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22 pags., 8 figs., 1 tab., Massive stars disrupt their natal molecular cloud material through radiative and mechanical feedback processes. These processes have profound effects on the evolution of interstellar matter in our Galaxy and throughout the universe, from the era of vigorous star formation at redshifts of 1-3 to the present day. The dominant feedback processes can be probed by observations of the Photo-Dissociation Regions (PDRs) where the far-ultraviolet photons of massive stars create warm regions of gas and dust in the neutral atomic and molecular gas. PDR emission provides a unique tool to study in detail the physical and chemical processes that are relevant for most of the mass in inter-and circumstellar media including diffuse clouds, proto-planetary disks, and molecular cloud surfaces, globules, planetary nebulae, and star-forming regions. PDR emission dominates the infrared (IR) spectra of star-forming galaxies. Most of the Galactic and extragalactic observations obtained with the James Webb Space Telescope (JWST) will therefore arise in PDR emission. In this paper we present an Early Release Science program using the MIRI, NIRSpec, and NIRCam instruments dedicated to the observations of an emblematic and nearby PDR: the Orion Bar. These early JWST observations will provide template data sets designed to identify key PDR characteristics in JWST observations. These data will serve to benchmark PDR models and extend them into the JWST era. We also present the Science-Enabling products that we will provide to the community. These template data sets and Science-Enabling products will guide the preparation of future proposals on star-forming regions in our Galaxy and beyond and will facilitate data analysis and interpretation of forthcoming JWST observations., Support for JWST-ERS program ID 1288 was provided through grants from the STScI under NASA contract NAS5-03127 to STScI (K.G., D.V.D.P., M.R.), Univ. of Maryland (M.W., M.P.), Univ. of Michigan (E.B., F.A.), and Univ. of Toledo (T.S.-Y.L.). O.B. and E.H. are supported by the Programme National “Physique et Chimie du Milieu Interstellaire” (PCMI) of CNRS/INSU with INC/INP co-funded by CEA and CNES, and through APR grants 6315 and 6410 provided by CNES. E. P. and J.C. acknowledge support from the National Science and Engineering Council of Canada (NSERC) Discovery Grant program (RGPIN-2020-06434 and RGPIN-2021-04197 respectively). E.P. acknowledges support from a Western Strategic Support Accelerator Grant (ROLA ID 0000050636). J.R.G. and S.C. thank the Spanish MCINN for funding support under grant PID2019-106110GB-I00. Work by M.R. and Y.O. is carried out within the Collaborative Research Centre 956, subproject C1, funded by the Deutsche Forschungsgemeinschaft (DFG)—project ID 184018867. T.O. acknowledges support from JSPS Bilateral Program, grant No. 120219939. M.P. and M.W. acknowledge support from NASA Astrophysics Data Analysis Program award #80NSSC19K0573. C.B. is grateful for an appointment at NASA Ames Research Center through the San José State University Research Foundation (NNX17AJ88A) and acknowledges support from the Internal Scientist Funding Model (ISFM) Directed Work Package at NASA Ames titled: “Laboratory Astrophysics—The NASA Ames PAH IR Spectroscopic Database.”

