Alloantigens In Mice Research Articles

Clodronate inhibits alloimmunization against distinct red blood cell alloantigens in mice.

Alloimmunization can be a significant barrier to red blood cell (RBC) transfusion. While alloantigen matching protocols hold promise in reducing alloantibody formation, transfusion-dependent patients can still experience RBC alloimmunization and associated complications even when matching protocols are employed. As a result, complementary strategies capable of actively preventing alloantibody formation following alloantigen exposure are warranted. We examined whether pharmacological removal of macrophages using clodronate may provide an additional strategy to actively inhibit RBC alloimmunization using two preclinical models of RBC alloimmunization. To accomplish this, mice were treated with clodronate, followed by transfusion of RBCs expressing the HOD (HEL, OVA, and Duffy) or KEL antigens. On days 5 and 14 post transfusion, anti-HOD or anti-KEL IgM and IgG antibodies were evaluated. Low dose clodronate effectively eliminated key marginal zone macrophage populations from the marginal sinus. Prior treatment with clodronate, but not empty liposomes, also significantly inhibited IgM and IgG anti-HOD alloantibody formation following transfusion of HOD RBCs. Similar exposure to clodronate inhibited IgM and IgG antibody formation following KEL RBC transfusion. Clodronate can inhibit anti-HOD and anti-KEL antibody formation following RBC transfusion in preclinical models. These results suggest that clodronate may provide an alternative approach to actively inhibit or prevent the development of alloantibodies following RBC transfusion, although future studies will certainly be needed to fully explore this possibility.

Mouse Alloantigen Ly10 Is Identical to Murine Fusion Regulatory Protein-1 (mFRP-1)/4F2/CD98: Aberrant Expression of mFRP-1/Ly10 Allotypes in Cells Derived from CDF1 Mice Due to the Gene Deletion

Murine FRP-1 (mFRP-1) and Ly10 antigens are alloantigens. Anti-Ly10.1, monoclonal antibody (mAb) reacts to HeLa cells stably expressing mFRP-1.1 (HeLa cells/mFRP-1.1), but does not react to HeLa cells/mFRP-1.2. On the other hand, anti-Ly10.2 mAb reacts to HeLa/mFRP-1.2 cells, but does not react to HeLa cells/mFRP-1.1. These findings indicate that Ly10.1 and Ly10.2 antigens are identical to the heavy chains of mFRP-1.1 and mFRP-1.2 molecules, respectively. Thymocytes and fibroblast cells obtained from CDF1 and BDF1 mice showed reactivity to both anti-Ly10.1 and anti-Ly10.2/FRP-2 antibodies, indicating that mFRP-1/Ly10 alloantigens are codominantly expressed in the F1 mice. Intriguingly, IMC carcinoma cells, derived from CDF1 mice, express mFRP-1.2, but do not express mFRP-1.1. Surprisingly, DBT cells, derived from CDF1 mice, consist of two different cell populations: one expresses both mFRP-1.1 and mFRP-1.2 antigens and the other expresses only mFRP-1.1 antigen. Consequently, we tried to isolate cloned DBT cell lines by limiting dilution. Finally, six cloned DBT cell lines were obtained, and three clones of these cloned DBT cells expressed both mFRP-1.1 and mFRP-1.2, and another three clones of these cloned DBT cells expressed both mFRP-1.1 and mFRP-1.2, and another three clones including clone 5 cells expressed only mFRP-1.1 antigen. No induction of mFRP-1.2/Ly10.2 by 5-azacytidine and sodiumn-butyrate could be detected in DBT clone 5 cells. Neither mFRP-1.2 mRNA nor the genomic cDNA clone encoding mFRP-1.2 could be detected in DBT clone 5 cells, nor could the genomic cDNA encoding mFRP-1.1 be detected in IMC cells, indicating that aberrant expression of mFRP-1/Ly10 allotypes in CDF1 mice-derived cells is due to the gene deletion.

