T and B lymphocytes in New Zealand Black mice: an analysis of the theta, TL and MBLA markers

The lymphocytes of thymus, blood, spleen, lymph nodes and bone marrow were studied in NZB mice between the ages of 1 and 14 months, and compared with lymphocytes of A and CBA strain mice of the same age. By standard cytotoxicity techniques, the proportion of cells possessing the θ, TL and MBLA markers was found to be similar in NZB in mice and controls. In 14-month old NZBs, whose spleen was largely replaced by reticulum cell sarcoma, and in younger recipients of the passaged tumour, there was a reduction in the percentage of θ and MBLA-positive cells in the spleen. In a few mice at 4 and 9 months, small numbers of MBLA-positive cells were present in the thymus and there was a corresponding decrease in θ-positive cells. TL-positive cells were not present outside the thymus, and θ-positive cells were not present in the bone marrow in unusual numbers. NZB peripheral lymphocytes appeared to have the same surface concentration of θ as those of A or CBA mice, as judged by anti-θ titration curves. The reticulum cell sarcoma was shown to be θ-negative and MBLA-negative, while an NZB thymoma was θ-positive, TL-positive and MBLA-negative. It was concluded that the peripheral lymphoid organs contain a large population of T lymphocytes of abnormal character.     

Natural cytotoxic autoantibody against thymocytes in NZB mice

NZB mice were found to produce natural thymocytotoxic autoantibody in high prevalence and antibody titre. This autoantibody in NZB mice was detectable by the cytotoxicity test at both 4°C and 37°C; the prevalence and antibody titre were generally higher at 4°C. Mice of other strains also produced natural thymocytotoxic autoantibody although in lower prevalence and antibody titre and in some instances the activity was greater at 37°C than at 4°C. Natural thymocytotoxic autoantibody in NZB mice reacted equally with the thymocytes of virtually all strains of mice tested but to a lesser degree with the thymocytes of SJL/J mice. A serum pool obtained from old NZB mice had an extremely high titre of natural thymocytotoxic autoantibody (1:1024 at 4°C). Nevertheless, the cells in lymph nodes, spleen and blood leucocytes were only partially sensitive to this serum pool, and bone marrow cells were for the most part negative. By absorption, the antigen reacting with natural thymocytotoxic autoantibody was found in thymus, lymph node, spleen and brain of adult mice, thymus of newborn mice and some leukaemias. Natural thymocytotoxic autoantibody in NZB mice was an IgM-globulin as determined by sensitivity to 2-mercaptoethanol treatment and by Sephadex G-200 column chromatography in contrast to other natural antibodies (antinuclear, antierythrocyte and G antibodies) of IgG-globulin class. NZB mice also produced natural antibodies against thymocytes of the rat and the hamster; these antibodies were species-specific and did not react with the thymocytes of any but the homologous species.     

Changes in lymphoid populations of ageing CBA and NZB mice

Changes in subpopulations of lymphoid cells of normal (CBA) and autoimmune (NZB) mice were studied as a function of age, by observing migration patterns of ⁵¹Cr labelled lymph node, spleen and thymus cells from donors aged 8 days to 12 months. The method permits analysis of the proportions and numbers of recirculating and non-recirculating lymphocytes in lymphoid compartments. Changes in the lymphoid populations of CBA mice were found, which could be attributed to the normal processes of maturation and senescence. In NZB mice relative and absolute decreases in the recirculating cell content of lymph node and spleen were observed which coincided with the time of development of autoimmunity. The significance of these results, in relation to altered immunocompetence with age, is discussed.     

Nomarski differential interference contrast studies of murine lymphocytes

Nomarski differential interference contrast microscopy (DIC) of lymphocyte surface morphology was combined with immunofluorescence studies of T and B cell markers on the thymus, lymph nodes, spleen, peripheral blood lymphocytes and thoracic duct lymph of female CBA mice. DIC identified smooth cells and several categories of villous cells; more extreme forms were present in lymph. Most B cells seemed to belong to the smooth group and most peripheral T cells to the villous group. Thymus cells were almost entirely smooth, but treatment with cortisone increased the proportion of villous cells to 50%. The surface morphology of lymphocytes was highly labile preventing direct identification or separation of T and B cells. In vivo removal of T cells by adult thymectomy, lethal irradiation and bone marrow reconstitution caused the villous cells to decrease. During recovery from irradiation, T lymphocytes tended to parallel villous cells, B lymphocytes smooth cells, but there were differences between the spleen and lymph nodes. Mice deprived of T1 cells by adult thymectomy showed a modest decrease of smooth cells in the spleen and blood; mice depleted of T2 cells by anti-lymphocyte serum, or which were naturally deficient in T2 cells, were markedly lacking in villous cells. Thoracic duct lymph, which is rich in T2 cells, had a high proportion of extremely villous lymphocytes. Exposure to lymph induced extreme villous features in lymph node cells, and it was found that the thoracic duct lymph was markedly hypertonic to serum, although varying in osmolarity throughout the day. It is suggested that the villous shape of T2 cells is a circulatory adaptation, necessitated by the peculiar character of the lymphatic system in mice.     

