Dr. Colvin showed that antibody is viral hepatitis (43). Th is collaboration also led to suffi cient to initiate chronic vascular rejection in Dr. Bhan’s participation in an NIH-sponsored mouse heart transplants, without a necessary par- multicenter clinical trial to evaluate therapy for ticipation of T cells (42). More recently, Dr. Col- hepatitis C. vin has collaborated with Drs. Sachs and Cosimi Th e development of knockout and transgenic on a series of preclinical trials of induction of tol- mice in the early 1990s revolutionized the way erance to kidney allografts by combined donor immunologically mediated reactions and diseases bone marrow transplantation. He also has an could be studied. Dr. Bhan’s collaboration with NIH-funded program on regulatory T cells in Dr. Peter Mombaerts in Dr. Susumu Tonegawa’s mouse kidney graft acceptance and in vivo imag- laboratory at MIT led to the recognition that ing of graft-infi ltrating cells. colitis could develop spontaneously in mice

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defi cient in T cells (44). Similar observations in investigator and continued his research in the new other immunodefi cient mice formed the basis immunopathology facilities on Warren 5 until for developing models of human infl ammatory 2002, when he returned to the Clinical Immu- bowel disease (IBD) and contributed to the for- nology Unit. He developed anti-CD3:anti-CD8 mulation of the widely accepted hypothesis that bifunctional antibodies to eliminate the replica- the two major forms of IBD, namely, Crohn’s tion component of virus and improvement of disease and ulcerative colitis, develop owing to immunologic defi cit in HIV-1 infected patients, dysregulated immune responses to normally resi- and as a therapeutic protocol for prolongation of dent enteric microfl ora. Dr. Atsushi Mizoguchi allograft survival with low systemic toxicity. Dr. and his wife, Dr. Emiko Mizoguchi, postdoctoral James A. MacLean, who had also been trained in fellows in Dr. Bhan’s laboratory, performed stud- Immunology/Allergy at MGH, participated in ies in these T cell receptor knockout mice, which these studies as a research fellow in the Immuno- resulted in long-term research support from pathology Unit from 1989 to 1992 (49). NIH. Th e most signifi cant fi ndings included the In 1994 the Residency Review Committee for recognition of a pathogenic role of cytokines in the Accreditation Council for Graduate Medi- colitis, the demonstration of a suppressive eff ect cal Education approved the Clinical Training of appendectomy on colitis development (45), Program in Immunopathology, directed by Dr. and the identifi cation of regulatory B cells and Bhan. Th e comprehensive training program their suppressive role in chronic mucosal infl am- included rotations through the Clinical Immu- mation (46). Dr. Bhan helped Dr. Daniel Podol- nology Laboratory under the supervision of Dr. sky, head of the MGH Gastrointestinal Unit, Kurt Bloch and, for molecular biologic training, establish an NIH-funded Center for the Study of the Pathology Research Laboratory at MGH Infl ammatory Bowel Disease (CSIBD) at MGH East under the supervision of Ivan Stamenkovic. in 1990. Th is is a nationally and internationally Th e fi rst trainee of the program was Dr. R. Neal recognized research center that helps investiga- Smith, who entered after his residency in Pathol- tors in their IBD-related research and provides ogy at MGH. He is now Associate Professor pilot feasibility grants. of Pathology; his primary interests are in renal Dr. Schneeberger’s research focused on two pathology and transplantation. As a research fel- themes supported by NIH grants. Th e fi rst low he had participated in the IBD studies in Dr. relates to examination of the molecular structure Bhan’s laboratory and helped in the identifi ca- and regulation of tight junctions as they per- tion of regulatory B cells. As a faculty member tain to permeability properties of the air/blood he became an investigator at the Harvard Center barrier in the lung (47). Th e second area is the for Islet Transplantation and provided pathology biology of dendritic cells in the lung (48). Th e services to the MGH Transplantation Biology studies include analysis of tricellulin in regulating Research Center. Dr. Shamila Mauiyyedi, who the migration of dendritic cells across airway and was a postdoctoral fellow with Dr. Colvin, work- alveolar epithelium in an ovalbumin-induced ing on research related to transplantation, com- model of asthma in mice. She also has partici- pleted immunopathology training in 1999, after pated in the diagnostic renal pathology service which she joined the faculty of the University of for many years. Texas at Houston Medical School. Dr. Johnson Wong, who had been trained in Dr. Stamenkovic, who had graduated from the Immunology/Allergy at MGH, joined the Immu- University of Geneva School of Medicine in 1978, nopathology Unit as Dr. Colvin’s research fellow and had been a research fellow in the Immunopa- from 1983 to 1985. He became an independent thology Unit and Molecular Biology Department

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Figure 23.8 Robert McCluskey’s eightieth birthday celebration, 2003. Left to right: Atul Bhan, Bernard Collins, Robert McCluskey, Eveline Schneeberger.

