MRC OX8 Research Articles

Alternative processing of RNA in lymphoid cells

In a recent paper Zamoyska etal. ~ have shown that there are at least two messenger (m)RNA species in thymocytes for the Lyt 2 cell surface marker antigen of mouse cytotoxic/suppressor cells (the equivalent antigen is CD8 in humans and MRC OX-8 in rats). The two mRNA species differ in that one has a 31 base pair deletion in the sequence coding for the cytoplasmic region which, with the shift in reading frame, results in a cytoplasmic tail of only 3 amino acids compared to 28 in the larger form. This provides a molecular explanation for the observation that two polypeptide chains c~ and c~' of the Lyt 2 antigen are found on thymocytes and is consistent with the biochemical studies of Luscher eta/. 2 which suggest that the smaller e' form, differs only in having a shorter cytoplasmic tail. Given the lack of understanding of the role of cytoplasmic tails of membrane glycoproteins in general, the functional significance of this is unclear. However, it is possible that the shorter form (c~') is not important in T-cell function as it and its mRNA are present in much lower amounts in mature T cells ~. Zamoyska et al. showed that the two mRNA species for Lyt 2 are derived from a single gene by alternative splicing at the RNA level 1. Mammalian genes are subdivided into exons which contain the coding and untranslated regions found in mRNA and these are separated by intervening sequences - introns. Both exons and introns are transcribed into a large nuclear RNA transcript and then the introns are spliced out to give the mature mRNA containing contiguous coding sequence, which is then translated in the cytoplasm. Introns vary greatly in size from about 50 to 10 000 or more base pairs but the positions of their boundaries with exons have characteristic sequences at which precise splicing occurs to give an mRNA containing all the required exons 3. However, alternative splicing can occur so that during the splicing event exons may be spliced out together with the introns resulting in a different protein product. This is what appears to happen in the Lyt 2 case described above, and many other examples of alternative splicing have been described in a number of tissues 4-6. One of the best examples is that of immunoglobulin heavy chains. B lymphocytes can express both IgM and IgD simultaneously at the cell surface. The initial RNA transcript runs from the leader and the rearranged V domain exons (V-D-J) through the C domain exons for both i and 6 chains 7-9. This is processed to give the V domain together with either the I~ or 6 C domains, i.e. to

T cells that help B cell responses to soluble antigen are distinguishable from those producing interleukin 2 on mitogenic or allogeneic stimulation.

An mAb MRC OX-22, reactive with the high molecular weight forms of the rat leukocyte-common antigen, has revealed a heterogeneity among CD4+ T cells in this species. Approximately two-thirds are CD4+, OX-22+, and one-third are CD4+, OX-22-. This phenotypic heterogeneity was found to be associated with a functional one. CD4+, OX-22+ cells proliferated well in mixed leukocyte culture, responded to the T cell mitogen Con A, and produced IL-2 on activation. In contrast, the CD4+, OX-22- cells performed poorly in these assays, but unlike CD4+, OX-22+ cells, did provide effective help for B cells. By sampling supernatants from cultures containing primed B cells and either of the two CD4+ T cell subsets, it was shown that, when specific antigen was included in the cultures, those containing the OX-22- subset of CD4+ cells produced high levels of antibody and some IL-2, whereas those containing the OX-22+ cells produced neither. In contrast, when specific antigen was replaced by Con A, the B cell cultures supplemented with CD4+, OX-22+ cells synthesized much higher levels of IL-2 than those containing CD4+, OX-22- cells, but only the latter cultures produced detectable levels of antibody. The data show that inducer/helper T cells comprise two functional subsets: one that, on appropriate stimulation, synthesizes high levels of IL-2, and may therefore be presumed to play an important role in cell-mediated immunity, and another that plays an essential role in humoral responses to soluble antigens. The significance of this functional heterogeneity, with regard to the possible independent regulation of cellular and humoral responses, is briefly considered.

Puromycin aminonucleoside nephropathy: ultrastructure, glomerular polyanion, and cell surface markers.

