Relationship Between Tuberculin Hypersensitivity and Cellular Immunity to Infection in Mice Vaccinated with Viable Attenuated Mycobacterial Cells or with Mycobacterial Ribonucleic Acid Preparations

The migration inhibition technique has been used to study delayed hypersensitivity in vitro by using peritoneal exudate cells and splenic lymphocytes from mice vaccinated with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis and from mice vaccinated with ribonucleic acid (myc RNA) preparations obtained from viable mycobacterial cells of the same strain. Inhibition of macrophage migration was noted when purified protein derivative (PPD) or viable H37Ra cells were added to peritoneal exudate cells obtained from mice immunized with viable H37Ra cells and not from mice immunized with myc RNA. Splenic lymphocyte cultures were exposed to the same antigens in vitro. Filtered supernatant fluids from these lymphocyte cultures, when added to peritoneal exudate cells obtained from nonimmunized mice, inhibited migration only when they were obtained from lymphocytes which came from mice immunized with viable H37Ra cells. Injection of PPD intravenously into vaccinated mice resulted in inhibitory supernatant fluids from splenic lymphocyte cultures only when the lymphocytes came from mice immunized with viable H37Ra cells. However, intravenous injection of either viable H37Ra cells or of myc RNA preparations into mice vaccinated with myc RNA occasionally produced inhibitory supernatant fluids when lymphocytes were obtained from these mice. On the other hand, mice vaccinated with myc RNA or viable H37Ra cell preparations were consistently and equally protected against intravenous challenge with the virulent H37Rv strain. Thus, although some evidence was obtained for a delayed type hypersensitivity in mice vaccinated with H37Ra cells or with myc RNA to ribosomal proteins or other proteins associated with the RNA preparation, no evidence of tuberculin hypersensitivity could be detected in any mice vaccinated with the myc RNA. These results argue against a role for tuberculin hypersensitivity in immunity to tuberculous infection.     

结核杆菌H37Ra免疫小鼠后脾淋巴细胞的杀伤活性及免疫机制的研究

目的探讨结核杆菌H37Ra免疫小鼠后,产生特异性的脾淋巴细胞杀伤活性及其免疫机制。方法(1)分别以结核杆菌H37Ra、BCG和PBS免疫小鼠后第30天及第60天,分离各组小鼠脾淋巴细胞作为效应细胞,以表达Ag85B分泌蛋白的Sp2/0细胞为靶细胞,用MTT比色法检测免疫小鼠脾淋巴细胞的杀伤率;(2)在免疫后第60天,小鼠脾淋巴细胞经PPD刺激后,以RT-PCR法检测穿孔素mRNA及颗粒酶BmRNA的表达。结果(1)结核杆菌H37Ra组脾淋巴细胞的杀伤率明显高于PBS对照组(P<0.05),略高于BCG组;(2)H37Ra组和BCG组穿孔素、颗粒酶mRNA的表达量均显著高于PBS组(P<0.05),H37Ra组穿孔素mRNA的表达量显著高于BCG组(P<0.05),但颗粒酶BmRNA的表达量两组无差异。结论结核杆菌H37Ra免疫小鼠后,能增强脾淋巴细胞的杀伤活性,其机制可能与增加穿孔素、颗粒酶B的表达有关。     

Relationship of macrophages to defective delayed-type hypersensitivity in B6/lpr mice.

These experiments were undertaken to clarify whether or not suppressor macrophages contribute to deficiency of delayed-type hypersensitivity induced by BCG CW immunization in B6/lpr mice. Addition of indomethacin in vitro did not increase MIF production by immunized lpr lymph node cells. Adherent cells in lymph node cell suspensions from lpr mice did not interfere with MIF production by nonadherent lymph node cells from immunized B6 mice. On the other hand, MIF production by nonadherent lymph node cells from immunized B6/lpr mice was not restored even if they were cultured with adherent lymph node cells from immunized B6 mice. In addition, macrophages from nonimmunized B6/lpr mice showed normal reactivity to MIF when lymphocytes coexisting with the macrophages in large proportions were depleted from PEC. These results suggest that macrophages make no contribution to the deficiency of DTH expression of B6/lpr mice.     