from 1985 to 1988, carried out work on CD44’s Dr. James Stone, who had been recruited from eff ect on tumor growth and migration (50) and the Brigham and Women’s Hospital to lead Cardio- role of CD22 in lymphocyte adhesion and activa- vascular Pathology (chapter 16), joined the group. tion (51). He was appointed Director of Pathol- In 2005 Drs. Bhan’s and Stone’s research labora- ogy Research at MGH East in 1994. He went to tories moved to the eighth fl oor of the Richard Switzerland in 2001 as Professor of Experimental B. Simches Research Center. Th is move allowed Pathology at the University of Lausanne, where Dr. Atsushi Mizoguchi to develop an indepen- he became chairman of the department in 2009. dent research laboratory. Dr. Colvin’s labora- In the late 1990s immunopathology research at tory expanded and, as the Immunopathology the Pathology research laboratory was carried Research Unit, moved to newly renovated space out by Drs. McCluskey (fi gure 23.8), Giuseppe on the eighth fl oor of the Th ier Building. Andres, and James Kurnick. Dr. Andres, a visiting Over this period, the Immunopathology Unit, Professor of Pathology, had a long collaborative with its busy immunohistochemistry, immu- research relationship with Dr. McCluskey dating nofl uorescence, fl ow cytometry, and ANCA from their days together in Buff alo (chapter 14). laboratories, became primarily engaged in diag- Th eir collaborations continued at MGH East and nostic testing. Th ese diagnostic activities grew involved the study of pathogenic mechanisms in dramatically in volume and sophistication. Th e a model of anti-GBM nephritis in rats. clinical volume of the Clinical Flow Cytometry In 2000 the Immunopathology Unit was Laboratory increased from 44 to 9,000 cases per moved to the renovated fi fth fl oor of the Warren year. Th ese studies include eight-color analyses Building. Dr. Schneeberger’s lab moved to the of lymphoid cells, leukocytes, and stem cells in Pathology Research Laboratory at Charlestown. normal, immunodefi cient, and malignant states.

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Th e immunofl uorescence studies, both direct leukoencephalitides with experimental allergic and indirect (for detection of circulating autoan- encephalomyelitis. J Neuropathol Exp Neurol tibodies), continued to expand; they now involve 21:491–518, 1962. more than 1,000 cases each year. Th e renal biopsy 4. Kosunen TU, Waksman BH, Flax MH, Tihen reference laboratory, under Dr. Colvins’s direc- WS. Radioautographic study of cellular mecha- tion and Collins’s technical supervision, provides nisms in delayed hypersensitivity. I. Delayed reactions to tuberculin and purifi ed proteins in the rat light, immunofl uorescence, and electron micros- and guinea pig. Immunology 6:276–290, 1963. copy services for 400–500 renal biopsies per year, 5. Flax MH. Experimental allergic thyroiditis in the and the unit performs serological testing on over guinea pig. II. Morphologic studies of develop- 8,000 ANCA and 800 anti-glomerular basement ment of disease. Lab Invest 12:199–213, 1963. membrane antibodies samples. Th e Immuno- 6. Dvorak HF, Simpson BA, Flax MH, Leskowitz S. histochemistry (Immunoperoxidase) Laboratory, Th e fate of antigen in delayed hypersensitivity skin under Dr. Bhan’s direction for most of this period, reactions. J Immunol 104:718–727, 1970. grew from a menu of fewer than 10 immunoper- 7. Dvorak