The 27 moons of Uranus (Figure 1) are enigmatic and remain poorly understood. Voyager 2 flew by the Uranus system in 1986, collecting fascinating images of its five largest, tidally-locked ‘classical’ moons (Figure 2), while also discovering a bevy of small moons nestled in its ring system (e.g., [1]) (Figure 3). The surfaces of Uranus’ classical moons Miranda, Ariel, Umbriel, Titania, and Oberon have been modified by endogenic activity, in particular Miranda and Ariel, which exhibit substantial evidence for geologic communication between their interiors and surfaces (e.g., [1-3]) (Figure 2). The available images therefore indicate that these classical moons are candidate ocean worlds, which have, or had, liquid H2O layers beneath their icy exteriors (e.g., [3-5]). Because the Voyager 2 flyby occurred near Uranus’ southern summer solstice (subsolar latitude ~81°S), the collected images are centered near the south poles of these moons, and their northern hemispheres were largely unobservable. Furthermore, only the classical moons and the largest ring moon Puck (Figure 3) were spatially resolved by Voyager 2. The other nine ring moons Cordelia, Ophelia, Bianca, Cressida, Desdemona, Juliet, Portia, Rosalina, and Belinda were not resolved. Another ring moon, Perdita, was discovered via reanalysis of Voyager 2 data [6], and two more ring moons, Cupid and Mab [7,8], were discovered by space-based telescope observations. All nine known irregular satellites, Francisco, Caliban, Stephano, Trinculo, Sycorax, Margaret, Prospero, Setebos, and Ferdinand, were not detected by Voyager 2 and were discovered later by ground-based observations (e.g., [9-11]). Voyager 2 was not equipped with a near-infrared (NIR) mapping spectrometer, and most of what we know about the compositions of Uranus’ moons has been determined using data collected by ground and space-based telescopes. The surfaces of Uranus’ classical moons are composed of H2O ice mixed with low albedo material that could be rich in organics and silicate minerals (e.g., [12-14]). Carbon dioxide (CO2) has been detected on the classical moons, primarily on their trailing hemispheres, in particular on Ariel [15,16] (Figure 4). Spectrally red material that could be rich in organics has been detected, primarily on the leading hemispheres of these moons (e.g., [17,18]) (Figure 4). Ammonia (NH3) has possibly been detected on the classical moons and may originate from their interiors [18,19]. Although useful, these prior observations are disk-integrated, limiting our ability to constrain the distribution of surface constituents and identify links between volatile species and geologic terrains. Much less is known about the surface compositions of Uranus’ 13 ring moons and nine irregular satellites, which are mostly too faint (Vmag 19.8 - 25.8) for spectroscopic analysis using existing facilities. Spectrophotometric datasets indicate that Uranus’ ring moons have dark surfaces that show hints of H2O ice features [6]. Uranus’ irregular satellites have dark, reddish surfaces (e.g., [20]) but little else is known about their surface compositions, except for Sycorax, which shows hints of H2O ice [21]. An orbiting spacecraft collecting data during close flybys of Uranus’ ring system and classical moons would reveal the surface geologies of these moons, including on their previously unobserved northern hemispheres, determine their surface compositions, and determine whether any of the classical moons are, or were, ocean worlds. Furthermore, an orbiter could spend time looking outward to characterize Uranus’ irregular satellites, providing new insight into these likely captured objects (e.g., Jewitt & Haghighipour 2007). By utilizing a Jupiter gravity assist (2030 - 2034 launch window), a mission could arrive at the Uranian system in the mid 2040’s (∼11 years flight time), using existing chemical propulsion technology [22]. This arrival time frame would allow us to observe these moons’ northern hemispheres. An orbiter making close flybys of the classical moons could search for evidence of ongoing geologic activity and characterize migration of CO2 in response to changes in subsolar heating as the Uranian system transitions into southern spring in 2050. To determine whether liquid H2O layers are present in the interiors of the classical moons, the highest priority instrument onboard an orbiter would be a magnetometer, which could detect and characterize induced magnetic fields emanating from briny subsurface oceans. Visible (VIS, 0.4 - 0.7 µm) and mid-infrared (MIR, 5 - 250 µm) cameras would also be vital to search for plume activity, hot spots, and other signs of geologic communication between the interiors and surfaces of these moons. A spectrometer (0.4 - 5 µm) would be critical for characterizing volatile species that might result from outgassing of material or recently exposed or emplaced surface deposits. The abundant evidence for geologic activity in the recent past on Ariel and Miranda likely makes them the highest priority targets for any mission that aims to characterize Uranus’ satellites. References: [1] Smith, B. A. et al. 1986, Science, 233, 43. [2] Schenk, P. M. 1991, JGR: Solid Earth, 96, 1887. [3] Beddingfield, C. B. & Cartwright, R. J. 2020, Icarus, 113687. [4] Hendrix, A. R. et al. 2019, Astrobiology, 19, 1. [5] Cartwright, R.J. et al. 2021. arXiv preprint arXiv:2105.01164. [6] Karkoschka, E. 2001, Icarus, 151, 51. [7] Showalter, M. R. & Lissauer, J. J. 2006, Science, 311, 973. [8] De Pater, I. et al. 2006, Science, 312, 92. [9] Gladman, B. J. et al. 1998, Nature, 392, 897. [10] Kavelaars, J. et al. 2004, Icarus, 169, 474. [11] Sheppard, S. S. et al. 2005, AJ, 129, 518. [12] Cruikshank, D. et al. 1977, AJ, 217, 1006. [13] Clark, R. N. & Lucey, P. G. 1984, JGR: Solid Earth, 89, 6341. [14] Brown, R. H. & Clark, R. N. 1984, Icarus, 58, 288. [15] Grundy, W. et al. 2006, Icarus, 184, 543. [16] Cartwright, R. J. et al. 2015, Icarus, 257, 428. [17] Buratti, B. J. & Mosher, J. A. 1991, Icarus, 90, 1. [18] Cartwright, R. J. et al. 2018, Icarus, 314, 210. [19] Cartwright, R. J. et al. 2020c, ApJL, 898, L22. [20] Maris, M. et al. 2007, A&A, 472, 311. [21] Romon, J. et al. 2001, A&A, 376, 310. [22] Hofstadter, M. et al. 2019, Planetary and Space Science, 177, 104680.

Up until 1980 the consensus answer to this question was no 1-4. Since 1983 the consensus answer from at least 4 separate experimental approaches is yes s-8. Most recently Desquenne-Clarke et al. 5 approached the question in tolerant rats and mice in which neonatal injections of lymphoid cells or bone marrow cells had induced the tolerant state. The question is fundamentally important, illustrating as it does the principle of symmetry between tolerance and immunity. It has been known since the fine experiments of Mike Bevan 9 that T-cell killing of targets expressing minor transplantation antigens is restricted by the MHC of the target and it is now well accepted that the T-cell receptor binds to determinants resulting from an association between the minor antigens and the class I MHC molecules on the target membrane. The receptor binds neither the minor antigen itself nor the MHC itself but only the associative complex. Is this also true of the interactions required for tolerance induction; i.e. does the sequence of events required for tolerance inductions require similar T-cell receptors? The answer to this question has important consequences for any putative mechanism of tolerance including self tolerance. Desquenne-Clark's experiments s were suggested by a reinterpretation of the results of previous experiments on the skin specific minor transplantation antigen (Skn). The observation was, that although both lymph node/spleen cell inocula.and bone marrow inocula from F~ hybrids would induce tolerance, as measured by persistent chimerism, only bone marrow cells procured the survival of skin grafts. This was originally interpreted as showing that bone marrow cells carry the Skn antigen whereas lymph node/spleen cells do not. These authors wondered whether what it really showed was a difference in the MHC restriction of bone marrow compared with mature lymphoid cells. Thus, bone marrow cells contain very few mature T cells but many T-cell precursors. These precursors, together with the host animal's own T-cell precursors, are processed in the host's thymus and their repertoire 'bent' to be MHC restricted 'to the host

