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The T cell response to allogeneic murine Mls determinants is not H-2 restricted but is dependent on H-2 class II molecules on the Mls-expressing stimulator cells. We have tested planar membranes containing H-2 class II I-E molecules alone or with I-A molecules for their ability to activate a panel of Mls1a-specific T hybrids. Despite the ability of the planar membranes to activate an alloreactive T hybrid and to present staphylococcal enterotoxins or an antigenic peptide to appropriately responsive T hybrids, they failed to stimulate the Mls1a-specific T hybrids. These findings, in the light of the various controls demonstrating sufficiency of the I-E molecules in the planar membranes, indicate that Mls1a determinants are not covalently bound to I-E molecules; the two molecular species are thus either not physically associated or are linked by a relatively weak interaction. In addition, our experiments show that isolated I-E molecules but not I-A molecules present staphylococcal enterotoxins A and B to two independently derived T hybrids expressing T cell receptor V beta 1, V beta 2 and V beta 6 elements.

TNF is a proinflammatory cytokine with opposing death/no-death effects in vivo and in vitro. Our studies showed that TNF regulates mouse thymocyte production, inducing both apoptosis and proliferation of the most immature CD3−CD4−CD8− triple negative (TN) subset within a broad range of dosages (101–105 pg/ml) in the presence of IL-7. TNF apoptosis affected only the TN3 (CD44−CD25+) and TN4 (CD44−CD25−) subsets that expressed both TNFR-p55 and -p75. Although each TNFR alone could mediate TNF apoptosis, maximal apoptosis was seen in C57BL/6J wild type, which expressed both TNFRs. TNF also induced proliferation of TN3 cells at higher doses (104–105 pg/ml) mediated only by TNFR-p75. Both anti-TNFR-p55 and -TNFR-p75 mAb inhibited apoptosis but only anti-p75 inhibited proliferation. TNF also regulated TN proliferation to IL-7 because TNFR knockout (KO), TNF KO, and TNF/lymphotoxin α and β triple KO mice showed 2- to 3-fold increased responses not seen in C57BL/6J wild type. In vivo, TNFR KO mice showed thymic hypertrophy with a 60% increase in total thymocytes, with no effect on the CD4/CD8 subsets. We conclude that TNF maintains homeostatic control of total thymocyte production by negative selection of TN3 and TN4 prothymocytes and down-regulation of their proliferation to endogenous IL-7.

Tumor necrosis factor (TNF), lyses a range of sensitive tumor targets and has been shown to be the mediator of natural cytotoxic (NC) activity first described in our laboratory. In this report, we identify two thymic lymphoma cell lines which lyse the prototype NC target WEHI-164 and share characteristics of NC. R1.1E and L5178-27av lyse the WEHI-164 sarcoma in 18-hr 51Cr release assays via a TNF-dependent, non-MHC-restricted (R1.1E) mechanism although they do not constitutively produce TNF. NC- and TNF-resistant variants of WEHI-164 are resistant to lymphoma-mediated lysis. Expression of the ganglioside GD3 by the lymphomas correlates with their relative levels of lysis. Thus, GD3, which is known to have a role in T cell activation may be involved in recognition or triggering for TNF-dependent cell-mediated lysis.

Natural cell-mediated cytotoxicity (NCMC), measured against a variety of tumors, is mediated by at least two sub-populations of effector cells: natural cytotoxic (NC) and natural killer (NK). The studies described in this report show that target lysis by NC cells requires a prolonged (18- to 24 h) assay period, whereas NC-susceptible targets can be lysed in short-term (4h) [3H]-proline assays using allo-sensitized CTL or mitogen-activated cytotoxic populations. NC cells are not "activated" during the long-term assay as indicated by: (1) demonstrating that lysis of NC-susceptible targets in long-term (20 h) 51Cr assays is still the function of a Qa-5- effector cell (NC) and (2) the fact that preincubation of the NC effector cell with susceptible targets for 18 h did not result in the activation of an NK-like population (kinetics of target lysis were comparable to those noted with fresh NC cell preparations). We show that NC cell activity is preserved in both the beige mutant and the PL/J mouse strains, both of which exhibit low NK cell activity, even in long-term assays. These combined studies support the view that NC and NK cell activities are the function of distinct cell types and not the property of a single cell class under different states of activation.

Culture of murine lymphoid cells without added antigen results in the generation of cells which suppress a variety of in vitro immune responses, such as the mixed lymphocyte response (MLR) and the generation of alloreactive cytotoxic T cells (CTL). The ontogeny of this phenomenon was studied. Cells which suppressed the MLR after preculture were isolated from spleens and hematopoietic livers of fetal and young (less than 1 wk old) mice. On the other hand, the generation of alloreactive CTL could be suppressed only by precultured spleen cells taken from 1-w-old or older mice. The parallel between the development of the suppressor functions and the maturation of the responses they regulate, suggests a possible biological significance of the effect.