HF, Dvorak AM, Simpson BA, Richer- oxidase stains in 1981 to over 200 stains in 2007, son HB, Leskowitz S, Karnovsky MJ. Cutaneous when the laboratory became fully automated. Th e basophil hypersensitivity. II. A light and electron most signifi cant eff ect of immunohistochemistry microscopic description. J Exp Med 132:558–582, on tumor diagnosis and classifi cation happened 1970. in the 1990s and led to numerous publications 8. Dvorak HF, Mihm MC Jr., Dvorak AM, Barnes BA, Manseau EJ, Galli SJ. Rejection of fi rst-set and presentations by both staff and trainees at skin allografts in man. Th e microvasculature is the national and international meetings. critical target of the immune response. J Exp Med Conclusion 150:322–337, 1979. 9. Dvorak HF, Dvorak AM, Churchill WH. Immu- Th e Immunopathology Unit has functioned for nologic rejection of diethylnitrosamine-induced more than 30 years as a center that has brought hepatomas in strain 2 guinea pigs. Participation of together physicians and researchers interested basophilic leukocytes and macrophage aggregates. not only in basic research but also in develop- J Exp Med 137:751–775, 1973. ing diagnostic tests. Perhaps its most important 10. Colvin RB, Johnson RA, Mihm MC Jr., Dvorak legacy has been the training of young patholo- HF. Role of the clotting system in cell-mediated gists and clinicians who have succeeded in both hypersensitivity. I. Fibrin deposition in delayed research and clinical practice. Th eir eff orts will skin reactions in man. J Exp Med 138:686–698, no doubt keep the fi eld of immunopathology a 1973. 11. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, vibrant component of patient care and medical Harvey VS, Dvorak HF. Tumor cells secrete a vas- inquiry for many years to come. cular permeability factor that promotes accumulation of ascites fl uid. Science 219:983–985, 1983. 12. Bhan AK, Schneeberger EE, Collins AB, McClus- References key RT. Evidence for a pathogenic role of a cell- 1. Gill TJ III, Ward PA. Immunopathology. Hyper- mediated immune mechanism in experimental sensitivity or tolerance? JAMA 238:1921–1923, glomerulonephritis. J Exp Med 148:246–260, 1978. 1977. 13. Raychowdhury R, Niles JL, McCluskey RT, Smith 2. Swartz MN, Dvorak HF. Obituary: Dr. Louis JA. Autoimmune target in Heymann nephritis is a Dienes. J Infect Dis 130:89–91, 1974. glycoprotein with homology to the LDL receptor. 3. Waksman BH, Adams RD. Infectious leu- Science 244:1163–1165, 1989. koencephalitis. A critical comparison of cer- 14. Niles JL, McCluskey RT, Ahmad MF, Arnaout tain experimental and naturally-occurring viral MA. Wegener’s granulomatosis autoantigen is a

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novel neutrophil serine proteinase. Blood 74:1888– of T lymphocytes propagated from human lung 1893, 1989. carcinomas. Clin Immunol Immunopathol 15. Niles JL, Pan GL, Collins AB, Shannon T, Skates 38:367–380, 1986. S, Fienberg R, Arnaout MA, McCluskey RT. 25. Stamenkovic I, Stegagno M, Wright KA, Krane Antigen-specifi c radioimmunoassays for anti-neu- SM, Amento EP, Colvin RB, Duquesnoy RJ, Kur- trophil cytoplasmic antibodies in the diagnosis of nick JT. Clonal dominance among T-lymphocyte rapidly progressive glomerulonephritis. J Am Soc infi ltrates in arthritis. Proc Natl Acad Sci 85:1179– Nephrol 2:27–36, 1991. 1183, 1988. 