Immunological Concepts Lead the Way in Unraveling Proliferative Diseases of the Immune System
Professor, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California
Davis, CA, USA
WSAVA/Waltham International Award for Scientific Achievement
The crux of investigation of immunopathological disease involves identification of the cells involved and the nature of the process, inflammatory (reactive) or neoplastic. Cell lineage determination in the immune system has advanced since the inception of the 'cluster of differentiation' (CD) antigen system. Precise cell identification can be achieved by the application of monoclonal antibodies (mAb) specific for leukocyte antigens to isolated cells, cell smears and tissue sections. In the case of lymphoid proliferation, assessment of B or T cell antigen receptor rearrangement by polymerase chain reaction (PCR) provides valuable information pertaining to antigen receptor diversity in the lesion. Specifically, the detection of a lymphocyte clonal expansion in lymphoproliferative disease, in the appropriate clinical and morphological context, is consistent with lymphoma. Furthermore, lymphocytes are mobile cells, which are able to adjust their migratory routes to enter non-lymphoid tissues. Discovery of molecular determinants of lymphocyte trafficking to mucosal and cutaneous sites has provided a paradigm for understanding clinical patterns of lymphoproliferative diseases.
II. Cell Lineages Revealed by Markers of Functional Significance
Very few leukocyte antigens are expressed only by a single lineage of cells. Cell lineage is best determined by evaluation of the expression pattern of leukocyte antigens that are functionally important for a particular cell. In this context, identification of T cells and important T cell subsets is best achieved by demonstration of components of the TCR/CD3 complex on the surface of the cell, in association with other functionally important TCR/CD3 co-receptor molecules such as CD4 (helper T cells) or CD8alpha (cytotoxic T cells)1. Similarly, B cells are identified by demonstration of the components of the B cell antigen receptor complex (specifically CD79a).
The identification of histiocytes in tissue sections also relies on the identification of functionally important molecules on these cells. Histiocytes include macrophages and dendritic cells (DC). Nomenclature of DC populations is based on the differentiation pathway followed by the cell (the cell lineage), and by the location in tissue. There are 3 DC lineages: interstitial DC, DC within epithelia (Langerhans cells or LC) and plasmacytoid DC.
Dendritic cells are the most potent antigen presenting cells. Hence, DC in humans and dogs are best defined by their abundant expression of molecules essential to their function as antigen presenting cells. These include major histocompatibility complex (MHC) molecules (presentation of peptides to T cells) and CD1 (presentation of glycolipids to T cells). Leukocyte adhesion molecules (beta-2 integrins, CD90 and E-cadherin)2-5 are also important in DC and macrophage function and are useful differentiation markers. DC arise in bone marrow and migrate through blood to a variety of epithelial sites (cutaneous and mucosal), where they take up residence either within epithelia, or in dermis and lamina propria. In these sites they function as antigen processing and ultimately antigen presenting cells, which interact with T cells. Migration of cutaneous DC (as veiled cells) via lymphatics to the paracortex of lymph nodes occurs following contact with antigen6. Dendritic cells differentiate into potent APC during this migration and change their surface phenotype accordingly.
Aspects of the developmental and migratory program of cutaneous DC are recapitulated in the DC proliferative disorders of canine skin. Canine cutaneous histiocytoma (CCH) is a LC disorder, in which tumor histiocytes express CD1, CD11c, MHC II and E-cadherin. The expected outcome in CCH is spontaneous regression and this happens most of the time if tumors are not excised. The ability to detect LC by immunohistochemistry led to the recognition of a spectrum of canine LC proliferative disorders ranging from single or multiple histiocytomas limited to skin with or without involvement of lymph nodes, to systemic Langerhans cell histiocytosis, which is invariably fatal4,5.
Successful interaction of DC and T cells in response to antigenic challenge also involves the orderly appearance of co-stimulatory molecules (B7 family) on DC, and their ligands (CD28 and CTLA-4) on T cells7,8. Defective interaction of interstitial DC and T cells appears to contribute to the development of reactive histiocytic proliferative diseases (cutaneous and systemic histiocytosis), which are related diseases arising out of disordered immune regulation. Recognition of this has led to the development of effective immunosuppressive treatment protocols for reactive histiocytic proliferative diseases.
