Lawrence L. Johnson

Use of monoclonal antibodies to analyse the expression of a multi‐tubulin family

We have used a panel of monoclonal antibodies in a study of the expression of multiple tubulins in Physarum polycephalum. Three anti‐β‐tubulin monoclonal antibodies, DM 1B, DM3B3 and KMX‐1 all reacted with the β1‐tubulin isotypes expressed in both myxamoebae and plasmodia. However, these antibodies showed a spectrum of reduced reactivity with the plasmodial β2‐tubulin isotype ‐ the competence of recognition of this isotype was graded DM1B > KMX‐1 > DM3B3. The anti‐α‐tubulin monoclonal antibody, YOL defined the full complement of Physarum α‐tubulin isotypes, whilst the anti‐α‐tubulin monoclonal antibody, KMP‐1 showed a remarkably high degree of isotype specificity. KMP‐1 recognises all of the myxamoebal α1‐tubulin isotypes but only recognises 3 out of the 4 α1‐tubulin isotypes expressed in the plasmodium (which normally focus in the same 2D gel spot). KMP‐1 does not recognise the plasmodial specific α2‐tubulin isotype. This monoclonal antibody reveals a new level of complexity amongst the tubulin isotypes expressed in Physarum and suggests that monoclonal antibodies are valuable probes for individual members of multi‐tubulin families.

Genetics of histocompatibility in mice

The H-Y antigen and minor H antigens defined by eight C57BL/6By (B6) congenic mouse strains were studied for their distribution among 13 different tissues. Antigen expression was detected by implanting B6 recipients with organ fragments from congenic strain donors that had been previously grafted with B6 bone marrow to minimize the number of allogeneic passenger leukocytes. Successfully immunized recipients were identified by the enlargement of their popliteal lymph nodes following footpad challenge with spleen cells of the donor type. No two strains had identical patterns of expression of their distinguishing antigens. The data provide evidence for differences in levels of minor H-antigen expression among different tissues.

Teratocarcinoma transplantation antigens are encoded in the H-2 region

Evidence is presented for the existence of teratocarcinoma transplantation antigens (Gt) encoded within the H-2 complex and present also on adult tissues. It has not been possible to separate these Gt loci from H-2 by recombination, and Gt factors map to each end of the H-2 complex. Previous reports indicating separation of all Gt loci from H-2 are reinterpreted. One class of such apparent recombinants has been shown to result from the outgrowth of tumor variants in mice of resistant genotype.

Genetics of histocompatibility in mice. II. Survey for interactions between minor (non-H-2) antigens by skin grafting.

Twenty-five congenic mouse strains differing at distinguishable minor (non-H−2) histocompatibility loci were paired in 71 different combinations. F1 offspring were used as skin-graft donors for more than 4000 recipients to test whether immune responses to parental strain antigens were statistically independent. Thirty-four (48 percent) of the 71 combinations were predicted adequately by an independent response hypothesis. A simple additive model was consistent with 39 (55 percent) of the observed responses, although 18 of these were among those in agreement with the independent hypothesis. A synergistic response faster than that predicted by either the independent or additive response model was seen in 12 (17 percent) of the combinations. The remaining 5 percent were not well described by any of these models. No strain was represented with unusual frequency among those involved in synergistic interactions.

Mice coisogenically immunized against H-2 class I antigens on transfected l cells reject transplanted embryonal carcinoma cells

Evidence is presented of the ability of H-2 class I antigens to function as teratocarcinoma transplantation (Gt) antigens. Coisogenic immunization against H-2 class I antigens expressed on transfected L cells is shown to induce resistance to embryonic carcinoma (EC) cell allografts. The Kb, Db, Dd, and, in appropriate recipients, Ld antigens can function as Gt antigens. The protocol presented may be useful for the molecular identification of other genes encoding histocompatibility antigens.

Tailskin grafts do not show accelerated rejection on splenectomized hosts.