The possibility of changes in immune reactivity during pregnancy was studied by measuring cellular immunity in vitro of the paraaortic (PA) lymph nodes, which drain the uterus in pregnant mice. The proliferation of PA lymph node cells from primiparous pregnant C57Bl/6J mice, in mixed lymphocyte cultures (MLC) against alloantigens, was lower in magnitude, but had the same kinetics, as the response of virgins. This was observed in syngeneic and allogeneic (to DBA/2J) pregnancies, and using the paternal as well as third party allogeneic stimulators. The response was depressed by day 8 of gestation and returned to normal two days after delivery. The decrease was not due to an active suppressor mechanism, as assayed by mixing experiments. Irradiation (1500R) of the lymphocytes from pregnant mice, prior to mixing with cells from virgins, did not reveal the presence of a radioresistant suppressor cell. In contrast to the MLC results, no differences were found between lymphocytes from pregnant and virgin mice in their ability to develop MLC-generated cell-mediated lympholysis (CML) against alloantigens, as measured in a chromium release assay. The PA lymph node cells from pregnant animals bearing allogeneic fetuses also did not show evidence of in vivo sensitization to the paternal alloantigens. Therefore, the local nodes draining the uterus from primiparous pregnant animals bearing syngeneic or allogeneic fetuses show a nonspecific decrease of MLC proliferation while retaining the capacity to generate normal CML activity.

Natural cell-mediated cytotoxicity (NCMC) capable of in vitro lysis of various lymphoid and non-lymphoid tumours has been described in mice and other species, including man. NCMC has been proposed as a first level of defence against tumour growth in vivo, one which does not need the priming of the conventional immunological response. The effector cells of NCMC seem to belong to a special category of lymphoid cells, being neither classical T or B cells nor macrophages; natural killer (NK) cells have been proposed as the prototype effector cell, although some heterogeneity among effector cells seems to exist, depending on the target cells used for testing. Two main subgroups of NCMC effector cells have been defined: NK cells directed against lymphoma targets and natural cytotoxic (NC) cells directed against solid non-lymphoid tumours. We describe here another distinction between the two systems: while NK activity is low in mice homozygous for the beige (bg) gene NC activity in spleen cell preparations from these animals is comparable with that observed in the appropriate controls (bg/+ and +/+ littermates). The bg syndrome of mice affects lysosome, melanosome and enzymatic functions and is a homologue of the Chediak--Higashi syndrome of man. Defective NK activity in blood lymphocytes has also been reported in patients with Chediak--Higashi syndrome. We also show that several mouse strains which have low NK activity, have normal or high levels of NC functions, expanding our previous observation that NC and NK cells are under distinct genetic control.

Lymphohemopoietic cells from normal mice show cell-mediated cytotoxicity (CMC) against syngeneic or allogeneic chemically induced fibrosarcomas, when tested in a 24-hr cytotoxicity assay with 3H-proline pre-labeled adherent target cells. When two BALB/c tumors, Meth A, and Meth 113 were studied, spleen cells from syngeneic normal donors showed high CMC against Meth A and no CMC toward Meth 113. However, Meth 113 was susceptible to natural CMC by spleen cells from other mouse strains or PHA-induced CMC by syngeneic spleen cells. Other solid tumors tested showed variable patterns of susceptibility-resistance to natural CMC, whereas normal fibroblasts were negative. Susceptibility to natural CMC does not correlate with expression of murine leukemia virus-related antigens. Established lines are more susceptible to natural CMC than secondary tumor cultures, although susceptibility-resistance appear as stable properties of the targets. All the mouse strains and sublines tested showed natural CMC against the Meth A target; however, only a few strains (A/J, CBA/HN, NZB, and C3H/He) showed natural CMC against Meth 113. Natural CMC against Meth 113 of (BALB/c × A/J) F1 spleen cells is dominant, and the hybrid behaves like the reactive A/J parent. Natural cytotoxic (NC) cells are detected in nude mice in a BALB/c or CBA/H background (and also in CBA/HN mice, which have a B cell defect). The tissue distribution of NC cells shows high activity in spleen and marrow, with lower activity in lymph nodes, peritoneal washings, and thymus. Peritoneal exudates induced by complete Freund's adjuvant or BCG show high NC cell activity. NC activity in spleen was detected from birth and does not seem to decay with age (even at 1 or 2 years of age). The described characteristics of the NC cell have some similarities (and some discrepancies) with the natural killer (NK) cell described against T lymphoma targets. The differences include: 1) strain distribution; 2) appearance at birth; 3) no decay with age, and 4) to some extent, tissue distribution (i.e. both systems show high activity in spleen; however, NC cells are detected in thymus whereas NK cells are not). The similarities include: 1) susceptibility not related to expression of murine leukemia virus antigens; 2) no H2 restriction of cytotoxicity; 3) activity present in nude mice (and CBA/HN); 4) reactivity dominant in F1 hybrids, and 5) increase activity in peritoneal exudates after BCG administration. It seems possible that these systems may act as anti-tumor surveillance devices.

Cytotoxic cells which lack major histocompatibility restriction for lysis are generated when murine spleen cells are cultured in the presence of FCS1 with or without allogeneic stimulation. The studies reported show that, following 3 days of culture with FCS, murine spleen cell cultures contain at least two cytotoxic populations. The first (AK-YAC), expresses an NK cell phenotype and target specificity while the second (AK-WEHI) shares the characteristics of the NC cell. Generation of the AK-YAC effector cell requires the presence of a pre-AK cytotoxic cell (Qa-5, LYT-2-plus;) and is dependent on the generation of interleukin-2 (IL-2) during the culture period, while the AK-WEHI effector is independent of IL-2 production. IL-2 production in the cultures is shown to require syngeneic la recognition by an Lyt-1+, L3T4+ T cell. These findings suggest a role for IL-2 in the in vivo regulation of NCMC and describe a mechanism for its production in the absence of antigenic stimulation.

