IL-12 prevents neonatal induction of transplantation tolerance in mice

We investigated the effect of IL-12 on the induction of transplantation tolerance by neonatal injection of allogenic cells. We first observed that injection of newborn BALB/c mice with IL-12 and (A/J × BALB/c) F1 spleen cells prevented the Th2 alloimmune response induced by neonatal inoculation of F1 cells alone and allowed the differentiation of T cells secreting high amounts of IL-2 and IFN-γ in mixed lymphocyte cultures with donor-type stimulators. Furthermore, IL-12 administration resulted in the emergence of anti-donor cytotoxic T lymphocyte responses although at lower levels than in control uninjected mice. In parallel, we found that mice injected at birth with IL-12 and F1 cells did not develop chimerism and were able to reject a donor-type skin graft as efficiently as control mice. We conclude that IL-12 inhibits the Th2 polarization of the newborn response to alloantigens and prevents thereby the establishment of transplantation tolerance.     

Different roles for LFA-1 and VLA-4 integrins in T–B-cell interactions in vivo

Adhesion molecules are critical in the cellular interactions involved in specific immune responses. They are used for homing, cell migration, cell-cell contact and, in some cases, for the delivery of costimulatory signals. Since the host-versus-graft (HVG) reaction represents a particular form of T-B-cell interaction, we have explored whether the inhibition of lymphocyte function-associated antigen-1/intracellular adhesion molecule-1 (LFA-1/ICAM-1) interactions and the signalling through very late activation antigen-4 (VLA-4) have any effect on the development of a lupus-like disease in BALB/c mice injected at birth with (BALB/cxC57BL/6)F1 spleen cells. In close association with the development of tolerance to donor allografts, these mice show a polyclonal activation of F1 donor B cells by alloreactive host CD4+ T cells, manifested by the production of autoantibodies (autoAbs) and the development of a mild glomerulonephritis. The dose of the monoclonal antibody (mAb) employed has been adjusted to block completely the molecule on the surface of peripheral lymphocytes without interfering with the induction of neonatal tolerance. Injection of saturating doses (100 microg/2 days) of either anti-LFA-1alpha or anti-ICAM-1 mAbs, but not anti-VLA-4alpha or anti-LFA-1beta mAbs, blocks the production of anti-ssDNA autoAbs and the thrombocytopenia characteristic of this HVG disease (HVGD). However, anti-VLA-4alpha treatment is only able to delay the production of autoAbs and the anti-LFA-1beta treatment, not to modify the evolution of the HVGD. These results point to the relevance of LFA-1/ICAM-1 interactions, but not of the VLA-4-mediated signal, in the polyclonal B-cell activation occurring during the allogeneic interactions between host T helper type 2 cells and donor B cells in HVGD.     

Autoimmune syndrome after induction of neonatal tolerance to alloantigens: analysis of the role of donor T cells in the induction of autoimmunity.

The injection of (C57BL/6 x BALB/c) F1 spleen cells into BALB/c newborn mice leads to activation of persisting F1 donor B cells and development of a lupus-like syndrome in tolerized BALB/c mice. This syndrome is characterized by hypergammaglobulinaemia, high levels of anti-DNA and anti-Sm antibodies, circulating immune complexes and deposits of immunoglobulin in renal glomeruli. The role of donor T cells in this model was investigated by injecting the newborn mice with F1 cells depleted in different T cell subsets by using specific monoclonal antibodies (MoAbs). Tolerance, as shown by an absence of H-2b-specific CTL alloreactivity and persistence of immunoglobulin bearing the donor allotype were observed in mice injected with F1 cells previously depleted in the CD4+ and/or CD8+ T cell subsets as well as in those which received Thy-1+-depleted F1 spleen cells. In these mice, a typical autoimmune syndrome was found, including splenomegaly and lymphadenopathy, anti-ssDNA and anti-aortic myosin IgG antibodies and renal deposition of immunoglobulin. However, some quantitative changes were seen: the levels of anti-aortic myosin antibodies were lower in mice tolerized with CD4+-depleted F1 cells than in those receiving untreated F1 cells. Conversely, higher levels of these autoantibodies were observed in mice tolerized with CD8+-depleted F1 cells. These results suggest that mature donor T cells are not necessary neither for the establishment of neonatal tolerance to alloantigens nor for the activation of F1 donor B cells in the production of the autoimmune syndrome in tolerant mice, but they may contribute in the regulation of the expression of autoreactive B cell clones.     

