Xenogeneic antisera against T-lymphocyte receptors recognizing allogeneic transplantation antigens: Preparation and properties

Antireceptor sera ARS-1 (anti-CBA-anti-C57BL/6) and ARS-2 (anti-C57BL/6-anti-BALB/c) were prepared in a xenogeneic system by immunizing rabbits with alloimmune CBA anti-C57BL/6 and C57BL/6 anti-BALB/c lymphocytes and subsequent exhaustive absorption of rabbit immune sera with red blood cells, lymphocytes, liver cells, and serum from intact mice. In the cytotoxicity test, both ARS sera lysed only activated T lymphocytes of corresponding, but no other specificities, and did not affect intact lymphocytes. They did not inactivate in vitro the PFC-producing antibodies to SRBC or inhibit the immune response to SRBC and Viantigen of Salmonella typhi in adoptive transfer experiments. Pretreatment of intact CBA lymphocytes in vitro with ARS-1 plus complement suppressed their ability to induce lethal GvH disease in irradiated (CBA × C57BL/6)F₁, but not (CBA × BALB/c)F₁ recipients; similar pretreatment of C57BL/6 lymphocytes did not lower lethality among irradiated (CBA × C57BL/6)F₁ recipients. This method of preparing ARS may help to obtain highly specific antireceptor sera for regulation of the immune response to different antigens in outbred animals and in humans.     

Immunogenetic differences between lymph node cell and bone marrow cell grafts in irradiated mice

C3H lymph node cell (LNC) grafts, but not bone marrow cell (BMC) grafts, were resisted by lethally irradiated NZB, (C57BL × NZB)F1, and (C57BL/6 × DBA/2)F1 mice. BALB/ c hosts did not resist C3H LNC, suggesting that Ir-like genes regulate resistance to such grafts. Cyclophosphamide, silica particles, and ⁸⁹Sr pretreatments of prospective host mice resulted in successful proliferation of C3H LNC in most instances. These agents were known to abrogate resistance to incompatible BMC grafts. The determinants for antigens recognized on LNC appear to map in or near the D region of H-2. LNC grafts of all H-2^k strains tested (C3H, CBA, C58, C57BR) were strongly resisted while A, C3H.A, B10.A(5R), A.TL, and A.Tla^b LNC grafts were not strongly resisted by NZB hosts. Grafts of H-2^b (C57BL/6, C57BL/10, 129) LNC, or BMC are resisted by NZB or (C57BL/6 × DBA/2)F1 hosts. (C3H × C57BL)F1 LNC but not BMC were resisted by similar hosts. (C57BL/6 × DBA/2)F1 mice were injected with C57BL/6 spleen cells four times to induce specific “unresponsiveness” to parental-strain Hemopoietic histocompatibility (Hh) antigens. Unresponsiveness was induced to C57BL/6 BMC, as expected, but C57BL/6 and C3H LNC grafts were resisted despite the spleen cell injections. The data suggest that the antigens recognized during rejection of C3H LNC are not expressed on C3H BMC. It is even conceivable that Hh antigens on C57BL/6 BMC and LNC have separate determinants. Alternatively, the injections of C57BL/6 spleen cells may have induced an anti-idiotypic response that was capable of eliminating C57BL/6 LNC by a different effector mechanism.     

Activation of T and B thymus cells to recognize histocompatibility antigens

Lethally irradiated (A × CBA) F₁ or (A × C57BL/6) F₁ mice were injected with 10⁷ A strain thymus cells in attempts to activate donor cells to recognize CBA or C57BL/6 histocompatibility antigens, respectively. Activation could be revealed by injecting activated thymus cells (day 5 irradiated F₁ hybrid spleen cells) into corresponding unirradiated F₁ hybrid hosts. The alloantibody titers formed by these cells and the antirecognition structure (anti-RS) antibody titers induced by them were similar to those observed after injection of normal parental strain spleen cells, indicating that thymus cells had become endowed with recognition structures (RS). Alloantibodies, but no anti-RS antibodies, were present in the serum of F₁ mice given activated thymus cells treated with anti-θ and complement. It, therefore, appeared that activated thymus cells contained sufficient B cells differentiated into antibody-forming cells to give a measurable alloantibody response. On the other hand, receptors responsible for anti-RS antibody induction presumably were located on T cells. Specificity and restriction of antigenic recognition were revealed by negative results obtained when activated thymus cells were injected into F₁ hosts not containing the antigens against which activation had been directed.     