We show that extremely fragile biomolecules such as DNA restriction and modifying enzymes can be dried in vitro in the presence of trehalose with no loss of activity, even after prolonged storage. A remarkable and unexpected property of the dried enzyme preparations is their ability to withstand prolonged exposure to temperatures as high as +70 degrees C. This stability is unique to trehalose and is not found with other sugars irrespective of their physical or chemical properties. The immediate significance of these observations is the ability to convert enzymes used in molecular biology into stable reagents. The indefinite stability and high temperature tolerance of these dried enzymes should permit the design of convenient formats that may be of particular significance in the automation of genome mapping and sequencing projects. The stabilization of a wide range of biomolecules by trehalose also has practical implications for a number of areas ranging from basic science, through healthcare and agriculture, to bio-electronics.

A new method of thymus transplantation is introduced, in which the graft is directly connected with the recipient's vascular system. This procedure was used both in euthymic rats and congenitally athymic nude rats. At all tested intervals after transplantation thymus grafts hardly differed from the recipient's own thymus in immunohistology and lymphocyte yield. In athymic nude rats, T cell-dependent immunity, tested by mitogen- and alloantigen-induced T cell responses, as well as by antibody production and delayed-type hypersensitivity after ovalbumin administration, showed that vascular thymus grafts could generate T cell functions to euthymic control levels. We conclude that the technique of vascular thymus transplantation represents a valuable tool, either in fundamental research on thymus function, or for the purpose of immune (re)constitution.

Trehalose, a non-toxic disaccharide of glucose, is responsible for the remarkable survival of a group of inconspicuous and little known desert organisms, called cryptobionts, which can desiccate totally during drought and yet recover completely when rehydrated. Our recent work has shown that this protection against desiccation damage can be reproduced in vitro when biomolecules are dried in the presence of this simple sugar. Trehalose drying technology has been utilised in a number of health care, pharmaceutical and biotechnological applications, where it has enabled the development of novel, robust and versatile formats.

Interactions between mouse and rat monoclonal antibodies (MAbs) in EIA have been detected using panels of mouse and rat MAbs of various isotypes. Mouse and rat antibodies of the IgG2a subclass were found to bind to each other most strongly. Immobilised IgG1 antibodies of the two species showed much less interaction. Rat IgG2b and IgG2c were intermediate in binding activity. We were unable to study examples of mouse IgG2b and IgG3 MAbs. By making use of the κ light chain allotype in the rat we were also able to show intraspecies interactions of rat MAbs with the IgG in rat serum. The molecular basis of these interactions has not been resolved but may involve the extended hinge region and flexible Fab arms of the IgG2a subclass of Ig permitting strong inter domain bonding between the constant region domains of adjacent molecules of this isotype.

We have identified an antigen present on the surface of lymphocytes in the rat which appears to play an important role in the preliminary stages of the immune response. This antigen, which we have called quiescent cell antigen 1 because of its apparent expression only on quiescent cells, is present on the majority of peripheral T and B cells and a small percentage of thymocytes which are located mainly in the medullary region. SDS-PAGE analysis of membrane molecules shows two bands on unreduced gels at approximately 43 and 47 kd. On reduction the bands ran at approximately 46 and 60 kd. When a monoclonal antibody against this antigen (HIS45) is present in an allogeneic mixed leukocyte reaction, it inhibits the proliferation of responding cells completely. When the antibody HIS45 is added to cytotoxic T lymphocyte mediated lysis assays it does not inhibit lysis nor does it affect the specificity of this lysis. Comparison with other antibodies which have been reported to affect lymphocyte function in rats and in other species fail to reveal any which have similar properties.

A monoclonal antibody-derived, dry blood grouping plate, based upon the simple disaccharide, trehalose, is described that is indefinitely stable at room temperature and was found to have a 99.8% accuracy when tested against a standard semiautomated assay. This plate can be used by personnel with no specific training to check the recorded A,B,O, and Rhesus blood type of potential transfusion recipients in the field and at the bedside. Trehalose-based reagents may be important for bedside testing and in developing countries where refrigeration is unreliable.

During treatment of heart-grafted rats with cyclosporine, an unusual large lymphocyte appears in the blood. These cells constitute up to 40% of the peripheral blood leukocyte population and carry both the T helper/DTH and T cytotoxic/suppressor differentiation antigens. They require both the allograft and CsA for their generation and are not recently thymus-derived. They gradually disappear after stopping CsA treatment, although the treated rats remain tolerant of the graft.