Most canine histiocytic sarcomas (HS) involve infiltration and expansion of cells of interstitial DC lineage9. These include localized and disseminated HS (malignant histiocytosis). However, proliferative disorders of macrophage lineage have also been recognized. The best example is the hemophagocytic histiocytic sarcoma (HS) (hemophagocytic malignant histiocytosis) of dogs10. In most instances this disorder is associated with expansion of splenic red pulp macrophages, often coincident with expansion of bone marrow macrophages, both of which manifest prominent erythrophagocytosis.
III. Lymphocyte Antigen Receptor Gene Rearrangement--An Aid to Diagnosis of Lymphoma
Lymphocyte antigen receptor gene rearrangement analysis by polymerase chain reaction (PCR) is a methodology used to detect clonality in B and T cell populations. This methodology provides the advantage of allowing sensitive detection of clonality in formalin-fixed paraffin embedded (FFPE) tissues11.
During T cell development in the thymus, T cells rearrange their antigen receptor genes TCRA, TCRB, TCRG and TCRD, and in the process create 2 lineages of T cells, alpha-beta and gamma-delta T cells. TCRG gene rearrangement occurs in the majority of T cells regardless of TCR lineage12,13. Recently, we characterized feline TCRG and developed PCR primers for determination of the clonality status of T cells in feline intestinal lymphoma. The assay is sensitive, reproducible, and discriminates between clonal, pseudoclonal and polyclonal TCRG gene rearrangements in DNA extracted from FFPE tissue14. In a companion paper, we also characterized the feline immunoglobulin heavy chain locus (IGH), and established its usefulness in the diagnosis of B cell neoplasia15. PCR based lymphocyte antigen receptor clonality assays represent important adjunctive tools in the diagnosis of feline lymphoma, when interpreted in the light of clinical, morphologic and immunophenotypic data from the feline patient. The role of immunophenotyping is critical for determination of cell lineage in lymphoma, since cross lineage rearrangement of antigen receptor genes limits its use for lineage determination.
Molecular clonality determination is indicated when morphological features of lymphocytes and immunophenotyping are inconclusive. These conditions are most often met in some lymphoid proliferations in gut and skin, or in organized lymphoid tissue when architecture is largely intact (for example: early marginal zone and T-zone lymphomas14,16. The relatively recent recognition of marginal zone lymphomas in dogs has complicated the interpretation of complex splenic lymphoid nodular lesions. Molecular clonality determination has become a decisive tool to unravel these complex lesions (Benak and Moore, unpublished data).
In cats, inflammatory bowel disease (IBD) and intestinal lymphoma are prevalent, and their distinction presents difficulties especially in endoscopic biopsy specimens. An understanding of intestinal lymphocyte trafficking patterns and their effects on mucosal architecture, coupled with assessment of lymphocyte antigen receptor gene rearrangement (molecular clonality) is vital to establishing the nature of the disease process in these instances.
The diffuse, mucosal-associated lymphoid tissue (MALT) of the small intestine, which consists of lamina proprial (LPC) and intra-epithelial compartments (IEC)), is populated largely by CD3+ T cells in normal cats17. Less than 5% of villous lamina proprial lymphocytes (LPL) are B cells and even fewer are found in the IEC. Plasma cells are concentrated in the intestinal crypt lamina propria. The inductive environment for adaptive immune responses is distributed in the distal small intestine where Peyer's patches are most numerous. Antigen stimulated B cells are induced to class switch to IgA and home to the intestinal crypts, and T cells are induced by interactions with DC to express cell surface molecules that enable them to traffic to the diffuse MALT18. Feline IEL have also been shown to express homologous cell surface molecules, hence the principles of intestinal mucosal lymphocyte trafficking established for humans and rodents likely apply to cats19.
Understanding of these small intestinal lymphocyte trafficking principles, coupled with the development of PCR primer sets capable of detecting clonal expansion of B and T cells, has led to the discovery of the high prevalence of mucosal-confined small T cell lymphoma in cats14.
