Immunology 1978 34 631

Immunobiology of congenitally athymic-asplenic mice M. E. GERSHWIN, A. AHMED, R. M. IKEDA, M. SHIFRINE & F. WILSON Section of Rheumatology, Clinical Immunology, and Laboratory of Radiobiology, University of California, School of Medicine, Davis, California 95616, and the National Naval Medical Research Institute, Bethesda, Maryland 20014 U.S.A.

Received 26 May 1977; acceptedfor publication 15 September 1977

growth and a lower inoculated cell threshold needed for successful transplantation of a human malignant melanoma. Finally, there was no evidence for autoantibody production in mice up to 9 months of age. Congenitally athymic-asplenic mice can be used for a variety of studies in which other immunologically deprived mouse mutants are desired.

Summary. Congenitally athymic-asplenic mice on an outbred N: NIH(S) background were produced by the mating of nude by hereditarily asplenic (Dh/+) mice. Athymic-asplenic mice survive for up to 9 months, under specific pathogen free conditions, with no evidence for increased risk of spontaneous neoplasia. Although lymphocyte surface markers and sera immunoglobulin levels of athymic-asplenic mice are similar to their nude and asplenic littermates, there are a number of significant differences. In particular, levels of sera IgA are higher than nude, but lower than either nu/ + or Dh/ + mice, related perhaps to the increased histiocytic engorgement of Peyer's patches. Athymic-asplenic mice have normal haematocrit, haemoglobin, and reticulocyte counts, but are markedly leucopenic, have a thrombocytosis and an increased number of bone marrow CFU-C. As expected, the response of the athymic-asplenic mice to the T cell mitogen PHA is markedly reduced. However, levels of Thy 1.2 bearing cells, while reduced compared to either nu/ + or Dh/ + littermates, are significantly higher than nude mice in both Peyer's patches and lymph nodes. Further, they, like their nude littermates, fail to respond to sheep red blood cell immunization. Nonetheless, athymic-asplenic mice appear more immunologically compromised than nude mice. Indeed, there is an elevated rate of


There have been two major congenitally immunologic deficient murine models described: the congenitally athymic (nude) mouse and the hereditarily asplenic mouse (Flanagan, 1966; Searle, 1969). Both of these lines were discovered as mutants in mouse colonies in Britain and have greatly served to advance our understanding of the interaction of the thymus and spleen with infectious agents autoimmune phenomena, and neoplasia (Lozzio & Wargon, 1974; Fletcher, Ikeda & Gershwin, 1977; Pantelouris, 1973). Thymic agenesis, in nude mice, is inherited as an autosomal recessive trait and is phenotypically identified by its association with hairlessness (Pantelouris, 1968). In contrast, the major defect in hereditarily asplenic mice is an age dependent retardation of T cell maturation as well as a decrease in total body B cells (Bucsi, Borek & Battisto, 1972; Wargon, Lozzio & Wust, 1975; Lozzio, 1972), both presumably secondary to the absence of

Correspondence: Dr M. Eric Gershwin, Section of Rheumatology-TB 192, School of Medicine, University of California, Davis, California 95616, U.S.A.

0019-2805/78/0400-0631 502.00 (©1978 Blackwell Scientific Publications



M. E. Gershwin et al.

a spleen. Asplenia is inherited as an autosomal dominant disorder, and is phenotypically identified by major abnormalities of hind limbs or hemimelia; hence, it is referred to as carrying the Dh gene (for dominant hemimelia). A feature of long-term studies of both nude and Dh/ + mice has been the low spontaneous incidence of neoplasia, observations which appear at variance with existing theories of immune surveillance (Rygaard & Poulsen, 1974; Fletcher, Ikeda & Gershwin, 1976). Because of the availability of both these mouse mutants under specific pathogen free conditions in our laboratory, we have intercrossed these colonies to produce outbred mice congenitally absent for both thymus and spleen. Such mice have previously been referred to as 'lasat' and have been used in transplantation studies of human myeloid leukaemias (Lozzio, Machado, Lozzio & Lair, 1976). We report herein a detailed survey of lasat mice and indicate that they too have a low spontaneous tumour incidence as well as several major immunologic differences which are distinctive from both nude and asplenic littermates.