Puromycin aminonucleoside nephropathy with heavy proteinuria and oedema was induced in rats by 10 consecutive daily subcutaneous injections of aminonucleoside (1.67 mg/100 g of body weight). The main ultrastructural lesions were vacuolation of podocytes and total fusion of foot processes with loss of colloidal iron-reactive polyanion layer on the epithelial surface adjacent to the basement membrane. On the other hand the outer surface of podocytes and intravacuolar granular substance stained with colloidal iron. In scanning electron microscopy of freeze-fractured tissue the swollen podocytes and the urinary spaces displayed granular and filamentous precipitates. Seven cell surface antigens were examined by indirect enzyme immunohistochemistry with a series of MRC OX monoclonal antibodies. Glomeruli of control rats exhibited rare isolated Ia- positive endocapillary cells, possibly monocytes; these elements were significantly reduced in puromycin aminonucleoside nephropathy but there was an increase in Ia- positive cells in the cortical interstitium. Control kidneys harboured scanty interstitial T lymphocytes. These latter, especially the T8- positive cytotoxic/suppressor subpopulation, were markedly augmented in puromycin aminonucleoside nephropathy. The expression of class I histocompatibility antigens and of differentiation antigens (Thy 1) was not altered by aminonucleoside.

The roles of interdigitating cells and natural killer cells in the rapid rejection of allogeneic lymphocytes.

The fate of radiolabeled allogeneic thoracic duct lymphocytes injected into congenitally athymic, nude rats was followed by autoradiography and electron microscopy. The allogeneic cells entered the host lymphoid organs at a normal rate, but once inside the lymphoid tissue they were rapidly phagocytozed by interdigitating cells (IDC) situated in the lymph node paracortex and the splenic periarteriolar lymphoid sheaths. Neither host nor donor T cells were required to initiate phagocytosis, as purified donor B cells were also avidly ingested by the athymic host IDC. Compared with fully allogeneic cells phagocytosis of semiallogeneic donor cells was much less efficient. When natural killer cell activity was blocked by preinjecting the recipients with antibodies against natural killer cells (anti-asialo GM1 or MRC OX-8) phagocytosis of the allogeneic cells was strongly reduced. As IDC did not bind these antibodies, the finding indicates that natural killer cells were needed to discriminate between own and foreign lymphocytes and to kill the allogeneic cells, which were then ingested by surrounding IDC. This was further supported by the observation that dendritic, constitutively Ia+ cells from peripheral lymph, phenotypically identical to IDC, did not lyse or phagocytoze allogeneic lymphocytes in vitro.

Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H.

The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.

Changes in brain serotonin levels affect leukocytes, lymphocytes, T-cell subpopulations and natural killer cell activity in rats

The intraventricular injection of 5,6-dihydroxytryptamine into desipramine-treated rats caused a marked reduction in serotonin levels 5 and 10 days after treatment without significantly affecting catecholamine levels. In these animals, total leukocytes were increased and lymphocytes decreased. The number of helper T-cells ( W3 25 + ) was significantly decreased after 5 days, whereas the numbers of total T-cells ( W3 13 + ) and suppressor T-cells (MRC OX8) were increased after 10 days. The percentages of total T-cells, helper T-cells and suppressor T-cells were significantly reduced after 5 days. Natural killer cell (NK) activity was markedly reduced after 5 days. This finding suggests that central serotonin-containing neurons might be involved in the regulation of certain parts of the peripheral immune system.

Interactions between T helper cells and dendritic cells during the rat mixed lymphocyte reaction.

This paper describes the interactions between dendritic cells (DC) and T helper (Th) cells in the rat mixed lymphocyte reaction (MLR). Th blasts that are actively proliferating were generated in a 5 d primary MLR; resting Th memory cells were derived from a 10-12 d MLR. The DC were purified from thoracic duct lymph derived from rats whose mesenteric lymph nodes had been removed. The results show that DC are the major stimulators in the primary MLR and also for the restimulation of Th blasts and Th memory cells. Th blasts rapidly formed large clusters when cultured with DC but not with Ia+ macrophages or B cells. This interaction was not dependent on a major histocompatibility complex (MHC) difference between the T blasts and the DC. Con A-activated T and B blasts also formed clusters when cultured with DC. Th memory cells formed small clusters with DC, but, in a different assay in which clusters are dispersed, we detected an antigen-specific interaction between Th memory cells and DC. The monoclonal antibodies W3/25 (anti-rat CD4) and MRC OX-6 (anti-MHC class II) blocked proliferation in the primary MLR and also inhibited the restimulation of Th memory cells. However, the restimulation of Th blasts in a secondary MLR was only blocked by MRC OX-6. These results suggest that there are different requirements for the restimulation of T blasts than for the activation of primary or memory Th cells. W3/25 and MRC OX-6 did not affect the clustering of T blasts with DC but they both inhibited the antigen-specific binding of Th memory cells to DC. The data suggest that the CD4 (W3/25) antigen is involved in antigen-specific interactions between Th cells and DC.

Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. Mediation of specific suppression by T helper/inducer cells.

DA rats grafted with major histocompatibility complex-incompatible PVG heart grafts and treated with cyclosporine (CY) for 10 d do not reject their grafts, and develop a state of specific unresponsiveness toward PVG allografts. Cells from these animals tested in an adoptive transfer assay were incapable of restoring PVG graft rejection, and capable of specifically inhibiting the capacity of adoptively transferred normal lymph node cells (LNC) to do so. They effected third party Wistar/Furth (W/F) graft rejection, however. Adoptive transfer assays with purified subpopulations of the lymphocytes that mediated this effect showed that W3/25+ T cells of the helper/inducer subclass, when injected alone, failed to restore rejection, and were also able, when injected with normal LNC or the W/25+ cells separated from them, to prevent these cells from effecting rejection. MRC OX8+ T cells of the cytotoxic/suppressor subclass, B cells, and serum from rats with long-surviving grafts all failed to inhibit the allograft responsiveness of normal LNC, and thus were not identified as mediators of the state of specific unresponsiveness. These results show that the specific unresponsiveness that develops in rats with long-surviving grafts, and which, in part at least, is responsible for prolonged graft survival, is due to an alteration in the alloreactivity of the helper/inducer subclass of T cells. These cells not only lack the capacity to initiate a rejection response against the alloantigens of the graft, but also have the ability to inhibit the capacity of normal W3/25+ cells to do so.

Immunohistologic Analysis of the T-Cell and Macrophage Infiltrate in 1,2-Dimethylhydrazine-Induced Colon Tumors in the Rat<xref ref-type="fn" rid="FN1">2</xref>

The infiltrating mononuclear cell (MNC) type, and the localization of major histocompatability class I and class II antigens within 1,2-dimethylhydrazine (CAS: 540-73-8)-induced colonic tumors and normal colonic mucosa in WF/Hsd BR rats were investigated by immunoperoxidase staining of frozen sections with the use of monoclonal antibodies. Infiltrating T-cells stained with monoclonal antibodies W3/13, W3/25, and MRC OX 8; W3/13+ cells were predominant. The most numerous and consistently observed infiltrating cell type was an la antigen-bearing (MRC OX 6+, MRC OX 17+) macrophage. A smaller subpopulation of macrophages, staining with W3/25, showed a similar distribution within tumors. In adenomas and in some well-differentiated adenocarcinomas, the infiltrating MNC were concentrated at the tumor periphery and were in close proximity to neoplastic epithelia, but without evidence of consequent tumor cell necrosis. In all other tumors, infiltrating host cells were confined to the connective tissue stroma dividing clusters of neoplastic glands. The extent of cellular infiltration and the phenotypes of infiltrating cells did not correlate with the degree of tumor differentiation or with tumor size. Expression of MHC class I antigens (MRC OX 18) by tumor cells did not differ from that of normal epithelia, and neoplastic epithelia were uniformly negative for class II antigens (MRC OX 6, MRC OX 17). The data do not support a role for cytotoxic macrophages or T-cells in the local response to colon tumors.

A novel cell-surface antigen expressed on most leukocytes and a minor cortisone-resistant population of thymocytes in rats: characterization by monoclonal antibodies.

A cell-surface antigen on rat lympho-hemopoietic cells was determined by using a monoclonal antibody, R2-1B3 (1B3). The 1B3 antibody, when tested for its reactivity with different hemopoietic cells by cytofluorography with a FACS analyzer, labeled more than 80% of lymph node, spleen, and bone marrow cells and 10-20% of thymus cells. Cytofluorographic analysis performed on purified rat T cells, B cells, macrophages, and granulocytes demonstrated that the antigen defined by 1B3 was readily detectable on all of these cell types, with the greatest expression on B cells. A minor population of thymocytes that were labeled by 1B3 appeared to be cortisone-resistant and were located mainly in the thymic medulla. These 1B3 positive thymic cells seemed to be functionally more mature than 1B3-negative thymus cells as suggested by the fact that the cytotoxic treatment of thymus cells with 1B3 antibody and complement (C) resulted in significant reduction of their responsiveness to phytomitogens and lymphokines derived from concanavalin A (con A) activated rat spleen cell cultures. Immunochemical data showed that 1B3 antibody recognized the broad ill-defined band with a molecular weight of 32K to 47K daltons as estimated by SDS-polyacrylamide gel electrophoresis. These data indicate that the 1B3 defined antigen is distinct from other, previously reported, antigens on rat lymphoid cells including leukocyte-common (L-C) and MRC OX-22 antigens, and that this 1B3 antibody is a useful reagent for analyzing the intrathymic differentiation of T cells in rats.