16. Clark RA, Lanigan JM, Della Pelle P, Manseau E, 26. Kradin RL, Kurnick JT, Lazarus DS, Preff er FI, Dvorak HF, Colvin RB. Fibronectin and fi brin Dubinett SM, Pinto CE, Giff ord J, Davidson E, provide a provisional matrix for epidermal cell Grove B, Callahan RJ, et al. Tumour-infi ltrating migration during wound reepithelialization. J lymphocytes and interleukin-2 in treatment of Invest Dermatol 79:264–269, 1982. advanced cancer. Lancet 333:577–580, 1989. 17. Cosimi AB, Colvin RB, Burton RC, Rubin RH, 27. Kurnick JT, Ramirez-Montagut T, Boyle LA, Goldstein G, Kung PC, Hansen WP, Delmonico Andrews DM, Pandolfi F, Durda PJ, Butera D, FL, Russell PS. Use of monoclonal antibodies to Dunn IS, Benson EM, Gobin SJ, van den Elsen T-cell subsets for immunologic monitoring and PJ. A novel autocrine pathway of tumor escape treatment in recipients of renal allografts. NEJM from immune recognition. Melanoma cell lines 305:308–314, 1981. produce a soluble protein that diminishes expres- 18. Bhan AK, Reinisch CL, Levey RH, McClus- sion of the gene encoding the melanocyte lineage key RT, Schlossman SF. T-cell migration into melan-A/MART-1 antigen through down-modu- allografts. J Exp Med 141:1210–1215, 1975. lation of its promoter. J Immunol 167:1204–1211, 19. Bhan AK, Reinherz EL, Poppema S, McCluskey 2001. RT, Schlossman SF. Location of T cell and major 28. Kradin RL, Divertie MB, Colvin RB, Ramirez J, histocompatibility complex antigens in the human Ryu J, Carpenter HA, Bhan AK. Usual interstitial thymus. J Exp Med 152:771–782, 1980. pneumonitis is a T-cell alveolitis. Clin Immunol 20. Bhan AK, Nadler LM, Stashenko P, McCluskey Immunopathol 40:224–235, 1986. RT, Schlossman SF. Stages of B cell diff erentiation 29. Xia W, Pinto CE, Kradin RL. Th e antigen- in human lymphoid tissue. J Exp Med 154:737– presenting activities of Ia+ dendritic cells shift 749, 1981. dynamically from lung to lymph node after an 21. Murphy GF, Bhan AK, Sato S, Harrist TJ, Mihm airway challenge with soluble antigen. J Exp Med MC Jr. Characterization of Langerhans cells by 181:1275–1283, 1995. the use of monoclonal antibodies. Lab Invest 30. Preff er FI, Colvin RB, Leary CP, Boyle LA, Tua- 45:465–468, 1981. zon TV, Lazarovits AI, Cosimi AB, Kurnick JT. 22. Harris NL, Nadler LM, Bhan AK. Immunohisto- Two-color fl ow cytometry and functional analy- logic characterization of two malignant lympho- sis of lymphocytes cultured from human renal mas of germinal center type (centroblastic/centro- allografts. Identifi cation of a Leu-2+3+ subpopula- cytic and centrocytic) with monoclonal antibod- tion. J Immunol 137:2823–2830, 1986. ies. Follicular and diff use lymphomas of small- 31. Preff er FI, Dombkowski D, Sykes M, Scadden cleaved-cell type are related but distinct entities. D, Yang YG. Lineage-negative side-population Am J Pathol 117:262–272, 1984. (SP) cells with restricted hematopoietic capacity 23. Kabawat SE, Bast RC Jr., Bhan AK, Welch WR, circulate in normal human adult blood. Immuno- Knapp RC, Colvin RB. Tissue distribution of a phenotypic and functional characterization. Stem coelomic-epithelium-related antigen recognized Cells 20:417–427, 2002. by the monoclonal antibody OC125. Int J Gynecol 32. Preff er F, Dombkowski D. Advances in com- Pathol 2:275–285, 1983. plex multiparameter fl ow cytometry technology. 24. Kurnick JT, Kradin RL, Blumberg R, Schnee- Applications in stem cell research. Cytometry Part berger EE, Boyle LA. Functional characterization B, Clinical Cytometry 76:295–314, 2009.