Orthotopic liver allografts in the rat survive indefinitely without immunosuppressive agents, despite incompatibility between donor and recipient for antigens of the major histocompatibility complex. This is strain-dependent. In the DA to PVG strain combination, liver grafts are never rejected. This is net due to failure of the recipient to mount an immune response against the donor tissue, because there is unequivocal histological evidence of a rejection response during the first few weeks after grafting. This response is moderate and disappears to leave a histologically normal liver, apart from mild bile duct proliferation. Liver function tests show evidence of damage during the phase of cellular infiltration, but these test results also return to normal levels within a few weeks. In the DA to BN strain combination liver grafts are rapidly rejected, and this process is accompanied by histological signs of a violent and progressive destructive cellular response with gross alterations in liver function test results that are progressive until the death of the recipients. F1 hybrid recipients between these two strains (BN x PVG)F1 show intermediate levels of both histological damage and elevated liver function values, but they do not reject their grafts. Recovery from the rejection episode appears to be complete, as judged histologically. However, biochemical values remain slightly elevated, indicating either that the original damage was so severe that it was inconsistent with complete functional recovery or that there is continuous damage that is not visible histologically.

T lymphocyte hybrid lines have been produced by fusion of the thymoma BW 5147 with spleen cells of C57BL/10 mice primed to sheep red blood cells (SRBC). The supernatant (culture fluid) of a T hybrid designated A 1 was able to suppress the primary (IgM) and secondary (IgM and IgG) antibody responses to SRBC in vitro. The suppressive activity of supernatants could be titrated to 50% end points at final dilutions of up to 1 : 270. The suppression affected only SRBC and haptens coupled to SRBC, except when used at high concentration when some nonspecific suppression was observed. Absorption of the A 1 supernatant with SRBC removed all activity, while several other species of red cells failed to do so. The suppressor factor present in A 1 supernatant was not removed by anti-Ig adsorbents, but was removed by anti-H-2 antibodies reacting specifically with the haplotype of the spleen cells used in the fusion (H-2b). The cellular target of action of the factor was apparently a B cell, based on absorption with different cell populations. No genetic restrictions in the activity of the factor were found. A 1 cells carried H-2 and Thy-1 alleles of both parental cells and formed rosettes with SRBC.

The present experiments provide the first evidence that congenic strains of rats, which differ only in the MHC, produce discriminably different urinary chemosignals. Urine from adult male PVG and PVG.R1 rats, which differ only in the A region (class 1) of the MHC, was used in a habituation-dishabituation task, with male PVG-RTlu, Wistar albino, and Lister hooded rats as subjects. Urine from PVG males was easily distinguished from that of PVG.R1 males by all three strains. Individual PVG males were not distinguished by their urine odours, but individual PVG.R1 males appeared to have discriminably different odours. A repetition of this experiment indicated that this discrimination may have been due to impurities in the urine. Odours from serum were not sufficient for discrimination between the two strains, nor was the class 1 molecule purified from the urine. Urine with the class 1 molecule removed (remainder fraction) could, however, be used to distinguish between the strains. The chemicals in the urine which give this distinctive odour may be fragments of the class 1 molecule or small molecules associated with the class 1 molecule. The MHC appears to control the odour cues which are used by mammals for individual recognition and may provide an olfactory basis for kin recognition but the mechanism by which the MHC controls these olfactory signals is unknown.

The classical class I antigens of the major histocompatibility complex (MHC) are cell-surface glycoproteins which were originally discovered because they cause rapid rejection of cells or tissues grafted between unrelated individuals. These molecules are encoded by the K, D and L loci of the mouse MHC (and analogous loci in other species) which show extreme species polymorphism and a large number of alleles. In an outbreeding population 3.6 X 10(9) unique MHC class I phenotypes can be encoded by the 100 alleles at each of the K and D loci and the 6 alleles at the L locus. This level of polymorphism ensures that the cells and tissues of each unrelated individual are uniquely identified by their class I membrane-bound antigens. Like other membrane bound proteins, these class I molecules are anchored in the lipid bilayer by a hydrophobic domain encoded by exon 5. However, there have been reports of the occurrence of classical class I molecules in true solution in the blood of humans, mice, and rats. We report here that classical polymorphic class I molecules in normal rats are constitutively excreted in the urine and that untrained rats can distinguish the smell of urine samples taken from normal donors that differ only at the class I MHC locus and therefore excrete different allelomorphs of class I molecules in their urine.

The nature of the cells required for first-set graft rejection in vivo was examined by using an adoptive transfer system to restore heart-graft rejection in irradiated rats. Highly purified inocula of peripheral T lymphocytes were shown to quantitatively account for the restorative ability of adoptively transferred cells. These T cells were shown to be long-lived small lymphocytes which are not recently derived from the thymus during adult life. They belong to the pool of T cells which constantly recirculate from blood to lymph as shown by their rapid appearance in the lymph of iradiated syngeneic rats after intravenous injection. Neither B lymphocytes nor antibodies in the circulation or in the graft itself are required for first-set graft rejection.

An adoptive transfer system was used to study the cellular basis of memory in animals immunized by grafting with major histocompatibility complex incompatible tissue. Memory was characterised by a large (greater than 100 fold) increase in the potency of lymphocytes to precure graft rejection. This increase in potency endured for at least 1 yr after sensitization. The memory cells were shown to be Ig-- small lymphocytes which were long lived and which did not recirculate from blood to lymph in normal recipients although they did home to lymphoid tissue from which they could be recovered several months later. The thymus was not required either for the generation of memory cells or their maintenance. Cells carrying memory for alloantibody synthesis did recirculate normally but alloantibody synthesis was shown not to be required for rejection.