Genetic control of “natural” killer lymphocytes in the mouse

Spleens from normal young mice contain lymphocytes that can kill certain in vitro grown Moloney lymphoma lines in a⁵¹Cr-release cytotoxicity test. A lymphoid cell without detectable T- or B-cell markers was previously shown to be responsible. Killing activity shows a marked dependence on the genotype of the donor mouse. When tested against a YAC line of strain A origin maintained in vitro spleens of A, A.CA, and A.SW mice had low activity, whereas CBA, C3H, C57L, and C57Bl spleens were highly active. In semisyngeneic F₁ crosses with strain A as one parent, reactivity resembled the opposite parental strain. Thus, (A×CBA)F₁, (A×C3H)F₁, (A×C57L)F₁, and (A×C57Bl)F₁ were reactive, whereas A×A.CA showed no significant activity. Analysis of the reactivity in (A×C57Bl)F₁×A backcross mice suggests that multiple genes are involved. Preliminary linkage analysis suggests at least oneH-2 linked factor. Another gene appears to be linked to theB (black) locus.     

Schistosoma japonicum: Infection characteristics in mice of various strains and a difference in the response to eggs

Female mice of 12 inbred strains were exposed to 20–25 cercariae of Schistosoma japonicum and infection status determined at day 40 by counting numbers of adult worms, eggs in faeces and eggs in a segment of liver. Most mouse strains appeared to be ‘permissive’ hosts although at least one strain (129/J) was shown to be relatively resistant in terms of day 40 adult worm numbers. In a radioisotopic lung assay for sensitivity to eggs, and developed as a rapid means of assessing granuloma formation, CBA/H mice were shown to differ from C57BL/6 mice in being non-responders. Histological examination of lungs of sensitized CBA/H and C57BL/6 mice injected intravenously with eggs established that granuloma formation was much more intense in C57BL/6 than CBA/H mice. Preliminary indications are that infected CBA/H mice are also low anti egg circumoval precipitin (COP) responders. Analysis of immune responses to isolated egg antigens in these two strains, and identification of the antigens of eggs to which such responses are directed in C57BL/6 mice, should provide insights into immunological disease processes (such as granulomatous inflammation) in this model system of japonicum schistosomiasis.     

Fate of H2-activated T lymphocytes in syngeneic hosts. III. Differentiation into long-lived recirculating memory cells.

Memory to H2 determinants was studied with an adoptive transfer system using a population of H2-activated blast T cells (T.TDL) obtained from thoracic duct lymph of irradiated F₁ hybrid mice injected with parental strain T cells. CBA T.TDL activated either to DBA/2 or C57BL determinants were transferred to syngeneic “B” mice. Thoracic duct lymphocytes (TDL) were obtained from the recipients 4–6 weeks later and tested for their capacity to produce (a) a graft-versus-host (GVH) reaction, (b) a mixed lymphocyte reaction (MLR) (measured by an in vivo technique) and (c) allograft rejection (suppression of the growth of allogeneic tumour cells in vivo). Control experiments involved testing the function of TDL obtained from “B” mice preinjected with TDL or no cells.TDL from “B” mice injected with TDL (passaged TDL) gave strong MLR and GVH reactions to both DBA/2 and C57BL determinants. Passaged T.TDL activated to C57BL antigens gave intermediate MLR and GVH reactions to the specific (C57BL) determinants but only very low responses to third-party (DBA/2) determinants; reciprocal results were obtained with passaged T.TDL activated to DBA/2 determinants. TDL from “B” mice given no cells failed to respond to either set of determinants.Since the responses by the passaged T.TDL did not exceed those by passaged TDL there was no evidence that adoptive transfer of T.TDL had conferred to the recipients a state of memory to either MLR or GVH determinants. Adoptive transfer did, however, lead to qualitative changes in the properties of T.TDL since, before transfer, they were unable to evoke GVH reactions or produce an MLR of normal kinetics.Passaged T.TDL were far superior to passaged TDL at suppressing the growth of allogeneic tumour cells. The protection was specific since protection against DBA/2 tumour cells was, cell for cell, 5–10 fold more effective with passaged T.TDL activated to DBA/2 determinants than with cells activated to C57BL determinants. No protection was observed with cells treated with anti-θ serum. The protective cells appeared to be precursors of effector cells rather than effector cells per se since they failed to lyse the tumour cells in vitro. These data suggest therefore that the descendants of T.TDL which survived after transfer to “B” mice were highly enriched in long-lived recirculating T lymphocytes reactive to determinants expressed by specific tumour allografts.     