Prolonged inhibition of an antigen-specific IgE response in vivo by monoclonal antibody against lymphocyte function-associated antigen-1

Intraperitoneal injection of ovalbumin (OVA) into BALB/c mice caused the induction of OVA-specific IgE production in vivo. However, administration of monoclonal antibody against lymphocyte function-associated antigen-1 (anti-LFA-1 mAb) at days 0 and 1 after OVA immunization resulted in an inhibition of OVA-specific primary and secondary IgE production in a dose-dependent manner. The inhibition of the antigen-specific IgE response due to anti-LFA-1 mAb was seen up to 8 weeks after anti-LFA-1 mAb administration. The OVA-specific IgG1 response was also blocked by anti-LFA-1 mAb. The spleen cells obtained from OVA-immunized mice showed enhanced proliferation against secondary stimulation with OVA in vitro. However, the spleen cells obtained from the mice treated with both OVA and anti-LFA-1 mAb revealed a markedly decreased proliferative responses to OVA, while they showed no reduced responses against keyhole limpet hemocyanin stimulation, indicating that anti-LFA-1 mAb might induce antigen-specific anergy in vivo. It was also demonstrated that treatment of the mice with anti-LFA-1 mAb significantly inhibited the interleukin-4-producing ability of OVA-immunized mouse spleen cells. These results demonstrated that LFA-1-dependent cell-cell interaction is essential for the production of IgE in vivo and may be important in IgE-dependent allergic disease.     

Autoimmune syndrome after neonatal induction of tolerance to alloantigens: analysis of the specificity and of the cellular and genetic origin of autoantibodies.

BALB/c mice neonatally injected with 10(8) semiallogeneic (C57BL/6 x BALB/c)F1 spleen cells become tolerant to the H-2b alloantigens, but also develop a wide range of autoimmune manifestations characteristic of systemic lupus erythematosus (SLE). Indeed, in these mice, the presence of a hypergammaglobulinaemia, autoantibodies--including anti-ssDNA, anti-platelet, thymocytotoxic and rheumatoid factor antibodies--circulating immune complexes, cryoglobulins as well as renal glomerular deposition of immunoglobulins have been observed. In this study, we have shown that the allogenic effect and B cell chimaerism which characterize these F1 cell-injected mice is associated with the expression of a large spectrum of autoantibodies, including anti-ssDNA and anti-cytoskeleton antibodies, and that these autoantibodies are not multispecific. We took advantage of the fact that, in this model, autoantibodies are exclusively produced by F1 donor B cells to inject newborn BALB/c mice with F1 Xid spleen cells lacking the CD5+ B cell subset. Injection of 2 x 10(8) F1 Xid spleen cells triggers the production of anti-ssDNA as well as anti-BrMRBC antibodies, and these mice developed tissue lesions. Finally, analysis of the VH gene family expressed by monoclonal autoantibodies derived from F1 cell-injected mice showed that they used the 2 largest families J558 and 7183. These results suggest that the allogenic effect and B cell chimerism which characterize the neonatal induction of tolerance to MHC alloantigens is associated with the selective triggering of autoreactive B cells producing monospecific IgG autoantibodies. They also imply that upon stimulation by persisting alloreactive CD4+ T cells, either CD5- B cells are able to produce autoantibodies or autoantibody-producing CD5+ B cells can differentiate from Xid spleen cells.     

CD4+ T cells determine the ability of spleen cells from F1 hybrid mice to induce neonatal tolerance to alloantigens and autoimmunity in parental mice