Nonspecific lymphocyte responses in F344 and LEW rats: susceptibility to murine respiratory mycoplasmosis and examination of cellular basis for strain differences

Mycoplasma pulmonis produces a mitogen which may play a role in the pathogenesis of murine respiratory mycoplasmosis in rats. Since LEW rats are more susceptible to this disease than F344 rats are, these two strains were used to examine a possible association between disease severity and the level of nonspecific lymphocyte stimulation by mitogens, including M. pulmonis membrane preparations. F344 and LEW spleen, lung, blood, and lymph node lymphocytes were exposed to various mitogens. LEW lymphocytes gave a significantly higher response to mitogenic stimulation, regardless of their anatomical source. These differences in lymphocyte responsiveness were primarily due to differences within the nonadherent cell population. Significantly higher numbers of W3/25+ (T helper) cells were found in LEW lymphoid populations, whereas no difference was found in MRC OX-8+ (T suppressor/cytotoxic) cells. These data suggest an association between disease severity and host responsiveness to nonspecific stimuli.

Evidence that rat renal allografts are rejected by cytotoxic T cells and not by nonspecific effectors.

Infiltrating cells were harvested on day 5 from rat renal allografts (LEW X BN into LEW) that were either rejecting (untreated) or healthy (cyclosporine [CsA]-treated or passively enhanced with LEW anti-BN serum). Similar numbers of cells were harvested from rejecting (mean 11 X 10(7), range 8-15 X 10(7], CsA-treated (6 X 10(7), 5-7 X 10(7], and enhanced (9 X 10(7), 7-10 X 10(7] grafts. These cells were a heterogeneous population of mononuclear cells. Fluorescence-activated cells sorter analysis after labeling with a range of monoclonal antibodies showed that they all labeled with MRC OX-1 (against the leukocyte-common antigen), and that about one-third of infiltrating cells bound MRC OX-19 (pan-T cells) and one-third bound MRC OX-12 (B cells), with little difference between the rejecting, CsA-treated or enhanced grafts. However, the ratio of the numbers of W3/25-positive cells (helper T cells and macrophages) to MRC OX8-positive cells (cytotoxic T cells and natural killer cells) was less in untreated (mean 0.6, range 0.5-0.6) than in CsA-treated (1.3, 0.8-1.6) or enhanced (2.1, 1.8-2.4) grafts. Using a 6-hr chromium-release assay, cells from untreated, CsA-treated, and enhanced grafts showed similar levels of nonspecific cytotoxicity (against Y3 myeloma cells), but only untreated grafts showed alloantigen-specific target cell lysis (against BN concanavalin A blasts). These results suggest that specific cytotoxic T cells rather than nonspecific responses play an essential role in allograft rejection in the rat.

Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes.

The molecular forms and antigenic heterogeneity of the leukocyte-common antigen (L-CA) of rat lymphocytes have been analyzed. Thymocytes show one main band at 180 kDa, T cells four bands at 180, 190, 200 and 220 kDa and B cells one broad band at about 240 kDa. T helper and T cytotoxic cell subsets show the same four bands with some differences in the proportion of each. Four mouse monoclonal antibodies (MRC OX-1, 28, 29 and 30) reacted with all molecular forms of L-CA and fell into two sets that were noncompetitive in binding to L-CA (MRC OX-1, 28, 29 vs. OX-30). The antigenic determinants seen by all these antibodies were lost when L-CA was reduced and alkylated. Three antibodies (MRC OX-22, 31 and 32) reacted selectively with B cells, T cytotoxic cells and about 2/3 of T helper cells. OX-22 and OX-31 competed for binding but were noncompetitive with OX-32. All these antibodies bound to a subfraction of the 190, 200 and 220-kDa forms of T cell L-CA but not at all to the 180-kDa form of T cells or thymocytes. One antibody bound to B cells only (MRC OX-33) and precipitated a subfraction of B cell L-CA. With all the antibodies that did not label thymocytes the antigenic determinants survived reduction and alkylation. Subsequent proteolysis with trypsin then destroyed all determinants except the one reacting with MRC OX-22 antibody. In this case tryptic peptides retained full antigenic activity which was, however, destroyed by further proteolysis with pronase.