Congenitally athymic (nude) as well as hereditarily asplenic (Dh/ +) mice on an N: NIH(S) background were maintained under specific pathogen free conditions at the University of California School of Medicine. The natural history and immunobiology of these colonies have been previously described in detail (Fletcher et al., 1977; Gershwin, Merchant, Gelfand, Vickers, Steinberg & Hansen, 1975; Gershwin, Merchant & Steinberg, 1977a). A colony of congenitally athymic-asplenic mice was prepared by the mating of Dh/ + females x nu/nu males. This mating resulted in offspring, all of which carried the nude gene and 50 % of which could be phenotypically identified by hind limb abnormalities as being asplenic. The male asplenic mice from these litters (i.e. nu/ + Dh/ + ) were then mated with either nu/nu or nu/ + females to produce litters composed of nu/nu, nu/ +, Dh/ +, and nu/nu Dh/ + mice. N : NIH(S) mice are extremely fertile and produce litters of 8-16 offspring. Therefore, appropriate numbers of mice on this outbred background can be produced without difficulty. Mice of both sexes were employed in the studies herein.

Natural history Over a period of 12 months, 79 athymic-asplenic (nu/nu Dh/ +) mice were studied. This group of mice included 51 who were followed for a total of 9 months. Comparable age- and sex-matched nu/nu, nu/ +, and Dh/ + mice were followed concurrently. At the time of death, vigorous autopsies and serial sections were prepared from visceral organs and lymph nodes. Haematologic characteristics Groups of 4-6 nu/nu, nu/ +, nu/nu Dh/ +, and Dh/ + mice were bled by orbital sinus puncture under ether anaesthesia. Haematocrit, haemoglobin, reticulocyte, white cell and platelet counts were determined. Additionally, other groups of mice were killed by cervical dislocation and their femora removed. Bone marrow cells were obtained from each femur, as described by injecting media through a 22-gauge needle inserted into the femoral cavity. A single cell suspension was prepared and the samples from each marrow handled individually and dispersed several times through a 27-gauge needle to prepare single cell suspensions (Garner, Gershwin & Steinberg, 1974). Viability was confirmed by the trypan blue dye exclusion technique. Quantitation of bone marrow CFU-C were performed by a modification of the methylcellulose culture system using 15 % methylcellulose in medium 199 supplemented with 5 % foetal calf serum, 2 mm glutamine, 100 units/ml penicillin, 100 grgm/ml streptomycin and 0-25 ugm amphotericin B (Wilson, O'Grady, McNeill & Munn, 1974). Cultures were initiated by adding 1 x 106 viable nucleated marrow cells/ml in methylcellulose and mixing the suspension on a magnetic stirrer. A 10% dilution of human leucocyte conditioned medium was used as a source of colony stimulating factor (Wilson et al., 1974). Cultures were incubated for 7 days at 370 in a 5 % CO2 humidified air atmosphere. In all experiments, colony counts were done following staining with 0-25 % new methylene blue. All counts were done at x 45 magnification using a dissecting microscope. Quantitative immunoglobulins Groups of 4-6 nu/nu, nu/ +, nu/nu Dh/ + and Dh/ + mice were bled by orbital sinus puncture and the sera allowed to clot. Levels of sera IgG1, IgG2, IgM, and IgA were quantitated by radial immunodiffusion using monospecific anti-immunoglobulin