Spleen cells from F₁ hybrid mice injected into newborn parental mice induce a state of cytolytic unresponsiveness to the corresponding alloantigens. However, these mice develop a transient autoimmune syndrome characterized by the production of multiple autoantibodies and glomerulonephritis. Previous reports indicated that the depletion of F₁ donor T cells, shortly prior the injection into parental mice, does not interfere with any of these events. Here, we have explored whether the continuous absence of T cells in F₁ mice influences the ability of their spleen cells to induce neonatal tolerance to alloantigens and the associated autoimmune manifestations. Our results revealed that spleen cells from athymic (BALB/c × C57BL/6) F₁ hybrid (CB6F₁) nulnu mice or from euthymic CB6F₁ mice depleted from birth of CD4⁺ T cells, but not of CD8⁺ T cells, are unable to induce neonatal tolerance to alloantigens and autoimmune manifestations. By contrast, the partial reconstitution of T cells in CB6F¹ nulnu mice, after the neonatal graft of a syngeneic thymus, restored the capacity of spleen cells from these mice to induce tolerance and autoimmunity when injected into newborn BALB/c mice. These results demonstrate that the functional defect of spleen cells from athymic CB6F₁ nulnu mice to induce neonatal tolerance to alloantigens is directly related to the long-term absence of mature CD4⁺ T cells. Interestingly, a new increase in the titers of anti-DNA Ab was observed when spleen cells from athymic CB6F₁ nulnu mice were injected into adult BALB/c mice that had been tolerized at birth with normal CB6F₁ spleen cells. This finding indicates that B cells from CB6F₁ nulnu mice recover their capacity to interact with alloreactive Th2 cells when they are placed into mice having functional CD4⁺ T cells. These data indicate that the continuous absence of CD4⁺ T cells causes a reversible functional defect in F₁ spleen cells that determines their inability to induce neonatal tolerance and autoimmunity.     

Persistence of anti-donor allohelper T cells after neonatal induction of allotolerance in mice

BALB/c mice rendered tolerant to A/J alloantigens by neonatal injection of 10(8) (A/J X BALB/c)F1 spleen cells develop an autoimmune disease associated with a polyclonal activation of donor B cells. To study the mechanisms leading to donor B cell activation in tolerant mice, we prepared mixed lymphocyte cultures (MLC) between splenic T cells from neonatally injected mice and donor-type (A/J X BALB/c)F1 or third-party (C57BL/6 X BALB/c)F1 B cells. T cells from tolerized mice were unable to generate cytotoxic T lymphocytes, to proliferate or to secrete interleukin (IL)2 after stimulation with donor alloantigens in MLC. These T cell responses were present after MLC with third-party antigens, but were of lower intensity than those generated by control BALB/c T cells. In contrast, T cells from tolerized mice stimulated immunoglobulin production by donor-type (A/J X BALB/c)F1 B cells much more powerfully than T cells from control BALB/c mice. The stimulation of donor-type (A/J X BALB/c)F1 B cells was polyclonal, as attested by the levels of anti-hapten and anti-DNA antibodies in the MLC supernatants. IgM was the dominant isotype secreted in vitro, but IgG1 and IgG3 were also produced in significant amounts. Lysis experiments indicated that the T cells responsible for F1 B cell stimulation in MLC were CD4+ host T cells. These T helper cells were alloreactive since they did not stimulate syngeneic BALB/c B cells, and their effect on donor B cells was specifically blocked by anti-donor Ia monoclonal antibodies. Addition of anti-IL 4 monoclonal antibody to MLC between T cells from tolerant mice and (A/J X BALB/c)F1 B cells almost completely abolished the production of IgG1, but not that of IgM or IgG3. Taken together, these findings indicate that neonatal injection of alloantigens in BALB/c mice induces a state of dissociated tolerance, with unresponsiveness of anti-donor T cells secreting IL 2 on the one hand, and persistence of T cells responsible for B cell help and IL 4 secretion on the other hand.     

Induction of Th2 cell tolerance to a soluble antigen by blockade of the LFA-1-dependent pathway prevents allergic inflammation

In this article, we show that induction of Th2 cell tolerance prevents antigen-induced eosinophil recruitment into the tissue and IgE antibody production, and that ICAM-1/LFA-1 interaction is involved as a costimulatory signal in inducing T cell tolerance to a soluble antigen. In vivo pretreatment with anti-ICAM-1 monoclonal antibody (mAb), anti-LFA-1 mAb, and a soluble antigen inhibited antigen-induced eosinophil recruitment into the airways and IgE antibody production in mice in an antigen-specific manner. In vitro antigen-induced IL-2, IL-4 and IL-5 production were decreased in spleen cells of the mice pretreated with the two mAbs and the antigen, indicating the induction of both Th1 and Th2 cell tolerance in vivo. These results suggest that the induction of antigen-specific Th2 cell tolerance by allergen immunotherapy with blockade of the ICAM-/LFA-1 interaction would be a rational therapeutic approach to allergic inflammation such as asthma.     