Influence of an M-Locus Difference on the Cellular and Humoral Immunological Reactivity Against H-2-Determined Antigens of the Mouse

Studies were initiated to determine whether an immune response to the Mls antigen of C3H mice could modify responses of CBA lymphocytes (H-2-compatible) to a foreign H-2 complex. CBA lymphocytes, partially tolerant to the C5H-determined Mls antigen, generated less effector cell activity against C57Bl cells (H-2-incompatible) Chan lymph node cells from normal CBA donors when infused into irradiated C.3H × C57Bl hosts. Effector cell activity was measured as the capacity of the cells in the irradiated spleens to inhibit CBA × C57Bl bone marrow proliferation. In contrast, immunization of CBA mice with C3H × C57Bl cells yielded lower antibody titers against C577Bl cells than immunization with CBA × C57Bl cells. Furthermore, preinjection of CBA mice with C3H × CBA cells strongly reduced the capacity of the animals to produce antibodies against C57Bl cells. Thus, these data support the conclusion that an immune response to a foreign Mls antigen may either enhance or suppress an immune response to H-2-incompatible cells.     

Antigen and mitogen induced production of macrophage migration inhibitory factor in the mouse.

Spleen cells of C57B1/6J mice immunized with complete Freund's adjuvant produced macrophage migration inhibitory factor (MIF) when incubated in vitro with tuberculin purified protein derivative (PPD). For optimal MIF production spleen cells were cultured for 48 h in a serum-free medium, at a concentration of 2 x 10(7) cells/ml. MIF was assayed in a xenogenic system, using oil-induced guinea pig peritoneal exudate cells as targets. MIF synthesis could also be induced by pulsing spleen cells for 2 h with concanavalin A, phytohemagglutinin, pokeweed mitogen or lipopolysaccharide, followed by culture in plain medium. No MIF secretion was induced by incubation of spleen cells with anti-theta or rabbit anti-mouse IgG sera. Cells producing MIF in response to PPD were characterized as B cells by virtue of being insensitive to anti-theta serum and complement, by being retained on nylon wool, glass bead and anti-Ig colums and by the presence of Fc receptors. PPD-stimulated T cells did not produce MIF. PPD-induced mouse spleen cell MIF demonstrated a moderate loss of activity by heating at 56 and 80 degrees C and was completely inactivated after digestion with chymotrypsin. By fractionation on Sephadex G-200, migration inhibitory activity was recovered in a molecular range of 100,000-12,400 daltons.     

Manipulation of macrophage migration inhibition/stimulation responses by adjuvants and interleukins

C57 B1/6 mice were immunized with bovine serum albumin (BSA) and Freund's complete and incomplete adjuvants in various concentrations. Spleen cells from these animals were subsequently stimulated with concanavalin A (Con A), purified protein derivative or BSA, and lymphokine responses were measured in one-stage migration assays. Con A consistently produced macrophage migration inhibition factor (MIF) responses in nonimmunized animals and those immunized with complete adjuvant. This was switched to migration stimulation factor (MStF) responses by prior immunization with incomplete adjuvant. Immunization with complete adjuvant and with BSA alone was followed by MIF responses to antigenic stimulation whereas incomplete adjuvant promoted MStF responses. The MStF responses to both mitogenic and antigenic stimulation were inhibited by the addition of affinity purified interleukin-1 to the spleen cells. Interleukin-1 also inhibited MStF responses and potentiated MIF responses to Con A stimulation by human mononuclear cells whereas interleukin-2 did not influence these responses.     

Preventive effect of a synthetic immunomodulator, 2-carboxyethylgermanium sesquioxide, on the generation of suppressor macrophages in mice immunized with allogeneic lymphocytes.