Sex differences in the regulation of experimentally induced autoantibodies in (NZB x NZW)F1 mice.

The ability to induce autoantibodies to erythrocytes in male and female (NZB x NZW)F1 mice was examined. Female (NZB x NZW)F1 mice were shown to produce significantly more autoantibody than the male (NZB x NZW)F1 mice. The regulation of this experimentaly induced autoantibody was studied by examining the ability of male and female (NZB x NZW)F1 mice to generate antigen-specific suppressor cells. A sex difference was found in the ability to generate these suppressor cells. Male mice generated antigen-specific suppressor cells in response to rat RBC which were capable to suppressing the experimental induction of red cell autoantibodies whereas female mice were unable to generate those antigen-specific suppressor cells.     

Perforin mRNA expression in the inflamed tissues of NZB/W F1 lupus mice decreases with methylprednisolone treatment.

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this study, the authors examined perforin mRNA levels in the kidney, spleen, liver, lung, heart, and brain of NZB/W F1 lupus mice and NZW mice. Perforin mRNA levels in the kidney, spleen, liver, and lung of NZB/W F1 mice increased significantly with age, whereas those in the heart and brain of NZB/W F1 mice showed little change between 2 and 10 months of age. In all tissues examined in NZW, control mice perforin mRNA levels showed little change during the experimental period. In addition, the authors examined the effect of methylprednisolone (MPSL) on perforin gene expression in the tissues of NZB/W F1 mice. MPSL ameliorated the increase in perforin mRNA levels in the kidney, spleen, liver, and lung of NZB/W F1 mice. These findings suggest that perforin may contribute to tissue injuries in autoimmune lupus mice and that MPSL may be effective in lupus partly by decreasing perforin expression.     

Inflamed kidneys of NZB / W mice are a major site for the homeostasis of plasma cells

(NZB x NZW)F1 (NZB / W) mice develop a disease similar to human systemic lupus erythematosus (SLE), including autoantibody production, hypergammaglobulinaemia and inflammation of the kidneys. It is known that large numbers of lymphocytes infiltrate the kidneys of these mice. Here, we compare the roles of bone marrow, spleen and inflamed kidneys of NZB / W mice in the activation of B cells and the persistence of antibody-secreting cells (ASC). ASC are present in the kidneys of NZB / W mice with full-blown disease, as many as in the spleen and bone marrow. The specificity of the ASC in the inflamed kidneys is not restricted to self-antigens. After immunization of NZB / W mice with ovalbumin (OVA) the OVA-specific ASC are found initially in the spleen. Weeks later, OVA-specific ASC are found in high numbers in the bone marrow and the kidneys of these mice, but no longer in the spleen. As determined by FACS, B cells with a germinal center phenotype (B220(+) / PNA(+)) are found only in very low numbers in the kidneys, but in high numbers in the spleen of NZB / W mice. Germinal centers could not be detected in the kidneys, but in the spleen, and plasma cells appear to be scattered over the tissue. These data suggest that in autoimmune NZB / W mice, plasma cells generated in immune reactions of secondary lymphoid organs, later accumulate and persist in the inflamed kidneys, were they enhance the local concentrations of Ab and immunocomplexes. These experiments identify the inflamed kidneys of NZB / W mice as a site of prime relevance for the homeostasis of plasma cells, irrespective of their specificity.     