The effects of monosaccharides on the cytotoxic activity of cytotoxic T lymphocytes (CTL) and three cloned long-term cytotoxic T-lymphocyte lines (CTLL) are compared. Uncultured CTL and clones CTLL-A2 and CTLL-A11 were derived from the peritoneal cavity of C57BL/6 mice immunized against the H-2D d determinants on the BALB/c sarcoma Meth A. Clone CTLL-R5 was derived from spleen of (BALB/c × C57BL/6)F 1 mice immunized against a unique determinant on the BALB/c radiation-induced leukemia RL♂1. The cell-surface phenotype of the clones is Lyt-1 + ,2 + ,3 + . Cytotoxic activity of CTLL-A2 and CTLL-R5 as determined by a 4-hr 51 Cr-release assay was inhibited over 50% by 1 m M 2-deoxy- d -glucose. CTLL-A11 and the uncultured cytotoxic T cells were more resistant to inhibition by 2DG (40% at 20 m M ). Surprisingly, it was found that the addition of d -mannose, d -galactose, d -glucose, l -fucose, α-methyl- d -mannose, and N -acetyl- d -glucosamine also inhibited, in a dose-related manner, the cytotoxicity of CTLL-A2 and CTLL-A11. CTLL-R5 showed a more restricted inhibition pattern: only d -mannose and d -galactose were inhibitory. The mechanism of inhibition remains to be clarified.

Ly-10.1 is a lymphocyte surface antigen controlled by a gene linked to the Ly-1.1 locus and expressed on activated T helper, T suppressor (Ts), and cytotoxic T lymphocytes (CTL). In this report, we describe the following: 1) Ly-10 is a heterodimeric glycoprotein consisiting of a 80 000 heavy and a 34 000 light chain. 2) Although mature CTL are Ly-10+ by negative selection with anti-Ly-10.1 and complement (C), CTL precursors reactive to allogeneic cells are Ly-10−. 3) Similarly, IL-2-producing effector T cells induced by MIs-incompatible cells and semiallogeneic stimulation are eliminated by anti-Ly-10.1 and C after activation but are not eliminated as precursors before activation. 4) In mixed lymphocyte culture with semiallogeneic cells, the frequency of Ly-10.1+ cells was highest on the 2nd to 5th day after stimulation, decreased by the 12th day, and increased after restimulation with fresh antigen as demonstrated by immunofluorescence, C-mediated lysis, and IL-2 production. 5) When spleen cells were treated with anti-Ly-10 and C before concanavalin A (Con A) activation, the suppressive activity in the Con A T blasts was reduced, suggesting that in normal mice, some Ts preexist in a Ly-10+ activated state. These results indicate that Ly-10 is a marker of activation of T cells, not expressed on precursor T cells and whose expression is both transient and dependent on the presence of antigen. The similarities in biochemical and cellular characteristics suggest that Ly-10 is a mouse homologue of the human lymphocyte activation marker 4F2.

The capacity to generate cytotoxic T cells (CTL) against allogeneic or hapten-modified syngeneic cells after in vitro sensitization was studied in C57BL/6J and CBA/H mice of different ages, beginning at 5 days of age. A differential maturation rate for allogeneic versus hapten-modified syngeneic CTL reactivity was observed in both strains, with alloreactivity appearing earlier than reactivity to hapten-modified self antigens. For example, in C57BL/6J mice, alloreactive CTLs were detected within the first week of life, reaching adult levels by the second-third week, while the response to modified-self appeared at the secon-third week and reached adult levels by the fourth-fifth week of life. Cross-reactive lysis of hapten-modified allogeneic targets followed the same maturation pattern as the modified-syngeneic response. The low CTL reactivity of 6-day-old spleens was not due to suppressor cells. Mixing Lyt-2− (Lyt-1+) and Lyt-2+ cells from 6-day-old and adult spleens showed that the main defect was in the T helper compartment. In addition, the capacity to produce interleukin-2 (IL-2) after mitogen or allogeneic stimulation was also defective in the young mice, appearing approximately by the second week of life and reaching adult levels by the fourth-fifth week. The IL-2-deficient production was the result of both T-cell immaturity (or low numbers) as well as macrophage-accessory cell deficiency. In summary, the inefficiency of CTL generation early during the postnatal development is complex and cannot be attributed to deficiencies of a single component. The present studies, however, cannot explain the reason for the earlier appearance of alloreactivity in ontogeny.

Tryptic peptide maps were prepared for four purified structural proteins derived from several murine leukemia viruses (MuLV's). Analyses of these peptide maps reveal that the p30 proteins of Rauscher, Moloney, and Gross MuLV's are very similar to each other, as are the p10's obtained from these three viruses. In contrast, the peptide maps of the individual p15's and p12's from the same viruses establish that each of these polypeptides is highly strain specific. For all four polypeptides studied, unique peptides appear in the Rauscher MuLV and Moloney MuLV tryptic profiles that are not present in the corresponding Gross MuLV profile. By this method of analysis it was possible to distinguish the p30's of N-tropic and B-tropic MuLV's derived from the same BALB/c mouse.