Cyclic AMP accumulation in cerebral cortex tissue from inbred strains of mice.

Large strain differences in neurohumorally induced increases in cyclic AMP can be observed in chopped cerebral cortex of genetically uniform strains of mice. Data from F₁ hybrids of C57BL/6J × DBA/2J and preliminary data from C57BL/6J × SEC/1ReJ suggests dominant transmission by C57 of a chemical factor favoring low accumulation of cyclic AMP in the first cross and recessive transmission of this factor in the second cross. These chemical observations correlate with previously observed transmission of “low active avoidance learning” in the same hybrid strains. Results of determinations of cyclic nucleotide phosphodiesterase activity in supernatant fractions from C57, DBA and the C57 × DBA cross provide a possible explanation for the accumulation differences in those strains.     

Murine responses to (Tyr-Glu-Ala-Gly)n: II. H-2 and non-H-2 gene effects

Mice of the H-2^b haplotype responded to the sequential polymer poly(Tyr-Glu-Ala-Gly) in the in vitro T-cell proliferative assay, irrespective of whether they were homozygous or heterozygous at the H-2^b locus. The antibody responses of the H-2^b congenic mice to this polymer were variable, with A.BY and BALB.B showing responses better than those of C57BL/6 and C57BL/10 strains. The antibody responses of the F₁ progeny of (responder × nonresponder) strains of mice to this polymer are generally lower than the responder parents. F₁ mice with C57BL/10 background were the poorest responders. Studies with F₂ mice and backcross progenies of selective breeding of high and low antibody responder (C57BL/6 × BALB/c) F₁ to high responder C57BL/6 mice indicated that both non-H-2 genes and H-2 gene dosage effects influenced the magnitude of the humoral antibody responses. Animals having low responder non-H-2 background and only half the dosage of the responder immune response genes has greatly diminished antibody responses.     

Separation of T and B lymphocytes from various mouse strains by density gradient electrophoresis

T and B mouse spleen lymphocytes were separated by density gradient electrophoresis on the basis of their surface charge. In all strains examined, the T lymphocytes were found in the high mobility fractions and the B in the low. The T and B cells were separated completely in most fractions, with some overlapping in the middle. Significant differences were found in the electrophoretic distribution profiles between the strains: C57BL/6j, C57BL/10j, (BALB/cXC57BL/6j)F₁, and all the following: B₆·C-H-2^d/cBy (congenic to C57BL/6j), BALB/c, CBA/H/T6j, C57BL/10Sn, and C3H. The C57BL/6j and the (BALB/cXC57BL/6j)F₁ cells appear more heterogeneous as far as electrophoretic mobility is concerned. Almost all the other strains give two major peaks. Moreover, the high mobility areas are less populated in the C57BL/6j and the (BALB/cXC57BL/6j)F₁ animals than in all the others. The above differences were found consistently when cells prepared by different methods were electrophoresed. It is concluded that the surface charge of lymphocytes may be genetically determined. Possible dependency on the H-2 complex or non-H-2 areas is discussed.     