Lack of high avidity IgM anti-ssDNA antibodies in cells from autoimmune-prone and autoimmune mice

Non-autoimmune prone CBA mice were compared with autoimmune prone NZB, NZW, and (NZB x NZW)F1 mice for the ability of their splenic cells to produce anti-ssDNA-forming cells spontaneously in vitro, measured in the plaque forming cell assay. The number of antibody forming cells was measured and the relative avidity of antibody produced determined using a plaque inhibition assay. Splenic lymphocytes from young animals of a non-autoimmune strain (CBA/J) were shown to be capable of generating anti-ssDNA IgM antibody-forming cells in culture which displayed a higher avidity for antigen than that from autoimmune-prone or frankly autoimmune mice. Since an increased switching from IgM to IgG autoantibody production and defects in Fc-mediated signalling by IgG antibody have been identified in autoimmunity, we suggest that the metabolic block, normally in force in non-autoimmune-prone animals, accounts for this elevated avidity of IgM autoantibody.     

Proliferation of Ly-1 B cells in autoimmune NZB and (NZB x NZW)F1 mice

Spontaneous autoimmune disease in NZB and (NZB x NZW)F1 (B/W) mice is associated with a spectrum of lymphoproliferative abnormalities, but the relationship between autoimmunity and lymphoproliferation is poorly understood. Lymphomas occur commonly in NZB mice, but they appear to be rare in B/W mice, perhaps because B/W mice die of murine lupus before the lymphomas are evident. We recently reported that autoimmune disease in B/W mice could be reversed by weekly treatment with monoclonal antibodies to the L3T4 antigen on "helper/inducer" T cells. This has enabled us to examine the evolution of lymphoproliferation in B/W mice that survive beyond the usual life span, both in long-term survivors of treatment with anti-L3T4 and in the occasional B/W mouse that spontaneously survives beyond 1 year of age. We find that all of these mice develop marked proliferation of a distinct subpopulation of B cells that express the Ly-1 antigen in low density. These Ly-1+ B cells account for a 2-10-fold increase in the number of splenic, lymph node and peripheral blood lymphocytes. The Ly-1 B cells in individual mice are restricted in their expression of immunoglobulin light chains, suggesting a clonal origin. NZB mice. develop similar proliferation of Ly-1 B cells, suggesting that this is due to underlying genetic and/or viral factors in NZB and B/W mice, and that it is not the result of treatment with anti-L3T4. Although recent studies have implicated Ly-1 B cells in the production of autoantibodies, proliferation of Ly-1 B cells in B/W mice was not associated with production of anti-DNA antibodies or with any paraprotein.     

Particles resembling murine leukaemia virus in New Zealand Black mice

Particles which morphologically resembled murine leukaemia virus were detected by electron microscopy in the tissues (spleen, thymus, inguinal lymph nodes, bone marrow or pancreas) but not in serum or plasma pellets of untreated conventional New Zealand Black (NZB) mice aged 1–82 weeks. They were also found in the corresponding tissues of NZB mice which had been thymectomized shortly after birth. The presence of similar particles in the spleen, thymus or pancreas of conventional NZB embryos and, additionally, in the lymph nodes of NZB mice which had originally been introduced into a germ-free environment by Caesarian section and fostering on germ-free mice of another strain, suggests that the virus is transmitted `vertically' through the germ cells or placenta. Preliminary investigations showed similar particles in the organs of conventional F₁ (NZB × NZW) hybrid and New Zealand White (NZW) mice.

Large numbers of particles also resembling murine leukaemia virus were found in the spleen and in plasma or serum pellets of young conventional NZB mice which had developed reticulum cell neoplasia following serial passage of lymphoid cell suspensions from ageing conventional NZB donors.

The possible relationship of these particles to the autoimmune reactions and malignant changes which occur spontaneously in conventional NZB mice is discussed.

     

Peripheral blood CD4+CD8+ lymphocytes in cynomolgus monkeys are of resting memory T lineage

In this study, we analyzed peripheral blood CD4+CD8+ double-positive (DP) lymphocytes in adult cynomolgus monkeys (Macaca fascicularis). Forty of 55 monkeys had > 5% of the peripheral blood DP subpopulation (9.3 +/- 5.9%; mean +/- SD) in peripheral blood lymphocytes (PBL) in contrast to a low percentage of peripheral blood DP cells in humans and mice. In a cross-sectional study, the peripheral blood DP cells were found to increase in proportion with age. To clarify whether peripheral blood DP lymphocytes were immature precursors released from thymus without prior differentiation, the expressions of CD8 chains and CD1b on peripheral blood DP lymphocytes were compared with those on thymocytes. The peripheral blood DP lymphocytes were CD8 alpha + beta- and CD1b-, while thymic DP lymphocytes were CD8 alpha + beta + and CD1b +, suggesting that the peripheral blood DP cells are extrathymic T lymphocytes. Furthermore, the peripheral blood DP lymphocytes exhibited a resting memory T cell phenotype with CD2hiCD3+CD28-CD29hiCD49dhiCD69- CD80lo. Taken together, adult cynomolgus monkeys possess a unique peripheral blood DP T cell subpopulation which expresses a resting memory T cell phenotype. In addition, similar phenotypic properties of DP lymphocytes were distributed in the spleen and lymph nodes, although the proportion was less in the spleen and much less in lymph nodes than in PBL.      