Cloned stocks of several murine leukemia viruses (MuLVs) were shown to be leukemogenic for susceptible mice after more than nine years of in vitro passaging in mouse embryo fibroblasts. Tissue culture-grown Rauscher (R-) MuLVs injected into newborn or young adult BALB/c mice induced lymphocytic leukemias in 100% of the animals beginning 80 days post-inoculation. No erythroblastic leukemia was observed even after passaging the tissue-culture-grown R-MuLVs twice through mice, indicating that the component responsible for that disease had been lost or attenuated during growth in fibroblasts. The tissue-culture-grown stock of Moloney (M-) MuLVs likewise induced lymphocytic leukemias in 94% of injected newborn BALB/c mice, and the tissue culture-grown Gross (G-) MuLVs induced lymphocytic leukemias in 42% of injected newborn C3Hf mice. The host range and neutralization characteristics of viruses recovered from animals that became leukemic after injection with the tissue-culture-maintained MuLVs were found to be identical with those of the injected viruses. These data implicate the injected MuLVs in the induction of the leukemias and suggest that the capacity to induce the disease is stably inherited as part of the viral genome even in the absence of expression. Activite Leucemogene Des Virus Murins De Type C Apres Passage A Long Terme in vitro Les auteurs ont montre que des stocks clones de plusieurs virus de la leucemie murine (MuLV) etaient leucemogenes pour les souris sensibles apres plus de neuf ans de passages in vitro dans des fibroblastes d'embryon de souris. Lorsque le MuLV souche Rauscher (R-) maintenu en culture de tissu a ete injecte a des nouveau-nees ou a de jeunes adultes BALB/c, il a induit des leucemies lymphocytaires chez 100% des animaux a partir de 80 jours apres l'inoculation. On n'a pas observe de leucemies erythroblastiques, měme apres deux passages de ce R-MuLV chez les souris, ce qui indique que l'element responsable de cette maladie a ete perdu ou s'est attenue pendant la multiplication dans les fibroblastes. Le stock de MuLV souche Moloney (M-) a egalement induit des leucemies lymphocytaires chez 94% des souris BALB/c nouveau-nees; quant au MuLV souche Gross (G-), il a induit des leucemies de ce type chez 42% des souris C3Hf nouveau-nees. On a constate que les hǒtes et les caracteristiques de la neutralisation des virus provenant des animaux devenus leucemiques apres injection des MuLV maintenus en culture de tissu sont identiques a ceux et a celles des virus injectes. Ces constatations indiquent que les MuLV injectes jouent un rǒle dans l'induction des leucemies et conduisent a penser que leur aptitude a induire cette maladie constitue un heritage stable qui fait partie du genome viral, et qui persiste měme en l'absence d'expression.

An autologous mixed lymphocyte reaction has been described in mice and humans in which T cells proliferate following stimulation with syngeneic non-T cells. This report demonstrates that in the murine syngeneic mixed lymphocyte reaction, the responder cell is an Ly1+, Ly23-, Qa-1-T cell. The stimulator is an Ia-bearing macrophage-like cell.

The ontogeny of interleukin 2 (IL 2) production in young CBA/HT6T6J mice was studied using both bulk culture and limiting dilution methods. The ability of spleen cells in bulk culture to produce IL 2 in response to concanavalin A (Con A) was found to rise through the first 2 weeks of life, from no production at day 1, through 20 units/ml at day 6, to 80-100 units/ml in adults. No evidence for suppression of IL 2 production by young spleen was found. Limiting dilution analysis of both young spleen and young lymph node (LN) shows that young spleen has a much lower complement of cells producing IL 2 in response to Con A or allogeneic stimulation than does adult spleen. The frequency of 6-day spleen cells producing IL 2 in response to Con A is 1/1000, while the adult frequency is approximately 1/50. Young LN, in contrast, has levels of IL 2-producing cells close to those of adult LN, with a frequency of responders to Con A of 1/20. No evidence was found for a deficiency in IL 2 production on a per cell basis, in either 6-day spleen or LN. In examining allogeneic reactivity, a high frequency of cells reacting to strong Mls stimulation was found in both young and adult spleen and LN.

T-cells develop as a consequence of intrathymic and postthymic events, in which hemopoietic progenitors are differentiated into precursors and effector cells. We are proposing that such process includes three integrated steps: (1) T-cell differentiation; (2) selection of the T-cell repertoire and (3) specialization into functional subsets of T-cells. Although there is evidence of specific and nonspecific humoral factors (i.e. thymic extracts, etc.) affecting T-cell differentiation, it is also proposed that the most critical component in this integrated process is the consequence of direct cell to cell interactions between precursor and inducer cells. Thus, the three processes are the consequence of the appropriate or condordant matching of precursor-inducer populations, both at intra- and extrathymic sites. It is also proposed that MHC determinants are critical in permitting the appropriate matching. The model can thus account for nonfunctional differentiation when the appropraite matching is not available and with intrathymic selection by excess cell production favoring the appropriate matching.

Natural cell-mediated cytotoxicity (NCMC) against a variety of tumor targets is mediated by a heterogeneous group of effector cells with the natural killer (NK) and natural cytotoxic (NC) cells being the predominant prototypes in mice. This report shows that non-lymphoid tumor targets, mostly derived from chemically induced fibrosarcomas, are susceptible to either (1) NK-mediated lysis with all the activity being the function of a poly-IC augmentable Qa-5+ effector cell; (2) NC-mediated lysis with all activity being the function of a Qa-5- cell not augmented by poly-IC; and (3) a combination of NK-and NC-mediated lysis with activity being the function of both Qa-5+ and Qa-5- cells, the NK (Qa-5+) augmented by poly-IC. These studies further support the view that murine NC and NK cells are distinct and collectively make up the NCMC system, and also that the previous association of NK cells with lymphoid tumor lysis and NC cells with non-lymphoid tumor lysis is not a valid one.