The kinetics of the popliteal lymph node response to an allograft of lymphoid cells injected into the foot pad were examined after challenge with strong (Ag-B) and weak (non Ag-B) transplantation antigens. The response to weak antigens was characterised by a lag period of 2 days. The response to strong antigens was more rapid in onset. Active immunisation against weak antigens accelerated the response so that it came to resemble the immediate response to strong antigens. Active immunisation against strong antigens depressed the response. This depression was reproduced by giving passive antiserum to normal recipients. Passive antiserum produced by immunisation against weak antigens was also suppressive. Adoptive transfer of immune lymphoid cells reproduced the effects seen with passive transfer of antiserum. These findings indicate that clonal expansion of antigen-sensitive cells is characteristic of immunity to weak and not to strong antigens and that antibody is produced in both situations and has a similar suppressive effect on the cell-mediated response in addition to its direct effect on the graft.

Classical class I transplantation antigens present in solution in the body fluids have been studied. These antigens have been found in a monomeric, soluble form in blood, lymph, and urine, and a major source is the hemopoietic system which gives rise to cells that secrete these molecules into the blood. The cell types most probably involved in their secretion are of the macrophage/dendritic cell lineage. The serum molecule is a heterodimer with a heavy chain of 39,000 mol wt associated noncovalently with beta 2-microglobulin and is present in serum at a concentration between 350 and 390 ng/ml. These molecules have a short half-life of 2.7 h and are excreted into the environment via the kidneys in the urine. In the urine, greater than 90% of the molecules are degraded into smaller fragments. This finding that normal metabolic processes lead to the excretion of classical highly polymorphic class I molecules in the urine provides a direct explanation in molecular terms of the ability of animals to identify individuals on the basis of urinary odor. Since intact class I molecules are unlikely to be the odoriferous component in the urine, two hypotheses have been suggested. Either small fragments of class I molecules are detected or the molecule acts as a carrier that transports volatiles from the serum into the urine where they are released, giving rise to the class I-associated odor.

Treatment of normal rats with certain monoclonal anti-CD4 antibodies, beginning on the day of grafting, prevents heart graft rejection across a full MHC-haplotype mismatch. Adequate doses of antibody MRC OX-35, which is very potent in vivo, led to the induction of authentic specific tolerance of the transplants. In the very stringent grafting test of transplantation of DA skin to high-responder LOU recipients, adjunctive therapy with cyclosporine, in addition to the anti-CD4 antibodies, is required to induce tolerance. Since tolerance persists indefinitely after a 30-day course of treatment and appears to involve T cell-mediated suppression, this form of treatment is of potential clinical interest.

A hybridoma producing the monoclonal antibody HIS42 was isolated from a fusion between spleen cells from a BALB/c mouse immunized with rat thymocytes and the fusion partner SP2/0. This antibody recognizes a minor subset of T cells in every haplotype of rat tested so far. The subpopulation of HIS42 positive T cells contains both CD4+ and CD8+ cells, in the same ratio as found in the peripheral T cell population. When bound to Sepharose beads, HIS42 induces T cell proliferation in the presence of interleukin-2. In contrast to lymph node T cells, a number of thymocytes were found to express HIS42 only in the cytoplasm or together with membrane expression. Most bright HIS42 surface labelled thymocytes were also positive for MRC OX-44, a marker predominantly identifying mature thymocytes. SDS-PAGE analyses of the membrane molecules immunoprecipitated by HIS42 show two bands on unreduced gels. One of these bands (85 kd) runs as two separate bands at 35 and 48 kd on reduction. The other much weaker broad band (approximately 100 kd) is hardly affected by reduced conditions. Taken together these data suggest that HIS42 is directed against a determinant on the rat T cell receptor for antigen, which is common to a small number of T cells.

In order to differentiate between donor (chimeric F1) cells and host cells as being responsible for suppression in transplantation tolerance (TT), cells from tolerant donors were first subject to negative selection through F1 hybrid intermediate host animals. This revealed that the suppressor cells was neither completely removed from the lymph nor recoverable from the lymphoid tissues of the filter rat when highly suppressive inocula were used, suggesting that suppression did not directly depend on cells with receptors for alloantigens. The phenotype of donor and host cells in the recirculating pool was studied with fluorescent antisera and showed that both host cells and chimeric F1 cells were in the thoracic duct lymph of tolerant rats and were capable of recirculation. T and B lymphocytes of both types were present but the IgG-positive, presumptive memory B cells were highly enriched in those rapidly recirculating cells, obtained by filtration of tolerant inocula through irradiated intermediate hosts. These cells were also highly enriched for suppressor function. Methods which selectively depleted either the chimeric T cells or the B cells were applied to tolerant inocula and on adoptive transfer of these inocula, suppression was eliminated only when chimeric T cells were eliminated. This strict dependence of suppression of F1 hybrid T cells is interpreted as evidence that these cells probably suppress directly, via an anti-idiotypic mechanism, the alloreactive cells bearing idiotype-positive major histocompatibility complex receptors.

