- Email: [email protected]
[4] Graft-versus-host reactions
28
SURGICAL TECHNIQUES
IN IMMUNOLOGY
[4]
to suture a thin skin graft to a bed on the superficial fascia of the lateral thoracic wall and leave it uncovered and thus visible from the day of grafting, until rejection. J8 However, this procedure requires trimming the teeth and toenails of the recipients to prevent the animals from destroying the graft, and the method is practicable probably only with mice or very young rats. The method of Bailey and Usama 19as modified by Bailey 2° is a simple, ingenious technique for grafting thin slices of tail skin orthotopically to the tail itself, where they are protected by a length of glass tubing and also visible at all times. This method is especially useful for histocompatibility testing or for comparing the survival of antigenically different test grafts on the same host because as many as 8 small (e.g., 5 × 2 mm) grafts can be placed on a single adult mouse tail. Obviously, the method is not appropriate for studies requiring larger grafts or other host sites. For skin grafting chickens or other birds, the article by Billingham 21 should be consulted. is p. H. Sugarbaker and A. E. Chang, J. Immunol. Methods 31, 167 (1979). ~9D. W. Bailey and B. Usama, Transplant. Bull. 7, 424 (1960). 20 D. W. Bailey, Tranplantation 1, 70 (1963). 2~ R. E. Billingham, in "Transplantation of Tissues and Cells" (R. E. Billingham and W. K. Silvers, eds.), p. 31. Wistar Inst. Press, Philadelphia, Pennsylvania, 1961.
[4] G r a f t - v e r s u s - H o s t R e a c t i o n s By DAVID STEINMULLER Introduction Graft-versus-host (GVH) reactions have been aptly defined by Elkins j as the immunological responses of donor lymphoid cells to foreign histocompatibility antigens expressed by the host. Thus, just as the ability to reject foreign skin grafts is a measure of immune competence at the organismic level, the ability to mediate GVH reactions is an in vivo measure of immune competence at the cellular level. In fact, GVH reactions may be considered a special form of adoptive immunity (see this volume [5]) in which cell-surface antigens of the host itself are the targets. Indeed, systemic GVH reactions can result in GVH d i s e a s e Y the complex syndrome W. L. Elkins, Prog. Allergy 15, 78 (1971). P. L. Weiden, in "Biology of Bone Marrow Transplantation" (R. P. Gale and C. F. Fox, eds.), p. 37. Academic Press, New York, 1980. 3 j. A. Hansen, J. M. Woodruff, and R. A. Good, in "Immunodermatology" (B. Safai and R. A. Good, eds.), p. 229. Plenum, New York, 1981.
METHODS IN ENZYMOLOGY, VOL. 108
Copyright © 1984 by Academic Press, inc. All rights of reproduction in any form reserved. ISBN 0-12-182008-4
[4]
GRAFT-VERSUS-HOSTREACTION
29
resulting from the combined effects of GVH reactions, secondary infectious complications, ~ and aberrations of the immune system, that is the major obstacle to the more widespread clinical application of bone marrow transplantation. The three prerequisites 4 for the induction of GVH reactions are (l) the graft must include a significant number of immunologically competent lymphocytes; (2) the host must express histocompatibility antigens foreign to the lymphocyte donor; and (3) the host must be, at least temporarily, incapable of rejecting the grafted cells. In the latter regard, permissive hosts are (1) embryonic and neonatal animals4 ; (2) Fi hybrids produced by mating two highly inbred strains, the lymphocyte donor being one of the parent strains4; (3) hosts with inherited or congenital immunologic deficiencies (e.g., athymic mice or children with severe combined immunodeficiency3); and (4) hosts deliberately immunosuppressed by total-body irradiation or cytotoxic drugs such as cyclophosphamide.2 The immunopathology of GVH disease (e.g., " r u n t " disease in neonates, "allogeneic" disease in FI hybrids, "secondary" disease in radiation chimeras, and acute and chronic GVH disease after clinical bone marrow transplantation) is reviewed extensively elsewhere. I-6 This chapter is concerned with the application of acute GVH reactions as ~'tools of re~ search," s specifically as in vivo measures of the immunologic competence of lymphoid cells from various sources.
