The introduction of foreign substances into animal tissues can have many effects. Many such substances can be filtered by the kidneys or removed by phagocytic cells such as liver or spleen cells. This is achieved by the interaction of the attached antibodies with a set of serum components, collectively known as the complement system (1). The model system I have studied is the mouse and rat Thy-1 or theta antigen. Several types of information revealed by analyzes of Thy-1 expression in different cell populations will be discussed in the following pages.
The genetic determination of Thy-1 antigenicity in mice, which is mainly treated in chapter 2, forms an underlying basis for the rest of the work. The various genetic, anatomical, developmental and phylogenetic factors that affect Thy-1 expression are discussed in chapter 4. Finally, chapter 5 shows that antibodies specific for Thyrl and at least one other genetically controlled mussel surface antigen can be produced in rats.
LOCATION OF THE GENE FOR THETA ANTIGEN IN THE MOUSE
During the course of our experiment, Itakura et al. '" reported linkage of the cytoplasmic malic enzyme locus, Mod-], to Thy-1 and d. Both H-14 and three-point assay markers on chromosome 9 Kleinn reported that the breakpoint location of one translocation [T(9;17) 138 Ca]* between curly mustache (cw) and short ear (se).
Using another translocation, T(9;19) 163H, Lyon et al." showed that the centromere is located at the end of the linkage group beyond cw. Segregation of the chromosome 9 markers Thy-I, Mod-I , and Trf were followed by means of backcrosses to the parental strains Order of loci in the second linkage group of the mouse with respect to the centromere.
Variations among Sublines of Inbred AKR Mice
ADDITIONAL DATA
The experiments described in the remainder of this chapter were designed to shed additional light on the issue of 0-R antigen expression by murine peripheral T cells. The first approach to this question was based on the hypothesis that anti-Thy-1.1 antibodies in the presence of complement may interfere with the peripheral function of murine T cells, even though they do not cause cytolysis. The results of an experiment using AKR/J immune PEC are shown in Figure 8, immune PEC pretreated with normal AKR/J mouse serum uniformly induced maximal tumor cell lysis.
Mouse PEC pretreated with AXR/cum anti-AKR/J thymocyte serum showed antitumor activity equal to those treated with normal serum, but pretreatment of these cells with anti-BN AKR/Cum spleen serum resulted in a complete inhibition of activity their cytotoxic. . Thus, the ability of these antisera to interfere with the function of WF mouse peritoneal cytotoxic cells is related to the presence of species-specific antibodies, rather than those directed against Thy-1.1. This is quite different from the pattern observed for AKR/J immune PECs, which are strongly inactivated by AKR/Cum anti-AKR/J thymocytes.
This serum also contains anti-Thy-1.1 antibodies in addition to antibodies directed against rat species-specific antigens. The effect of a similar set of antisera on WF rat PEC viability was determined in a separate experiment, one-tenth ml aliquots of PEC were pretreated with antibody and complement as described above. The viability of the cells in each group was then determined by the Trypan Blue exclusion method. It is clear from the data presented in Table 4 that the inactivation of rat PEC by AKR/Cum anti-BN rat spleen serum is the result of complement-mediated cytolysis. The partial decrease in viability of cells treated with either AKR/Cm normal serum or anti-Thy-1.1 antibody5 also correlates well with the partial loss of effector cell activity observed in similarly treated cell populations. On the basis of these data plus those presented in Table 2, it seems unlikely that anti-Thy-1.1 reagents can be used for the 2, it seems unlikely that anti-Thy-1.1 reagents can be used for selective elimination of peripheral rat T cells from mixed lymphoid cell populations, as has been done successfully in mice.
The final approach taken in researching 8-R in mouse peripheral T cells was based on the hypothesis that small amounts of this material might be immunogenic even though they did not present an adequate. The results of direct complement-dependent cytotoxicity tests of these sera against thymocytes from mice of the A K R/C~~, AKR/J, and C3H/HeJ strains are shown in Figure 9. Anti-AKR/J thymocyte serum instead of AKR -Cum anti&m/J thymocyte serum was used in this experiment because of its higher titer.
Expression of Thy-1.1 antigen by WF rat and AKR/J mouse lymphoid cells. WF rat cells and AKR/J mouse cells.
40 FIGURE 8
Although anti-mouse thjrm~cytc antibodies were demonstrated by dye-exclusion microcytotoxicity assays2 at the end of the 1st week of immunization, subsequent bleeds showed much higher titers and were pooled for further experiments. One milliliter of each absorbed antiserum was then absorbed four times with pooled lymph node, spleen, and thymus RF/J (a/a) cells. Each absorption was performed for 30 min at t 0 C and used either 0.3 ml of packed, washed erythrocytes or one quarter of pooled lymph node, spleen and thymus cells from a single donor.