Regulation of the immune response in experimental models of autoimmune disorders: resistance of (NZB X NZW)F1 mice to tolerance induction in vivo

Studies were carried out to test whether tolerance to alloantigens and to heterologous proteins could be induced in (NZB X NZW)F1 (B/W) female mice, compared with females of various other mouse strains, including BALB/c, C3H/eb, C57Bl/Ka and (BALB/c X C57Bl/6)F1. Untreated BALB/c and B/W mice were resistant to tolerance induction by deaggregated BSA, while all other strains were susceptible, as indicated by their lack of response to antigen challenge. Tolerance induction to BSA was further potentiated in all mouse strains including BALB/c with the exception of B/W, following prior conditioning of the mice with total lymphoid irradiation (TLI). Similarly, specific and permanent tolerance to H-2 incompatible alloantigens was successfully induced in TLI conditioned BALB/c, C3H/eb, (BALB/c X C57Bl/6)F1 injected with bone marrow cells, however, B/W mice were resistant. Stable chimeras could be established in TLI treated B/W mice only across a semi-allogeneic combination (BALB/c--greater than B/W). No graft vs host disease (GVHD) was observed in any of the chimeras including B/W mice. We conclude that B/W mice are resistant to tolerance induction to heterologous proteins and alloantigens, even after TLI conditioning. We postulate that this phenomenon is a function of both the intrinsic properties of the haemopoietic stem cells, including their differentiated progeny, as well as characteristics of their cellular microenvironment.     

Completely allogeneic spleen cells induced cytolytic neonatal tolerance to alloantigens,but failed to establish allo-helper interactions with host T cells.

The injection of spleen cells from F1 mice into-newborns from a parental strain results in the establishment of cytolytic tolerance to donor alloantigens and the development of a lupus-like disease. This syndrome is the consequence of the recognition by alloreactive host CD4+ T cells of discordant major histocompatibility complex (MHC) class II antigens on semi-allogeneic donor B cells. We have analysed whether completely allogeneic spleen cells are as able as semi-allogeneic spleen cells to induce cytolytic tolerance to donor alloantigens and to co-operate with alloreactive T cells for autoantibody production. BALB/c mice were injected at birth with Thy-1-depleted spleen cells from (C57BL/6 x BALB/c)F1 or C57BL/6 mice, either alone or in combination. Cytolytic tolerance was always induced, as manifested by persistence of chimerism and acceptance of skin allografts. However, only F1 semi-allogeneic B cells were activated by alloreactive host T cells to produce anti-DNA IgG antibody. The deficient co-operation between BALB/c CD4+ T cells and completely allogeneic C57BL/6 B cells was confirmed after neonatal injection of (C57BL/ 6 x BALB/c)F1(Igha) spleen cells together with C57BL/6(Ighb) spleen cells. These mice developed anti-DNA antibodies bearing only the Igha allotype. Similar results were observed in experiments of allogeneic interaction in vitro, in which BALB/c CD4+ T cells were cocultured with either (C57BL/6 x BALB/c)F1 or C57BL/6 B cells. The present results demonstrate that completely allogeneic spleen cells efficiently induced cytolytic unresponsiveness to donor alloantigens, but B cells contained in this spleen cell population were unable to establish allo-helper interactions with alloreactive CD4+ T cells, suggesting that cytolytic and helper T-cell interactions involved in alloreactivity may be different.     

Characterization of host CD4+ T lymphocytes in mice neonatally tolerized to alloantigens

BALB/c mice injected at birth with semi-allogeneic F1 spleen cells become tolerant to alloantigens as shown by their CTL unresponsiveness to the corresponding alloantigen and the persistence of donor F1 cells into the BALB/c host. Moreover, these mice develop a transient systemic lupus erythematosis-like autoimmune syndrome characterized by splenomegaly, glomerulonephritis, thrombocytopenia and abnormal serological findings, such as several autoantibodies and IgG1 hypergammaglobulinemia. Recent studies done in our laboratory have shown that donor F1 B cells persisting in the host are responsible for the production of autoantibodies and must be activated in vivo by the host CD4⁺ T lymphocytes in a MHC class II-restricted fashion. In the present work, we have focused our attention on the ability of splenic CD4⁺ T cells recovered at different periods from BALB/c mice injected at birth with (CBA/Ca × BALB. Igh^b) Fl spleen cells to interact with and activate F1 semi-allogeneic spleen cells in vitro. We show that (i) only CD4⁺ T cells from 2- and 3-week-old tolerant BALB/c mice preferentially produce IL-4 and IL-5 in response to a F1 semi-allogeneic in vitro stimulation, (ii) CD4⁺ T cells purified from 3-week-old tolerant BALB/c mice are able to induce in vitro IgG and IgM production by F1 B cells. Taken together, these results strongly suggest that host CD4+ T cells, belonging to the TH2 subset progressively lose their reactivity towards the F1 semi-allogeneic persistent B cells, reaching a state of unresponsiveness that correlates with the disappearance of serum autoantibodies and autoimmune pathology.     