Sources of Effector Cells Because acute GVH reactions are mediated by thymus-dependent lymphocytes, 7 the GVH potency of a lymphoid-cell inoculum depends on its content of mature T cells. 8 Thus, mouse bone marrow, which, in contrast to human bone marrow, contains very few T cells, s is a poor source of GVH-inducing cells compared to splenic or lymph node cells, whereas fatal GVH disease can be produced with cells obtained from the thoracic duct, a rich source of T cells, even with only minor histocompatibility differences. 9 Methods of preparing single-cell suspensions from various lymphoid organs and the thoracic duct are described elsewhere in this volume [6]. The appropriate dose is an empirical matter depending on the type of assay (systemic versus regional, see below), the cell source 4 R. E. Billingham, Science 130, 947 (1959). M. Simonsen, Prog. Allergy 6, 349 (1962). S. C. Grebe and J. W. Streilein, Adv. lmmunol. 22, 119 (1976). 7 A. P. Dalmasso, C. Martinez, and R. A. Good, Proc. Sot'. Exp. Biol. Med. 110, 205 (1962). D. A. Vallera, A. Filipovich, C. C. B. Soderling, and J. H. Kersey, Clin. Immunol. Imrnunopathol. 23, 437 (1982). 9 R. Korngold and J. Sprent, J. Exp. Med. 148, 1687 (1978).
30
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[4]
(the proportion of mature T cells, as just indicated), the immune status of the donor (whether the donor is "normal" or has been specifically immunized against host histocompatibility antigens~), and the nature and extent of donor-host genetic disparity (much higher doses of cells usually are required to induce GVH reactions with minor histocompatibility differences~), and an experimental protocol may require testing different dosages. However, in general, GVH reactions usually can be elicited with 107 or fewer unseparated spleen or lymph node cells, at least when major histocompatibility differences are involved. J.5.6
Systemic GVH Assays Mortality and Gross Pathology. When appropriate numbers of allogeneic T cells are injected intravenously or intraperitoneally into permissive perinatal or adult hosts (see above), the simplest way of assaying the consequences is to record the mortality and the well-known pathological changes associated with GVH disease, such as runting or weight loss and the development of skin lesions, hunched posture, and diarrhea. ~°-~2 However, this relatively crude way of measuring GVH reactions is subject to numerous variables, such as the overall health of the hosts to start with, the fastidiousness of animal care and management, and, especially, complications of infectious diseases. ~3 The Discriminant Spleen Assay. This more precise and objective assay was designed by Simonsen 5 to take advantage of the marked splenomegaly that characteristically precedes the atrophy of lymphoid tissue when adult T cells are injected into allogeneic embryos or neonates. To conduct the assay with newborn mice or rats, litters 1-10 days old that are being well cared for by their mothers are selected. Half the litter is used as test animals and half as uninjected controls (one group can be marked by cauterizing the tips of their tails with an electrocauterizer or with the end of a scalpel heated with a Bunsen burner). Cells (107) (or some other desired number) are injected intraperitoneally with a 25-gauge f-in. needle. Even this small volume cannot be injected directly into the tight belly of a newborn rodent without some of the fluid escaping, so the tip of the needle has to be inserted through an arm or leg muscle and be pushed subcutaneously until it passes into the peritoneal space. To do this, the newborns are carefully anesthetized with ether or some other inhalant 10 R. E. Billingham, V. Defendi, W. K. Silvers, and D. Steinmuller, J. Natl. Cancer Inst. (U.S.) 28, 365 (1962). 11 p. Stastney, V. A. Stembridge, and M. Ziff, J. Exp. Med. 118, 635 (1963).
~2D. W. van Bekkumand M. J. de Vries, "RadiationChimaeras." AcademicPress, New York, 1967. i3 D. Keast and M. N. I. Waiters,Immunology 15, 247 (1968).
[4]
GRAFT-VERSUS-HOSTREACTION
31
(see below) or very young litters, which are poikilothermic, are placed in a refrigerator for a few minutes. Splenomegaly peaks 8-10 days after donor-cell injection. ~4The entire litter is sacrificed at this time by cervical dislocation or by overanesthetizing it. First each entire animal is weighed to the nearest milligram; then the spleen is excised and weighed, being careful first to remove all adherent fat. The spleen weight is expressed as a percentage of the body weight for each test and control host. An index of spleen enlargement for each test host is then calculated by dividing its spleen/body weight ratio by the mean ratio for the littermate controls. This index permits comparing results between litters because the variation in spleen weight between litters is greater than that within litters.~4 Inhibition of Colony-Forming Units (CFU). A disadvantage of the Simonsen assay is that it requires newborn hosts. However, systemic GVH reactivity can also be measured in terms of the ability of aliogeneic lymphoid cells to inhibit hematopoietic CFUs in lethally irradiated adult hosts. 15 The assay was designed for inbred animals where one can use syngeneic bone marrow (i.e., marrow from other, histocompatible donors of the same strain) as a source of hematopoietic stem ceils, but one can perform it also with outbred animals if one obtains bone marrow cells from the host before irradiation for later injection. The assay is conducted by exposing hosts to a lethal dose of total-body, gamma irradiation (750900 rads for mice or rats) and then giving them 2 x 10 6 syngeneic (or autologous) bone marrow cells intravenously alone (the control) or with the desired number of test lymphoid cells. Six days later 0.2/xCi of ~9Fe citrate is injected intraperitoneally into the hosts. After 17 hr, the hosts are sacrificed, their spleens are removed, and the radioactivity present is determined in a liquid scintillation counter, using a scintillation cocktail appropriate for counting aqueous samples.
