Transfer Agent of Immunity

Antibody formation in vitro was studied using erythrocytes (RBC) as antigen and immunocytoadhesion as the technique for detection of antibody-forming cells. Spleen cells (SPC) of nonimmune mice gained the ability to produce antibody after treatment with ribonucleic acid (RNA) preparation extracted from allogeneic mice immunized with xenogeneic or allogeneic RBC. It was also found that a small proportion of SPC from individual mice of certain strains formed antibody against autologous RBC when the cells were treated in vitro with RNA preparation obtained from the spleen of an allogeneic mouse immunized with RBC of that individual. No converting ability was observed in the RNA preparation from spleen of nonimmune autologous or allogeneic mice. The converting activity of immune RNA preparation was shown to be sensitive to ribonuclease treatment. These evidences exclude the possible contribution of antigen or fragments thereof in the RNA preparation to the induction of antibody formation in RNA recipient cells.     

Human autologous rosette-forming cells. III. Binding of erythrocytes from different species to the T-cell receptors for autologous red blood cells

Spontaneous rosette formation in humans is restricted to a subpopulation of the circulating T cells. We have previously shown that the interaction between lymphocytes and autologous red blood cells (auto-RBC) is not mediated by a self-recognition mechanism, since allogeneic (allo-) RBC bind to T cells through the same receptors. In this work, we have extended these observations to thymocytes. Using a mixed-rosette assay in which one type of erythrocyte was identified by FITC labeling, we have shown that almost all the thymocytes which attached auto-RBC could also fix allo-RBC. However, as for the peripheral blood lymphocytes (PBL), binding of human RBC to thymocytes occurred with varying affinities according to the erythrocyte's origin. In order to further study the specificity of the erythrocyte to lymphocyte binding in rosette formation, PBL were mixed with auto-RBC and erythrocytes of xenogeneic (xeno-) origin. Although very disparate incidences of rosettes were found according to the species from which the RBC were derived, most of the autorosetting lymphocytes also had receptors for xeno-RBC. In addition, preincubation of PBL with monoclonal antibody OKT11A (directed against the sheep RBC receptors on T cells) completely abrogated rosette formation with all the erythrocytes tested (human auto- and allo-, sheep, pig, and rabbit) except mouse RBC. Taken together these data strongly suggest that human auto- or allo-, as well as sheep or some other xeno-RBC, bind to T lymphocytes by a single receptor and that the combining sites are expressed with different densities or varying affinities depending upon the RBC's origin. Therefore, spontaneous autorosettes may represent T lymphocytes having high-affinity receptors for sheep RBC.     

Role of hemoglobin oxygenation in the modulation of red blood cell mechanical properties by nitric oxide

It has been previously demonstrated that both externally generated and internally synthesized nitric oxide (NO) can affect red blood cell (RBC) deformability. Further studies have shown that the RBC has active NO synthesizing mechanisms and that these mechanisms may play role in maintaining normal RBC mechanical properties. However, hemoglobin within the RBC is known to be a potent scavenger of NO; oxy-hemoglobin scavenges NO faster than deoxy-hemoglobin via the dioxygenation reaction to nitrate. The present study aimed at investigating the role of hemoglobin oxygenation in the modulation of RBC rheologic behavior by NO. Human blood was obtained from healthy volunteers, anticoagulated with sodium heparin (15 IU/mL), and the hematocrit was adjusted to 0.4 L/L by adding or removing autologous plasma. Several two mL aliquots of blood were equilibrated at room temperature (22 ± 2 °C) with moisturized air or 100% nitrogen by a membrane gas exchanger, The NO donor sodium nitroprusside (SNP), at a concentration range of 10^?7–10^?4 M, was added to the equilibrated aliquots which were maintained under the same conditions for an additional 60 min. The effect of the non-specific NOS inhibitor l-NAME was also tested at a concentration of 10^?3 M. RBC deformability was measured using an ektacytometer with an environment corresponding to that used for the prior incubation (i.e., oxygenated or deoxygenated). Our results indicate an improvement of RBC deformability with the NO donor SNP that was much more pronounced in the deoxygenated aliquots. SNP also had a more pronounced effect on RBC aggregation for deoxygenated RBC. Conversely, l-NAME had no effect on deoxygenated blood but resulted in impaired deformability, with no change in aggregation for oxygenated blood. These findings can be explained by a differential behavior of hemoglobin under oxygenated and deoxygenated conditions; the influence of oxygen partial pressure on NOS activity may also play a role. It is therefore critical to consider the oxygenation state of intracellular hemoglobin while studying the role of NO as a regulator of RBC mechanical properties.     