EIVDENCE THAT EPIDERMAL ALLOANTIGEN EPA-1 IS AN IMMUNOGEN FOR MURINE HEART AS WELL AS SKIN ALLOGRAFT REJECTION

Epa-1 is a non-H-2 mouse alloantigen defined by MHC-restricted, CD8+ cytotoxic T cells. In vitro it is a strong determinant for the lysis of epidermal cells, fibroblasts, and macrophages but not lymphocytes, and in vivo it functions as a target for skin allograft rejection and cutaneous graft-versus-host reactions. Genetically, Epa-1 appears to be the nonpolymorphic manifestation of a loss mutation. The establishment of C3H.Epa-1 (Epa), an Epa-1+ congenic strain on the Epa-1- C3H/HeJ (C3H) inbred strain background, facilitated the investigation of the role of Epa-1 in skin and heart allograft rejection. C3H females and males rejected first-set Epa skin grafts with median survival times (MSTs) of 20 and 30 days, respectively. However, there was a strong factor of immunization, because all second-set skin allografts were rejected by hosts of both sexes within 10 days. In contrast, all Epa hosts of both sexes permanently accepted C3H skin allografts, consistent with Epa-1 arising from a loss mutation. C3H hosts of both sexes rejected primarily vascularized first-set Epa heart allografts in similar tempo to first-set Epa skin allografts, with MSTs of about 30 days. However, in contrast to the accelerated rejection of skin allografts, sensitized C3H hosts rejected Epa heart allografts in chronic fashion, with some transplants showing very prolonged survival. Thus, Epa-1 is a relatively strong determinant of skin allograft rejection but a weaker determinant of heart allograft rejection.

Neonatal tolerance induction by class II alloantigens activates IL-4-secreting, tolerogen-responsive T cells.

Neonatally induced tolerance of class II MHC alloantigens in mice of the A/J strain background is achieved without evident clonal deletion, because lymphoid cells from tolerant animals proliferate in mixed lymphocyte reactions against tolerogen-bearing stimulators. However, the pattern of lymphokines produced in bulk cultures by these tolerogen-reactive cells is unusual in that, unlike lymphocytes from nontolerant normal mice, lymphocytes from tolerant mice produce measurable amounts of IL-4 when stimulated with tolerogen. Using limit dilution analysis, we have determined that the ability to detect IL-4 in bulk cultures is caused by a high frequency of precursors of Ag-specific IL-4 secreting (pIL-4) cells among tolerant, but not normal, responder lymphocytes. Moreover, after repeated in vitro exposure to class II alloantigens, and after in vivo grafting with class II-disparate skin, the frequency of pIL-4 cells rises among lymphocytes from normal mice, eventually reaching levels similar to those found in class II tolerant mice. Because in normal mice a high frequency of class II-reactive pIL-4 cells reflects the "primed" state, we conclude that a subset of T cells that resemble Th2 cells is similarly primed in neonatally tolerized mice. We propose that this priming is achieved by the neonatal inoculation of semiallogeneic bone marrow cells. Because no expansion in the clone size of tolerogen-specific precursors of Ag-specific IL-2-secreting T cells (pIL-2) is observed among tolerant animals, and because the pIL-2 cells from tolerant mice are incapable of differentiating into cytotoxic and delayed hypersensitivity effector cells, we further propose that the precociously primed pIL-4 cells function as "suppressor" cells, helping to maintain the tolerant state.

Lipopolysaccharide-induced suppression of DTH-reactivity to histocompatibility antigens. I. Kinetic aspects and specificity

The effects of bacterial lipopolysaccharide (LPS) on the development of DTH-reactivity to alloantigens in mice were investigated. DTH to a particular set of alloantigens could be suppressed by treatment of responder mice with a single intravenous (i.v.) injection of 100 [tg LPS and a simultaneous subcutaneous (s.c.) injection of the appropriate allogeneic spleen cells. The suppression lasted at least 60 days and affected the afferent limb of the DTH response as well as the efferent limb. The suppression could be adoptively transferred to naive syngeneic recipient mice by spleen cells, but not by immune serum, proved to be antigen-specific. In spite of this specificity, the DTH-response against unrelated «third-party» alloantigens could be suppressed as well, provided the latter were presented during the induction-phase of DTH together with the alloantigens that had been used for the induction of suppression.