Immunobiology of congenitally athymic-asplenic mice impregnated plates (Gershwin et al., 1975). The plates were prepared by mixing rabbit monospecific anti-mouse immunoglobulin sera with 1-5 % agarose in phosphate buffered saline. Known positive reference sera were included in each plate. Surface markers Nu/nu, nu/+, nu/nu Dh/+ and Dh/+ mice, 6-8 weeks of age, in groups of 3-5, were killed by cervical dislocation and their thymus, lymph nodes, spleen, bone marrow and Peyer's patches removed asceptically. Thymus and spleen were also removed from those animals containing such organs. Single cell suspensions were prepared and the number of lymphocytes bearing either theta or Ig surface markers were determined as follows. Theta bearing cells were assayed by trypan blue dye exclusion using AKR anti-Thy 1.2 antisera, previously known to be cytotoxic for approximately 50 % of C3H thymocytes at a 1: 1024 dilution (Gershwin, Chused & Steinberg, 1974). This anti-Thy 1.2 sera was produced by a modification of the technique of Reif & Allen (Parker, Chused & Steinberg, 1974). All assays were done in duplicate and the values between samples differed by less than 5 %. Complement alone provided less than 3 % cytotoxicity. Surface immunoglobulin was detected by immunofluorescence using a rabbit polyvalent fluorescein conjugated anti-mouse immunoglobulin. This antisera was centrifuged at 40,000 g immediately prior to use and gave similar results to F(AB)2 prepared conjugates. Response to mitogens Lymph nodes from groups of 5-7 nu/nu, nu/+, nu/nu Dh/ + and Dh/ + mice 4-6 weeks of age were obtained following cervical dislocation. Single cell suspensions were prepared in RPMI 1640 media containing 20 mm HEPES, 2 mm L-glutamine, 100 units/ml Penicillin, 100 pg/ml streptomycin, and 5% heat inactivated foetal calf serum (Rehautin, Phoenix, Arizona). The cells were cultured in U bottom microtitre plates. To sextuplicate cultures from each animal were added either 100 p1 of phytohaemagglutinin-P, 0-2 % final concentration (Difco Labs, Detroit, Michigan) or Escherichia coli lipopolysaccharide 50 pg per culture (Difco Labs, Detroit, Michigan) or RPMI 1640 media alone. These mitogen concentrations were previously found to be optimal (Kramer & Gershwin, 1976). Cultures were incubated for 72 h at 370 in 5 %


carbon dioxide, 95 % humidified air atmosphere. Four hours before harvest, each culture received 1 pCi of 3H-methylthymidine (3H-TdR) (1 9 Ci/mM, New England Nuclear, Boston, Massachusetts in 10,p1 of media). Cultures were harvested with a multiple automated sample harvester (Strong, Ahmed & Thurman, 1973). Spontaneous antibodies Groups of 6-9 nu/nu, nu/ +, nu/nu Dh/ + and Dh/ + mice ranging in age from 6 weeks to 9 months of age were bled by orbital sinus puncture. The sera were removed and a modified Farr ammonium sulphate microprecipitation method performed to quantitate antibodies to both native DNA ('4C-KB DNA) and the synthetic double stranded RNA, Poly I. Poly C. ("4C-Poly I. Poly C, Miles Labs, Elkhart, Indiana) (Gershwin, Blaese, Steinberg, Wistar & Strober, 1976). The same sera were quantified for antinuclear antibodies by indirect immunofluorescence. A direct Coombs' test was performed on the washed red cells (Gershwin & Steinberg, 1975). Red cells from 1-year-old NZB mice served as positive controls. Finally, levels of thymocytotoxic antibodies were measured in all mice using sera from 6-month-old NZB males as positive controls (Shirai, Yoshiki & Mellors, 1972). Response to sheep red cells Groups of 5-7 nu/nu, nu/ +, nu/nu Dh/ +, and Dh/ + mice were immunized with 2 x 108 washed SRBC i.p. Ten days later mice were bled by orbital sinus puncture, the sera collected, and the haemagglutination titre determined in microtitre plates. They were then boosted with 2 x 108 SRBC and the titre repeated 10 days thereafter.

Growth of heterotransplanted tissue Groups of nu/nu and nu/nu Dh/ + mice were injected subcutaneously with 105, 106, or 107 viable cells from a human malignant melanoma (Gershwin, Ikeda, Kayakami & Owens, 1976). The success for transplantation and subsequent growth rate was compared between the two groups. Ratio of delta to mu Cell membrane proteins of lymph node cells from nu/nu, nu/+, nu/nu Dh/+ and Dh/+ mice were labelled by the lactoperoxidase iodination technique as previously described (Finkelman, Shevach, Vitetta, Green & Paul, 1976).