Regional GVH Assays lntracutaneous. One can obtain a semiquantitative measure of GVH reactivity in most laboratory animals by injecting a few million allogeneic lymphocytes directly into the skin and scoring the resultant reactions like delayed-type hypersensitivity tests. 6 The best sites for injection are the flank for guinea pigs, the belly for rats, and the back for mice. First the hair is removed with electric clippers (Oster small animal clippers with size 40 cutting blades are recommended); then the area is shaved with a razor or depilated with a cream such as Nair (Carter Products, NY). In ~4 M. Simonsen, J. Engelbreth-Holm, E. Jensen, and H. Poulsen, Attn. N. Y. A c , d . Sci. 73, 834 (1958). ~s H. Blomgren and B. Andersson, Cell. lrnrnunol. 3, 318 (1972).
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[4l
rats and guinea pigs, the desired dose (e.g., 107 cells) is injected intraderreally in 0.1 ml using a 27-gauge needle with an intradermal bevel; with mice 30/.Li is injected using a 30-gauge needle and a Hamilton No. 705 syringe (Hamilton Co., Reno, NV). The cells must be injected intradermally, not subcutaneously, by inserting the needle very superficially bevel-up. If a bleb does not form by pushing the syringe plunger the needle is in the subcutaneous tissue. Replicate injections are given each host at 3-4 separate sites. The reactions peak at 2-3 days, and the injection sites should be assessed at 24-hr intervals for erythema, induration, and necrosis (the latter may occur only with presensitized cells), assigning scores according to size and intensity, such as + / - for a small, erythematous spot without swelling and 5+ for a large indurated and necrotic reaction. Streilein and Billingham j6 recommend the following scale for mice: 0, no response; 1+, barely perceptible swelling; 2+, swelling 3-4 mm in diameter, site soft to palpation; 3+, swelling 5 mm or more in diameter, site firm; 4+, large reaction with indurated core; 5+, site necrotic or ulcerated. At least 3 sites on each animal should be read daily and the results expressed as a mean score. Moreover, because of the subjective nature of the assay, it is wise to code-number the subjects and score them "blindly" in experiments involving different dosages and treatment groups. A disadvantage of the assay is that if the hosts are able to respond to donor histocompatibility antigens, the skin reactions may include a host-versus-graft component. One can preclude this with inbred animals by using Fj hybrid hosts, as discussed later for the PLN enlargement assay. lntrarenal. The injection of lymphoid cells from inbred, parent-strain donors beneath the renal capsule of adult F~ hybrid hosts results in a marked local, invasive-destructive lesion, j7 There is an initial burst of donor-cell proliferation in the injected kidney that peaks at 6 days, which can be measured by the incorporation of r25IUdR,~8 followed by an increase in renal weight that peaks at 14 days. For the assay, the injected and the uninjected contralateral (control) kidneys either are weighed or the incorporation of ~25IUdR is determined with a gamma counter, or both, and the results expressed as ratios of injected to control values. J8 The intrarenal GVH reaction is of great interest to transplantation immunologists because its histopathology mimics that of acute renal allograft rejection. 6 However, the assay is difficult because the kidney must be surgically exteriorized for inoculation, jv and it is not as sensitive as the PLN enlargement assay described below. 16 j. W. Streilein and R. E. Billingham, J. Exp. Med. 131, 409 (1970). i7 W. L. Elkins, J. Exp. Med. 12,0, 329 (1964). is W. L. Elkins, Transplantation 9, 273 (1970).