The lymphocytes from neonatally tolerant rats which adoptively transfer tolerance to sublethally irradiated recipients do so by specifically suppressing the regeneration of alloreactivity which normally occurs after irradiation. Although tolerant cells will only partially suppress normal alloreactive cells when the two are mixed in near equivalent numbers, experiments in which the interval between injection of tolerant and normal cells into irradiated recipients was gradually extended, indicated that total suppression of normally alloreactive cells was achieved after 8 weeks of prior residence of tolerant cells in the adoptive host. Further evidence that tolerant cells would only suppress if present in excess of normal cells was obtained by reducing the tolerant cell population in tolerant donor rats by whole body irradiation. These animals then lost their ability to suppress normal alloreactive cells administered to them. The immune status of adoptively tolerized animals did not mimic that of the donors of the tolerant cells. Even where full tolerance, as measured by skin graft survival, failure to synthesize alloantibodies, and capacity to further transfer skin graft tolerance to secondary recipients, was evident the lymphocytes of these animals showed considerable graft-versus-host (GVH) reactivity. The persistence of tolerance through repeated adoptive transfers was correlated with themore » persistence of donor (chimeric) cells and the indicator skin graft on adoptive recipients only amplified tolerance expression where the inocula of tolerant cells given was weakly suppressive. Finally, removal of the minor population of chimeric cells from tolerant inocula using cytotoxic alloantisera abolished the capacity to transfer tolerance. These results imply an active role for chimeric cells which is best understood as an immune response involving proliferation driven by the idiotypes of the alloreceptors on host cells.« less

Using PVG-RT1av1 neonatal heart grafts transplanted to the plantar space in irradiated PVG recipients, and adoptive transfer of normal and immune cells, we have analyzed the role of lymphocyte subsets in graft rejection. This assay was found to be reproducible and to show good dose-response characteristics, permitting a precise analysis of the potency of the cells transferred. Immune cells were 36 times more potent than normal cells and all of their activity was in the T cell fraction. The increase in potency of immune populations was entirely within the CD8+ population defined by the mouse monoclonal antibody MRC OX-8. The CD4+ population, defined by the antibody W3/25, in both normal and immune populations, restored first-set rejection. All the graft rejection activity of CD4+ cells could be ascribed to the MRC OX-22- subset of these cells.

Allograft rejection in the rat has been shown to be under stringent immune response (Ir) gene control using major histocompatibility complex recombinant animals as donors. Presentation of an isolated class I antigenic difference to high responder recipients results in rapid graft rejection, but low responders fail to reject. This striking qualitative difference is also seen in some liver grafting experiments in which the donor presents a full MHC haplotype and minor antigen mismatch to the responders. Grafts of other organs, however, do not discriminate qualitatively between high and low responders when a full haplotype mismatch exists. We have used the canonical high and low-responder animals, (PVG X PVG-RT1u)F1 and PVG to examine whether any qualitative difference in responsiveness can be detected against the a haplotype using a variety of organ grafts. We have confirmed a qualitative difference between high and low responders using PVG.R1 donors presenting an isolated class I (Aa) difference. Rapid rejection by high responders contrasted with complete failure to reject by the low responders. No difference in rejection tempo was found when a full a haplotype mismatch was introduced. This could have reflected vigorous responses to I and C region differences, because rapid rejection through these regions was demonstrated using the PVG.r1 (AaIcCc) and PVG.r8 (AaIuCu) recombinants. The feeble immunogenicity of the Aa antigen for PVG animals was revealed by priming and cross-priming experiments showing not only that r1 failed to prime for subsequent r1 graft rejection, but that the Aa antigen presented in concert with Ia and Ca also failed to prime. An unexpected result was that the Aa antigen of r1 actually suppressed responsiveness, especially when delivered by a heart graft. This suppression not only extended to subsequent r1 grafts (for example, skin rafts) but also to subsequent grafts of a tissue. The mechanism of this suppression remains unclear but preliminary experiments argue in favor of enhancement rather than active suppression.

Summary A new transplantable lymphocytic leukaemia of the inbred Hooded Oxford strain of rat is described. Fewer than 10 cells will transfer disease to syngeneic recipients. Leukaemic cells bear the surface markers of thymus-derived (T) cells and recirculate from blood to lymph. In contrast to the usual thymic lymphomas of rodents the pathophysiology of the disease-bears a close resemblance to human acute lymphoblastic leukaemia.

Efferent lymph collected from a thoracic duct fistula initially contains no macrophages. However, the surgical procedures used to insert plastic cannulae into efferent lymphatics incite a vigorous foreign body reaction leading to the contamination of collected lymph with significant numbers of these cells. A sensitive and specific assay for the presence of macrophages in lymphocyte populations was used to quantitate the degree of contamination in rats bearing thoracic duct cannulae. The origin of some of these contaminant cells from the peritoneal macorphage population was established by adoptive transfer of labelled peritoneal cells to cannulated recipients.

SEVERAL cellular mechanisms have been postulated to account for the phenomenon of transplantation tolerance in animals injected at birth with allogeneic or semiallogeneic cells. These include deletion of the clone of specific alloantigen sensitive cells1, continuous production of conventional blocking alloantibody2 or anti-receptor antibody3 and the action of suppressor cells4. Although these phenomena have been demonstrated in tolerant animals, it has never been shown that either alone or in combination they are actually capable of inducing true transplantation tolerance. In the course of experiments designed to prove formally that recirculating small lymphocytes are responsible for first-set rejection of skin grafts, we obtained results suggesting that rapidly recirculating cells from tolerant donors might be capable of actively transferring a state of transplantation tolerance (TT) to irradiated recipients5,6. These results, which suggested that the transfer of TT was due to humoral or cellular suppressor mechanisms, were not consistent with the clonal deletion theory of tolerance. The present experiments were designed to confirm that TT is not merely the absence of reactivity to a given set of allo-antigeneic determinants but an active suppression of reactivity to those determinants, and to determine whether T or B cells mediate this suppression.