Two distinct populations of peripheral lymphocytes in mice distinguishable by immunofluorescence

Immunofluorescent staining has been used to detect antigenic determinants on the surface of living lymphoid cells of mice. It has been possible to distinguish two distinct populations of peripheral lymphocytes: one population carries the theta isoantigen and is thymus-dependent; the other has naturally-occurring immunoglobulin determinants on the cell surface and appears to be thymus-independent. The relative proportions of these two populations of cells is characteristic for each type of lymphoid tissue and these proportions have been determined for thymus, lymph node, spleen, and thoracic duct cells.     

Genetic control of the spontaneous activation of CD4+ Th cells in systemic lupus erythematosus-prone (NZB x NZW) F1 mice

The F(1) hybrid of autoimmune hemolytic anemia-prone NZB and nonautoimmune NZW strains of mice has been studied as a murine model of systemic lupus erythematosus. Both NZB and F(1) hybrid mice show age-dependent spontaneous activation of peripheral CD4(+) T cells as reflected by the elevated frequencies of CD4(+) T cells positive for CD69 early activation marker. Both strains also show age-dependent abnormal decrease of the frequencies of CD62L(+) naive CD4(+) T cells and/or NTA260(+) memory CD4(+) T cells in the spleen. We studied the multigenic control of these abnormal features of peripheral CD4(+) T cells in (NZB x NZW) F(1) x NZW backcross mice by quantitative trait loci mapping and by association rule analysis. The abnormally elevated frequencies of CD69(+)CD4(+) T cells and decreased frequencies of CD62L(+) naive and/or NTA260(+) memory CD4(+) T cells were under the common genetic control, in which the interaction between MHC and a hitherto unknown locus, designated Sta-1 (spontaneous T-cell activation) on chromosome 12, plays a major role. The allelic effects of these loci likely predispose CD4(+) T cells to the loss of self-tolerance, and are responsible for the accelerated autoimmune phenotypes of (NZB x NZW) F(1) hybrid mice.     

Surface properties of lymphocyte subpopulations in autoimmune NZB/NZW F1 hybrid mice: alterations correlated with the immunodeficiency of aging

Partitioning in a two-polymer aqueous phase system was used to probe the surface properties of lymphoid cell subpopulations in aged male NZB/NZW F1 hybrid (B/W) mice, an important model of autoimmunity, immunodeficiency, and lymphoid malignancy. Spleen cells were fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged dextran-polyethylene glycol system. CCD of spleen cells from young, clinically normal male B/W mice yielded several broad distribution patterns which frequently had two or more peaks. Analysis of differentiation antigens and functional properties of cells from different parts of the distribution revealed a subfractionation of the three major lymphocyte subpopulations. B lymphocytes had a low partition coefficient (K); T cells had an intermediate K and null cells had the highest K. To examine the partitioning behavior of T lymphocytes, spleen cells which were nonadherent to nylon wool columns were subjected to CCD. Nonadherent cells from young B/W mice consistently gave a single peak with high K. Aged mice (18 months) usually had nonadherent cells with a predominantly low K. In some experiments a systematic increase in the number of these cells could be demonstrated with increasing mouse age. An analysis of the adherence and partitioning behavior of lymphocyte subpopulations revealed no change in the adherence properties or proportions of B lymphocytes in aged mice. The large proportion of cells having a low partition coefficient in the nonadherent spleen cell population of old mice appears to be due to an increase in the number of null cells and in a decrease in the K of some T lymphocytes.     

Loss of lymphocyte chalone activity in mice with autoimmune disease.