Murine natural killer (NK) and natural cytotoxic (NC) cells showed different patterns of augmentation of lytic activity after preincubation for 24 h with either poly-IC, interleukin 2 (IL-2), or interleukin 3 (IL-3): (a) Poly-IC augmented only NK cells, with no effect on NC activity, as we have previously observed (4); (b) IL-2 augmented both NK and NC activity; and (c) IL-3 augmented only NC lysis, without affecting NK activity. In addition, both precursor and the augmented effector cells showed differences in expression of the Qa-5 surface marker: NK precursors and effectors are Qa 5+, whereas NC precursors and effector cells are Qa-5-.

Nude (nu/nu) mice in a CBA/H background show an age-dependent susceptibility to tumor development after polyoma virus infection (strain LID-1) when compared with nu/+ or CBA/H mice, which is apparent when 15- or 30-day-old mice are used: tumor incidence was 83 to 90% in nudes and 0 to 10% in controls. Latent periods for tumor development were also shortened in nudes. However, with increasing age nude mice become partially resistant and only 25% develop tumors when infected at 120 days of age. This partial resistance could be transferred with spleen cells to newborn mice. The cells in spleen responsible for this transfer can be eliminated by lysis with anti-Ig and complement or by pre-treatment of the donor with 100 mg/kg of cyclophosphamide and were not affected by treatment in vitro with anti-Thy.1.2 or procedures that remove adherent cells and/or macrophages. When the cells in 15-day-old nu/+ spleen were studied, both anti-Ig or anti-Thy.1.2 treatment eliminated tranfer of resistance to newborn. Virus replication in tissues of nude mice was increased 5 days after infection when compared with nu/+ but became comparable by day 15 after infection. Hemagglutination-inhibition antibodies in serum of nude and nu/+ had comparable titers when measured early after infection but higher titers were observed in nu/+ later after infection.

Normal T and immune B lymphocytes interact in a fashion that leads to suppression of the immune response. Normal spleen cells added to cultures of primed spleen cells specifically suppressed both the IgM and IgG secondary antibody response of the primed cells to less than 30% of the response of the immune cells cultured alone. Cell crowding as a possible in vitro artifact was ruled out. The suppression was specific for the priming antigen, even when the specific and nonspecific antigens were included in the same cultures. Suppression required both normal T and immune B cells to be present in culture. We suggest that the immune population produces a signal that can induce normal T cells to become specific suppressor cells. This form of interaction may represent an important regulatory (homeostatic) mechanism in the immune system.

Feedback regulatory signals to an intermediary suppressor cell may provide the mechanism of the suppression of antibody responses which has been described in cocultures of primed (immune) and unprimed (normal) spleen cells. The active cell(s) in the unprimed spleen population is nonadherent to nylon wool, Sephadex G-10 and glass beads. Lymph node cells and cortisone-resistant thymocytes from normal animals act similarly to normal spleen cells in this coculture system. Spleen cells from homozygous nude mice, unlike their heterozygous thymus-bearing littermates, do not produce a high degree of suppression in coculture with immune spleen cells. These data strongly suggest that the normal cell which interacts with primed cells in the cocultures to produce suppression is of thymic origin. However, spleen cells from neonatally thymectomized mice are suppressive in the presence of spleen cells immune to sheep red blood cells. The unprimed cell(s) active in the suppression are sensitive (in the presence of complement) to antisera directed against the surface markers, Thy-1, Qa-1, Lyt-1, Lyt-2, and Ia. Most of the antiserum treatments abrogated the suppressive capacity of the normal spleen cells only partially. Only treatment with anti-Qa-1 and complement routinely eliminated the ability of these cells to suppress in the cocultures suggesting either low concentration of or inaccessible surface alloantigen on the active cells. Alternatively, more than one Qa-1-positive cell set from the unprimed population may be involved. It is postulated that there is at least one subset of Qa-1-positive T lymphocytes present in unprimed spleen and lymph node cell populations capable of participating in the suppression of specific secondary antibody responses when cocultured with spleen cells from specifically primed animals.

The following characteristics of the effector cells for natural cytotoxicity (NC) against BALB/c Meth A tumor cells in BALB/c and other mouse strains were observed: 1) time course studies showed that most of NC was completed by 12 to 24 hr of incubation; 2) pre-incubation at 37°C for as long as 6 days had no effect on NC activity; 3) pre-treatment with ammonium chloride had no effect on NC activity; 4) NC cells were not adherent to plastic surfaces; 5) NC cells showed some degree of adherence to nylon wool fibers and activity was recovered from the adherent population; 6) NC cells were partially retained by G10-Sephadex columns; 7) by using velocity sedimentation at unit gravity in Ficoll gradients, NC cells were homogeneous having an average sedimentation velocity of 4.3 to 4.5 mm/hr; 8) treatment with carbonyl iron and magnetism had no effect on NC activity; 9) treatment with various antisera + C including, anti-Thy 1, anti-Lyt 1, anti-Lyt 2, anti-Ala 1, anti-NK, rabbit anti-mouse brain, and rabbit anti-mouse Ig, had no effect on NC activity; 10) heat-aggregated Ig had no effect on NC-mediated CMC; 11) trypsin treatment decreased NC activity but the effect was reversible, dependent on trypsin dose, and detected only in short-term CMC assay. These properties were similar for NC cells obtained from normal or nude mice, as well as peritoneal exudates obtained 5 days after i.p. BCG. When compared to the properties of the NK cell, many similarities were evident although differences in sensitivity to pre-incubation at 37°C and in the relative degree of adherence to nylon fibers were found. It is proposed that both the NC and NK cells belong to a family of effector cells with similar functions.

