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Canine X-linked severe combined immunodeficiency
Veterinary Immunology and Immunopathology 69 (1999) 127±135
Canine X-linked severe combined immunodeficiency Peter J. Felsburga,*, Brian J. Hartnetta, Paula S. Henthorna, Peter F. Mooreb, Steven Krakowkac, Hans D. Ochsd a
Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA b Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA, USA c Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA d Department of Pediatrics, School of Medicine, University of Washington, Seattle, WA, USA
Abstract Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gc) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors. The most striking clinical feature is a failure to thrive or `stunted' growth. Recurrent or chronic infections begin at the time of decline of maternal antibody, usually between six and eight weeks of age. Affected dogs rarely survive past three to four months of age. The major pathologic feature of canine XSCID is a small, dysplastic thymus. Grossly identifiable lymph nodes, tonsils, and Peyer's patches are absent in XSCID dogs. During the neonatal period, XSCID dogs have few, if any, peripheral T cells and increased number of peripheral B cells. Some XSCID dogs do develop phenotypically mature, nonfunctional T cells with age, however, the absolute number of peripheral T cells remain significantly decreased compared to age-matched normal dogs. An interesting finding is that as soon as T cells begin to appear in XSCID dogs they rapidly switch from a CD45RA (naive) phenotype to a CD45RAÿ (activated or memory phenotype). One of the characteristic findings in XSCID dogs is an absent or markedly depressed blastogenic response of T cells in response to stimulation through the T cell receptor and when the necessary second messengers for cellular proliferation are directly provided that by-pass signals delivered through ligand±receptor interaction. The proliferative defect is due to the inability of T cells to express a functional IL-2 receptor. Canine XSCID B cells do not proliferate following stimulation with T cell-dependent B cell mitogens, however, they proliferate normally in response to T cell-independent B cell mitogens. Canine XSCID B cells are capable of producing IgM but are incapable of class-switching to IgG antibody production following immunization with the T cell-dependent neoantigen, bacteriophage * Corresponding author. Tel.: +1-215-898-6678; fax: +1-215-573-8183 E-mail address: [email protected] (P.J. Felsburg) 0165-2427/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 9 9 ) 0 0 0 4 9 - 5
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X174. The number of thymocytes in the XSCID thymus is 0.3% of the thymocytes present in the thymus of age-matched normal dogs. The proportion of CD4ÿCD8ÿ thymocytes in XSCID dogs is increased 3.5-fold and the CD4CD8 population is decreased 2.3-fold. These findings demonstrate that (1) a functional gc is required for normal B and T cell function, (2) early T cell development is highly dependent upon a functional gc, and (3) B cell development can occur through a gc-independent pathway. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Canine; Immunode®ciency; B cells; T cells; Common gamma chain
1. Introduction Severe combined immunodeficiency (SCID) represents a heterogeneous group of genetic disorders characterized by the absence of T and B cell function which usually results in death during infancy (Rosen et al., 1995). In the past 10 years, the genes responsible for most forms of SCID have been identified, cloned and their function and expression characterized. The most common form of SCID is X-linked SCID (XSCID) which is due to mutations in the common gamma (gc) subunit of the receptors for IL-2, IL-4, IL-7, IL-9 and IL-15 (Noguchi et al., 1993; Leonard, 1996). Thus, the XSCID phenotype is the complex result of multiple cytokine defects. The shared usage of the gc by receptors for growth factors that are essential for normal B and T cell development and function explains the profound immunologic abnormalities and clinical severity of the disease. XSCID boys present in the first year of life because of severe, recurrent or persistent infections that generally begin between three and six months of age, at a time when maternally derived antibody has virtually disappeared. The most striking clinical feature is a failure to thrive. The immunologic abnormalities have been recently reviewed (Rosen et al., 1995; Conley et al., 1990; Gougeon et al., 1990; Conley, 1991; Buckley et al., 1993; Matthews et al., 1996). At the time of diagnosis, affected boys have markedly reduced or absent T cells that fail to proliferate in response to stimulation. Peripheral B cells are present in normal or increased numbers and have a virgin (IgM) phenotype, but they fail to mature and function normally. The primary B cell defect is assumed to be the inability to class-switch from IgM to IgG since bone marrow transplanted XSCID boys who do not engraft donor B cells fail to produce IgG antibody following immunization with bacteriophage X174 (Buckley et al., 1993). Although a mutant gc has a profound effect on early human T cell differentiation, the observation that XSCID boys have normal or elevated numbers of IgM B cells and that peripheral IgM B cells from carrier females exhibit random X-chromosome inactivation (Conley et al., 1988) suggests that a mutant gc does not interfere with the early stages of human B cell development. The purpose of this paper is to review a naturally-occurring animal model of XSCIDÐ canine X-linked severe combined immunodeficiency that was identified by our laboratory. Canine XSCID is a naturally occurring disease that results in a clinical and immunologic phenotype virtually identical to human XSCID and is due to mutations in the gc gene making it a true homologue of the human disease (Jezyk et al., 1989; Felsburg