Lymphocyte chalone from the spleens of old BALB/c, young BALB/c and young NZB mice caused significant suppression of the proliferative response of BALB/c and NZB spleen cells to T and B mitogens, whereas lymphocyte chalone from old NZB spleen did not suppress. Lymphocyte chalone from young and old NZB mice was tested using different ages of NZB/NZW responding spleen cells; at all ages concanavalin A- and lipopolysaccharide-induced proliferation was suppressed less by the chalone from old NZB mice than from that of young NZB mice. The responding NZB/NZW cells were suppressed equivalently at all ages studied. The basis for the loss of lymphocyte chalone activity in old NZB mice remains unknown; however, it appears likely that this event has a role in the disturbance of the negative feedback control system which contributes to NZB autoimmune disease.     

CD4−8− T-cells increase in MRL/lpr mice treated with thymic factors

The in vivo effect of thymic factors on immature lymphocytes was analysed in MRL/lpr mice. This strain carries a genetic defect that causes during their life cycle a block of T-cell differentiation and abnormal proliferation of CD4⁻8⁻ (double-negative, DN) T-lymphocytes. In vivo administration of four preparations of thymic factors, thymopentin (TP-1), thymopoietin (TP-5), thymolymphotropin (TLT), and thymomodulin (TMD) into young (2-month-old) MRL/lpr mice induced a significant increase of DN T-cells both in the thymus and in the peripheral lymph nodes, with a concomitant decrease of double-positive (DP) T-cells in the thymus and of single-positive (SP) T-cells in the lymph nodes. The level of DNA fragmentation measured as propidium iodide fluorescence was increased in the thymus population of young mice and in the lymph node population of old mice treated with TLT. SCID mice transplanted with lymph node cells from MRL/lpr donors (MRL→SCID) developed graft versus host (GvH) reaction due to the activation of MRL CD8⁺ alloreactive T-cells. This model was used to analyse the effect of TMD/TLT in vivo on MRL cell proliferation and expansion; in fact, spleen cells from MRL→SCID mice after treatment with TMD/TLT showed an increased cell proliferation, and an expansion of DN T-cells with a concomitant decrease of SP cells (both CD4⁺ and CD8⁺ cells). Decreased SP cell numbers in this context could explain why TMD/TLT treatment of SCID mice engrafted with MRL cells increased their survival compared to untreated MRL→SCID mice.     

Enhanced response of autoantibody-secreting B cells from young NZB/NZW mice to T-cell-derived differentiation signals

Spleen cells from young NZB/NZW mice spontaneously produce IgM antihistone and anti-DNA antibodies in culture, and this in vitro autoantibody production is T-cell dependent. In the present studies, we investigated the response of young autoantibody-producing NZB/NZW B cells to various T-cell-derived signals. Stimulation with unprimed allogeneic T cells resulted in a 10- to 20-fold increase in IgM antihistone and anti-DNA antibody production compared with cultures of B cells alone. The responding cells were found in the large B-cell fraction after separation on Percoll gradients. Allo-stimulated B cells from nonautoimmune mice produced much lower absolute amounts of IgM autoantibodies as well as total IgM compared with NZB/NZW cells. Marked IgM antinuclear antibody and total IgM production was also observed when NZB/NZW B cells were cultured with supernatants from TH2 but not TH1 T-helper clones. Although B cells from nonautoimmune mice produced high levels of autoantibodies after stimulation with lipopolysaccharide, only minimal levels were secreted in response to the active supernatants. These results suggest that young NZB/NZW mice have IgM autoantibody-producing B cells that are more sensitive to certain T-cell-derived signals compared with B cells from normal mice. Although these hyperresponsive NZB/NZW cells appear to be in an advanced stage of activation, they require additional T-cell signals to express this abnormality.     

Differential sensitivity of lymphocyte subsets to corticosteroid treatment.

Corticosteroid treatment of SJL mice produced a marked decrease in the number of viable lymphocytes obtained 48 hr later from the thymus, spleen and lymph node but no change in peripheral blood. Within the residual lymphocyte population there was a fall in the relative number of splenic B cells with increasing dose; in contrast the proportion of B cells increased in the lymph nodes. The most marked change however was a dose-related increase inthe Lyt-2+ population in all of the lymphoid organs examined including the thymus though, in this organ alone, the lowest dose caused a pronounced reduction in the Lyt-2+ population, since most immature thymocytes are Lyt-2+. These findings support the concept that mature thymocytes have phenotypic identity with peripheral T cells and provide a basis for the immunosuppressive action of corticosteroids.