Changes in lymphocyte subsets during the course of experimental allergic neuritis.

The percentages of various leucocyte subsets in the blood, spleen and popliteal lymph nodes of animals with experimental allergic neuritis (EAN) were determined at various times between the initiation of the disease and recovery. The total cell yield from these tissues and the weights of the spleens and lymph nodes were recorded. Clinical and histological signs of disease were assessed at the same time points. Normal and adjuvant control animals were also studied. The most marked change observed was that the percentage of MRC OX 8+ ('suppressor/cytotoxic') cells in the blood of animals with EAN was significantly below the normal value during the most severe clinical disease, and then returned to normal during recovery. Adjuvant controls did not show this change. No significant differences were observed in the other lymphocyte subsets studied (surface Ig+, Ia+, W3.13+ and W3.25+ cells) or in the total white cell count in the blood. There were no changes from normal values in any of the parameters examined in the spleen. The popliteal lymph nodes were enlarged in both adjuvant controls and animals with EAN and both showed a decrease in the percentage of W3.25+ lymphocytes from normal unenlarged nodes.

The response of gut-associated T lymphocytes to intestinal viral immunization.

The cell-mediated immune response by the gut-associated lymphoid tissues to antigens within the intestinal tract is poorly understood. Our objective was to investigate the antigen-specific T cell proliferative response and cytotoxic T lymphocyte (CTL) response of cells from the GALT after enteric immunization with vaccinia virus. Lymphocytes able to proliferate in the presence of vaccinia virus in vitro were found in large numbers in mesenteric lymph nodes (MLN) 6 days after the injection of vaccinia virus into the lumen of the small bowel. The MLN at this time also contained vaccinia-specific CTL, but unlike the proliferating cells, which were found for several weeks after immunization, the CTL were demonstrable in the MLN for only a few days. Peyer's patches were found to contain neither antigen-stimulated proliferating cells nor CTL. The viral-specific proliferating lymphocytes from the MLN 10 days after immunization were sIg-, monoclonal antibody W3/25+, MRC OX-8- large lymphoblasts. The vaccinia-specific CTL were also large lymphoblasts, but they belonged to the W3/25-, OX-8+ subset. Thus, a strong T helper and cytotoxic T lymphoblast response is generated within the MLN after viral challenge of the gut.

Membrane antigen phenotype of sensitized T lymphocytes mediating tuberculin-delayed hypersensitivity in rats

The membrane antigen phenotype of immune lymph node cells (LNC) which mediate tuberculin-delayed hypersensitivity (DH) in Lewis rats was examined. The results show that the T-cell population which expresses the RT7.1 alloantigen defined by the BC 84A monoclonal antibody contained cells capable of transferring DH. Separation of the RT7.1-positive T-cell population with the monoclonal antibodies W3/25, MRC OX-8, or DS 4.23 (which defines the RT6.1 alloantigen) revealed that either the W3/25-positive or the RT6.1-negative T-cell subpopulations contained DH effector cells, whereas the corresponding MRC OX-8-positive or RT6.1-positive T-cell subpopulations did not. Moreover, when the W3/25-positive T-cell subpopulation was divided into either RT6.1-positive or RT6.1-negative T-cell subsets, only the W3/25-positive, RT6.1-negative subset transferred DH. These results indicate that the effector cells that mediate tuberculin DH are contained within the immune T-cell subset which bears both the RT7.1 and the W3/25 markers, but lacks both the MRC OX-8 and the RT6.1 markers.

The mediators of acquired resistance to Listeria monocytogenes are contained within a population of cytotoxic T cells

T cells from peritoneal exudates induced in rats convalescing from a recent infection of Listeria monocytogenes were fractionated into two subsets based on their ability to bind monoclonal antibodies to cell-surface determinants that are expressed on some but not all peripheral T cells. Two phenotypically distinct subsets, one recognized by the antibody MRC OX8 and the other by W3/25, were assayed for their protective capacity in Listeria-challenged recipients, and for their ability to kill unmodified syngeneic fibroblasts in vitro. The two activities were mediated by the OX8 + subset which comprised approximately half the T cells in the exudates.

MRC OX-19: a monoclonal antibody that labels rat T lymphocytes and augments in vitro proliferative responses.