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Popliteal Lymph Node (PLN) Enlargement Assay Introduction. The injection of parent-strain lymphoid cells below the hind footpads of F~ hybrid rats 19or mice 2° results in an enlargement of the single lymph node draining the site, the PLN, which can be weighed to provide a simple, reliable measurement of regional GVH reactivity. The assay is easier and more sensitive than the intrarenal assay, ~9 and more objective than the intracutaneous assay, its only disadvantage is that it is applicable only to inbred animals where F~ hybrids can be produced by mating genetically homozygous parents. Such hybrids are genetically tolerant of parent-strain cells; if they are not used, host-versus-graft reactions contribute to the reaction. 6 One cannot suppress the host component with radiation, for example, because radiosensitive host lymphocytes are required for the full expression of PLN enlargement. 6 Injecting the Host. With rats, the desired number of cells (e.g., 10 7 donor lymph node or spleen cells) is injected in 0.1 ml using a 25-gauge, ~in. needle attached to a I ml or smaller syringe. With mice, the cells are injected in 50 /zl using the same size needle but a Hamilton No. 705 syringe. The animal is anesthetized by placing it in a closed glass jar or other container with a wad of cotton or gauze pads wet with ether or, preferably, a nonflammable, nonexplosive inhalant anesthetic such as methoxyflurane, enflurane, or halothane. The hind foot of the anesthetized animal is grasped by the toes and the tip of the needle is inserted just in front of the footpads. The tip of the needle is pushed 6-10 mm toward the heel before delivering the dose. The needle should be in the subcutaneous, not the intradermal, tissue because the former injection site gives the best results. 19 When the needle is withdrawn, a brief pressure is applied at the puncture site with a cotton applicator or piece of gauze to prevent leakage. Although one can inject both hind feet (e.g., with different cell populations or dosages~9), it is useful to leave one foot uninjected as a control (see below). Harvesting the Node. PLN enlargement usually peaks 7 days after inoculation of the footpad in both rats and mice and this is the optimal time for weighing the nodes, though one can weigh them earlier if one wants time-course data. The PLN is a spherical node embedded in adipose tissue in the middle of the popliteal fossa :f (Fig. 1). To remove it, the animal is sacrificed with an overdose of anesthesia (or with mice, by cervical dislocation). The animal is pinned belly-down on an operating board and the legs are rotated so that the soles face upward. The hair is t~ W. L. Ford, W. Burr, and M. Simonsen, Transplantation 10, 258 (1970). z0 V. W. Twist and R. D. Barnes, Transplantation 15, 182 (1973). 2~ y . Kawashima, M. Sugimura, Y. C. Hwang, and N. Kudo, Jpn. J, Vet. Res. 12, 69 (1964).
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FiG. 1. Location of the popliteal lymph node in rats and mice (after Kawashima the node actually is more medial than suggested by this sketch).
[4]
e t a l . z~ ;
removed from the site with electric clippers and the area is soaked with 70% alcohol. A superficial incision is made with blunt-tipped scissors (the tip of the scissors should run just under the skin) from mid-thigh nearly to the heel, passing over the middle of the popliteal region, and the skin from the incision is retracted to reveal the node, which is quite obvious on the injected side (Fig. 2). The incision is kept open by pinning-down or clamping the retracted skin, and the node is removed with smooth-tipped, fine forceps (curved jeweler's forceps are highly recommended; e.g., Storz Instrument Co., St, Louis, MO, No. E-1947-7). The node must be freed of
FIG. 2. The enlarged left popliteal lymph node of an Ft hybrid mouse 7 days after the injection of 107 allogenic spleen cells below the left footpad (the node was freed from the surrounding fat for the photograph).
[5]
ADOPTIVE
TRANSFER
35
fat before placing it in saline (0.9% sodium chloride) in a small petri dish. If the node does not sink, fat is still attached to it. If necessary, the node can be excised from an anesthetized, living animal by making a smaller incision and suturing the retracted skin together after removing the node. Weighing the N o d e . It is very important to remove excess moisture before weighing. This can be done by gently rolling the node on a stack of 2 or more pieces of hard filter paper (e.g., Whatman No. 50). Each node is weighed to 0.01 mg with an analytical balance. A convenient way of doing this is to first weigh an empty piece of weighing paper, then place the nodes one by one on the paper and record the change in weight each time. The results for each injected host are expressed as the P L N enlargement ratio calculated by dividing the weight of the node from the injected side by that of the node from the uninjected, control side. One should test at least 3 hosts with each dose and type of cells and calculate the mean enlargement ratio -+ the standard error or standard deviation. With organ enlargement assays in general, the significance of differences between test and control groups is best determined by analysis of variance. G V H Reactions in Birds
GVH reactions in chickens and other birds can be assayed by measuring spleen enlargment 5 or by counting and measuring the white " p o c k s " that appear on the chorioallantoic membrane (CAM) following inoculation of the embryo with allogeneic blood or lymphoid cells. See Longenecker et al. 22,z3 for details of the CAM assay. .,2 B. M. Longenecker, S. Sheridan, G. R. J. Law, and R. F. Ruth, Transplantation 9, 544 (1970). 2~ B. M. Longenecker, F. Pazderka, G. R. J. Law, and R. F. Ruth, Cell. hnmunoL 8, I (1973).
[5] Adoptive Transfer By DAVID STEINMULLER
The term, adoptive immunity, was coined by Billingham et al. ~in 1954 to describe the transfer of immunity from one animal to another by live, immunocompetent cells as opposed to immune serum or preformed antibody (the term passive transfer should be reserved for the transfer of t R. E. Billingham, L. Brent, and P. B. Medawar, Proc. R. Sot'. London, Ser. B 143, 58 (1954).