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et al., 1992; Snyder et al., 1993; Somberg et al., 1994; Henthorn et al., 1994; Somberg et al., 1996; Felsburg et al., 1998). The mutation in our colony is a 4-bp deletion in the signal peptide region resulting in a frame-shift with a premature termination codon in exon 1. The predicted product is a truncated protein of 21 aa instead of the normal 373 aa, in essence representing a naturally occurring gc `knock-out'. A breeding colony of XSCID dogs was developed from a single carrier female resulting in all affected dogs having the same genetic defect. 2. Clinical features The most striking clinical feature of canine XSCID is a failure to thrive or `stunted' growth. Affected puppies have reduced growth rates as evidenced by their size and weight compared to normal littermates. The size difference is less apparent in pups less than three weeks of age, but becomes more obvious as the pups become older. Problems with infections in the neonatal period are rare due to the presence of maternal antibody. Recurrent or chronic infections begin to appear between six and eight weeks of age, about the time maternal antibody begins its rapid decline. Clinical signs include superficial pyoderma, diarrhea and respiratory infections. These infections, usually of bacterial origin, are nonresponsive to antibiotic therapy and become generalized resulting in severe pneumonia or overwhelming, systemic infections. XSCID dogs reared in a conventional environment rarely survive past three to four months of age, and usually die of generalized Staphylococcal infections. The natural history of the disease has probably been modified by the infectious disease precautions taken in our closed colony thereby limiting the viral infections to which these dogs are highly susceptible. Several XSCID dogs inadvertently vaccinated with a modified-live canine distemper virus vaccine died two to three weeks later of vaccine-induced distemper. One XSCID dog raised in a gnotobiotic environment for 20 months developed acute monocytic leukemia (Felsburg et al., 1994). 3. Pathologic features We have previously reported the pathologic feature of 52 XSCID dogs free of any clinical or histologic evidence of infection (Snyder et al., 1993). The major pathologic feature of canine XSCID is a small, dysplastic thymus. The thymus in XSCID dogs is 10% the weight of thymi from age-matched normal dogs. Within this population of XSCID dogs with the same genetic defect, three histologic patterns of thymic dysplasia were observed: 27 dogs exhibited a simple dysplasia with varying numbers of thymocytes, no corticomedullary demarcation, and an absence of Hassall's corpuscles; 21 dogs had dysplastic thymi consisting of varying numbers of thymocytes, no corticomedullary demarcation, but containing varying numbers of Hassall's corpuscles; and four dogs had relatively normal looking thymi consisting of a corticomedullary demarcation and numerous Hassall's corpuscles, however, the lobules were extremely small and the subcapsular cortical region was devoid of lymphocytes.
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Grossly identifiable lymph nodes are absent in the majority of XSCID dogs. When present, these small nodes show little organization and few, if any, typical small lymphocytes. All XSCID dogs lack grossly identifiable tonsils. Histologically, the tonsilar crypts reveal a profound lymphoid hypoplasia with a paucity of small to medium lymphocytes. Peyer's patches cannot be identified grossly or histologically. 4. Immunologic abnormalities Profound lymphopenia is not a prominent feature of canine XSCID. Although XSCID dogs have reduced lymphocyte counts, the majority of dogs have lymphocyte counts >1000/ml. FACS analysis reveals that newborn XSCID dogs have peripheral blood lymphocyte phenotypes that are identical with those of the majority of human XSCID patients, characterized by normal or elevated proportions of B cells and markedly reduced or absent T cells (Fig. 1). We have also shown that some XSCID dogs are capable of developing increased proportions of phenotypically mature T cells ca. 8±10 weeks of age (Somberg et al., 1996). Even in XSCID dogs that had relatively normal percentages of peripheral T cells, the absolute number averaged only 30% of normal, including three XSCID dogs raised in a gnotobiotic environment for three years. Fig. 1 also shows that as soon as T cells begin to appear in XSCID dogs they rapidly switch from a CD45RA (naive) phenotype to a CD45RAÿ (activated or memory
Fig. 1. Proportion of peripheral B and T cells in XSCID dogs.