Streilein and Wiesner (1977) have recently reported that skin grafted onto previously splenectomized mice shows a shortened survival time. Specifically, these authors have found that removing the spleen from C57BL/6 female mice seven days prior to grafting them with syngeneic male skin results in graft median survival times diminished from 16.5 days for controls to nine days for a splenectomized group. Another study seems to conflict with this finding, however. Nemec and coworkers (1974) observed no significant differences in the time of onset, the duration, or the end of the rejection process between controls and mice splenectomized 14 days prior to skin grafting. These authors found, in addition, that hosts splenectomized only two days prior to grafting supported viable grafts significantly longer than did their controls. These two studies differ, however, in at least three important experimental aspects: the histocompatibility loci defining the antigenic stimuli (H-Y versus H-I), the site of graft placement on the host (body versus tail), and the time interval between splenectomy and grafting (seven days versus 14 or two days). We have conducted a set of grafting experiments which bears on the effects caused by the first two of these differences. Female C57BL/6By mice were either splenectomized or sham operated under sodium pentobarbital anesthesia. Seven days after surgery these mice received two orthotopic tailskin grafts (Bailey and Usama 1960). The donors of both of the grafts were either C57BL/6By males or B 6 . C H 1 b females (synonym HW80). Thus, the grafting scheme in this case can be represented as H 1 b ~ H 1 c, whereas the Nemec group (1974) used H U ~ 1-1-1 a. All hosts also received a single isograft. Starting one week after grafting, observations were made on alternate days for at least 40 days and twice weekly thereafter. Scoring was based on visual examination of the graft for its epithelial scale pattern, pigmentation, and hair. In the case of the more vigorously rejected male skin grafts, the survival endpoint chosen was the first day of a detectable difference between the condition of the test graft and an isograft, or for brevity, FSR (first sign of rejection). Typically, a brown discoloration accompanied by dermal disintegration was seen. A much weaker rejection of the HW80 grafts occurred. In most cases, one or more transient periods of epidermal flaking, mild discoloration, and hair loss was seen. Therefore, the parameters chosen to describe these grafts were both the first sign of difference between the appearance of the test grafts and the isograft and also

H-2 class I and Gt (H-2) antigens are identical: Evidence from H-2 mutant mice

Target (Gt) antigens involved in the rejection of embryonal carcinoma (EC) transplants map to both the K and D regions of H-2 and elsewhere (Johnson et al. 1983a, b). Paradoxically, EC cells do not appear to express H-2 class I antigens (Artzt and Jacob 1974, Morello et al. 1978, 1982, Croce et al. 1981, Maher and Dove 1984). It has recently been reported that mice coisogenically immunized against H-2 class I antigens on transfected L cells reject transplanted EC cells (D6mant and Oudshoorn-Snoek 1985, Moser et al. 1985). From these results, we have concluded that either the EC cells express H-2 class I antigens or the Gt (H-2) antigens share cross-reactive determinants with the H-2 molecules encoded by the transfected genes. We now report experiments performed to distinguish between these possibilities. We find that mice bearing mutations in the H 2 K b gene reject implants of the EC line PCC3/A/1 (PCC3, genotypically H-2 b) after immunization with C57BL/6J (H-2 b) spleen cells. Thus, we conclude that PCC3 cells express H-2 b determinants that are missing, or altered, in at least two different K b mutations. This strongly suggests that the Gt antigens reside on H-2Kb-encoded molecules themselves, rather than simply sharing an antigenic determinant. The experimental strategy for the present experiments depends on the following previously reported observations (Tyan and Cole 1963, Johnson et al. 1983a, b, Moser et al. 1985). Mice immunized against various transplantation antigens and then sublethally irradiated reject subsequently administered grafts which express those antigens. In contrast, irradiated control mice pre-immunized against extraneous antigens do not reject such grafts, or do so only belatedly. Thus, the capacity of irradiated recipients to reject these grafts is an assay for the expression of antigens on a challenge

Male-specific transplantation antigen expression by XY teratocarcinomas PCC7 and 7'.

Male-specific antigen expression by XY teratocarcinomas PCC7 and 7′ is demonstrated first by the rejection of tumors by female but not by male mice following challenge with these cell lines. Male-specific antigen expression is confirmed by an indirect method in which females are immunized against H-Y antigen by male skin grafts. A variant of PCC7 lacking male-specific antigen expression is described. Analysis of the karyotype and of the DNA from this variant indicate that the loss of male-specific antigen expression is a result of the loss of the Y chromosome. The ability to recover variants that have lost expression of male-specific antigen opens the possibility of their selection after mutagenesis.

A protocol for detection of H-Y antigenic variants

A skin grafting protocol is described for finding H-Y antigenic variants. The method is applicable regardless of the location of the structural gene(s) for this antigen (X, Y, or autosomal). Use of this protocol revealed no evidence for H-Y antigenic variation between C57BL/6J and strains 129/J, A.BY/SnJ, C3H.SW/SnJ, and LP/J.