Absorbed antisera do not kill thymocytes from congenic A/Thy-1 stock mice. Although this latter finding illustrates the presence of antibodies specific for the Thy 1.2 antigen, it does not prove the absence of other antibodies because the gene(s) for the corresponding antigen (~) may not be present in the A/J background. These classes exactly match the results of typing 0 ie. Thy-1.2-positive cells are rapidly killed, but Thy-1.2-negative cells are not.
On the basis of its apparent strain distribution (ie, presence in CBA/J but absence in AKR/J and RF/J), the antigen giving rise to this effect cannot be identified with any of the excised specificities checked of the Tla, Ly-2 or Ly-3 loci (3, 4). The evidence therefore suggests that a reagent specific for Thy 1.2 can be prepared by appropriate absorption from rat anti-AKR/Cum serum. In addition to the antigen-antibody systems already discussed, the properties of the absorbed rat antisera described in this report can also be distinguished from H-2 (in d.
Although antisera raised in rabbits against mouse thymocyte membranes (5) or brain tissue (6, 9) have been reported to show specificity for thymus-derived lymphocytes, direct cytotoxicity and absorbance assays of these sera generally failed to detect antibodies specific for the Thy-1.1 or Thy-1.2 antigens (5, 9. These investigators also observed that rabbit antirat brain sera contain antibodies reactive with one or more antigenic components (possibly equivalent to Thy-1 . 1) that are present in the brain and thymus present from AKR/J mice (a/a) but not from eight b/b strains The strain distribution and genetic segreg-a t' ion data presented here show that reagents specific for the Thy-1.2 -antigen (0 C3H) of the mouse can be prepared by the absorption of heteroantisera raised in rats.
In any case, rat antisera now appear to be a ready source of reagents for the detection of at least one class of lymphocyte alloantigens in mice.
If this association is not purely coincidental due to the limited number of strains tested, it may reflect either linkage disequilibrium between Thy-1 and leukemia susceptibility factors or direct participation of the Thy-1 system itself in the process of leukemogenesis. Historically, the genetic determinants of alloantigen systems were first described as simple di- or triallelic sequences, as these systems have been analyzed more closely, additional genetic complexity has often been discovered (33). An example of this is the = region of the mouse which has been described as "one of the most complex genetic systems known" (34).
Data indicating the occurrence of new Thy-1 alleles in inbred strains of mice has recently been presented (3 5). If these observations are correct, our understanding of Thy-1 may already be on the path to complexity. Thy-1.2 has only been detected in mice, and neither antigen has been found in hamsters (27) or guinea pigs. This pattern of species specificity has made it possible to use defined mouse alloantisera to study the rat 0-R antigen. also underpins the successful efforts to produce antisera specific for Thy-1.2 in rats. To date, it has not been possible to raise rat antibodies against 8-R; neither qualitative nor quantitative variation in the expression of this antigen has been found among inbred rat strains. If so, the specialized functions of the cells carrying the antigen may provide valuable clues about the physiological role of the antigen itself. Second, the chemical analysis of Thy-1 antigen molecules resolved from cell membranes is beginning to progress (39). -41).
A RAT ANALOG OF THE MOUSE THETA ANTIGEN
A Rat Analog of the Mouse Theta Antigen
Anti-8 sera have been widely used for the selective elimination of thymus-dependent lymphocytes from heterogeneous cell population^.^ The failure to observe significant killing of rat spleen and lymph node cells by anti-8-AKR sera sug-. say that this strategy may not be of use with rat cells. The absorptive capacity of a given sample was defined as the reciprocal of the number of milligrams of brain homogenate (G) required to produce a 50% inhibition of cytotoxicity using this standard serum and AKR/J target cell^.^^*' ^ As can be seen in Fig. 4, the 6-absorptive capacity of WF rat brain increases rapidly from birth (day 0) to approximately day 20 of postnatal life.
The reduced expression of 8-R in the spleen, lymph nodes, and bone marrow cells relative to thymocytes also resembles the distribution of 8. Finally, the kinetics of 8-R development in rat brain closely parallel those reported for 8 in the mouse. These characteristics of 8-R clearly place it in the category of "differentiation antigens"15 and point to the potential utility of this system for studies of neurophysiological and immunological development in the mouse.