M. E. Gershwin et al.

Figure 1. Congenitally athymic-asplenic (lasat) mouse. Note the hairlessness as well as bilateral deformed hind legs.

Frequency of Ig positive cells of varying density using the fluorescent cell sorter Single cell suspensions of lymph nodes from nu/nu, nu/ +, nu/nu Dh/ + and Dh/ + mice were prepared at 5 x 106 per ml. The cell suspensions were stained with the use of either a fluorescent polyvalent antisera or a fluorescent anti-p sera. Use was made of a fluorescent activated cell sorter (FACS-2) and the frequency of total Ig positive cells determined. Further, fluorescent Ig positive cells were subdivided into 2 subpopulations; one that possessed a low to intermediate level of Ig and the other consisting of cells with dense surface Ig. RESULTS Natural history Congenitally nu/nu Dh/ + mice on a N: NIH(S) background maintained under the conditions outlined above, and without the need for antibiotics, survive 7-9 months without difficulty, with less than a 5 % monthly mortality until age 6 months (Fig. 1). Thereafter, the mortality increases to 10% per month. The perinatal mortality of these mice, however, is slightly higher than nude mice and appears due to occasional imperforate anus and short gastro-intestinal tracts. This increase of perinatal

mortality is similar to conventional Dh/ + mice (Searle, 1959). Additionally, over the 9-month time interval studied herein, not a single tumour was observed in either the nu/nu, nu/ +, nu/nu Dh/ + or Dh/ + group. As expected on the basis of the phenotypic markers hairlessness and hemimelia, nu/nu Dh/ + mice had neither a thymus or spleen at autopsy. Histologic examination of lymph nodes and Peyer's patches revealed striking abnormalities. The lymph nodes of nu/nu Dh/ + mice were depleted of paracortical areas with poor follicle formation and absence of germinal centres. Such features are similar to lymph nodes from nu/nu mice. However, in contrast to nude mice, both lymph nodes and Peyer's patches from nu/nu Dh/ + mice were strikingly engorged with histiocytic-like elements and were grossly larger than all other groups (Fig. 2). Lymph nodes and Peyer's patches from Dh/ + mice are normal and similar to nu/+ mice. Haematology The haematocrit and reticulocyte count of all groups of mice studied were approximately the same (Table 1). In contrast, the mean white cell count of nu/nu and nu/nu Dh/ + mice was markedly diminished, 2900-3200 compared to 7600/mm in nu/+ controls.

Immunobiology of congenitally athymic-asplenic mice


Figure 2. Lymph nodes from athymic-asplenic mouse. (a) Note gross absence of germinal centres or follicles as well as pseudocyst formation in the medulla (H & E, x 40). (b) Under higher magnification the extent of depletion of the lymph node T-cell dependent area is evident (H & E, x 150). (c) The lymph node is diffusely infiltrated with mononuclear and histiocytic cells (H & E, x 450).

M. E. Gershwin et al.


Table 1. Haematologic characteristics of athymic-asplenic mice


nu/nu nu/+ nu/nu Dh/ + Dh/ +



46 48 49 48

2 2 2 2

WBC (cells/mm) 3200t 7600 2900t


Platelets Femur cellularity ( x 106) (x 106/mm)

0-8t 0-8$ 1-2 1-3

13-5 ± 1-1 12-2±3-0 11-5 ±2-1 10-8 ±2-7



1167 ± 85t 940±130 1152 ± 110$ 890 ±93

158 ± 18t 114±20 131 ±20t 96 ±29

* Mean ± s.e.m. t P < 0001 compared to nu/ + or Dh/ + group. $ P < 001 compared to nu/ + or Dh/ +.

Of interest was that the white cell count of Dh/ + mice was significantly increased, with levels of approximately 13,500/mm (P< 0)01) (Table 1). Additionally, the platelet count of nu/nu Dh/ + and Dh/ + mice were increased compared to nu/nu and nu/+ mice (Table 1) (P

Immunobiology of congenitally athymic-asplenic mice.

Immunology 1978 34 631 Immunobiology of congenitally athymic-asplenic mice M. E. GERSHWIN, A. AHMED, R. M. IKEDA, M. SHIFRINE & F. WILSON Section of...
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