Freshly obtained normal lymphoid cells kill certain tumor target cells in vitro. Using peripheral blood lymphocytes (PBLs) and the human tumor target cell line BT-20, we have defined a tumor necrosis factor (TNF)-dependent, cell-mediated cytotoxic mechanism that is homologous to the murine natural cytotoxic (NC) cell activity. Human NC cell activity was detected in freshly isolated PBLs and was augmented by short in vitro pulses with recombinant human interleukin-2 but not with recombinant human alfa interferon. Monoclonal anti-TNF antibodies inhibited the killing of the target cells. The independence of interferon and the mediation of killing by TNF distinguish human NC cell activity from natural killer and lymphokine-activated killer cell activities.

T cells (Ts) from BALB/c mice bearing 6-day-old syngeneic Meth A tumors can mediate suppression of the efferent limb of the delayed-type hypersensitivity (DTH) response elicited by immunization with irradiated Meth A cells as well as the augmented response obtained when the immunogen is inoculated into BCG-primed sites. Ts in the spleen are Lyt 1 − 23 + and bear I-J determinants. Their induction is thymus dependent. Hydrocortisone depletes the thymus of Ts but spares Ts in the spleen. Ts are metabolically active and dividing as evidenced by their sensitivity to low doses of cyclophosphamide (CY) (20 mg/kg body wt), a 15-hr pulse with vinblastine (Vbl), and irradiation with 600 rad or more. Ts adhere to Sephadex G-10 columns but are unaffected by treatment with carbonyl iron and magnetism. Their phenotype and mechanism of action appear to be comparable to those of Ts 2 cells defined in models of DTH to simple haptens.

Summary Reversal of post-thymectomy wasting in mice has been achieved by subcutaneous (s.c.) or intraperitoneal (i.p.) grafting of five thymuses from syngeneic, hemiallogeneic or allogeneic donors. The i.p. route was more effective in salvaging the wasted animals. In some instances a single i.p. graft proved to be effective. The reconstituted animals were able to reject skin homografts from third party strains. When syngeneic thymus donors were used, the spleen cells of restored animals had significant graft-vs.-host capability. When hemiallogeneic or allogeneic donors were used, the pattern of immunologic reconstitution varied with the strain combination and the site of thymus implants. If skin of the donor strain was rejected, the thymus grafts were usually not viable at autopsy, and the graft-vs.-host reactivity was appreciable and of host type. When skin of the donor strain was accepted, viable thymus grafts were noted at autopsy, and graft-vs.-host reactivity of host type was significant but reduced, presumably a reflection of the chimeric state. In certain allogeneic combinations, both donor and host components were detected in discriminating spleen assays.

SUMMARY A parent to F1 hybrid mouse graft-versus-host assay was used to measure the inhibiting effects of parent antihybrid sera on the reaction. Either no influence or complete inhibition of the graft-versus-host reaction could be achieved by varying the dosage of antibody used. Specific antibody of IgG2a or IgG3 class produced the maximum inhibitory activity. A minimal accentuation of the reaction was obtained with IgM antibodies.

The fetal thymus at 13 days of gestation withstands transplantation and develops normally under the renal capsule of a syngenic host. Distinct differences were observed between the fetal thymus grafts and grafts from neonatal or adult thymus donors. The fetal thymus graft did not undergo the rapid and severe necrosis observed when adult thymus was grafted. Furthermore, when thymuses were transplanted into allogenic recipients, rejection was delayed. The fetal thymus was as effective as the adult thymus in restoring syngenic neonatally thymectomized mice and far superior to adult thymus when grafted into allogenic recipients. These observations seem relevant to clinical efforts to restore immunocompetence in patients with congenital absence of the thymus.

The immune functions of neonatally thymectomized C3Hf mice exposed only temporarily to thymus function show a progressive decay with time in the absence of the thymus. The immune responses studied at different ages in the range of 100–600 days were: first-set rejection of H-2-compatible and incompatible skin allografts, second-set rejection of skin allografts, capacity of spleen cells to produce graft-versus-host reactions in F1 hybrids, resistance to infection with mouse hepatitis virus, and response of spleen cells to phytohemagglutinin in vitro. These long-term studies had the purpose of determining the duration of the restoration induced by thymus function when the mice were exposed only temporarily to it. Different models were used but the two basic ones were: (a) mice grafted intraperitoneally at 15 days of age with a syngeneic thymus that was removed surgically at 10, 20, or 30 days after grafting, and (b) mice grafted at 15 days of age with allogeneic strain A thymoma or C57BL thymus, these representing situations in which there is spontaneous rejection of the restoring graft. In all the experimental models used, the animals were restored when tested at 100 days of age, but progressively became immunologically incapacitated at 200–300 days of age. From the more controlled experiments in which the restoring thymus graft was removed surgically, the following conclusions can be drawn. (a) A short exposure to a thymus graft can produce restoration of immune functions in neonatally thymectomized mice, but this restoration is not self-sustaining in the absence of the thymus and declines progressively with age. The decline usually starts at 200–300 days of age. (b) This was especially clear in experiments in which the same animal was tested twice in its lifetime for capacity to produce graft-versus-host reactions; these animals were competent at 100 days and became incompetent at 400 days of age. (c) The shortest period of thymic exposure studied was 10 days; if vascularization of the graft is taken into account, 2–3 days of thymic function are sufficient to produce restoration. (d) The immune decay observed in the thymectomized animals exposed temporarily to thymus was more profound than the physiological decay of immunity observed in control animals of similar age. (e) Of all the tests studied, the response of spleen cells to phytohemagglutinin was to be preserved the longest in animals exposed only temporarily to thymic function. The present results were interpreted in accordance with our previous findings indicating that a population of postthymic cells can be developed by temporary exposure of neonatally thymectomized animals to thymic function, but that this population is not self-sustaining in the absence of thymus and progressively decays by physiological attrition.