Modulation of murine host-versus-graft disease by anti-CD3 monoclonal antibody.

BALB/c mice made tolerant to A/J alloantigens by neonatal injection of (A/J x BALB/c)F1 spleen cells develop a host-versus-graft (HVG) disease due to the activation of donor B cells by a subset of host alloreactive helper T cells. We have investigated the effects of a single neonatal injection of the 145-2C11 anti-mouse CD3 monoclonal antibody (MoAb) on the establishment of allotolerance and on the development of the immunopathological features of HVG disease. First, this treatment did not modify the specific anti-donor cytotoxic T lymphocyte (CTL) unresponsiveness or the persistence of circulating immunoglobulins bearing donor allotype. Second, the hyper IgE, the hyper IgG1 and the increased expression of Ia antigens on B cells found in untreated HVG mice were not observed after injection of the 145-2C11 MoAb. Likewise, treated mice displayed lower levels of anti-DNA IgG antibodies and less glomerular immune deposits as compared with untreated HVG mice. We conclude that the administration of the anti-CD3 MoAb did not interfere with the induction of allotolerance but exerts a pronounced inhibitory effect on the associated immunopathological syndrome.     

CD2/LFA-3 or LFA-l/ICAM-1 but not CD28/B7 interactions can augment cytotoxicity by virus-specific CD8+ cytotoxic T lymphocytes

It is well established that adhesion molecules are required for interaction between cytotoxic T lymphocytes (CTL) and target cells. Two adhesion pathways, CD2/LFA-3 and LFA-l/ICAM-1 can support cytotoxicity by allospecific CD8+ CTL. In this study, it was investigated whether these adhesion pathways can be utilized independently by influenza virus-specific HLA-A2-restricted CTL clones. It was furthermore examined whether the CD28/B7 pathway can augment virus-specific CTL activity. To this end, seven CD8⁺ CTL clones were established that were specific for a peptide encompassing positions 59 to 68 (p[59-68]) of the influenza virus matrix protein. These seven clones apparently originated from different precursors, as they utilized different Vα and Vβ or Jα gene segments. Six of seven clones were able to lyse mouse L cells co-transfected with HLA-A2 and either LFA-3 (LA2/LFA-3) or ICAM-1 (LA2/ICAM-1) in the presence of p[59-68] but did not lyse Lcells that expressed only HLA-A2 and peptide. Three of the most cytotoxic clones were selected for further analysis. The cytotoxicity of the clones against LA2/LFA-3 cells was blocked by anti-LFA-3 and anti-CD2 monoclonal antibodies (mAb), while these antibodies did not affect cytotoxicity against LA2/ICAM-1 cells. Likewise, the activity against LA2/ICAM-1 was blocked only by anti-LFA-1 and ICAM-1 mAb. These clones were unable to lyse Lcells co-transfected with HLA-A2 and B7, the counter structure of CD28, despite the fact that these clones expressed CD28. These data indicate that CD8⁺ virus-specific CTL can utilize either the CD2/LFA-3 or the LFA-l/ICAM-1 adhesion pathway. The CD28/B7 pathway seems not to be required for cytotoxicity mediated by activated virus-specific CTL.     

Susceptibility of neonatal T cells and adult thymocytes to peripheral tolerance to allogeneic stimuli

We studied the tolerization of neonatal thymocytes (NT), neonatal splenocytes (NS) and adult thymocytes (AT), transferred to syngeneic nude (nu/nu) hosts previously injected with semi-allogeneic splenocytes, without any supportive immunosuppressive treatment. This protocol allows the study of peripheral tolerance in the absence of the thymus. BALB/c neonatal T cells and ATs were able to expand in syngeneic BALB/c nu/nu mice and functionally reconstituted an allogeneic response, rejecting (BALB/c x B6.Ba) F1 splenocytes transferred 3-4 weeks after injection of BALB/c cells. However, if (BALB/c x B6.Ba) F1 cells were injected into BALB/c nude hosts 30 days before transfer of NT, NS or AT cells, the F1 population was preserved and specific tolerance to B6 allografts was established. Furthermore, transfer to lymphopenic F1 nu/nu showed that tolerance could be established only for neonatal populations, showing that unique properties of neonatal T cells allow their tolerization in both lymphopenic and non-lymphopenic conditions, in the absence of suppressive immunotherapy. These results bring empirical support to the possibility of T-cell engraftment in immunodeficient patients showing partial identity with donor major histocompatibility complex (MHC) genes; the manipulation of immunological maturity of donor T cells may be the key for successful reconstitution of immunocompetence without induction of graft-versus-host disease.     