The effect of 2-carboxyethylgermanium sesquioxide (Ge-132) on the generation of splenic suppressor macrophages (S-M phi) in C3H/He mice (H-2k) immunized with allogeneic spleen cells from C57Bl/6 mice (H-2b) was investigated. We have previously demonstrated that S-M phi expressing I-J antigen, which appeared during alloimmunization, inhibited cytotoxic T lymphocyte (CTL) generation in the MLR and the elimination of these S-M phi before subjection to the MLR resulted in more effective generation of CTL. The CTL activity, which was determined in vivo by the Winn's test, was markedly enhanced when immunized mice received a 100 mg/kg dose of Ge-132. The compound was found to be the most efficacious when injected simultaneously with the immunization. The activity of allospecific CTL co-cultured with M phi fractions obtained from immunized mice in a 4-h 51Cr-release assay was shown to be 31% lysis of the target cells as compared with 90% lysis of the target cells in effector cells co-cultured with normal M phi fractions. In contrast, effector cells co-cultured with M phi fractions from Ge-132-treated immunized mice lysed 95% of the target cells. Analysis of the level of I-J antigen expression on macrophages (M phi) obtained from mice 7 days after immunization revealed a > 2.5-fold increase, whereas I-A antigen expression remained constant when compared with splenic M phi from naive mice. In contrast, the opposite effect on I-J and I-A antigen expression was observed in splenic M phi from alloimmunized mice treated with Ge-132. These results suggest that Ge-132 could regulate CTL generation in alloimmunized mice by preventing the generation of I-J+ S-M phi.     

Macrophage activation during Plasmodium chabaudi AS infection in resistant C57BL/6 and susceptible A/J mice.

Macrophage activation was examined in resistant C57BL/6 and susceptible A/J mice during the course of blood-stage infection with Plasmodium chabaudi AS. Three parameters of macrophage activation (lipopolysaccharide [LPS]- and malaria antigen-induced tumor necrosis factor [TNF] production in vitro, phorbol myristate acetate [PMA]-induced production of oxygen metabolites in vitro, and Ia antigen expression) were assessed during infection in populations of peritoneal and splenic macrophages recovered from infected mice of the two strains. The peak level of LPS-induced TNF production in vitro by splenic macrophages from both infected C57BL/6 and infected A/J mice occurred on day 7, which was 3 days before the peak of parasitemia. Although the kinetics of TNF production in vitro in response to either LPS, soluble malaria antigen, or intact parasitized erythrocytes varied in some of the other macrophage populations during infection, there was no significant difference in the peak level of production. Peritoneal and splenic macrophages from infected C57BL/6 mice exhibited significantly increased PMA-induced production of H2O2 in vitro on day 7. Peritoneal macrophages from infected A/J mice also exhibited significant PMA-induced H2O2 production on day 7, while production by splenic macrophages from these hosts was not increased in comparison with production by cells from normal animals. Only peritoneal macrophages from infected C57BL/6 mice produced significantly increased levels of O2-, and this occurred on day 7 postinfection. Ia antigen expression by both peritoneal and splenic macrophages from resistant C57BL/6 and susceptible A/J mice was significantly increased during P. chabaudi AS infection. However, the percentage of Ia+ peritoneal macrophages on days 8 and 10 postinfection and Ia+ splenic macrophages on day 3 postinfection was significantly higher in C57BL/6 than in A/J mice. Thus, these results demonstrate that macrophages from P. chabaudi AS-infected A/J mice exhibit defects in oxygen metabolism and Ia antigen expression which may contribute to the susceptibility of these hosts to this intraerythrocytic parasite. The cause-and-effect relationship between these defects and the susceptibility of A/J mice to P. chabaudi AS is unknown.     

Transfer factor: a murine model.

Transfer factor has been studied extensively in humans, but a satisfactory subprimate model has not been established. Using BALB/c mice immunized with complete Freund adjuvant, we show that a low-molecular-weight substance derived from disrupted spleen cells transferred sensitivity to purified protein derivative (PPD) to recipient nonimmunized BALB/c mice. Transfer was confirmed by footpad swelling to PPD in vivo and by splenic lymphocyte transformation to PPD in vitro. In recipients of transfer factor, an inverse correlation was noted between the splenic lymphocyte response to PPD and to concanavalin A. Material obtained from spleens of saline-treated BALB/c mice did not transfer sensitivity to PPD to recipient mice.     

In vitro and in vivo autoimmune response to enamel matrix proteins.

Enamel matrix proteins taken from neonatal C57Bl/6 mice were shown to be able to elicit in vitro proliferative responses in C57Bl/6 splenic lymphocytes taken from animals which had not been exposed to exogenous enamel proteins. Animals which had been injected with enamel matrix proteins in Freund's complete adjuvant made IgG antibodies against enamel proteins which were detected by indirect immunofluorescence. Spleen cells from the immune animals gave an augmented in vitro proliferative response to enamel proteins, while spleen cells from C57Bl/6 nu/nu mice or anti-Thy 1 and complement-treated normal C57Bl/6 spleen cells were incapable of responding to enamel proteins in vitro. Thus, enamel matrix proteins appear to be T-cell dependent autoantigens. The natural history of enamel matrix proteins is reviewed, and it is suggested, based on the anatomic details of enamel synthesis, secretion, and maturation, that enamel matrix proteins are autoantigenic because they are anatomically sequestered.     