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Fig. 2. Proliferative response of XSCID peripheral blood lymphocytes.
phenotype), a phenomenon that is reported to be proliferation-dependent in normal humans (Johannisson and Festin, 1995). Although it is clear that the early T cells observed in XSCID dogs are recent thymic emigrants, it is unclear whether the increased proportions seen in older XSCID dogs are due to increased release of thymocytes that rapidly switch their CD45RA phenotype or due to extra-thymic expansion of the few CD45RA peripheral T cells present during the first few weeks of life. One of the characteristic findings in XSCID dogs is an absent or markedly depressed lymphocyte blastogenic response to T cell mitogens and specific antigens. Fig. 2 illustrates that canine XSCID peripheral blood lymphocytes (PBL) exhibit a significant lack of proliferation in response to stimulation through the T cell receptor (PHA) and when the necessary second messengers for cellular proliferation are directly provided by the combination of phorbol ester, PMA, and calcium ionophore, CI, that by-pass signals delivered through ligand±receptor interaction. These data suggest that the proliferative defect resides distal to the generation of second messengers associated with activation through the T cell receptor. The addition of exogenous human recombinant IL-2 does not increase the proliferative response of XSCID PBL. The poor proliferative response of XSCID T cells can partially be attributed to the defective expression of functional IL-2 receptors (Fig. 3). XSCID dogs are hypogammaglobulinemic. Serum IgM concentrations may be normal, but IgG is dramatically reduced and IgA is usually absent (Jezyk et al., 1989). XSCID B cells are capable of proliferation when stimulated with formalin-fixed, heat-killed Staphylococcus aureus, a T-independent, B cell mitogen (Fig. 2). Polyclonal activation of canine XSCID B cells with the T cell-dependent, B cell mitogen pokeweed mitogen results in the production of IgM, but little or no IgG or IgA suggesting that partially functional B cells are present in XSCID dogs, but isotype switching does not occur
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Fig. 3. IL-2 receptor expression on XSCID resting and activated peripheral bood lymphocytes.
Fig. 4. Secondary antibody response in XSCID dogs following immunization with bacteriophage X174.
(Felsburg et al., 1992). To further address the functional capability of XSCID B cells, we immunized gnotobiotic normal and XSCID dogs with the T-cell dependent neoantigen, bacteriophage X174 at approximately one year of age. Fig. 4 illustrates the total antibody and IgG specific antibody titers following secondary immunization. Although XSCID dogs can produce minimal amounts of specific antibody, it is almost exclusively
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Fig. 5. Block in B cell differentiation in canine XSCID.
IgM. On the other hand, the specific antibody response in normal dogs is primarily IgG. Based on the presence of normal proportions of peripheral B cells and in the ability of XSCID B cells to produce IgM antibody but failure to class-switch to IgG or IgA, the block in B cell differentiation in canine XSCID appears to be relatively late in B cell differentiation pathway (Fig. 5). 5. Defective thymocyte development in XSCID dogs XSCID appears to be a disease primarily affecting the differentiation and maturation of the T cell lineage because of the paucity of peripheral T cells. A major issue that has not been previously addressed is what is occurring in the XSCID thymus. During human thymopoiesis, thymocytes undergo an ordered expression of cell surface antigens on developing thymocytes as well as a controlled mitogenesis of thymocytes at discrete stages of development (Terstappen et al., 1992; Spits et al., 1992). Proliferation is one of the most important events occurring during early thymocyte development. The differentiation of CD4ÿCD8ÿ (DN) thymocytes to CD4CD8 (DP) thymocytes is proliferation dependent, whereas the maturation of DP thymocytes to mature CD4CD8ÿ or CD4ÿCD8 (SP) thymocytes is proliferation independent. A steady expansion in the number of thymocytes takes place from the DN stage to the CD3DP stage. Proliferation ceases in the DP population following the surface expression of CD3. We have studied the thymi of 24 XSCID dogs and 24 age-matched normal littermates between 4 and 10 weeks of age (Somberg et al., 1994). The XSCID thymus is 8% the weight of age-matched normal dogs. The total number of thymocytes in XSCID dogs (0.63 108) is 0.3% of the number of thymocytes present in age-matched normal dogs (203 108). In normal dogs, the thymocyte subsets were found in proportions similar to those described in humans suggesting that the same ordered maturation process occurs in the dog thymus (Table 1). However, in the postnatal XSCID thymus, a mutated gc severely affects thymocyte development. The proportion of DN thymocytes in XSCID dogs is increased 3.5-fold and the DP population is decreased 2.3-fold. Interestingly, there is no significant difference in the proportion of SP thymocytes. When one takes into
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Table 1 Phenotypic characterization of XSCID thymocytes Subset ÿ
Normal ÿ
CD4 CD8 CD4CD8 CD4CD8ÿ CD4ÿCD8 a
XSCID a
13.1 6.1 68.7 12.4 11.6 7.8 2.9 1.8
45.6 22.2 30.7 20.6 10.5 8.6 2.2 4.4
Percent SD.