A mouse monoclonal antibody, designated MRC OX-19, has been prepared that binds to virtually all rat thymocytes, T lymphocytes and a maximum of 2% B lymphocytes. Immunoperoxidase studies on tissue sections showed no reactivity with nonlymphoid cells in intestine, thymus, spleen and lymph node. The antigen recognized by MRC OX-19 antibody was identified by metabolic and cell surface labeling of thymocytes followed by immunoprecipitation and sodium dodecyl sulfate gel electrophoresis. It is a surface glycoprotein of Mr = 69 000. When included in in vitro assays for T cell functions, MRC OX-19 increased proliferation stimulated by allogeneic spleen cells or the lectins phytohemagglutinin and concanavalin A. The antibody itself was not mitogenic and its stimulatory effect could be correlated with an increase in interleukin 2 production. Taken together the data suggest that MRC OX-19 could be the equivalent of mouse Ly-1 antigen and human T1 antigen.

The cellular basis of allograft rejection in vivo. III. Restoration of first-set rejection of heart grafts by T helper cells in irradiated rats.

An adoptive transfer model was used to examine the subpopulations of lymphocytes required to effect first-set rejection of directly vascularized heart allografts. PVG heart grafts are not rejected in irradiated DA hosts for at least 50 days. The adoptive transfer of 5 X 10(7) syngeneic lymph node cells (LNC) restores rejection to 14.4 +/- 2.4 days (mean +/- SD). Subpopulations of LNC, were separated by an indirect "panning" technique using the mouse antirat monoclonal antibodies W3/13, MRC OX8, or W3/25 to deplete the unwanted subsets of cells. Each subpopulation was tested, in a number equivalent to the number present in 5 X 10(7) normal LNC, for its ability to cause the rejection of heart grafts. Whole T cells (W3/13+) or helper/inducer T cells (W3/25+) restored graft rejection to 16.4 +/- 3.8 d and 16.0 +/- 2.4 days, respectively. Neither cytotoxic/suppressor T cells (MRC OX8+) nor B cells (Ig+) restored rejection. Indirect immunoperoxidase stains of the grafts showed that although W3/25+ cells predominated in the rejected tissue, MRC OX8+ cells were also present even in grafts from rats restored with inocula that contained less than 1% MRC OX8+ cells. Examination of lymphoid tissues suggested that the MRC OX8+ cells might be of host origin. By the time the grafts were rejected in irradiated hosts, significant thymic regeneration had occurred and there were large numbers of MRC OX8+ cells present in the thymus, as well as some in lymph nodes and spleen.

Leukocyte subsets in first-set rat cardiac allograft rejection. A serial immunohistologic study using monoclonal antibodies.

For an immunohistochemical analysis of the cells infiltrating rat cardiac allografts undergoing unmodified first-set rejection, functioning Wistar Furth (RT1u) cardiac allografts transplanted to Lewis (RT1(1)) recipients were arbitrarily removed at days 3-6 posttransplantation, and were evaluated histologically using a series of monoclonal antibodies (MAbs) of known cellular distribution, the binding of which was visualized by an indirect immunoperoxidase technique. Sequestered cells present in cryostat sections were segregated numerically on the basis of staining for MAbs detecting T cells, some NK cells, and neutrophils (W3/13); cytotoxic suppressor T cells and most NK cells (MRC OX8); helper T cells and all macrophages (W3/25); B cells and a macrophage subpopulation (MRC OX6, Ia common part determinant); immature T and B cells (MRC OX7, Thy 1.1); and all leukocytes (MRC OX1, LCA). The proportion of cells stained for each MAb was expressed as a percentage of all estimated leukocytes as determined directly on the hematoxylin counterstained sections. Macrophages expressing the W3/25 and MRC OX6 antigens were enumerated on the basis of intracytoplasmic accumulation of previously administered colloidal carbon. There was a significant fall in the relative proportion of W3/25+ carbon- cells within allografts as a function of time. The proportion of mononuclear cells staining for W3/13 and for MRC OX8 showed no statistically significant variation during the period of study. There was a correlation at all periods between the proportional sum of W3/25+ carbon- cells and MRC OX8+ cells and the proportion of W3/13+ mononuclear cells present. The W3/25+ carbon-:MRC OX8+ ratio showed a significant linear decrease with duration of allograft placement. The proportion of carbon+ cells expressing the MRC OX6 and the W3/25 antigens showed a highly significant rise as a function of time. Nonspecific esterase-stained cells showed a similar sequential pattern. These data provide well defined immunohistologic parameters of leukocyte subsets present in unmodified first-set cardiac allograft rejection in the rat.