HIV-specific cytotoxic T lymphocytes against alveolar macrophages: specificities and downregulation

To analyse the evolution of alveolar-lymphocyte-mediated cytotoxic activity directed against autologous alveolar macrophages (AM), cytotoxic assays against various HIV⁺ target cells were performed in a cohort of 75 patients with HIV-associated lymphoid interstitial pneumonitis (LIP) studied at distinct stages of HIV infection. Our data confirm that alveolar HIV-specific cytotoxic T lymphocytes (CTL) against AM were detectable before AIDS in patients with CD8⁺ LIP. Mild CD8⁺ lymphocytic alveolitis occurs silently in 62% of stage II and III patients with no respiratory symptoms. In these cases, the lack of spontaneous alveolar-lymphocyte-mediated cytotoxic activity against autologous AM may contrast with the detection of primary alveolar CTL specific for HIV proteins such as nef. In AIDS patients, the alveolar CTL lytic efficiency against both AM- and HIV-antigen-expressing cells can be inhibited by a suppressore factor produced by alveolar CD8⁺ CD57⁺ cells. Therefore, spontaneous CTL lysis of AM may be (1) limited to a subgroup of patients with active LIP and (2) controlled by distinct mechanisms, including suppressor phenomenons, and HIV replication levels in AM.     

Aggregation behavior and electrophoretic mobility of red blood cells in various mammalian species

Differences of red blood cell (RBC) aggregation among various mammalian species has been previously reported for whole blood, for RBC in autologous plasma, and for washed RBC re-suspended in polymer solutions. The latter observation implies the role of cellular factors, yet comparative studies of such factors are relatively limited. The present study thus investigated RBC aggregation and RBC electrophoretic mobility (EPM) for guinea pigs, rabbits, rats, humans and horses; RBC were re-suspended in isotonic 500 kDa dextran solutions for the EPM and aggregation measurements, with aggregation studies also done in autologous plasma. Salient results included: (1) species-specific RBC aggregation in both plasma and dextran (horse > human > rat > rabbit approximately = guinea pig) with a significant correlation between aggregation in the two media; (2) similar EPM values in PBS for rat, human and horse, a lower value for guinea pig, and a markedly reduced EPM for rabbit RBC; (3) EPM values in dextran with a rank order identical to that for cells in PBS; (4) relative EPM results indicating formation of a polymer-poor, low viscosity depletion layer at the RBC surface (greatest depletion for horse RBC). EPM-aggregation correlations were evident and generally consistent with the Depletion Model for aggregation, yet did not fully explain differences between species; additional studies at various ionic strengths and with various dextran fractions thus seem warranted.     

Assessment of the hemorheological profile of koala and echidna

Koala, a marsupial, and echidna, a monotreme, are mammals native to Australia. Blood viscosity (62.5–1250 s^?1), red blood cell (RBC) deformability, RBC aggregation, aggregability and surface charge, and hematological parameters were measured in blood samples from six koalas and six echidnas and compared to adult human blood. Koala had the largest RBC mean cell volume (107.7±2.6 fl) compared to echidna (81.3±2.6 fl) and humans (88.4±1.2 fl). Echidna blood exhibited the highest viscosity over the entire range of shear rates. Echidna RBC were significantly less deformable than koala RBC but more deformable than human RBC. Echidna RBC had significantly lower aggregability (i.e., aggregation in standardized dextran medium) than koala or human RBC, while aggregation in autologous plasma was similar for the three species. Erythrocyte surface charge as indexed by RBC electrophoretic mobility was similar for human and echidna cells but was 40% lower for koala RBC. Data obtained during this preliminary study indicate that koala and echidna have distinct hemorheological characteristics; investigation of these properties may reveal patterns relevant to specific behavioral and physiological features of these animals.     

New method to quantify erythrophagocytosis by autologous monocytes.

BACKGROUND: Anemia is the net result of decreased red blood cell (RBC) production and increased removal of RBCs. Replication and maturation of erythroid precursors and RBC lysis can be measured by standardized in vitro methods and surrogate markers, respectively. In contrast, erythrophagocytosis by autologous phagocytes is more difficult to quantify. METHODS: We developed a method to assess erythrophagocytosis by autologous monocytes from 5 ml of whole blood. RBCs were labeled with carboxyfluorescein-diacetate-succinimidyl ester (CFDA-SE) and subsequently coincubated with autologous CD14(+) monocytes. Phagocytosis was quantified using flow cytometry. After standardization, the assay was validated in patients with severe malarial anemia (SMA), a condition that is associated with increased erythrophagocytosis. RESULTS: After labeling, CFDA-SE was stably incorporated into RBCs and no significant leakage leading to contamination of nonlabeled cells was observed. Monocytes ingested opsonized, labeled RBCs seven times more than nonopsonized controls. Erythrophagocytosis was significantly higher in SMA than in healthy controls. CONCLUSIONS: The established assay showed enhanced autoerythrophagocytosis associated with SMA and hence was able to detect clinically relevant erythrophagocytosis. This novel assay is well suited for rapid quantification of in vitro erythrophagocytosis by autologous monocytes.     