CROSSREACTIONS BETWEEN AN I-A ALLOSPECIFICITY AND THE CYTOSKELETON OF GLOMERULAR EPITHELIUM AND OF VASCULAR SMOOTH MUSCLE1

During studies to investigate the localization of Ia antigens in normal mouse kidney, a monoclonal antibody (Mab) specific for I-Ab by microcytotoxicity criteria was found to crossreact with a tissue antigen present in the glomeruli and vasculature of murine tissues, irrespective of their Ia haplotype, as well as in human kidneys. Immunofluorescence and immunoperoxidase studies of frozen tissue sections revealed that the antigen responsible for the crossreaction was present in the cytoplasm of glomerular epithelial cells and of smooth muscle of blood vessels. The immunofluorescence pattern of primary cultures of human glomerular epithelial cells and of a glioblastoma cell line indicated that the crossreactive antigen forms part of the cytoskeleton, and resembled the pattern observed with a monoclonal against vimentin, one of the intermediate filaments. Thus this Mab, which is directed against mouse alloantigen Ia.20 in microcytotoxicity was found to crossreact with a cytoskeletal component present in the mouse, as well as in human glomerular epithelial and smooth muscle cells.

An in vivo and in vitro analysis of systemic immune function in mice with histologic evidence of neural transplant rejection

Histologic and immunocytochemical analyses of fetal neocortical tissue transplanted to the lateral ventricle of inbred adult mice indicate that this tissue survives transplantation well if the donor and host are isogeneic. The major histocompatibility complex (MHC) of the mouse is known as the H-2 locus. H-2-incompatible neural transplants (allografts), unlike their H-2-identical counterpart (isografts), are characterized by the presence of T cells comprising both major T-cell subsets and macrophages, and by a marked increase in the expression of both class I and class II (Ia) MHC antigens. These findings suggest a recognition of H-2 alloantigens by the host's immune system followed by an appropriate effector response. We report here our attempts to demonstrate systemic host sensitization to alloantigens in mice bearing H-2-incompatible intraventricular neural transplants. We measured the time to rejection of orthotopic skin grafts subsequent to neural transplantation, splenocyte proliferative responses to alloantigens in mixed lymphocyte cultures (MLC), and class I-restricted antigen-specific cytolytic T lymphocyte (CTL) activity. No significant differences were found in any of these tests of host systemic sensitization between mice with allogeneic neural transplants and those with isogeneic transplants or control animals. We conclude that intraventricular neural transplants, while recognized and affected by cells of the host's immune system, do not elicit a detectable systemic sensitization to class I H-2 alloantigens. Rejection of neural transplants may depend on sensitization to class II H-2 alloantigens, to so-called minor histocompatibility antigens, or some combination thereof.

Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins.

A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.

Delayed-type hypersensitivity reaction to alloantigens in mice

A new scheme of induction of delayed hypersensitivity (DH) to alloantigens in mice is described. The optimal conditions for DH development are intraperitoneal injection of 1 X 10(7) cells of the allogeneic spleen to normal mice (not treated with cyclophosphamide) 5 days before the challenge with 5 X 10(6) spleen cells. s.c. The maximal response was seen 24 h after resolution. The reaction was specific, being mediated by T lymphocytes; it could not be transferred to intact recipients by allogeneic serum. However, DH was found to be transferrable by s.c. injection of 5 X 10(6) lymphocytes from sensitized donors. Such a modification of the scheme of DH induction may appear useful for studies of the animals' immunity, particularly for studies of the tolerance to allografts.

Evidence for expression of Ly-6.2 on non-bone marrow-derived cells in kidney, skin and connective tissues.