Antibodies against cellular antigens appear during aging in some strains of mice but not others. In susceptible strains, manifestations of autoimmunity include development of Coombs positive hemolytic anemia, circulating anti-DNA and anti-desoxynucleoprotein antibodies, positive LE cells, and immunologically based renal and cardiac lesions. Neonatal thymectomy may accelerate development of these autoimmune phenomena. The autoimmune susceptible strains are prone to lose immune capacities (particularly cell-mediated immune responses) with aging. Thymectomy accelerates the development of immune deficiency and autoimmunity. Reconstitution with a sufficient number of postthymic immune competent cells can reverse not only the evidence of immune deficiency, but also wasting and autoimmune phenomena. In some strains not susceptible to development of autoimmunity with aging, there is increased evidence of autoimmunity of lesser degree and frequency than that observed in autoimmune strains after thymectomy. These findings suggest that involution of the thymic system is related to autoimmunity and that genetic factors are involved in the involution of the thymus dependent system. This hypothesis is supported by experiments in which spleen cells from old mice of autoimmune susceptible strains have decreased ability to reconstitute immunologically neonatally thymectomized mice; spleen cells from old mice of autoimmune nonsusceptible strains still can reconstitute neonatally thymectomized mice.

A progressive decrease of the restoring effectivity of syngeneic or allogeneic thymus and functional thymoma grafts was observed when the treatment of neonatally thymectomized mice was delayed. Early treatment (5–20 days postthymectomy) was effective, while the number of restored animals was markedly decreased after late treatment (30–50 days postthymectomy). Similar results were obtained with subcutaneous or intraperitoneal thymus grafts and with thymus grafts within cell-impenetrable diffusion chambers. After the onset of the postthymectomy-wasting syndrome the only successful treatment was the implantation of multiple thymus grafts. On the other hand, single thymus grafts, thymoma grafts, or thymus or thymoma within diffusion chambers were ineffective. When spleen cells from 5-day old or 45-day old neonatally thymectomized animals were given in association with thymoma grafts, only the cells derived from the 5-day old thymectomized mice proved effective in restoring wasted thymectomized hosts. These results suggest that a population of cells sensitive to the action of the thymus decreases progressively with time in the absence of thymic function.

It has been proposed that the murine cell surface antigen Lyt 2 could serve to distinguish cytotoxic T lymphocytes (CTLs) and their immediate precursors, which bear Lyt 2, from helper T cells, which do not1. Later work, however, has demonstrated that Lyt 2+ cells can ‘help’ B cells mature into antibody-secreting cells, provided that the B cells differ from the allo-helper T cells at the K or D region of the H–2 major histocompatibility complex (MHC)2. Furthermore, a T-cell line has recently been described which is Lyt 2− but specifically cytotoxic for H–2I region determinants3,4. These new findings have prompted an alternative hypothesis, that Lyt 2 antigen does not discriminate between cytotoxic and non-cytotoxic (helper) T cells, but rather between I-region- and K/D-region-specific cells5. In this new view, the apparent association between presence of Lyt 2 antigen and killer T-cell function merely reflects the tendency, by no means absolute6–8, for CTLs to recognize K- or D- rather than I-region determinants9. We show here that I-region-specific murine CTLs, generated in primary in vitro culture, do in fact bear the Lyt 2 antigen, as do CTLs directed against the K region of H–2.

Athymic-nude (nu/nu) mice and normal (nu/+) mice showed no differences in either latent period or incidence of local sarcomas or lung adenomas within 120 days after administration of 3-methylcholanthrene at birth. However, nu/nu mice were incapable of rejecting allogeneic skin grafts for the duration of the experiment. These results argue against an active role of thymus-dependent immunity as a surveillance mechanism preventing tumor development.