METHODS IN ENZYMOLOGY, VOL. 108
Copyright ~t~ 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182008-4
SURGICAL TECHNIQUES
IN IMMUNOLOGY
[4]
to suture a thin skin graft to a bed on the superficial fascia of the lateral thoracic wall and leave it uncovered and thus visible from the day of grafting, until rejection. J8 However, this procedure requires trimming the teeth and toenails of the recipients to prevent the animals from destroying the graft, and the method is practicable probably only with mice or very young rats. The method of Bailey and Usama 19as modified by Bailey 2° is a simple, ingenious technique for grafting thin slices of tail skin orthotopically to the tail itself, where they are protected by a length of glass tubing and also visible at all times. This method is especially useful for histocompatibility testing or for comparing the survival of antigenically different test grafts on the same host because as many as 8 small (e.g., 5 × 2 mm) grafts can be placed on a single adult mouse tail. Obviously, the method is not appropriate for studies requiring larger grafts or other host sites. For skin grafting chickens or other birds, the article by Billingham 21 should be consulted. is p. H. Sugarbaker and A. E. Chang, J. Immunol. Methods 31, 167 (1979). ~9D. W. Bailey and B. Usama, Transplant. Bull. 7, 424 (1960). 20 D. W. Bailey, Tranplantation 1, 70 (1963). 2~ R. E. Billingham, in "Transplantation of Tissues and Cells" (R. E. Billingham and W. K. Silvers, eds.), p. 31. Wistar Inst. Press, Philadelphia, Pennsylvania, 1961.
[4] G r a f t - v e r s u s - H o s t R e a c t i o n s By DAVID STEINMULLER Introduction Graft-versus-host (GVH) reactions have been aptly defined by Elkins j as the immunological responses of donor lymphoid cells to foreign histocompatibility antigens expressed by the host. Thus, just as the ability to reject foreign skin grafts is a measure of immune competence at the organismic level, the ability to mediate GVH reactions is an in vivo measure of immune competence at the cellular level. In fact, GVH reactions may be considered a special form of adoptive immunity (see this volume [5]) in which cell-surface antigens of the host itself are the targets. Indeed, systemic GVH reactions can result in GVH d i s e a s e Y the complex syndrome W. L. Elkins, Prog. Allergy 15, 78 (1971). P. L. Weiden, in "Biology of Bone Marrow Transplantation" (R. P. Gale and C. F. Fox, eds.), p. 37. Academic Press, New York, 1980. 3 j. A. Hansen, J. M. Woodruff, and R. A. Good, in "Immunodermatology" (B. Safai and R. A. Good, eds.), p. 229. Plenum, New York, 1981.
METHODS IN ENZYMOLOGY, VOL. 108
Copyright © 1984 by Academic Press, inc. All rights of reproduction in any form reserved. ISBN 0-12-182008-4
[4]
GRAFT-VERSUS-HOSTREACTION
29
resulting from the combined effects of GVH reactions, secondary infectious complications, ~ and aberrations of the immune system, that is the major obstacle to the more widespread clinical application of bone marrow transplantation. The three prerequisites 4 for the induction of GVH reactions are (l) the graft must include a significant number of immunologically competent lymphocytes; (2) the host must express histocompatibility antigens foreign to the lymphocyte donor; and (3) the host must be, at least temporarily, incapable of rejecting the grafted cells. In the latter regard, permissive hosts are (1) embryonic and neonatal animals4 ; (2) Fi hybrids produced by mating two highly inbred strains, the lymphocyte donor being one of the parent strains4; (3) hosts with inherited or congenital immunologic deficiencies (e.g., athymic mice or children with severe combined immunodeficiency3); and (4) hosts deliberately immunosuppressed by total-body irradiation or cytotoxic drugs such as cyclophosphamide.2 The immunopathology of GVH disease (e.g., " r u n t " disease in neonates, "allogeneic" disease in FI hybrids, "secondary" disease in radiation chimeras, and acute and chronic GVH disease after clinical bone marrow transplantation) is reviewed extensively elsewhere. I-6 This chapter is concerned with the application of acute GVH reactions as ~'tools of re~ search," s specifically as in vivo measures of the immunologic competence of lymphoid cells from various sources.
Sources of Effector Cells Because acute GVH reactions are mediated by thymus-dependent lymphocytes, 7 the GVH potency of a lymphoid-cell inoculum depends on its content of mature T cells. 8 Thus, mouse bone marrow, which, in contrast to human bone marrow, contains very few T cells, s is a poor source of GVH-inducing cells compared to splenic or lymph node cells, whereas fatal GVH disease can be produced with cells obtained from the thoracic duct, a rich source of T cells, even with only minor histocompatibility differences. 9 Methods of preparing single-cell suspensions from various lymphoid organs and the thoracic duct are described elsewhere in this volume [6]. The appropriate dose is an empirical matter depending on the type of assay (systemic versus regional, see below), the cell source 4 R. E. Billingham, Science 130, 947 (1959). M. Simonsen, Prog. Allergy 6, 349 (1962). S. C. Grebe and J. W. Streilein, Adv. lmmunol. 22, 119 (1976). 7 A. P. Dalmasso, C. Martinez, and R. A. Good, Proc. Sot'. Exp. Biol. Med. 110, 205 (1962). D. A. Vallera, A. Filipovich, C. C. B. Soderling, and J. H. Kersey, Clin. Immunol. Imrnunopathol. 23, 437 (1982). 9 R. Korngold and J. Sprent, J. Exp. Med. 148, 1687 (1978).