Mouse alloantigen Ly-6.2 is detectable in various non-lymphoid tissues such as kidney, but it is not clear whether or not this expression is due to bone-marrow derived passenger leukocytes. To determine whether non-marrow derived cells express Ly-6.2, we examined the expression of this antigen in kidney and on isolated connective tissue and epidermal cells. Studies in radiation chimeras demonstrated that the kidney did not become Ly-6.2 positive when negative animals were reconstituted with positive marrow. Thus, passenger leukocytes cannot account for the renal expression of Ly-6.2, indicating that most of this antigen is on non-marrow-derived (parenchymal) cells in kidney. Various isolated cell types--fibroblasts, osteocytes, chondrocytes and skin epidermal cells--were found to be Ly-6.2 positive. Indeed, absorption and cytotoxicity results suggested that the amount of Ly-6.2 on fibroblasts exceeded the amount of an H-2 antigen on these cells. Comparison of fibroblasts to lymphocytes indicated that fibroblasts had 13--60 times more Ly-6.2 than spleen cells and three times more than PHA blasts. The results indicate that the Ly-6.2 detected in non-lymphoid tissues is predominantly on the parenchymal or connective tissue elements of those tissues.

Dissociation of biological activities of Corynebacterium parvum by chemical fractionation

Fractionation of Corynebacterium parvum (C. parvum) by phenol-water extraction resulted in components which differentially affected the cell-mediated cytotoxic response to tumor alloantigens in mice. The residue of the extraction (Fraction C) and its light component (Fraction D) retained all of the activities of the whole micro-organism, i.e. inhibition of primary spleen cell cytotoxicity, inhibition of in vitro memory cell-mediated cytotoxicity (CMC) and generation of macrophage-like suppressor cells. Administration of the phenol phase extracted material (Fraction A) inhibited both the primary and memory cytotoxic responses, but did not elicit suppressor macrophages. Destruction of carbohydrate components of Fraction D significantly reduced its ability to inhibit the memory response and to elicit suppressor macrophages. In contrast, its ability to inhibit the primary cytotoxic reponse was unaffected. This study clearly demonstrates that the several effects of C. parvum administration on the cell-mediated cytotoxic response to tumor alloantigens can be associated with different components of the organism. Furthermore, the dissociation of the effects indicate that the generation of primary cytotoxic lymphocytes, the priming of memory cytotoxic lymphocytes, and the induction of regulatory macrophages are not necessarily linked during the generation of a normal cytotoxic response.

Dissociation of the Humoral and Cell‐mediated Responses to Alloantigens in Mice by Sublethal Whole‐Body Irradiation

Spleen cells from mice exposed to 500 rad sublethal whole-body irradiation before immunization with 100 x 10(6) allogeneic spleen cells exhibited an enhanced cytolytic T-lymphocyte (CTL) activity. In contrast, this treatment completely abrogated the humoral alloantibody response, as measured by complement-mediated lysis. In addition to the observed dissociation between primary humoral and cell-mediated responses, the character of the in vitro secondary CTL response of spleen cells obtained from mice sublethally irradiated before alloimmunization was also modified. Although these spleen cells were perfectly capable of mounting a secondary response upon restimulation in vitro with intact allogeneic spleen cells, they were no longer capable of responding to subcellular alloantigen preparations. These results suggest that the cellular requirements for the induction of secondary CTL responses in vitro depend upon the nature of the antigen used for restimulation.

An Ly-like specificity with extensive nonlymphoid expression

The characteristics of a strong mouse alloantigen with renal, bone marrow, and lymphoid expression were studied. This antigen is probably identical to that currently designated Ly-6.2. It was defined by the high-titered (1:1000) cytotoxic activity of three different antisera against peripheral lymphocyte target cells from DBA/2, DBA/1, and a variety of other strains. In the F(2) and four backcross generations the genetic control of this specificity segregated as a single autosomal dominant gene. In lymphoid tissues the predominant expression was on T cells but 10-30% of B cells were lysed by these antisera. The specificity was expressed strongly in kidney, as shown by sequential absorption, in amounts equal to or greater than the amount in lymphoid tissues. Comparison to the rate of absorption of H-2 by kidney indicated that this antigen may be expressed in amounts comparable to an H-2 antigen in kidney. Immunization with kidney tissue resulted in a strong cytotoxic antibody response. The number of bone marrow cells expressing this antigen (40-50%) was well beyond what could be accounted for by T lymphocytes in bone marrow. In addition, a nonlymphoid tumor, the P815Y mastocytoma, was positive by cytotoxicity and by absorption. The extensive nonlymphoid expression and antigenic strength of Ly-6.2 raises the possibility that this serologically defined lymphocyte alloantigen will have histocompatibility effects when allografts of the appropriate tissues are examined.