Interaction of thymus lymphocytes with histoincompatible cells. IV. Mixed lymphocyte reactions of activated thymus lymphocytes

CS7BL-activated CBA T cells (T.TDL) were obtained by thoracic duct cannulation of (CBA × C57BL)F₁ mice 4 days after heavy irradiation and injection of CBA thymus cells. T.TDL behaved differently from the TDL of normal CBA mice in unidirectional mixed lymphocyte culture in a number of respects: (a) the response of T.TDL was directed specifically against C57BL antigens, whereas normal TDL responded to both C57BL and BALB/c antigens; (b) the response of T.TDL was rapid but transient compared to that of TDL; (c) whereas only approximately 3% of TDL synthesized DNA specifically in response to C57BL antigens, as many as 25% of C57BL-activated T.TDL responded to these antigens. Evidence is presented which suggests that the T.TDL have a very limited capacity to proliferate. Most of the cells which responded to antigen synthesized DNA without subsequently entering mitosis.     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

Influence of acetylcysteine on cytogenetic effects of etoposide in mouse oocytes

The influence of N-acetylcysteine (ACC) on the cytogenetic effects of etoposide in F₁ CBA × C57BL/6 mice was studied. Etoposide introduced intraperitoneally in doses of 10, 20, 40, and 60 mg/kg has a dose-dependent clastogenic activity and has an aneugenic effect with the induction of mainly hypohaploid oocytes. ACC significantly decreases the aneugenic and clastogenic activity of etoposide (20 mg/kg) in oocytes of 6-, 9-, and 12-week-old mice during triple introduction at a dose 200 mg/kg per os. The most pronounced anticlastogenic ACC activity (an 80% decrease) was registered in 9-week-old females; a 100% decrease in aneugenesis was detected in 6-week-old female mice.     

RNA in the periphery of rapidly proliferating mouse lymphoid cells

RNA in the peripheries of various populations of lymph node cells (LNC) has been evaluated by measuring the electrophoretic mobilities of cells, before and after treatment with active or inactivated ribonucleases. Three different populations of LNC were studied: (1) “resting” normal age control LNC; (2) “syngeneic” LNC from irradiated (C3H × C57BL)F₁ or C3H mice four to six days following transplantation of syngeneic spleen cells; such cells were progeny of lymphopoietic progenitor cells of the spleen; and (3) “allogeneic” LNC from irradiated (C3H × C57BL)F₁ mice four to six days after grafting C3H (parental) spleen cells; such cells were progeny of lymphopoietic progenitor cells, but also alloantigen-sensitive cells of the spleen which proliferate in response to the host's alloantigens (a “graft-versus-host” immunological reaction). Whereas the normal LNC had no detectable peripheral RNA, the allogeneic and syngeneic LNC did, i.e., ribonuclease reduced their mean electrophoretic mobilities by 13.6 and 9.2 per cent, respectively. Since both allogeneic and syngeneic LNC had peripheral RNA, no specific correlation could be made with immunological activity. ³H-uridine and ¹⁴C-thymidine incorporation into lymph nodes was greatest in allogeneic, intermediate in syngeneic and least in age control lymph nodes, indicating a “population shift” in the spleen cell chimeras toward relatively immature, rapidly proliferating cells, which had a relatively high rate of RNA synthesis. Thus, rapidly proliferating lymphoid cells do have RNA in their peripheries, but its relation to specific immunological function has yet to be ascertained.     

Teratocarcinoma transplantation rejection loci: AnH-2-linked tumor rejection locus

Embryoid bodies (ascites tumor) from a 129/Sv transplantable teratocarcinoma produce tumors (100%) in syngenic 129/Sv mice but fail to form tumors (3–6%) in BALB/c mice, C3H/He mice and C57BL/6 mice, in spite of the fact that the malignant stem cells of this tumor do not express detectable H-2 antigens. The available evidence indicates that this allogeneic tumor restriction has an immunological basis; 100% of the F₁ hybrid mice between 129/Sv and the three other inbred mouse strains accept the 129/Sv teratocarcinoma. The backcross and F₂ mice segregate the BALB/c, C3H/He and C57BL/6 tumor transplantation rejection loci in a manner that indicates that each of these inbred strains of mice contain one to two major transplantation rejection loci. A linkage analysis in the BALB/c and C3H/He backcross and F₂ generations indicates that these mice have a teratocarcinoma transplantation rejection locus on chromosome 17, about eight to nine recombination units from theH- 2 complex. An F₁ complementation analysis between allogeneic mice that each reject teratocarcinomas tumors (BALB/c × C57BL/6 and C3H/He × C57BL/6), indicates that the C57BL/6 mice have the 129/Sv tumor-accepting (sensitive) allele at theH-2-linked locus but reject teratocarcinomas because of antigenic differences at a second locus.While these major teratocarcinoma transplantation rejection loci determine the acceptance or rejection of a tumor by a mouse injected with high doses of tumor tissue (750 g of tumor protein), evidence is presented for a number of minor genetic factors that can (1) affect the efficiency of tumor rejection and (2) cause complete tumor rejection at lower tumor doses (7.5–75 g of tumor protein).     