Fig. 6. Block in T cell differentiation in canine XSCID.
consideration the size and cellularity of the XSCID thymuses, one can appreciate that there is a paucity of all thymocyte subsets in the XSCID dogs. These results suggest a block at a very early stage in T cell differentiation which occurs in the XSCID thymus (Fig. 6). In addition to the significantly reduced cellularity in the postnatal XSCID thymus, we have shown that there is an increased proportion of DN cells (2.5-fold) that appear to be in the thymoblast stage of development (Somberg et al., 1994). These results suggest that postnatal XSCID thymocytes have a reduced ability to enter the mitotic phase of the cell cycle. The dramatic reduction in cellularity and the alteration in the proportion of DN and DP thymocytes in the XSCID thymus is highly suggestive of a proliferation defect limiting the expansion of the pool of thymocytes and impeding their differentiation. It appears that the transition of DP to SP cells, which is proliferation independent, occurs normally in XSCID dogs in the absence of a functional gc. Acknowledgements This work was supported by National Institutes of Health Grants AI 26103, HL 52971, AI 43745, and RR 12211 (PJF); and AI 33177 (PSH).
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References Buckley, R., Schiff, S., Schiff, R., Markert, M.L., Williams, L., Ochs, H., 1993. B cell function after haploidentical stem cell transplantation in human severe combined immunodeficiency. FASEB J. 7, 95A. Conley, M., Lavoie, A., Briggs, C., Brown, P., Guerra, C., Puck, J.M., 1988. Nonrandom X chromosome inactivation in B cells from carrier of X chromosome-linked severe combined immunodeficiency. Proc. Nat. Acad. Sci. 85, 3090±3094. Conley, M., Buckley, R.H., Hong, R., Guerra-Hanson, C., Roifman, C.M., Brochstein, J.A., Pahwa, S., Puck, J.M., 1990. X-linked severe combined immunodeficiency. Diagnosis in males with sporadic severe combined immunodeficiency and clarification of clinica findings. J. Clin. Invest. 85, 1548±1554. Conley, M., 1991. X-linked severe combined immunodeficiency. Clin. Immunol. Immunopathol. 61, S94±S99. Felsburg, P.J., Somberg, R.L., Perryman, L.E., 1992. Domestic animal models of severe combined immunodeficiency: canine X-linked severe combined immunodeficiency and severe combined immunodeficiency in horses. Immunol. Rev. 3, 277±303. Felsburg, P.J., Somberg, R.L., Krakowka, G.S., 1994. Acute monocytic leukemia in a dog with X-linked severe combined immunodeficiency. Clin. Diag. Lab. Immunol. 1, 379±384. Felsburg, P.J., Somberg, R.L., Hartnett, B.J., Henthorn, P.S., Carding, S.R., 1998. Canine X-linked severe combined immunodeficiency. A model for investigating the requirement for the common gamma chain (gc) in human lymphocyte development and function. Immunol. Res. 17, 63±73. Gougeon, M.L., Drean, G., LeDeist, F., Dousseau, M., Fevrier, M., Diu, A., Theze, J., Griscelli, C., Fischer, A., 1990. Human severe combined immunodeficiency disease. Phenotypic and functional characteristics of peripheral B lymphocytes. J. Immunol. 145, 2873±2879. Henthorn, P.S., Somberg, R.L., Fimiani, V.M., Puck, J.M., Patterson, D.F., Felsburg, P.J., 1994. IL-2Rg gene microdeletion demonstrates that canine X-linked severe combined immunodeficiency is a homologue of the human disease. Genomics 23, 69±74. Jezyk, P.F., Felsburg, P.J., Haskins, M.E., Patterson, D.F., 1989. X-linked severe combined immunodeficiency in the dog. Clin. Immunol. Immunopathol. 52, 173±189. Johannisson, A., Festin, R., 1995. Phenotype transition of CD4 T cells from CD45RA to CD45RO is accomnpanied by cell activation and proliferation. Cytometry 19, 343±352. Leonard, W.J., 1996. Dysfunctional cytokine signaling in severe combined immunodeficiency. J. Invest. Med 44, 304±311. Matthews, D.J., Clark, P.A., Herbert, J., Morgan, G., Armitage, R.J., Kinnon, C., Minty, A., Grabstein, K.H., Caput, D., Ferrara, P., Callard, R., 1996. Function of the interleukin-2 (IL-2) receptor g-chain in biologic responses of X-linked severe combined immunodeficient B cells to IL-2, IL-4, IL-13 and IL-15. Blood 85, 38±42. Noguchi, M., Yi, H., Rosenblatt, H.M., Filipovich, A.H., Adelstein, S., Modi, W.S., McBride, O.W., Leonard, W.J., 1993. Interleukin-2 receptor g chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 73, 147±157. Rosen, F.S., Cooper, M.D., Wedgewood, R.J.P., 1995. The primary immunodeficiencies. N. Engl. J. Med. 333, 431±440. Snyder, P.W., Kazacos, E.A., Felsburg, P.J., 1993. Histologic characterization of the thymus in canine X-linked severe combined immunodeficiency. Clin. Immunol. Immunopathol. 67, 55±67. Somberg, R.L., Robinson, J.P., Felsburg, P.J., 1994. T lymphocyte development and function in dogs with Xlinked severe combined immunodeficiency. J. Immunol. 153, 4006±4015. Somberg, R.L., Tipold, A., Hartnett, B.J., Moore, P.F., Henthorn, P.S., Felsburg, P.J., 1996. Postnatal development of T cells in dogs with X-linked severe combined immunodeficiency. J. Immunol. 156, 1431± 1435. Spits, H., Lanier, L.L., Phillips, J.H., 1992. Development of human T and natural killer cells. Blood 85, 2654± 2670. Terstappen, L.W.M.M., Huand, S., Picker, L.J., 1992. Flow cytometric assessment of human T cell differentiation in thymus and bone marrow. Blood 79, 666±677.