Effects of anti-B7 monoclonal antibodies on humoral immune responses

The costimulatory interaction between CD28 on T cells and B7-related molecules on antigen presenting cells plays an important role in a broad range of functions of the immune system, including protective immunity, tolerance induction, allograft rejection, and the development of autoimmune diseases. Monoclonal antibodies to B7-1 and B7-2 have been used in vivo to examine the mechanisms underlying these processes and to evaluate costimulation antagonism as an approach to treatment of chronic autoimmune diseases. To determine whether anti-B7 mAb might elicit, or inhibit, a host immune response that could influence the effects of these antibodies in vivo, we assessed the immune response to rat anti-B7-1 and anti-B7-2 mAb in healthy (BALB/c) mice and in lupus-prone NZB/NZW F1(B/W) mice. In BALB/c mice, low doses (1-10 microg) of mAb to B7-1 and mAb to B7-2 elicited brisk immune responses that occurred earlier and were significantly greater than the immune response to an isotype-matched control rat mAb to ovalbumin. In contrast, at higher doses (100-500 microg), both anti-B7 mAb, but not the control mAb, blocked the mouse anti-rat response. No such blockade occurred in B/W mice, who generated a significant mouse anti-rat response even at very high doses of anti-B7 mAb (1,000-4,000 microg). Blockade of the immune response to the anti-B7 mAb in BALB/c mice apparently did not reflect generalized immune suppression, because high doses of these mAb had little, if any effect on the humoral immune response to another antigen. These findings indicate that: (1) mAb to B7-1 and B7-2 can elicit either a potent immune response or no immune response at all depending upon the dose administered; (2) blockade of the immune response to anti-B7 mAb may be more difficult in the setting of autoimmunity; and (3) neither anti-B7-1 nor anti-B7-2 causes generalized suppression of humoral immunity.     

Cyclosporin alters the induction of allospecific tolerance in vivo

CBA mice were made hyporesponsive to A/J alloantigens by either neonatal inoculation of (CBA X A)F1 hybrid lymphoid cells or by intravenous injection of adult mice with A/J bone marrow cells. Specific alloreactivity was assessed in vitro by induction of anti-A/J cytotoxic T lymphocytes (CTL) or in vivo by skin graft rejection. Cyclosporin A given at the same time as the tolerance-inducing regimen of F1 (or parental) lymphoid cells abolished the hyporesponsiveness normally induced by these injections.     

Cytotoxic T cell clone-specific monoclonal antibodies used to select clonotypic antigen-specific cytotoxic T cells

Two rat monoclonal antibodies (mAb) have been produced which recognize a clone-specific determinant on the alloreactive cytotoxic T lymphocyte (CTL) clone 3 F9. CTL clone 3F9 of BALB/c origin is specific for H-2Db and can be grown by weekly restimulation with irradiated stimulator spleen cells expressing H-2Db in the presence of interleukin 2. Two mAb against T cell clone 3F9, 44-22-1(IgG2a) and 46-6 B5(IgM), have been proven to be clone specific: they inhibit cytotoxic activity of 3F9 only and bind specifically to 3 F9 when compared in a panel of different CTL clones, or cells from different mixed lymphocyte cultures (MLC), BALB/c thymus and spleen cells. The mAb 44-22-1 has been used to sort cells from a primary MLC BALB/c anti-H-2Db by fluorescence-activated cell sorter (FACS) to select CTL expressing 3 F9 clonotype-specific determinants. The lymphocytes reactive with 44-22-1 represent a minor subpopulation of the CTL of the primary MLC. The specific alloreactive cytotoxicity of unsorted lymphocytes of the bulk primary MLC could not be inhibited by the mAb 44-22-1 and 46-6 B5 whereas the sorted 3 F9 clonotype-positive cultures could be inhibited very effectively. All the CTL clones derived from the FACS-sorted clonotype-positive culture show all the same properties and are identical with clone 3 F9 with respect to antigen-specific cytotoxicity, inhibition of cytotoxicity by the mAb and surface markers.     