30
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[4]
(the proportion of mature T cells, as just indicated), the immune status of the donor (whether the donor is "normal" or has been specifically immunized against host histocompatibility antigens~), and the nature and extent of donor-host genetic disparity (much higher doses of cells usually are required to induce GVH reactions with minor histocompatibility differences~), and an experimental protocol may require testing different dosages. However, in general, GVH reactions usually can be elicited with 107 or fewer unseparated spleen or lymph node cells, at least when major histocompatibility differences are involved. J.5.6
Systemic GVH Assays Mortality and Gross Pathology. When appropriate numbers of allogeneic T cells are injected intravenously or intraperitoneally into permissive perinatal or adult hosts (see above), the simplest way of assaying the consequences is to record the mortality and the well-known pathological changes associated with GVH disease, such as runting or weight loss and the development of skin lesions, hunched posture, and diarrhea. ~°-~2 However, this relatively crude way of measuring GVH reactions is subject to numerous variables, such as the overall health of the hosts to start with, the fastidiousness of animal care and management, and, especially, complications of infectious diseases. ~3 The Discriminant Spleen Assay. This more precise and objective assay was designed by Simonsen 5 to take advantage of the marked splenomegaly that characteristically precedes the atrophy of lymphoid tissue when adult T cells are injected into allogeneic embryos or neonates. To conduct the assay with newborn mice or rats, litters 1-10 days old that are being well cared for by their mothers are selected. Half the litter is used as test animals and half as uninjected controls (one group can be marked by cauterizing the tips of their tails with an electrocauterizer or with the end of a scalpel heated with a Bunsen burner). Cells (107) (or some other desired number) are injected intraperitoneally with a 25-gauge f-in. needle. Even this small volume cannot be injected directly into the tight belly of a newborn rodent without some of the fluid escaping, so the tip of the needle has to be inserted through an arm or leg muscle and be pushed subcutaneously until it passes into the peritoneal space. To do this, the newborns are carefully anesthetized with ether or some other inhalant 10 R. E. Billingham, V. Defendi, W. K. Silvers, and D. Steinmuller, J. Natl. Cancer Inst. (U.S.) 28, 365 (1962). 11 p. Stastney, V. A. Stembridge, and M. Ziff, J. Exp. Med. 118, 635 (1963).
~2D. W. van Bekkumand M. J. de Vries, "RadiationChimaeras." AcademicPress, New York, 1967. i3 D. Keast and M. N. I. Waiters,Immunology 15, 247 (1968).
[4]
GRAFT-VERSUS-HOSTREACTION
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(see below) or very young litters, which are poikilothermic, are placed in a refrigerator for a few minutes. Splenomegaly peaks 8-10 days after donor-cell injection. ~4The entire litter is sacrificed at this time by cervical dislocation or by overanesthetizing it. First each entire animal is weighed to the nearest milligram; then the spleen is excised and weighed, being careful first to remove all adherent fat. The spleen weight is expressed as a percentage of the body weight for each test and control host. An index of spleen enlargement for each test host is then calculated by dividing its spleen/body weight ratio by the mean ratio for the littermate controls. This index permits comparing results between litters because the variation in spleen weight between litters is greater than that within litters.~4 Inhibition of Colony-Forming Units (CFU). A disadvantage of the Simonsen assay is that it requires newborn hosts. However, systemic GVH reactivity can also be measured in terms of the ability of aliogeneic lymphoid cells to inhibit hematopoietic CFUs in lethally irradiated adult hosts. 15 The assay was designed for inbred animals where one can use syngeneic bone marrow (i.e., marrow from other, histocompatible donors of the same strain) as a source of hematopoietic stem ceils, but one can perform it also with outbred animals if one obtains bone marrow cells from the host before irradiation for later injection. The assay is conducted by exposing hosts to a lethal dose of total-body, gamma irradiation (750900 rads for mice or rats) and then giving them 2 x 10 6 syngeneic (or autologous) bone marrow cells intravenously alone (the control) or with the desired number of test lymphoid cells. Six days later 0.2/xCi of ~9Fe citrate is injected intraperitoneally into the hosts. After 17 hr, the hosts are sacrificed, their spleens are removed, and the radioactivity present is determined in a liquid scintillation counter, using a scintillation cocktail appropriate for counting aqueous samples.