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33. Cerf-Bensussan N, Guy-Grand D, Lisowska- alloantibody-mediated renal allograft rejection Grospierre B, Griscelli C, Bhan AK. A monoclonal in Cynomolgus monkeys. Am J Transplantation antibody specifi c for rat intestinal lymphocytes. J 8:1662–1672, 2008. Immunol 136:76–82, 1986. 42. Hirohashi T, Uehara S, Chase CM, Della Pelle P, 34. Russell GJ, Parker CM, Cepek KL, Mandelbrot Madsen JC, Russell PS, Colvin RB. Complement DA, Sood A, Mizoguchi E, Ebert EC, Brenner independent antibody-mediated endarteritis and MB, Bhan AK. Distinct structural and functional transplant arteriopathy in mice. Am J Transplan- epitopes of the alpha E beta 7 integrin. Eur J tation 10:510–517, 2010. Immunol 24:2832–2841, 1994. 43. Wands JR, Dienstag JL, Bhan AK, Feller ER, Issel- 35. Maruiwa M, Mizoguchi A, Russell GJ, Narula N, bacher KJ. Circulating immune complexes and Stronska M, Mizoguchi E, Rabb H, Arnaout MA, complement activation in primary biliary cirrho- Bhan AK. Anti-KCA-3, a monoclonal antibody sis. NEJM 298:233–237, 1978. reactive with a rat complement C3 receptor, dis- 44. Mombaerts P, Mizoguchi E, Grusby MJ, Glim- tinguishes Kupff er cells from other macrophages. cher LH, Bhan AK, Tonegawa S. Spontaneous J Immunol 150:4019–4030, 1993. development of infl ammatory bowel disease in T 36. Colvin RB, Bhan AK, McCluskey RT. Diagnostic cell receptor mutant mice. Cell 75:274–282, 1993. Immunopathology, 2nd ed. New York: Raven Press, 45. Mizoguchi A, Mizoguchi E, Chiba C, Bhan AK. 1995. Role of appendix in the development of infl am- 37. Sobel RA, Blanchette BW, Bhan AK, Colvin RB. matory bowel disease in TCR-alpha mutant mice. Th e immunopathology of experimental aller- J Exp Med 184:707–715, 1996. gic encephalomyelitis. I. Quantitative analysis of 46. Mizoguchi A, Mizoguchi E, Takedatsu H, Blum- infl ammatory cells in situ. J Immunol 132:2393– berg RS, Bhan AK. Chronic intestinal infl amma- 2401, 1984. tory condition generates IL-10-producing regula- 38. Colvin RB, Cohen AH, Saiontz C, Bonsib S, tory B cell subset characterized by CD1d upregula- Buick M, Burke B, Carter S, Cavallo T, Haas M, tion. Immunity 16:219–230, 2002. Lindblad A, Manivel JC, Nast CC, Salomon D, 47. Hou J, Renigunta A, Konrad M, Gomes AS, Weaver C, Weiss M. Evaluation of pathologic cri- Schneeberger EE, Paul DL, Waldegger S, Good- teria for acute renal allograft rejection. Reproduc- enough DA. Claudin-16 and claudin-19 interact ibility, sensitivity, and clinical correlation. J Am and form a cation-selective tight junction com- Soc Nephrol 8:1930–1941, 1997. plex. J Clin Invest 118:619–628, 2008. 39. Collins AB, Schneeberger EE, Pascual MA, Said- 48. Schneeberger EE, Vu Q, LeBlanc BW, Doerschuk man SL, Williams WW, Tolkoff -Rubin N, Cosimi CM. Th e accumulation of dendritic cells in the AB, Colvin RB. Complement activation in acute lung is impaired in CD18-/- but not in ICAM-1-/- humoral renal allograft rejection. Diagnostic sig- mutant mice. J Immunol 164:2472–2478, 2000. nifi cance of C4d deposits in peritubular capillar- 49. MacLean JA, Su Z, Guo Y, Sy MS, Colvin RB, ies. J Am Soc Nephrol 10:2208–2214, 1999. Wong JT. Anti-CD3:anti-IL-2 receptor bispecifi c 40. Mauiyyedi S, Della Pelle P, Saidman S, Collins monoclonal antibody. Targeting of activated T AB, Pascual M, Tolkoff -Rubin NE, Williams cells in vitro. J Immunol 150:1619–1628, 1993. WW, Cosimi AB, Schneeberger EE, Colvin RB. 50. Bartolazzi A, Peach R, Aruff o A, Stamenkovic Chronic humoral rejection. Identifi cation of anti- I. Interaction between CD44 and hyaluronate body-mediated chronic renal allograft rejection by is directly implicated in the regulation of tumor C4d deposits in peritubular capillaries. J Am Soc development. J Exp Med 180:53–66, 1994. Nephrol 12:574–582, 2001. 51. Sgroi D, Koretzky GA, Stamenkovic I. Regula- 41. Smith RN, Kawai T, Boskovic S, Nadazdin O, tion of CD45 engagement by the B-cell receptor Sachs DH, Cosimi AB, Colvin RB. Four stages CD22. Proc Natl Acad Sci 92:4026–4030, 1995. and lack of stable accommodation in chronic

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