Tolerance induction to an alloepitope involves antibodies interacting with the T cells activated by the alloepitope.

Hyperimmunization of BALB/c mothers with Ab1 (BALB/c anti-C57BL/6) antibody can alter the T-cell repertoire of the syngeneic offspring called BALB/c-p-Ab1. The latter are rendered tolerant to specific alloantigens and are therefore resistant to graft-versus-host disease (GVHD), when challenged 24-48 hours after birth with allogeneic spleen cells. These offspring appear to express cell structures recognized by hyperimmunized mothers and which are presumably the T-cell counterparts of Ab2. Our data suggest that tolerance induction in our mouse model may be modulated by an idiotype network acting during foetal life. The factor modifying the foetal cell repertoire is identified as Ab2 (BALB/c anti-Ab1) antibody transplacentally transmitted from mother to foetus, which seems to carry an "image" of the GVHD-inducing alloepitope and can induce a "GVHD-like" syndrome when passively injected intraperitoneally in normal BALB/c newborn mice.     

Increased expression of Ia antigens on B cells after neonatal induction of lymphoid chimerism in mice: role of interleukin 4

BALB/c mice rendered chimeric at birth by injection of 10(8) (A/J X BALB/c)F1 spleen cells develop a lupus-like autoimmune disease linked to the activation of donor B cells by host T cells. As in vitro studies previously indicated that interleukin 4 (IL4) was a mediator of the interactions between T and B cells, we analyzed the intensity of Ia antigen expression on B cells of chimeric mice. Flow cytometric analysis with anti-Ia monoclonal antibodies (mAb) revealed that B cells from spleens and lymph nodes of 2-week-old chimeric BALB/c mice displayed a two- to threefold increase in membrane Ia antigen expression, this increase still being present in spleens of 30-week-old animals. An increase in Ia antigen expression was also found in the small number of donor B cells detected in spleens and lymph nodes of chimeric mice. IL4 was the major stimulus leading to increased B cell Ia antigen expression, as this phenomenon was substantially prevented by in vivo treatment of chimeric mice with the anti-IL4 11B11 mAb. In vitro experiments revealed that host splenic T cells of chimeric mice, while unable to generate anti-donor cytotoxic T lymphocytes, secreted significant amounts of IL 4 when stimulated in mixed lymphocyte cultures (MLC) with donor alloantigens. This IL4 secretion led to an increased expression of Ia antigens on donor-type F1 B cells present in MLC. No significant increase in Ia antigen expression was found on syngeneic BALB/c B cells co-cultured with T cells from chimeric mice unless A/J B cells were added to the cultures. Taken together, these findings indicate that increased Ia antigen expression on donor B cells is induced by IL4 secreted by anti-donor T cells. IL4 released in this setting also leads to increased Ia antigen expression on host B cells through a bystander effect.     

T-cell tolerance induction is normal in the (NZB x NZW)F1 murine model of systemic lupus erythematosus

The (New Zealand black (NZB) x New Zealand white (NZW))F1 (NZB/W) mouse strain spontaneously develops an autoimmune disease characterized by anti-dsDNA antibody production and glomerulonephritis. Although evidence suggests that production of pathogenic autoantibodies is T-cell dependent, the immunological defects that lead to activation of these autoreactive T cells are unknown. In particular, it has not been resolved whether autoreactive T cells become activated in these mice because of a generalized defect in T-cell tolerance induction. Previous work has demonstrated that thymic and peripheral tolerance to strongly deleting antigens are intact in NZB/W mice. In this study we investigate whether these mice possess a more subtle T-cell tolerance defect. To this end, we have produced NZB/W mice carrying a transgene encoding beef insulin (BI) which is expressed at levels close to the threshold for T-cell tolerance induction. In BALB/c mice this transgene produces a profound but incomplete state of BI-specific T-cell tolerance, mediated predominantly by clonal anergy. Comparison of BI-specific tolerance in NZB/W, major histocompatibility complex (MHC)-matched (BALB/c x NZW)F1, and BALB/c BI-transgenic mice clearly demonstrates that T-cell tolerance induction is normal in NZB/W mice. The data suggest that the loss of T-cell tolerance that ultimately supports nephritogenic autoantibody production in NZB/W mice does not result from a generalized defect in T-cell tolerance, and by extension likely results from aberrant activation of specific autoreactive T cells.