A new immunomodulator, LF 1695--II. Effects on allogenic and antigenic responses.

LF 1695, a chemically defined immunomodulator has been shown to induce in vitro T-cell differentiation and to potentiate mitogenic proliferation. The effects of LF 1695 on antigenic and allogenic responses were studied in vitro and lymphocyte proliferation was estimated by 3H-thymidine uptake for 5 and 6 days, respectively. Human peripheral blood lymphocytes (PBL) from tuberculin sensitive donors were significantly stimulated by purified protein derivative (PPD) in the presence of LF 1695. Optimal lymphocyte proliferation with PPD was obtained at 0.5 micrograms/ml LF 1695. The ability of LF 1695 to enhance proliferation of PBL in an allogenic reaction was also examined. LF 1695 (0.2 micrograms/ml) enhanced the proliferation of PBL from two individuals with different HLA DR loci in the bilateral mixed lymphocyte reaction (MLR). Moreover, LF 1695 was tested in vivo in the experimental graft vs host reaction (GvHR) in mice. CBA, H2k mice receiving a lethal dose of irradiation were injected with spleen cells from LF 1695-treated or untreated C57 Bl/6, H2b mice via different routes. CBA mice were found to have a spleen weight x 1000/body ratio up to 1.5 when injected with cells from untreated C57 Bl/6 mice. Mice treated intraperitoneally with LF 1695 at 2.5 or 5 mg/kg/day showed an increase of GvHR intensity with splenic index of 1.71 and 1.80, respectively. When the animals were treated continuously through drinking water containing LF 1695 (4 mg/kg, 10 mg/kg or 100 mg/kg), these ratios were: 1.82, 1.10 and 1.37, respectively. Finally, LF 1695 enhanced GvHR significantly when used at low doses, while high doses induced a decrease of GvHR intensity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in migration inhibitory factor production by C57Bl/6 and BALB/c mice in allergic and irritant contact dermatitis

Two strains of mice, BALB/c and C57Bl/6, which are known to differ in their inflammatory responsiveness to allergens, were analyzed regarding their expression of macrophage migration inhibitory factor (MIF). Allergic contact dermatitis to 2,4-dinitro-1-fluorobenzene and irritant contact dermatitis to croton oil were studied immunohistologically at designated time intervals after elicitation. BALB/c mice presented a significantly more intense ear swelling response than C57Bl/6 mice and showed a strong endothelial MIF expression in the early phase of inflammation in both allergic and irritant contact dermatitis. Endothelial MIF expression was much weaker in C57Bl/6 mice. Furthermore, the infiltration rate of inflammatory cells (MIF+ and BM8+ macrophages, Lyt2+ and L3T4+ T cells) was significantly higher in BALB/c than in C57Bl/6 mice. We conclude that genetically determined differences of susceptibility to allergens and irritants in BALB/c and C57Bl/6 mice are reflected by the intensity of MIF expression in the microvascular endothelium and immigrating inflammatory cells. MIF seems to appear as first molecular equivalent of developing inflammation and probably determines the degree of cellular infiltration.     

Development of the cellular immune response to tuberculin in mice of different genotypes

Inbred CBA and C57BL mice and (CBA×C57BL)F₁ hybrids were immunized intraperitoneally with Freund's complete adjuvant and the production of macrophagal migration inhibiting factor (MIF) by lymphocytes in different situations was investigated. Lymphocytes were activated to produce MIF in a certain order: cells of the peritoneal exudate first, then lymph node cells and, finally, spleen cells. Maximal MIF production by lymphocytes of the peritoneal exudate was obtained in C57BL mice, and it occurred earlier (on the 3rd day after immunization). Increased spontaneous migration of macrophages also was observed after immunization and it was more marked in the CBA mice.Department of Immunology, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. V. Lopukhin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 972–974, August, 1976.     