Hapten-Sandwich Labeling

Abstract Bifunctional reagents have been prepared which permit extensive coupling of haptens to antibodies with retention of antibody-binding capacity. This modification procedure increases the sensitivity with which cell-surface antigens can be labeled for fluorescent or electron microscopy by the hapten-sandwich method to a level comparable to or greater than may usually be achieved by conventional indirect techniques. With two different haptens, and amplifying with anti-hapten antibodies bearing fluorescent or other markers, different antigens can be distinguished readily on a single cell or on separate cells. Mouse alloantigens detected only with difficulty by conventional fluorescence can be discerned clearly and with high specificity. The reagent, either methyl hydroxybenzimidate (HB) or methyl 3,5-dihydroxybenzimidate (DHB), is first reacted with a diazonium phenyl hapten. Since the resulting azoderivative retains the imido ester function, the reaction solution can be used directly to amidinate antibodies. Both HB and DHB are easily prepared and may be stored for use as needed. With these reagents, the hapten-sandwich procedure may be applied to proteins other than immunoglobulins, e.g., hormones and mitogens. In addition to labeling antigens for fluorescent or electron microscopy, haptenamidinated antibodies or other ligands may be used in conjunction with appropriately modified antihapten amplifiers for a variety of purposes in cell biology, such as radioisotope studies, selective cell killing or suppression, and isolation of membrane antigens.

Genetic regulation of the antibody response to H-2Db alloantigens in mice. III. Inhibition of the IgG Response to noncongenic cells by preimmunization with congenic cells.

When B10.A (5R) mice (H-12i5) are immunized with spleen cells from congenic B10 mice (H-12b), they respond to alloantigens of the H-2Db region by producing antibodies of only IgM type. In contrast, they produce both IgM and IgG antibodies when immunized with A.BY cells (H-2b) that carry other foreign cell surface antigens (non-H-2) in addition to H-2Db. Preimmunization of 5R mice with two injections of congenic cells leads to an H-2Db specific inhibition of the IgG response to a subsequent immunization with A.BY cells. It is concluded that congenic B10 cells fail to activate helper T cells which are necessary to induce the switch from IgM to IgG production. Instead T- or B-cell tolerance may be induced with prohibits the subsequent IgG response to A.BY cells, possibly by way of suppressor T cells which may act either on B cells directly or via helper T cells.

The use of cross-reacting microbial antigens to abolish tolerance produced to transplantation mouse antigen

Immunization of CC57BR mice tolerant toCandida albicans antigens and to related C3H mouse transplantation antigens and to group A (type I)Streptococcus containing antigens cross-reacting with the donor's tissues partially abolished tolerance to C3H mouse alloantigens. This was manifested as shortening of the survival of skin allografts and an increase in the rate of elimination of51Cr-labeled donor's lymphocytes from the lymph nodes and spleen. The immune response toC. albicans antigens was not restored.

Genetic regulation of the antibody response to H-2Db alloantigens in mice. II. Tolerance to non-H-2 determinants abolishes the antibody response to H-2Db in B10.A(5R) mice.

B10.A(5R) mice (H-2i5), immunized with spleen cells from congenic B10 mice (H-2b), responded to alloantigens of the H-2Db region by producing antibodies of only IgM type. In contrast, they produced both IgM and IgG antibodies when immunized with noncongenic H-2b cells that carry other foreign cell surface antigens (non-H-2) in addition to H-2Db. A hypothesis was proposed comparing the H-2Db antigen on a congenic cell to a hapten on a nonimmunogenic carrier which fails to induce T-cell helper function responsible for the switch from IgM to IgG secretion in B cells. Data presented here confirmed this hypothesis. 5R mice rendered tolerant to the relevant non-H-2 antigens were unable to mount the anti-H-2Db IgG response in a noncongenic immunization. Tolerance induction did not lead to abrogation of the T-cell mediated cytotoxicity.