Canine X-linked severe combined immunodeficiency Peter J. Felsburga,*, Brian J. Hartnetta, Paula S. Henthorna, Peter F. Mooreb, Steven Krakowkac, Hans D. Ochsd a
Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA b Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA, USA c Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA d Department of Pediatrics, School of Medicine, University of Washington, Seattle, WA, USA
Abstract Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gc) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors. The most striking clinical feature is a failure to thrive or `stunted' growth. Recurrent or chronic infections begin at the time of decline of maternal antibody, usually between six and eight weeks of age. Affected dogs rarely survive past three to four months of age. The major pathologic feature of canine XSCID is a small, dysplastic thymus. Grossly identifiable lymph nodes, tonsils, and Peyer's patches are absent in XSCID dogs. During the neonatal period, XSCID dogs have few, if any, peripheral T cells and increased number of peripheral B cells. Some XSCID dogs do develop phenotypically mature, nonfunctional T cells with age, however, the absolute number of peripheral T cells remain significantly decreased compared to age-matched normal dogs. An interesting finding is that as soon as T cells begin to appear in XSCID dogs they rapidly switch from a CD45RA (naive) phenotype to a CD45RAÿ (activated or memory phenotype). One of the characteristic findings in XSCID dogs is an absent or markedly depressed blastogenic response of T cells in response to stimulation through the T cell receptor and when the necessary second messengers for cellular proliferation are directly provided that by-pass signals delivered through ligand±receptor interaction. The proliferative defect is due to the inability of T cells to express a functional IL-2 receptor. Canine XSCID B cells do not proliferate following stimulation with T cell-dependent B cell mitogens, however, they proliferate normally in response to T cell-independent B cell mitogens. Canine XSCID B cells are capable of producing IgM but are incapable of class-switching to IgG antibody production following immunization with the T cell-dependent neoantigen, bacteriophage * Corresponding author. Tel.: +1-215-898-6678; fax: +1-215-573-8183 E-mail address: [email protected] (P.J. Felsburg) 0165-2427/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 9 9 ) 0 0 0 4 9 - 5
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X174. The number of thymocytes in the XSCID thymus is 0.3% of the thymocytes present in the thymus of age-matched normal dogs. The proportion of CD4ÿCD8ÿ thymocytes in XSCID dogs is increased 3.5-fold and the CD4CD8 population is decreased 2.3-fold. These findings demonstrate that (1) a functional gc is required for normal B and T cell function, (2) early T cell development is highly dependent upon a functional gc, and (3) B cell development can occur through a gc-independent pathway. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Canine; Immunode®ciency; B cells; T cells; Common gamma chain
1. Introduction Severe combined immunodeficiency (SCID) represents a heterogeneous group of genetic disorders characterized by the absence of T and B cell function which usually results in death during infancy (Rosen et al., 1995). In the past 10 years, the genes responsible for most forms of SCID have been identified, cloned and their function and expression characterized. The most common form of SCID is X-linked SCID (XSCID) which is due to mutations in the common gamma (gc) subunit of the receptors for IL-2, IL-4, IL-7, IL-9 and IL-15 (Noguchi et al., 1993; Leonard, 1996). Thus, the XSCID phenotype is the complex result of multiple cytokine defects. The shared usage of the gc by receptors for growth factors that are essential for normal B and T cell development and function explains the profound immunologic abnormalities and clinical severity of the disease. XSCID boys present in the first year of life because of severe, recurrent or persistent infections that generally begin between three and six months of age, at a time when