Tolerance to the major transplantation alloantlgens, coded by the major histocompatibility gene complex, is readily induced in newborn rats by the intravascular injection of bone marrow cells from immuno-competent allogeneic donors, usually F1 hybrids carrying one haplotype identical with that of the newborn strain. Analysis of the alloreactive repertoire of lymphocytes from adult tolerant animals of this provenance has shown that, not only are their cells unreactive in all in vivo and in vitro assays against the tolerated alloantigens but they actively and specifically suppress recovery of alloreactivity against these antigens on adoptive transfer to irradiated syngeneic animals. This suppression is mediated by a lymphocyte with the property of rapid recirculation from blood to lymph. These cells are not found in organs deficient in recirculating cells, such as thymus and bone marrow. Inocula of recirculating cells from tolerant donors have been treated with specific alloantisera and monoclonal antibodies to define the membrane antigen phenotype and receptor repertoire of suppressor cells. They are inactivated by treatment with alloantisera of specificity restricted to the unshared haplotype on chimaeric cells. Immunofluorescent studies have shown that chimaeric cells are present in very small numbers throughout the lymphoid tissues of tolerant rats and adoptive recipients of their cells. There is a clear correlation between survival of chimaeric cells and persistence of tolerance on transfer; however the chimaeric cell responsible for suppression is a highly differentiated functional subtype of T lymphocyte. The mode of action of these cells is unknown but for genetic reasons can be inferred to involve reactivity against the idiotypes on receptors for allo-antigen in the tolerant host. A model for self-sustaining chimaerism, suppression, clonal inactivation and tolerance which incorporates these findings is presented.

ALTHOUGH the phenomenon of memory is central to the adoptive immune response to antigenic stimulation, the magnitude of the memory effect in graft rejection has never been quantified. We have directly compared the potency of naive and memory cells in two quantitative adoptive transfer assays, one measuring graft rejection and the other graft-versus-host activity. While the well known lack of a significant factor of immunisation in the latter was confirmed, memory cells showed a dramatic, long term increase in their potency to reject heart grafts.

In order to test the hypothesis that commensal bacteria influence the urinary odors of individuality, we collected urine from PVG and PVG.R1 male rats born by cesarian section and reared in a germ-free environment. Using the habituation-dishabituation test with PVG.RT1 u and Lister hooded rats as subjects, we found that urine from the germ-free rats was not discriminated, while urine from conventionally housed rats of the same strains could be discriminated (experiment 1). When the germ-free rats were moved to a conventional animal house after recolonization with commensural flora and their urine collected, it was discriminated, indicating an essential role of bacteria in determining the unique urinary odors of MHC-congenic rats (experiment 2). The conventionally housed and germ-free rats did not differ in the amount of class I antigen in their urine (experiment 3). Finally, urines of PVG and PVG.R1 donors inoculated with a defined and highly restricted flora to render them specific-pathogen-free (SPF) could not be discriminated. Urine from SPF donors moved to a conventional animal house could be discriminated (experiment 4). These results indicate that commensal bacteria are essential for the production of the unique individual odor of the urine of MHC-congenic strains of rats.

Previous studies have shown that the Ir-gene-controlled rejection of rl tissues by c/u responder and non-rejection by c low responders does not extend to tissues expressing a full a haplotype mismatch. However, antibody responses and liver graft rejection are both defective in low responders, even across a full haplotype barrier. We have therefore used a titrated adoptive transfer assay to search for quantitative differences in the responsiveness of c and c/u animals to a organ grafts. We first established that a heart graft rejection could be ablated in both recipient strains with whole-body irradiation and could be restored with syngeneic cells. Titration of restorative cells revealed that 5 times as many c cells were required to restore graft rejection in c recipients as c/u cells were required in c/u recipients. Use of cells from primed donors showed that in both c/u and c animals these cells had undergone about a 5-fold increase in potency, showing that there was no failure of proliferation and differentiation in the low responder after contact with antigens. Cross-transfer experiments were done to attempt to localize the defect in low-responder animals either to a failure of low-responder antigen-presenting cells (APC) to trigger a response or a defect in the responsiveness of alloreactive cells toward the a antigens. In these experiments c cells were obtained from radiation chimeras of the c----c/u type. These cells were used to restore graft rejection in c/u irradiated recipients. Similar experiments employing c/u cells obtained from c/u----c chimeras and given to irradiated c recipients were also done. These showed that c cells from chimeras were marginally less potent than c/u cells from chimeras. In contrast when cross-transfer of c/u cells to c animals bearing a nonrejected rl heart was done, no rejection was seen even when antigen presenting cells were cotransferred. The conclusions from this series of experiments were that quantitatively small defects were present in both repertoire and antigen presentation, and that these quantitative defects in aggregate were probably sufficient to explain the documented low responsiveness of c animals to the a haplotype. The failure of high-responder c/u cells to secure rejection of rl tissues in the low-responder c environment suggests that presentation of isolated class I differences in host APCs is mandatory for rejection to occur and is highly defective in the c animal.