Adjuvant activity of mycobacterial fractions. III. Adjuvant effect of cell wall of Mycobacterium bovis BCG on cell-mediated cytotoxicity in mice.

A quantitative assay and characterization of oil-attached cell wall of Mycobacterium bovis BCG (BCG-CWS) which stimulates cell-mediated immunity of spleen cells to alloantigens in mice were carried out by an in vitro cell-mediated cytotoxicity test using 51Cr-labeled target cells. C57BL/6J mice (H-2b) were immunized intraperitoneally with mastocytoma cells (H-2d) with or without oil-attached BCG-CWS. The cytotoxicity, comparable to that of spleen cells from mice immunized with mastocytoma cells (3 X 10(7)), could be induuced in spleens of mice immunized with a mixture of mastocytoma cells (10(4)) and oil-attached BCG-CWS. The enhancing effect persisted from 55 days or more after the alloantigenic immunization. Oil-attached BCG-CWS enhanced cell-mediated cytotoxicity of T cells in the spleen and the mesenteric lymph node, but not in the thymus. The cytotoxicity showed specificity toward the alloantigen used for immunization. In addition to BCG-CWS, the cell walls of Nocardia rubra and Corynebacterium diphtheriae PW8 and the peptidoglycolipids of Mycobacterium tuberculosis Aoyama B were found to be potent stimulants of cell-mediated cytotoxicity in mice. Oil-attached BCG-CWS did not enhance humoral response to mastocytoma cells but enhanced cell-mediated cytotoxicity when viable mastocytoma cells were used as antigen. The above result was supported by the fact that anti-hapten antibody response induced by viable trinitrophenyl (TNP)-mastocytoma cells (10(4)) plus oil-attached BCG-CWS did not increase to the maximum levels as was observed in mice immunized with a larger number of mastocytoma cells (3 X 10(7)) alone, while cell-mediated cytotoxicity induced by the same treatment increased to the maximum level obtained by immunization with mastocytoma cells (3 X 10(7)) alone.

Human lymphocytes recognise mouse alloantigens.

H-2 LD antigens1 of the mouse major histocompatibility complex and the lymphocyte-activating determinants of the Mls (ref. 2) locus induce vigorous lymphocyte proliferation and enhance generation of cytotoxic effector cells in allogeneic combinations (ref. 3 and D. J. Schendel, and F. H. Bach, unpublished). These antigens do not serve well as targets for cell-mediated lympholysis (CML)—a function which in the mouse is performed by the classical H-2 determinants, the SD antigens4,5. A similar differential function of LD and SD antigens is found in human allograft reactions in vitro6.

New lymphocyte antigen system (Lna) controlled by the Ir region of the mouse H-2 complex.

A new system of lymphocyte alloantigens in mice is described. This Lna (lymph-node antigen) system is associated with the Ir region of the H-2 (histocompatibility-2) gene complex. It has the following distinctive characteristics: (1) The gene or genes controlling these antigens has been mapped in the Ir (immune response) region between H-2K and Ss-Slp. (2) The antigens are most readily detectable on lymph-node cells, although they are also expressed on peripheral blood lymphocytes, splenic lymphocytes, and thymocytes. (3) Cytotoxicity against only about half of lymph-node cells is consistently observed. (4) Cytotoxic antibody titers against these antigens are strikingly high-more than 2000 by (51)Cr-release and up to 100,000 in the microcytotoxic test. (5) At least two, probably allelic, forms of the antigen(s) have been defined, one associated with the H-2(k) haplotype and one with the H-2(a) haplotype. (6) Antisera against Lna contain multiple antibody specificities that can be fractionated by absorption either with certain recombinants or with other H-2 halotypes that have crossreactive antigens. The antisera against Lna may be of value for definition and characterization of the products of the Ir and MLR (mixed lymphocyte reaction stimulatory) genes associated with the H-2 complex.