Regional GVH Assays lntracutaneous. One can obtain a semiquantitative measure of GVH reactivity in most laboratory animals by injecting a few million allogeneic lymphocytes directly into the skin and scoring the resultant reactions like delayed-type hypersensitivity tests. 6 The best sites for injection are the flank for guinea pigs, the belly for rats, and the back for mice. First the hair is removed with electric clippers (Oster small animal clippers with size 40 cutting blades are recommended); then the area is shaved with a razor or depilated with a cream such as Nair (Carter Products, NY). In ~4 M. Simonsen, J. Engelbreth-Holm, E. Jensen, and H. Poulsen, Attn. N. Y. A c , d . Sci. 73, 834 (1958). ~s H. Blomgren and B. Andersson, Cell. lrnrnunol. 3, 318 (1972).
32
SURGICAL TECHNIQUES IN IMMUNOLOGY
[4l
rats and guinea pigs, the desired dose (e.g., 107 cells) is injected intraderreally in 0.1 ml using a 27-gauge needle with an intradermal bevel; with mice 30/.Li is injected using a 30-gauge needle and a Hamilton No. 705 syringe (Hamilton Co., Reno, NV). The cells must be injected intradermally, not subcutaneously, by inserting the needle very superficially bevel-up. If a bleb does not form by pushing the syringe plunger the needle is in the subcutaneous tissue. Replicate injections are given each host at 3-4 separate sites. The reactions peak at 2-3 days, and the injection sites should be assessed at 24-hr intervals for erythema, induration, and necrosis (the latter may occur only with presensitized cells), assigning scores according to size and intensity, such as + / - for a small, erythematous spot without swelling and 5+ for a large indurated and necrotic reaction. Streilein and Billingham j6 recommend the following scale for mice: 0, no response; 1+, barely perceptible swelling; 2+, swelling 3-4 mm in diameter, site soft to palpation; 3+, swelling 5 mm or more in diameter, site firm; 4+, large reaction with indurated core; 5+, site necrotic or ulcerated. At least 3 sites on each animal should be read daily and the results expressed as a mean score. Moreover, because of the subjective nature of the assay, it is wise to code-number the subjects and score them "blindly" in experiments involving different dosages and treatment groups. A disadvantage of the assay is that if the hosts are able to respond to donor histocompatibility antigens, the skin reactions may include a host-versus-graft component. One can preclude this with inbred animals by using Fj hybrid hosts, as discussed later for the PLN enlargement assay. lntrarenal. The injection of lymphoid cells from inbred, parent-strain donors beneath the renal capsule of adult F~ hybrid hosts results in a marked local, invasive-destructive lesion, j7 There is an initial burst of donor-cell proliferation in the injected kidney that peaks at 6 days, which can be measured by the incorporation of r25IUdR,~8 followed by an increase in renal weight that peaks at 14 days. For the assay, the injected and the uninjected contralateral (control) kidneys either are weighed or the incorporation of ~25IUdR is determined with a gamma counter, or both, and the results expressed as ratios of injected to control values. J8 The intrarenal GVH reaction is of great interest to transplantation immunologists because its histopathology mimics that of acute renal allograft rejection. 6 However, the assay is difficult because the kidney must be surgically exteriorized for inoculation, jv and it is not as sensitive as the PLN enlargement assay described below. 16 j. W. Streilein and R. E. Billingham, J. Exp. Med. 131, 409 (1970). i7 W. L. Elkins, J. Exp. Med. 12,0, 329 (1964). is W. L. Elkins, Transplantation 9, 273 (1970).