AM630 is a competitive cannabinoid receptor antagonist in the guinea pig brain

AM 630 has been demonstrated to be a cannabinoid receptor antagonist in the mouse brain and vas deferens. Conversely, it was recently reported that AM630 acts as a cannabinoid agonist in the guinea pig ileum. This research was designed to determine whether the difference in the action of AM630 is species specific. Studies conducted in guinea pig brain reveal that AM630 antagonizes the stimulatory effect of the cannabinoid agonist WIN 55,212-2 on [³⁵S]GTPγS binding suggesting that difference in AM630 activity in different tissues is not due to species Variation.     

Modulation of natural killer-mediated lysis by red blood cells

Natural killer (NK)-mediated cytotoxicity is significantly enhanced in the presence of red blood cells (RBC). The enhancement is dose dependent on the number of RBC present and can be induced by autochthonous, allogeneic, or xenogeneic RBC. The enhancement was demonstrated in cytotoxicity against two different NK-sensitive tumor target cell lines, K562 and U937, and by three different assay systems, chromium release, lactate dehydrogenase release, and inhibition of thymidine incorporation. RBC directly enhance the cytotoxic activity of NKH-1/Leu19+ large granular lymphocyte NK cells. Intact RBC have to be present during the cytotoxicity assay to induce the enhancement, which probably occurs at a postbinding, preprogramming phase. The anti-CD2 antibody Leu5 cannot block the enhancement at a concentration inhibitory to lymphocyte rosetting with sheep RBC, suggesting that the enhancement is not induced by interaction through the CD2 antigen. These results indicate that RBC are a potent modulator of NK cytotoxicity and suggest that in vitro NK studies using purified lymphocytes may not always truly reflect NK activity in the blood stream.     

Early evaluation of mixed leukocyte interactions in mice and guinea pigs using measurements of protein synthesis

A rapid microassay for mixed lymphocyte interactions (MLI) in the mouse and the guinea pig has been developed. This assay is based on the stimulation of protein synthesis (SPS) that occurs as soon as 11 hr after initiation of the culture and can be measured with high precision. The cells are cultured in leucine-free medium without serum in microtiter plates in 0.2 ml volumes using 2 × 10⁶ cells of each cell type. Stimulation of protein synthesis as measured by increased incorporation of a pulse of ³H-leucine into protein occurs in allogeneic MLI's in which cells differ at major and minor, as well as at only minor histocompatibility loci, but does not occur in xenogeneic MLI's. Unidirectional MLI's can also be performed using either Xirradiated cells or cells from F₁-hybrid animals. The sum of the two one-way MLI's in each case equals that of the two-way MLI. The advantage of this assay are: (1) it can be completed in less than 24 hr; (2) serum is not required in the culture medium; (3) relatively few cells and small amounts of reagents are required; (4) it is highly reproducible.     

Effect of enhanced red blood cell aggregation on blood flow resistance in an isolated-perfused guinea pig heart preparation

The role of red blood cell (RBC) aggregation as a determinant of in vivo blood flow is still unclear. This study was designed to investigate the influence of a well-controlled enhancement of RBC aggregation on blood flow resistance in an isolated-perfused heart preparation. Guinea pig hearts were perfused through a catheter inserted into the root of the aorta using a pressure servo-controlled pump system that maintained perfusion pressures of 30 to 100 mmHg. The hearts were beating at their intrinsic rates and pumping against the perfusion pressure. RBC aggregation was increased by Pluronic (F98) coating of RBC at a concentration 0.025 mg/ml, corresponding to about a 100% increment in RBC aggregation as measured by erythrocyte sedimentation rate. Isolated heart preparations were perfused with 0.40 l/l hematocrit unmodified guinea pig blood and with Pluronic-coated RBC suspensions in autologous plasma. At high perfusion pressures there were no significant differences between the flow resistance values for the two perfusates, with differences in flow resistance only becoming significant at lower perfusion pressures. These results can be interpreted to reflect the shear dependence of RBC aggregation: higher shear forces associated with higher perfusion pressures should have dispersed RBC aggregates resulting in blood flow resistances similar to control values. Experiments repeated in preparations in which the smooth muscle tone was inhibited by pre-treatment with papaverine indicated that significant effects of enhanced RBC aggregation could be detected at higher perfusion pressures, underlining the compensatory role of vasomotor control mechanisms.     

Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: A model for utilization of primordial oocytes

Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1^nu). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electrostimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 14.3%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion.     

Direct activation of tumoricidal properties in rat alveolar macrophages by Nocardia rubra cell wall skeleton

Summary Alveolar macrophages (AM) lavaged from lungs of normal F344 rats were rendered tumoricidal following their direct interaction with squalene-treated Nocardia rubra cell wall skeleton (N-CWS) present in the culture medium. Maximum tumoricidal activity was obtained by incubating AM with 1 g N-CWS/ml for a 24-h period. These AM were cytolytic to syngeneic, allogeneic, and xenogeneic tumorigenic cells. Tumoricidal activity following interaction with N-CWS decreased gradually and was lost completely by 96 h. A second in vitro exposure to N-CWS reactivated AM to their full tumoricidal potential. The present studies suggest that N-CWS can directly activate AM to render them tumoricidal.     

Cell surface changes accompanying aging in human diploid fibroblasts : II. Two types of age-related changes revealed by concanavalin A-mediated red blood cell adsorption

Age-related changes in cell surfaces of human diploid fibroblasts (TIG-1) were investigated using the concanavalin A (ConA)-mediated red blood cell (RBC) adsorption assay. When ConA-coated RBCs were adsorbed to fibroblasts (RBC coating method), the amount of RBCs adsorbed per mg of fibroblast protein increased continuously from the early phases of cell passage up through cell senescence. On the other hand, when RBCs were adsorbed to ConA-coated fibroblasts (fibroblast coating method), RBC adsorption did not occur throughout phase II and increased with the advance of phase III. [³H]ConA binding to fibroblasts, however, did not change with aging to the extent that could explain the observed changes in RBC adsorption. These age-related characteristics in RBC adsorption and [³H]ConA binding were also observed for WI38 and IMR-90 cells. In addition, SV40- and ⁶⁰Co-transformed WI38 cells showed a close resemblance in their RBC adsorption capacity to early phase III cells.RBC adsorption with both the RBC and fibroblast coating methods was not a function of cell cycle phase and time spent in culture (metabolic time). Co-culturing of young cells with old or transformed cells did not affect the RBC adsorption capacity of respective cells. These results suggest that RBC adsorption with the RBC and fibroblast coating methods may represent cell surface markers for division age and senescence of aging human diploid cells.     

Human autologous rosette-forming cells: II. Specificity of the binding between lymphocytes and erythrocytes

The relationship between autorosettes and allorosettes was investigated using a mixed rosette assay in which the origin of the erythrocytes was assessed by the fluorescein isothiocyanate (FITC) labeling of one type of erythrocyte. The data show that auto- and allorosettes belong to the same T-cell subset: (1) in most of the subjects, the percentages of T cells binding autologous red blood cells (auto-RBC) are equivalent to those binding allogeneic RBC (allo-RBC); (2) the percentage of rosettes formed after the simultaneous addition of auto- and allo-RBC is similar to that of autorosettes alone or allorosettes alone; and (3) nearly 80% of the resetting cells bind both types of RBC as directly visualized in the mixed rosette assay. The experiments in which the lymphocytes are resetted first with one type of RBC, and then with the other type support the finding that auto- and allo-RBC may bind to the lymphocytes through a single receptor which exhibits a varying affinity for RBC according to their origin.     

Aberrancy in immunogenicity and cell-surface expression of H-2 antigens on erythrocytes

Immunogenicity for T cell-independent B-cell response assessed by splenic plaque-forming cell (PFC) response and cell-surface expression measured by laser flow cytometry of various class I H-2 antigens on mouse red blood cells (RBC) were compared. It was found that the order of magnitude of both immunogenicity and cell-surface expression on RBC is H-2D^d H-2D^b > H-2K^d, H-2K^b. Furthermore, H-2^d public antigens and H-2L^d antigens were neither immunogenic nor easily demonstrable on RBC. These findings contrasted with poor immunogenicity for PFC response (Nakashima et al. 1982, 1983) and proportionally strong expression of H-2 antigens on lymphoid cells. Immunogenicity and cell-surface expression of H-2D^d antigen on RBC were not shown to be controlled by the action of genes outside H-2D. It was therefore suggested that a number of H-2 antigens, including H-2K^d private, H-2K^b private, and H-2^d public specificities are at least functionally defective on RBC. This is possibly due to the structural characteristics of the antigens. Since immunogenicity and cell-surface expression were in parallel, the expression of H-2 antigens on RBC must be dictated by a subset of B cells whose activity was assessed by PFC response. This finding supports the view that the H-2 molecules display a new category of activity which is different from their ability to activate T cells and depends on their expression on RBC.     