maternally derived antibody has virtually disappeared. The most striking clinical feature is a failure to thrive. The immunologic abnormalities have been recently reviewed (Rosen et al., 1995; Conley et al., 1990; Gougeon et al., 1990; Conley, 1991; Buckley et al., 1993; Matthews et al., 1996). At the time of diagnosis, affected boys have markedly reduced or absent T cells that fail to proliferate in response to stimulation. Peripheral B cells are present in normal or increased numbers and have a virgin (IgM) phenotype, but they fail to mature and function normally. The primary B cell defect is assumed to be the inability to class-switch from IgM to IgG since bone marrow transplanted XSCID boys who do not engraft donor B cells fail to produce IgG antibody following immunization with bacteriophage X174 (Buckley et al., 1993). Although a mutant gc has a profound effect on early human T cell differentiation, the observation that XSCID boys have normal or elevated numbers of IgM B cells and that peripheral IgM B cells from carrier females exhibit random X-chromosome inactivation (Conley et al., 1988) suggests that a mutant gc does not interfere with the early stages of human B cell development. The purpose of this paper is to review a naturally-occurring animal model of XSCIDÐ canine X-linked severe combined immunodeficiency that was identified by our laboratory. Canine XSCID is a naturally occurring disease that results in a clinical and immunologic phenotype virtually identical to human XSCID and is due to mutations in the gc gene making it a true homologue of the human disease (Jezyk et al., 1989; Felsburg
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et al., 1992; Snyder et al., 1993; Somberg et al., 1994; Henthorn et al., 1994; Somberg et al., 1996; Felsburg et al., 1998). The mutation in our colony is a 4-bp deletion in the signal peptide region resulting in a frame-shift with a premature termination codon in exon 1. The predicted product is a truncated protein of 21 aa instead of the normal 373 aa, in essence representing a naturally occurring gc `knock-out'. A breeding colony of XSCID dogs was developed from a single carrier female resulting in all affected dogs having the same genetic defect. 2. Clinical features The most striking clinical feature of canine XSCID is a failure to thrive or `stunted' growth. Affected puppies have reduced growth rates as evidenced by their size and weight compared to normal littermates. The size difference is less apparent in pups less than three weeks of age, but becomes more obvious as the pups become older. Problems with infections in the neonatal period are rare due to the presence of maternal antibody. Recurrent or chronic infections begin to appear between six and eight weeks of age, about the time maternal antibody begins its rapid decline. Clinical signs include superficial pyoderma, diarrhea and respiratory infections. These infections, usually of bacterial origin, are nonresponsive to antibiotic therapy and become generalized resulting in severe pneumonia or overwhelming, systemic infections. XSCID dogs reared in a conventional environment rarely survive past three to four months of age, and usually die of generalized Staphylococcal infections. The natural history of the disease has probably been modified by the infectious disease precautions taken in our closed colony thereby limiting the viral infections to which these dogs are highly susceptible. Several XSCID dogs inadvertently vaccinated with a modified-live canine distemper virus vaccine died two to three weeks later of vaccine-induced distemper. One XSCID dog raised in a gnotobiotic environment for 20 months developed acute monocytic leukemia (Felsburg et al., 1994). 3. Pathologic features We have previously reported the pathologic feature of 52 XSCID dogs free of any clinical or histologic evidence of infection (Snyder et al., 1993). The major pathologic feature of canine XSCID is a small, dysplastic thymus. The thymus in XSCID dogs is 10% the weight of thymi from age-matched normal dogs. Within this population of XSCID dogs with the same genetic defect, three histologic patterns of thymic dysplasia were observed: 27 dogs exhibited a simple dysplasia with varying numbers of thymocytes, no corticomedullary demarcation, and an absence of Hassall's corpuscles; 21 dogs had dysplastic thymi consisting of varying numbers of thymocytes, no corticomedullary demarcation, but containing varying numbers of Hassall's corpuscles; and four dogs had relatively normal looking thymi consisting of a corticomedullary demarcation and numerous Hassall's corpuscles, however, the lobules were extremely small and the subcapsular cortical region was devoid of lymphocytes.