Long-lived, immunologically vigorous (C3H f × A f )F 1 hybrids were produced after lethal irradiation by administration of spleen cells from C3H f or syngeneic donors. Further, neonatally thymectomized C3H f or A f strain donors reconstituted irradiated C3H f or (C3H f × A f )F 1 hosts. In addition, C3H f spleen cells from nonthymectomized 10- to 15-day-old donors protected irradiated hybrid mice, but A f cells of young mice as well as of older mice produced graft-versus-host reaction and early death in irradiated C3H f or (C3H f × A f )F 1 hybrids. Abrogation of secondary disease by treatment of irradiated mice with spleen cells from allogeneic neonatally thymectomized mice is possibly attributable to diminished immunologic competence of the cells grafted, followed by the development of immunological tolerance of the donor cells. Donor cells, receiving thymus influence in the recipient host after transplantation, could explain the long-lived immunologically vigorous radiation chimeras that did not experience graft-versus-host reactions. The findings of this study help to understand the differential susceptibility of A f and C3H f mice to development of tolerance to one another's antigens observed in prior investigations. It appears that, in these mice, the host thymus influences the maturation of the spleen cells from young mice or from neonatally thymectomized mice. However, this influence was often greater in mice given 767 rads than in those given 1046 rads. This differential influence is possibly attributable to irradiation damage to the thymus produced by the higher dose of irradiation. Spleen cells from neonatally thymectomized mice can be differentiated and expanded by the thymus of the host. The differential susceptibility of T 1 , early differentiation stages, of thymus-dependent lymphocytes and T 2 , late differentiation stages, of thymus-dependent lymphocytes to tolerance induction and immunostimulation, respectively, are proposed as the bases for these otherwise paradoxical influences.

The in vitro generation of cytotoxic T lymphocytes (CTL) specific for 2, 4, 6-trinitrophenyl (TNP)-modified syngeneic spleen cells is found to be almost invariably depressed in apparently healthy 18-month-old mice of the long-lived (BALB/c × C57BL/6)F1 hybrid strain. Studies of CTL production from Lyt-2+ thymus cells have suggested that pre-killer cells may require, for maturation into effectors, the presence of a soluble helper factor, interleukin 2 (IL2), produced by Lyt-2− cells which are themselves devoid of pre-CTL activity. We have therefore developed a petri-dish adherence technique for separating spleen cells into Lyt-2+ and Lyt-2− populations in order to test for helper and pre-killer activity independently. Pre-CTL function is measured by stimulating Lyt-2+ cells in the presence of exogenous IL2. Helper cell activity is tested by adding Lyt-2− cells to “indicator” populations of Lyt-2+ pre-CTL. Estimation of IL2 levels in medium conditioned by unfractionated, TNP-self-stimulated splenocytes provides a second measurement of helper cell function. Mice 18 months of age, when compared to 4 month-old controls, are found to retain nearly all of their pre-CTL activity, but to have lost sufficient helper cell activity to account for the decline in unseparated spleen cell cultures. Older mice also produce lower IL2 levels.

Natural cytotoxic (NC) and natural killer (NK) cells have been defined by their ability to lyse certain solid or lymphoid tumor targets in vitro, without prior sensitization. Our present studies describe an attempt to characterize the structures involved in the effector-target recognition leading to tumor cell lysis. Addition of the monosaccharide D-mannose to the NC cell assay significantly blocked cytotoxicity of the fibrosarcoma Meth A target by the effector cells at 50 mM and lower concentrations. D-Galactose showed blocking activity in one of five experiments, only at 50 mM. L-Fucose, D-glucose, and N-acetyl-D-glucosamine did not affect NC cell cytotoxicity at similar concentrations. All of the sugars tested inhibited NK cell lysis of the lymphoma YAC-I target. None of the sugars affected killing of the appropriate target by allosensitized cytotoxic T lymphocytes. The blocking of NC-mediated cytotoxicity was not due to a direct toxic action of the sugars on the effector cells. These findings suggest that, in the NC system, recognition involves lectin-like structures with a specificity for D-mannose (or D-galactose, or both), whereas, in the NK system, such lectin-like structures are less restricted. Such structures appear not to be involved in the specific cytotoxicity mediated by T cells.

CELL-MEDIATED CYTOTOXICITY (CMC) against C3H/Umc (C3H) syngeneic mammary tumours measured in vitro, after in vivo immunisation, is mediated by cytotoxic T lymphocytes (CTL) and is directed mostly against cross-reacting antigens related to the mouse mammary tumour virus (MTV)1–3. The long-term CMC assay required for detecting this response against adherent target cells has complex kinetics and at least two superimposing components: (1) an initial early peak of cytotoxicity, detectable at approximately 6 h and accounting for 20–30% of the CMC, and (2) a later peak, detectable after 18–20 h and representing an additional 40–50% of the cytotoxic effect3. Both peaks are mediated by CTL with the Ly23 phenotype (that is, Ly1−Ly2,3+); however, non-cytotoxic T cells of Ly1 and probably Ly123 phenotype (that is, Ly1+Ly2,3− and Ly1+Ly2,3+, see refs 3 and 4 for nomenclature and functional characteristics of these T-cell subsets) are required for the expression of the second cytotoxic peak3. On the other hand, the short-term assays of CTL-mediated CMC show single-hit kinetics5, perhaps comparable to part of the early CMC peak in our system. During our studies of immunity to syngeneic mammary tumours, we observed that when C3H or C3Hf immune CTL were tested against allogeneic MTV-induced mammary tumour targets, a relative degree of H–2 restriction of CMC was observed2, although not of the magnitude detected in other systems using mostly short-term CMC assays6–11. The experiments reported here show that when the kinetics of the CMC response against adherent syngeneic and allogeneic mammary tumour targets were compared, some differences became apparent: whereas the early CMC peak showed absolute H–2 restriction, no restriction was observed in the late CMC peak, although both events were mediated by Ly23 CTL (ref. 3).