[4]
GRAFT-VERSUS-HOSTREACTION
33
Popliteal Lymph Node (PLN) Enlargement Assay Introduction. The injection of parent-strain lymphoid cells below the hind footpads of F~ hybrid rats 19or mice 2° results in an enlargement of the single lymph node draining the site, the PLN, which can be weighed to provide a simple, reliable measurement of regional GVH reactivity. The assay is easier and more sensitive than the intrarenal assay, ~9 and more objective than the intracutaneous assay, its only disadvantage is that it is applicable only to inbred animals where F~ hybrids can be produced by mating genetically homozygous parents. Such hybrids are genetically tolerant of parent-strain cells; if they are not used, host-versus-graft reactions contribute to the reaction. 6 One cannot suppress the host component with radiation, for example, because radiosensitive host lymphocytes are required for the full expression of PLN enlargement. 6 Injecting the Host. With rats, the desired number of cells (e.g., 10 7 donor lymph node or spleen cells) is injected in 0.1 ml using a 25-gauge, ~in. needle attached to a I ml or smaller syringe. With mice, the cells are injected in 50 /zl using the same size needle but a Hamilton No. 705 syringe. The animal is anesthetized by placing it in a closed glass jar or other container with a wad of cotton or gauze pads wet with ether or, preferably, a nonflammable, nonexplosive inhalant anesthetic such as methoxyflurane, enflurane, or halothane. The hind foot of the anesthetized animal is grasped by the toes and the tip of the needle is inserted just in front of the footpads. The tip of the needle is pushed 6-10 mm toward the heel before delivering the dose. The needle should be in the subcutaneous, not the intradermal, tissue because the former injection site gives the best results. 19 When the needle is withdrawn, a brief pressure is applied at the puncture site with a cotton applicator or piece of gauze to prevent leakage. Although one can inject both hind feet (e.g., with different cell populations or dosages~9), it is useful to leave one foot uninjected as a control (see below). Harvesting the Node. PLN enlargement usually peaks 7 days after inoculation of the footpad in both rats and mice and this is the optimal time for weighing the nodes, though one can weigh them earlier if one wants time-course data. The PLN is a spherical node embedded in adipose tissue in the middle of the popliteal fossa :f (Fig. 1). To remove it, the animal is sacrificed with an overdose of anesthesia (or with mice, by cervical dislocation). The animal is pinned belly-down on an operating board and the legs are rotated so that the soles face upward. The hair is t~ W. L. Ford, W. Burr, and M. Simonsen, Transplantation 10, 258 (1970). z0 V. W. Twist and R. D. Barnes, Transplantation 15, 182 (1973). 2~ y . Kawashima, M. Sugimura, Y. C. Hwang, and N. Kudo, Jpn. J, Vet. Res. 12, 69 (1964).
34
SURGICAL TECHNIQUES IN IMMUNOLOGY
FiG. 1. Location of the popliteal lymph node in rats and mice (after Kawashima the node actually is more medial than suggested by this sketch).
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e t a l . z~ ;
removed from the site with electric clippers and the area is soaked with 70% alcohol. A superficial incision is made with blunt-tipped scissors (the tip of the scissors should run just under the skin) from mid-thigh nearly to the heel, passing over the middle of the popliteal region, and the skin from the incision is retracted to reveal the node, which is quite obvious on the injected side (Fig. 2). The incision is kept open by pinning-down or clamping the retracted skin, and the node is removed with smooth-tipped, fine forceps (curved jeweler's forceps are highly recommended; e.g., Storz Instrument Co., St, Louis, MO, No. E-1947-7). The node must be freed of
FIG. 2. The enlarged left popliteal lymph node of an Ft hybrid mouse 7 days after the injection of 107 allogenic spleen cells below the left footpad (the node was freed from the surrounding fat for the photograph).
[5]
ADOPTIVE
TRANSFER
35
fat before placing it in saline (0.9% sodium chloride) in a small petri dish. If the node does not sink, fat is still attached to it. If necessary, the node can be excised from an anesthetized, living animal by making a smaller incision and suturing the retracted skin together after removing the node. Weighing the N o d e . It is very important to remove excess moisture before weighing. This can be done by gently rolling the node on a stack of 2 or more pieces of hard filter paper (e.g., Whatman No. 50). Each node is weighed to 0.01 mg with an analytical balance. A convenient way of doing this is to first weigh an empty piece of weighing paper, then place the nodes one by one on the paper and record the change in weight each time. The results for each injected host are expressed as the P L N enlargement ratio calculated by dividing the weight of the node from the injected side by that of the node from the uninjected, control side. One should test at least 3 hosts with each dose and type of cells and calculate the mean enlargement ratio -+ the standard error or standard deviation. With organ enlargement assays in general, the significance of differences between test and control groups is best determined by analysis of variance. G V H Reactions in Birds
GVH reactions in chickens and other birds can be assayed by measuring spleen enlargment 5 or by counting and measuring the white " p o c k s " that appear on the chorioallantoic membrane (CAM) following inoculation of the embryo with allogeneic blood or lymphoid cells. See Longenecker et al. 22,z3 for details of the CAM assay. .,2 B. M. Longenecker, S. Sheridan, G. R. J. Law, and R. F. Ruth, Transplantation 9, 544 (1970). 2~ B. M. Longenecker, F. Pazderka, G. R. J. Law, and R. F. Ruth, Cell. hnmunoL 8, I (1973).
[5] Adoptive Transfer By DAVID STEINMULLER
The term, adoptive immunity, was coined by Billingham et al. ~in 1954 to describe the transfer of immunity from one animal to another by live, immunocompetent cells as opposed to immune serum or preformed antibody (the term passive transfer should be reserved for the transfer of t R. E. Billingham, L. Brent, and P. B. Medawar, Proc. R. Sot'. London, Ser. B 143, 58 (1954).
METHODS IN ENZYMOLOGY, VOL. 108
Copyright ~t~ 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182008-4