The discovery of CD molecules and their functions in leukocytes have made it possible to precisely identify the lineages of infiltrative cells in diseased tissues. This coupled with the analysis of lymphocyte antigen receptor gene rearrangement in lymphoproliferative diseases, has greatly expanded our abilities to diagnose proliferative diseases of the immune system. However, the interrogative techniques of immunohistochemistry and molecular clonality determination, while powerful, should only be used as adjunctive aids coupled with careful clinical assessment of the patient and morphologic assessment of lesional tissue.
1. Janeway C, Travers P, Walport M, Shlomchik M. Immunobiology: The immune system in health and disease. 5th. ed. New York: Garland Publishing; 2001.
2. Danilenko DM, Moore PF, Rossitto PV. Canine leukocyte cell adhesion molecules (LeuCAMs): characterization of the CD11/CD18 family. Tissue Antigens 1992;40(1):13-21.
3. Danilenko DM, Rossitto PV, Van der Vieren M, Le Trong H, McDonough SP, Affolter VK, et al. A novel canine leukointegrin, alpha d beta 2, is expressed by specific macrophage subpopulations in tissue and a minor CD8+ lymphocyte subpopulation in peripheral blood. J Immunol 1995;155(1):35-44.
4. Moore P, Affolter V, Olivry T, Schrenzel M. The use of immunological reagents in defining the pathogenesis of canine skin diseases involving proliferation of leukocytes. In: Kwotchka K, Willemse T, von Tscharner C, editors. Advances in Veterinary Dermatology. Oxford: Butterworth Heinmann; 1998. p. 77-94.
5. Moore PF, Schrenzel MD, Affolter VK, Olivry T, Naydan D. Canine cutaneous histiocytoma is an epidermotropic Langerhans cell histiocytosis that expresses CD1 and specific beta 2-integrin molecules. Am J Pathol 1996;148(5):1699-708.
6. Cyster JG. Chemokines and cell migration in secondary lymphoid organs. Science 1999;286(5447):2098-102.
7. Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 1995;3(5):541-7.
8. Walunas TL, Lenschow DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM, et al. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1994;1(5):405-13.
9. Affolter VK, Moore PF. Localized and disseminated histiocytic sarcoma of dendritic cell origin in dogs. Vet Pathol 2002;39(1):74-83.
10. Moore PF, Affolter VK, Vernau W. Canine hemophagocytic histiocytic sarcoma: a proliferative disorder of CD11d+ macrophages. Vet Pathol 2006;43(5):632-45.
11. van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003;17(12):2257-317.
12. Theodorou I, Raphael M, Bigorgne C, Fourcade C, Lahet C, Cochet G, et al. Recombination pattern of the TCR gamma locus in human peripheral T-cell lymphomas. J Pathol 1994;174(4):233-42.
13. Thompson SD, Manzo AR, Pelkonen J, Larche M, Hurwitz JL. Developmental T cell receptor gene rearrangements: relatedness of the alpha/beta and gamma/delta T cell precursor. Eur J Immunol 1991;21(8):1939-50.
14. Moore PF, Woo JC, Vernau W, Kosten S, Graham PS. Characterization of feline T cell receptor gamma (TCRG) variable region genes for the molecular diagnosis of feline intestinal T cell lymphoma. Vet Immunol Immunopathol 2005;106(3-4):167-78.
15. Werner JA, Woo JC, Vernau W, Graham PS, Grahn RA, Lyons LA, et al. Characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of B-cell neoplasia. Vet Pathol 2005;42(5):596-607.
16. Valli VE, Vernau W, de Lorimier LP, Graham PS, Moore PF. Canine indolent nodular lymphoma. Vet Pathol 2006;43(3):241-56.
17. Roccabianca P, Woo JC, Moore PF. Characterization of the diffuse mucosal associated lymphoid tissue of feline small intestine. Vet Immunol Immunopathol 2000;75(1-2):27-42.
18. Cheroutre H, Madakamutil L. Acquired and natural memory T cells join forces at the mucosal front line. Nat Rev Immunol 2004;4(4):290-300.
19. Woo JC, Roccabianca P, van Stijn A, Moore PF. Characterization of a feline homologue of the alpha E integrin subunit (CD103) reveals high specificity for intra-epithelial lymphocytes. Vet Immunol Immunopathol 2002;85(1-2):9-22.