In vitro spleen cell responsiveness to various analogs of MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine), a synthetic immunoadjuvant, in MDP high-responder mice

N-Acetylmuramyl-l-alanyl-d-isoglutamine (MDP), a synthetic immunoadjuvant, was incubated with spleen cells of DBA/2 or Balb/c mice and optimal responses were obtained after 4 or 5 days of culture in a serum-free medium supplemented with 2-mercaptoethanol. In contrast, lymphocytes of (C57B1/6 × AKR)F₁ hybrids responded weakly under the same conditions. The results reported here show that like in the case of DBA/2 and Balb/c strains, spleen cells of Swiss mice and of inbred AKR and CBA mice could be stimulated in vitro whereas C57B1/6 and LPS-refractory C3H/He mice did not respond. Fourteen synthetic MDP analogs (eight known to be adjuvant active and six devoid of activity) were tested in DBA/2 high-responder mice. A good correlation was observed between in vitro stimulation and the presence or absence of adjuvant activity in vivo of these compounds.     

Development of in vivo somatic mutation system using antibody against hemoglobin Preparation and use of an anti-hemoglobin antibody for identifying C57BL/6 red cells in artificial mixture of DBA/2 and C57BL/6 red cells

A cellular specific-locus mutation test is described for detecting mutant cells in mammals. The test is based upon the use of specific anti-C57BL/6J mouse hemoglobin antibody that binds hemoglobin “single” (hemoglobin s, present in C57BL/6J mouse) and not hemoglobin “diffuse” (hemoglobin d, present in DBA/2J mouse). Attempts to purify such antibody from pony and rabbit antisera through cross-absorption were unsuccessful. Immunization of LP/J mouse with C57BL/6J hemoglobin produced antiserum that reacted with s hemoglobin but not with d hemoglobin. In a fluorescent antibody technique, this antibody was found to label fixed red blood cells from C57BL/6J mice but not from DBA/2J mice. In a mixture of C57BL/6J and DBA/2J red cells, the C57BL/6J cells could be differentiated by their bright fluorescence from the non-fluorescent DBA/2J cells. Reconstruction experiment with artificial mixtures of DBA/2J and C57BL/6J cells showed that s hemoglobin bearing cells could be detected in DBA/2J red cells at frequencies as small as 0.4×10^?6. Thus, the system is sensitive enough to detect d → s mutation in DBA/2J mice. Amino acid comparison of the globin chains of s and d hemoglobins shows that our antibody can probably detect mutations leading to a substitution of serine or proline by alanine at β²⁰ position and/or a substitution of threonine by alanine at β¹³⁹ position.     

Studies on the mechanism of transplantation tolerance: I. Do suppressor cells play a role in the induction and maintenance of neonatal transplantation tolerance?

Neonatal transplantation tolerance was induced in CBA (H-2^k) mice to A (H-2^a) mice by injection of (CBA × A)F₁ spleen cells. Animals carrying an A-skin test allograft for more than 4 months without any visible sign of rejection were considered to be permanently tolerant. Permanently tolerant CBA mice were given normal syngeneic spleen cells to abrogate the state of tolerance. Abrogation of tolerance was greatly facilitated by antithymocyte serum (ATS) treatment of tolerant mice prior to the normal syngeneic cell transfer. Survival of A allografts on normal, adult, ATS-treated CBA mice was significantly prolonged (and in many cases “adult” tolerance was achieved) by transfer of spleen cells of syngeneic mice made permanently tolerant at neonatal age. The possible role of the F₁-cell “contamination” in the tolerance-inducing effect of the transferred “tolerant” spleen cells was excluded. The results indicate that ATS-sensitive suppressor cells play a definite role in the induction, maintenance, and transfer of neonatally induced transplantation tolerance.     