Demonstration in tissue culture of lymphocyte-mediated immunity to tuberculosis

We have shown that splenic lymphocytes from mice immunized with viable attenuated mycobacterial cells (H37Ra) or ribonucleic acid prepared from these cells will bring about inhibition of multiplication of virulent tubercle bacilli (H37Rv) within normal mouse peritoneal macrophages in tissue culture. Apparently these lymphocytes, when stimulated with viable virulent tubercle bacilli, elaborate a filterable substance(s) which is responsible for this intracellular growth inhibitory effect.     

Relationship of anti-tuberculous protection to lung granuloma produced by intravenous injection of synthetic 6-O-mycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine with or without specific antigens.

Intravenous administration of 6-O-mycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (mycol-MDP) together with a specific antigen, PPD, in a water-in-oil emulsion was found to produce lung granuloma and to provide a low but significant grade of protection in mice against tuberculous infection within 4 weeks. However, these products, when given in an oil-in-water emulsion did not produce granuloma. Mycol-MDP alone produced comparable lung granuloma in both C57Bl/6 mice, high responders to BCG cell walls (CW), and C3H/He mice, low responders, 1 week after the injection, and when challenged at this time by aerosol containing virulent bovine tubercle bacilli, they showed significantly increased resistance. The present results confirmed the close relationship between lung granuloma and protection against aerosol challenge with Ravenel and revealed that the extent of lung granuloma at the time of aerosol challenge is crucial for the development of protection in mice immunized with mycol-MDP plus PPD as it is in mice immunized with BCG CW. However, these findings are not always the case for lung granuloma induced with mycol-MDP alone.     

Modulation of Myelopoiesis In Vivo by Chemically Pure Preparations of Cell Wall Components from Gram-Negative Bacteria: Effects at Different Stages

Modulation of myelopoiesis by chemically pure preparations of different cell wall components from gram-negative bacteria was investigated in vivo. The effects of lipid A, outer membrane lipoprotein, and murein were evaluated at several distinct stages: induction of colony-stimulating activity (CSA) in the serum, increase in the number of committed splenic precursor cells (CFU-C) forming granulocyte-macrophage colonies in vitro, and triggering into the cell cycle of noncommitted hemopoietic stem cells (CFU-S) from bone marrow. The results reveal different patterns of activity of the bacterial cell wall components (BCWC) tested. (i) In C57Bl/6 mice and C3H/Bom mice, all three preparations were potent inducers of CSA. In C3H/HeJ mice, CSA was only induced by lipoprotein and murein and not by lipid A. After injection of lipid A or lipoprotein, but not murein, the number of CFU-C in spleens of C57Bl/6 mice was increased up to 100-fold. In C3H/Bom and C3H/HeJ mice, not only murein but also lipoprotein were much less potent in this respect. (iii) In C57Bl/6 mice, both lipid A and lipoprotein, but not murein, were capable of inducing the proliferation of CFU-S, as demonstrated by a hot thymidine cytocide technique. Thus, induction of CSA and changes in the pool size of splenic CFU-C after administration of BCWC may be unrelated events. On the other hand, the increase of CFU-C might reflect the mitogenicity of BCWC for CFU-S.     

Evidence for a Dual Effect of Acebutolol, a Beta Blocker, on the Mouse Humoral Immune Response

Acebutolol induces transient polyclonal B cell activation in C57B1/6 mice but down-modulates the spontaneous polyclonal activation of NZBxNZW lupus mice. The immunomodulatory effects of this beta-blocker were studied in C57B1/6 mice injected with LPS or immunized with sheep red blood cells. The effect of acebutolol on the polyclonal activation of lymphocytes induced by LPS was also investigated in heterozygous and nu/nu C57B1/6 mice. Finally, the direct effet of acebutolol on spleen cells was studied in vitro. Acebutolol treatment for 15 days (50mg/kg/day) inhibited the polyclonal activation of lymphocytes induced by LPS in C57B1/6 and in C57B1/6 nu/nu mice, but increased the humoral response to sheep red blood cells in C57B1/6 mice. Moreover, spleen cells from C57B1/6 mice treated for 15 days with acebutolol showed an increased number of CD5⁺ and CD4⁺ lymphocytes, as well as an increased reactivity to concanavalin A but not to LPS. In vitro, acebutolol at 10^-5 10^-7 M induced an increased reactivity of spleen cells from naive mice to concanavalin A, whereas it did not affect the B cell responsiveness to LPS. These results indicate that acebutolol modulates both T-cells and non T-cells in the immune system.     