The transplantation of organs between individuals of a species normally has two main consequences: (1) the tissue is rejected unless the individuals are matched for identity of transplantation antigens, especially those encoded by the major histocompatibility complex (MHC), and (2) the recipient is sensitized to the transplantation antigens of the donor so that second-set grafts are rejected in a more violent manner1. These rules of transplantation are not obeyed by liver grafts. Orthotopic transplants of liver are never rejected by many strains of rat even though the MHC barrier is crossed2. We have recently shown that instead of sensitizing the recipient, these enduring liver grafts induce a state of donor-specific unresponsiveness in which subsequent grafts of other organs, such as skin, are accepted permanently. It is possible that many strains simply cannot mount a sufficiently vigorous destructive immune response to outstrip the liver's great capacity to repair immune damage and that the systemic unresponsiveness observed is due to diversion of alloreactive lymphocytes with donor specificity into the liver allograft. Manipulations which increase the vigour of the immune response might therefore tip the balance in favour of liver graft rejection and away from induction of unresponsiveness. We have previously measured the degree to which the immune response can be increased by previous exposure to MHC antigens in a quantitative adoptive transfer system3. In the strain combination DA (MHC haplotype RT-1a) grafted to PVG (RT-1c), the lymphocytes of immunized animals are 1,000 times more potent than those of non-immune animals in procuring graft destruction when transferred to irradiated hosts. Unexpectedly, we have now found that liver grafts transplanted into previously immunized rats not only fail to reject but convert the state of heightened reactivity to donor grafts characteristic of immune recipients into one of non-reactivity characteristic of tolerant animals.

The major histocompatibility complex (MHC) of the rat has three regions--A (class I), B/D (class II), and C/E (class I)--and congenic strains are available which differ in each of these regions. We used the habituation-dishabituation procedure to examine the ability of PVG-RT1u male rats to discriminate between the urinary odors of congenic rat strains which differ genetically only at certain individual regions of the MHC. The results of five experiments indicate that discrimination can be made between urine from rats which differ in all three regions of the MHC (PVG vs. PVG-RT1av1 donors), only in the class I A region (PVG vs. PVG.R1 donors), only in the class I C/E region (PVG.R19 vs. PVG-RT1av1 donors), only in the class II B/D region (PVG.R1 vs. PVG.R19 donors), and in all regions except the classical class IA locus (PVG-RT1av1 vs. PVG.R1 donors). These results indicate that all of the MHC regions may contribute to the individual odors of rats.

Several simple procedures for experimentation on specific-pathogen-free rats and mice are described which combine the technique of plastic-film-isolator containment and a laminar flow sterile environment. These permit the full range of cellular immunology experiments to be performed without compromising the microbiological barrier.

Neonatally tolerant rats can be “immunized” by the intravenous injection of small numbers (3 × 107) of T lymphocytes. The effect of this priming injection is to render the tolerant animals highly suppressive so that tolerance can no longer be broken with normal alloreactive cells. Negative selection experiments to remove certain sets of alloantigen receptors from the priming cells showed that the suppressor response was against the idiotype of the T helper cell receptors for the tolerated class II antigens. The chimeric donor hemopoietic cells of tolerant rats were found to be secreting monomeric class I donor antigens into the circulation of the tolerant rats. The characteristics of these soluble antigens were studied and their possible role in suppressing class I reactive cytotoxic cells discussed.

Membrane antigens on PVG leukaemic cells have been compared with those on normal PVG peripheral T cells by studying the capacity of the cells to bind antibodies against these cell types. The antibody bound has been quantitated by secondary binding of a radiolabelled anti-immunoglobulin. It was shown that the leukaemic cell lacks the peripheral thymus-derived lymphocyte antigens which the normal PVG peripheral T lymphocyte carries. In this respect, in its reduced distribution of the W3/13 antigen compared with T cells, as revealed by binding of the hybridoma monoclonal antibody W3/13, and in its abundance of Thy 1.1 antigen, the leukaemic cell resembles the immature thymocyte, rather than the mature T lymphocyte. This antigenic profile represents an illustration of de-differentiation of tumour cells at the molecular level. Two antigens shared by PVG leukaemic cells, normal PVG peripheral T cells and thymocytes were indicated by absorption studies with antisera against the leukaemic cell. In an immunotherapy model, treatment with AUG cells sensitized against the PVG leukaemia cell resulted in elimination of leukaemic cells from PVG rats. The peripheral T cell antigens clearly cannot be antigen(s) recognized by the AUG rat on the leukaemic cell, since the present studies indicate the absence of peripheral T cell antigens on this cell.

Small animals have been of limited use as models for liver preservation and subsequent transplantation because the surgery has been technically difficult. A cuff technique for the portal vein anastomosis was specially developed1, which simplified vascular anastomosis after the isolated liver had been subjected to continuous perfusion. As a perfusate, fluorocarbon (FC) emulsion (Fluosol-43; Green Cross Co., Japan) was added to the basic perfusate2, containing plasma protein fraction, which has produced the best results experimentally and clinically in our laboratory.

Summary The karyotype of a transplantable T cell leukaemia which arose originally in a PVG strain rat undergoing chronic internal β irradiation of the spleen was examined and a marker chromosome was found. Although the leukaemia progressively became more acute during 4 years of continuous passage the marker did not change. Cytogenetic analysis of normal rats given an acute exposure to external irradiation revealed, among other abnormalities, a marker of the same morphology; indicating that the original marker was probably radiation induced. This is the first description of a stable marker chromosome in an experimental animal leukaemia.