Synergistic cytotoxicity. I. Characterization of a heat labile plasma fraction that induces nonspecific cytotoxicity by human mononuclear cells.

The mechanism by which non-immune mononuclear cells recognize invading foreign material and are activated for cytotoxic attack was studied in a model system employing human mononuclear cells, fresh plasma, and 51Cr-labeled xenogeneic target erythrocytes. In these experiments, fresh antibody-depleted plasma or mononuclear leukocytes alone were poorly cytotoxic to xenogenic erythrocytes. However, these target cells were rapidly lysed when both fresh antibody-depleted plasma and mononuclear cells were present in the assay. The plasma factor could not be removed by extensive absorption with the target cells, was present in plasma from hypogammaglobulinemic patients, was heat labile, and was sensitive to incubation with zymosan and cobra venom factor. The "antigen" specificity of this reaction was directed by the serum factor inasmuch as target cells autologous to the effector cells could be killed in the presence of antibody-depleted xenogeneic plasma, but not autologous plasma. These data suggest that an important mechanism for the recognition of "foreigness" by non-immune mononuclear cells is via interaction with a plasma component, possibly a factor related to serum complement.     

HVJ infection in alveolar macrophages of various laboratory animals

Alveolar macrophages (AM) in bronchoalveolar lavage derived 6 species of laboratory animals (mouse, rat, Syrian hamster, Mongolian gerbil, guinea pig, and rabbit) were cultivated in vitro and were infected with HVJ, parainfluenza virus type 1, for the purpose of studying virus reproduction in these cells. Using infectivity assay, fluorescent antibodies and hemadsorption techniques, three categories were proposed depending on the potential of virus replication and antigen production. Category 1 (mouse, Syrian hamster, and guinea pig AM) was the most susceptible group to HVJ infection. All AM became antigen positive, and more than 10(6) PFU/ml of virus infectivity in the culture fluids was noted at 4 days after the virus infection. Rat and Mongolian gerbil AM were grouped into Category 2. Their AM showed a moderate virus yield (average 10(5) PFU/ml of infectivity) and antigen production. On the other hand, Category 3 (rabbit AM), exhibited no production of an infectious virus. It was likely that the virus penetrated into the AM but did not replicate complete virus. However, antigen positive cells ranging from 10 to 50% depended on the multiplicity of the infection to the AM. These findings suggest that HVJ infection in the rabbit AM was abortive (incomplete virus replication), since trypsin treatment of the culture fluids, which may contain incomplete HVJ particles, did not induce infectivity.     

Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference

ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [¹²⁵I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC₅₀ value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.     

Histocompatibility typing by cellular radioimmunoassay

A quantitative cellular radioimmunoassay (CRIA) for histocompatibility typing is described. Chicken red blood cells (RBC) were incubated in microtiter plates with specific anti-MHC (B) alloantisera and the alloantibody bound measured indirectly by a second binding step with¹²⁵I-labeled rabbit anti-chicken IgG. The assay is objective, highly consistent, and three to four orders of magnitude more sensitive than conventional hemagglutination assays. The new CRIA was used to detect minor subpopulations of cells in artificial cell mixtures; as few as 1% of relevant cells were easily detected. Erythrocyte chimerism was induced following the injection of B²/B² chicken embryos with B¹⁵/B²¹ embryonic stem cells. Five weeks after hatching, erythrocyte chimerism was precisely quantitated by comparing the reaction of RBC from the putative chimeras with artificial cell mixtures using specific anti-B₁₅/B₂₁ alloantisera. The percent varied from 13–40% in 13 chimeric animals. The new CRIA was also used for the sensitive detection of tumor-specific antigens on a T-cell lymphoma. An unexpected finding was that anti-B₁₅ alloantibody bound almost as well to B¹⁵/B²¹ heterozygous RBC as to B¹⁵/B¹⁵ homozygous cells, suggesting that either the concentration or the steric arrangement of B₁₅ alloantigen at the erythrocyte surface may not conform to conventional expectations.