Natural killer cell activity and macrophage-dependent inhibition of growth or killing of Mycobacterium avium complex in a mouse model

Natural killer (NK) cells from spleens of normal and Mycobacterium avium complex (MAC)-infected C57 black mice (C57 BL/6 bg/+) were examined for their capacity to activate splenic and peritoneal macrophages from beige mice to inhibit or kill intracellular MAC. Peritoneal and splenic macrophages from beige mice were exposed in vitro to NK cells obtained from MAC-infected and uninfected black mice. NK cells from uninfected black mice were also treated in vitro with recombinant interleukin-2 (IL-2) for 48 h before incubation with macrophages. While control macrophages supported intracellular growth of MAC, macrophages exposed to unactivated NK cells inhibited growth of the intracellular bacteria, as determined 4 days after infection. IL-2 stimulated NK cells, and NK cells obtained from MAC-infected animals were able to activate murine macrophages in vitro to inhibit growth or kill 40.0 +/- 5% and 61.3 +/- 6% of the intracellular bacteria, respectively. In other experiments, beige mice (C57 BL/6 bg/bg) were treated intraperitoneally with NK cells obtained from MAC-infected and uninfected C57 black mice. Peritoneal macrophages harvested from beige mice treated with NK cells activated in vitro with IL-2 killed 24.4 +/- 4% of intracellular bacteria by day 4 after infection. Macrophages obtained from animals treated with NK cells harvested from MAC-infected black mice killed 58.8 +/- 7% of intracellular bacteria by 4 days after infection, in contrast with intracellular growth observed in macrophages obtained from untreated animals and from animals treated with Hanks' solution or unactivated NK cells. These crossover studies suggest that NK cells may be important in host defense against MAC.     

实验性自身免疫性脑脊髓炎小鼠脾脏和肝脏中NKT细胞调变的研究

目的:观察NKT细胞在实验性自身免疫性脑脊髓炎(EAE)小鼠脾脏和肝脏中所占百分比的变化特点,探讨NKT细胞在EAE模型中的免疫调节作用.方法:以MOG35-5521肽诱导C57BL/6小鼠建立EAE模型并进行临床评分.于发病高峰期处死小鼠,分离脾脏和肝脏淋巴细胞,采用免疫荧光染色和流式细胞术(FCM)分析,观察EAE小鼠与正常小鼠脾脏和肝脏中NKT细胞在全部淋巴细胞中所占百分率的变化.结果:在EAE小鼠不同器官中,NKT细胞占淋巴细胞的百分率均较正常小鼠减少.脾脏NKT细胞百分率(%)从正常组2.22±0.14下降到EAE模型组1.94±0.07(P<0.05),肝脏NKT细胞百分率(%)从正常组5.52±2.17下降到2.67±1.41(P<0.05).结论:NKT细胞在EAE模型C57BL/6小鼠脾脏和肝脏中增殖受抑,提示EAE发病可能通过对NKT细胞数量的调节进而影响其对免疫应答的调节.     

H9/25 monoclonal antibody recognizes a new allospecificity of mouse lymphocyte subpopulations: Strain and tissue distribution

C3H/He-mg mice were immunized with C57 BL/10 (B10) spleen cells and the immune spleen cells were fused with BALB/c myeloma cells (NS1). One of the monoclonal antibodies (H9/25 antibody) produced by the hybrid cells was studied. It reacts with subpopulations of B 10 lymphocytes as well as some lymphoid tumor lines including some of the Abelson virus-induced leukemias. The antigen recognized by H 9/25 antibody is expressed on lymphocytes from all the B 10 congeneic mice tested as well as some other strains of mice. No linkage between genes coding for the antigen and H-2 loci was found as judged by its presence on cells of the B 10 strains regardless of H-2 type and the distribution of the antigen on Bailey recombinant inbred mice. The antigen is expressed on subpopulations of lymph node cells, spleen cells, thymocytes and bone marrow cells. The strain distribution of the H9/25 antigen seems to be identical to that of Ly-6, Ly-8 and Ala-1 antigens. However, the tissue distribution of the antigen recognized by H9/25 antibody, while similar to these alloantigens, is unique and the antigen may be distinct from the other alloantigens.