H-gene (histocompatibility) mutations induced by triethylenemelamine in the mouse

Based on a simple dermal-graft procedure, the H-test for histo-compatibility mutations screens the mouse “H-system” of not less than 29 and perhaps considerably more than 100 loci that are scattered throughout the genome. Graft-tests with normally heterozygous or hemizygous loci permit scoring mutations as gains, losses, and gains+losses. Tests with normally homozygous loci screen only for gains, but further analysis can detect accompanying losses. Assayed by the H-test, triethylenemelamine (TEM) increased the spontaneous mutation rate per generation by approximately four- to five-fold in the case of BALB/c spermatogonia and F₁ hybrid oocytes (BALB/c females × C57BL/6 males), but had a much smaller effect, if any, on the rate of C57BL/6 spermatogonia. Single intraperitoneal doses of 2.4–4.0 mg/kg were given.     

Quantitative trait loci in a bacterially induced model of inflammatory bowel disease

Inflammatory bowel diseases (IBDs) are complex disorders caused by a combination of environmental, microbial, and genetic factors. Genome-wide association studies in humans have successfully identified multiple genes and loci associated with disease susceptibility, but the mechanisms by which these loci interact with each other and/or with environmental factors (i.e., intestinal microbiota) to cause disease are poorly understood. Helicobacter hepaticus-induced intestinal inflammation in mice is an ideal model system for elucidating the genetic basis of IBD susceptibility in a bacterially induced system, as there are significant differences in H. hepaticus-induced disease susceptibility among inbred mouse strains. Infected A/J mice develop acute overexpression of proinflammatory cytokines followed 2?C3?months later by chronic cecal inflammation, whereas infected C57BL/6 mice fail to develop cecal inflammation or increased cytokine expression. The goal of this project was to use quantitative trait locus (QTL) mapping to evaluate genetic factors that contribute to the differential disease susceptibility between these two mouse strains. Using acute cecal IL-12/23p40 expression as a biomarker for disease susceptibility, QTL analysis of H. hepaticus-infected F₂ mice revealed involvement of multiple loci. The loci with the strongest association were located on Chromosome 3 and Chromosome 17, with logarithm of odds (LOD) scores of 6.89 and 3.09, respectively. Cecal expression of IL-12/23p40 in H. hepaticus-infected C57BL/6J-Chr3^A/J/NaJ chromosome substitution mice had an intermediate phenotype, significantly higher than in resistant C57BL/6 but lower than in susceptible A/J mice, confirming the importance of this locus to the immune response to H. hepaticus infection.     

T-cell neoplasm induced by subcutaneous transplantation of a plasmacytoma: characterization of tumor cells.

Thymocytes, isolated 6 days following subcutaneous (sc) transplantation of BALB/c MOPC-315 plasmacytoma into F₁ (BALB/c × C57BL) hybrid mice, when injected sc into normal syngeneic mice, caused the development of a solid sc tumor. The cells of the newly developed tumor were of a mixed population of θ⁺F₁ (BALB/c × C57BL) and θ^? BALB/c cells (approximately 1:1), which represents a new type of mixed T cell-plasma cell neoplasm. Efforts were made to isolate the transformed thymocytes (θ⁺) from the plasmacytoma (θ^?) cells in the new tumor, exploiting differences in their surface properties. Treatment of the mixed tumor cell population with peanut agglutinin (PNA) revealed that only the T tumor cells were agglutinated. The agglutinated cells were recovered after dispersing the clumps with d-galactose (0.15 M) and consisted of 95% θ⁺ cells. The PNA-agglutinated cells were found to induce a similar tumor (85% θ⁺ cells) when injected sc into F₁ (BALB/c × C57BL) mice.