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Grossly identifiable lymph nodes are absent in the majority of XSCID dogs. When present, these small nodes show little organization and few, if any, typical small lymphocytes. All XSCID dogs lack grossly identifiable tonsils. Histologically, the tonsilar crypts reveal a profound lymphoid hypoplasia with a paucity of small to medium lymphocytes. Peyer's patches cannot be identified grossly or histologically. 4. Immunologic abnormalities Profound lymphopenia is not a prominent feature of canine XSCID. Although XSCID dogs have reduced lymphocyte counts, the majority of dogs have lymphocyte counts >1000/ml. FACS analysis reveals that newborn XSCID dogs have peripheral blood lymphocyte phenotypes that are identical with those of the majority of human XSCID patients, characterized by normal or elevated proportions of B cells and markedly reduced or absent T cells (Fig. 1). We have also shown that some XSCID dogs are capable of developing increased proportions of phenotypically mature T cells ca. 8±10 weeks of age (Somberg et al., 1996). Even in XSCID dogs that had relatively normal percentages of peripheral T cells, the absolute number averaged only 30% of normal, including three XSCID dogs raised in a gnotobiotic environment for three years. Fig. 1 also shows that as soon as T cells begin to appear in XSCID dogs they rapidly switch from a CD45RA (naive) phenotype to a CD45RAÿ (activated or memory
Fig. 1. Proportion of peripheral B and T cells in XSCID dogs.
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Fig. 2. Proliferative response of XSCID peripheral blood lymphocytes.
phenotype), a phenomenon that is reported to be proliferation-dependent in normal humans (Johannisson and Festin, 1995). Although it is clear that the early T cells observed in XSCID dogs are recent thymic emigrants, it is unclear whether the increased proportions seen in older XSCID dogs are due to increased release of thymocytes that rapidly switch their CD45RA phenotype or due to extra-thymic expansion of the few CD45RA peripheral T cells present during the first few weeks of life. One of the characteristic findings in XSCID dogs is an absent or markedly depressed lymphocyte blastogenic response to T cell mitogens and specific antigens. Fig. 2 illustrates that canine XSCID peripheral blood lymphocytes (PBL) exhibit a significant lack of proliferation in response to stimulation through the T cell receptor (PHA) and when the necessary second messengers for cellular proliferation are directly provided by the combination of phorbol ester, PMA, and calcium ionophore, CI, that by-pass signals delivered through ligand±receptor interaction. These data suggest that the proliferative defect resides distal to the generation of second messengers associated with activation through the T cell receptor. The addition of exogenous human recombinant IL-2 does not increase the proliferative response of XSCID PBL. The poor proliferative response of XSCID T cells can partially be attributed to the defective expression of functional IL-2 receptors (Fig. 3). XSCID dogs are hypogammaglobulinemic. Serum IgM concentrations may be normal, but IgG is dramatically reduced and IgA is usually absent (Jezyk et al., 1989). XSCID B cells are capable of proliferation when stimulated with formalin-fixed, heat-killed Staphylococcus aureus, a T-independent, B cell mitogen (Fig. 2). Polyclonal activation of canine XSCID B cells with the T cell-dependent, B cell mitogen pokeweed mitogen results in the production of IgM, but little or no IgG or IgA suggesting that partially functional B cells are present in XSCID dogs, but isotype switching does not occur
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Fig. 3. IL-2 receptor expression on XSCID resting and activated peripheral bood lymphocytes.
Fig. 4. Secondary antibody response in XSCID dogs following immunization with bacteriophage X174.
(Felsburg et al., 1992). To further address the functional capability of XSCID B cells, we immunized gnotobiotic normal and XSCID dogs with the T-cell dependent neoantigen, bacteriophage X174 at approximately one year of age. Fig. 4 illustrates the total antibody and IgG specific antibody titers following secondary immunization. Although XSCID dogs can produce minimal amounts of specific antibody, it is almost exclusively
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Fig. 5. Block in B cell differentiation in canine XSCID.
IgM. On the other hand, the specific antibody response in normal dogs is primarily IgG. Based on the presence of normal proportions of peripheral B cells and in the ability of XSCID B cells to produce IgM antibody but failure to class-switch to IgG or IgA, the block in B cell differentiation in canine XSCID appears to be relatively late in B cell differentiation pathway (Fig. 5). 5. Defective thymocyte development in XSCID dogs XSCID appears to be a disease primarily affecting the differentiation and maturation of the T cell lineage because of the paucity of peripheral T cells. A major issue that has not been previously addressed is what is occurring in the XSCID thymus. During human thymopoiesis, thymocytes undergo an ordered expression of cell surface antigens on developing thymocytes as well as a controlled mitogenesis of thymocytes at discrete stages of development (Terstappen et al., 1992; Spits et al., 1992). Proliferation is one of the most important events occurring during early thymocyte development. The differentiation of CD4ÿCD8ÿ (DN) thymocytes to CD4CD8 (DP) thymocytes is proliferation dependent, whereas the maturation of DP thymocytes to mature CD4CD8ÿ or CD4ÿCD8 (SP) thymocytes is proliferation independent. A steady expansion in the number of thymocytes takes place from the DN stage to the CD3DP stage. Proliferation ceases in the DP population following the surface expression of CD3. We have studied the thymi of 24 XSCID dogs and 24 age-matched normal littermates between 4 and 10 weeks of age (Somberg et al., 1994). The XSCID thymus is 8% the weight of age-matched normal dogs. The total number of thymocytes in XSCID dogs (0.63 108) is 0.3% of the number of thymocytes present in age-matched normal dogs (203 108). In normal dogs, the thymocyte subsets were found in proportions similar to those described in humans suggesting that the same ordered maturation process occurs in the dog thymus (Table 1). However, in the postnatal XSCID thymus, a mutated gc severely affects thymocyte development. The proportion of DN thymocytes in XSCID dogs is increased 3.5-fold and the DP population is decreased 2.3-fold. Interestingly, there is no significant difference in the proportion of SP thymocytes. When one takes into
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Table 1 Phenotypic characterization of XSCID thymocytes Subset ÿ
Normal ÿ
CD4 CD8 CD4CD8 CD4CD8ÿ CD4ÿCD8 a
XSCID a
13.1 6.1 68.7 12.4 11.6 7.8 2.9 1.8
45.6 22.2 30.7 20.6 10.5 8.6 2.2 4.4
Percent SD.
Fig. 6. Block in T cell differentiation in canine XSCID.
consideration the size and cellularity of the XSCID thymuses, one can appreciate that there is a paucity of all thymocyte subsets in the XSCID dogs. These results suggest a block at a very early stage in T cell differentiation which occurs in the XSCID thymus (Fig. 6). In addition to the significantly reduced cellularity in the postnatal XSCID thymus, we have shown that there is an increased proportion of DN cells (2.5-fold) that appear to be in the thymoblast stage of development (Somberg et al., 1994). These results suggest that postnatal XSCID thymocytes have a reduced ability to enter the mitotic phase of the cell cycle. The dramatic reduction in cellularity and the alteration in the proportion of DN and DP thymocytes in the XSCID thymus is highly suggestive of a proliferation defect limiting the expansion of the pool of thymocytes and impeding their differentiation. It appears that the transition of DP to SP cells, which is proliferation independent, occurs normally in XSCID dogs in the absence of a functional gc. Acknowledgements This work was supported by National Institutes of Health Grants AI 26103, HL 52971, AI 43745, and RR 12211 (PJF); and AI 33177 (PSH).
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