Immunobiology and immunotherapeutic implication of syneneic/autologous graft-versus-host disease

Summary: Administration of the immunosuppressive drug cyclosporine (CsA) after syngeneic/autologous bone marrow transplantation (BMT) elicits an autoimmune syndrome with pathology virtually identical to graft-vs-host disease (GVHD). The induction of this syndrome, termed syngeneic/autologous GVHD, is a two-tiered process requiring both the active inhibition of thymic-dependent clonal deletion and the elimination of mature T cells that have an immunoregulatory effect. Eradication of the peripheral immunoregulatory compartment by the preparative regimen provides a permissive environment for the activation of the syngeneic/autologous GVHD effector T cells. Although the repertoire of autoreactive effector T lymphocytes is highly conserved, these T cells promiscuously recognize MHC class II determinants. This novel specificity of the autoreactive lymphocytes appears to he dependent on the peptide derived from the MHC class 11 invariant chain. Recent studies also suggest that these promiscuous autoreactive T cells can effectively target and eliminate MHC class Il-expressing tumor cells. Administration of cytokines that upregulate the target antigen or expand the effector population can potentiate the antitumor activity of syngeneic/autologous GVHD, Although the induction of syngeneic/autologous GVHD is an untoward effect of CsA immunosuppression, mobilization of these autoimmune mechanisms provides a promising immunotherapeutic approach for certain neoplastic diseases.     

A novel experimental model of giant cell myocarditis induced in rats by immunization with cardiac myosin fraction

It is suspected that autoimmune disease processes are involved in the pathogenesis of a part of giant cell myocarditis. However, evidence for autoimmunity has rarely been demonstrated in clinical investigations. In this study, we have demonstrated a new animal model of autoimmune myocarditis characterized by the appearance of multinucleated giant cells. Lewis rats were immunized twice with human cardiac myosin fraction in complete Freund's adjuvant. Cardiac myosin fraction was prepared from the ventricular muscle of human hearts. Three weeks after the first immunization, acute and severe myocarditis was elicited in all rats. This myocarditis was characterized by massive pericardial effusion, enlargement of the heart, and gray discoloration of the cardiac muscle. Microscopically, there was marked cellular infiltration consisting of mononuclear cells, neutrophils, fibroblasts, and a considerable number of multinucleated giant cells. Extensive myocardial necrosis was also present. The heart weights increased from the third week to the fourth week and then gradually decreased. The titer of anti-myosin antibodies began to elevate from the second week and remained high until the sixth week. In the sixth week, inflammation became smoldering and the multinucleated giant cells disappeared. These findings indicate that the cardiac myosin fraction contains myocarditogenic antigen and that giant cell myocarditis can be induced by autoimmune involvement. To our knowledge, this is the first report of experimental giant cell myocarditis, which is closely similar to human giant cell myocarditis in its histology and clinical course.     

Increased expression of class II major histocompatibility complex molecules on B cells in rats susceptible or resistant to HgCl2-induced autoimmunity

Administration of HgCl2 to the susceptible Brown-Norway (BN) rats induces an autoimmune disease characterized by a T-dependent polyclonal activation of B cells responsible for a dramatic increase in serum IgE concentration. The resistant Lewis (LEW) rats injected with HgCl2 do not exhibit such autoimmune manifestations. We show here that, upon HgCl2 injections, major histocompatibility complex (MHC) class II molecule expression is increased very early in lymph nodes and spleen B cells from both strains. So far, it is the earliest marker (day 3) of the effect of HgCl2 on the immune system. In both strains this enhancement is transient, but regulatory mechanisms are much more efficient in the resistant LEW strain than in the susceptible BN strain. In addition, we observed that MHC class II molecule expression on B cells differs according to the organ and the rat strain tested. All these findings are discussed in an attempt to underline the role of MHC class II molecule expression in the occurrence of mercury-induced autoimmunity.     

Differential regulation of expression of the MHC class II molecules RT1.B and RT1.D on rat B lymphocytes: effects of interleukin-4, interleukin-13 and interferon-gamma.

Susceptibility to induction of both T helper 1- (Th1) and Th2-mediated autoimmunity is multifactorial and involves genetic linkage to the major histocompatibility complex (MHC) class II haplotype. Brown Norway (BN) rats exposed to mercuric chloride develop a Th2-dependent systemic autoimmunity, whereas Lewis rats, which are highly susceptible to Th1-mediated autoimmunity, develop immune suppression after mercuric chloride exposure. Exposure to mercuric chloride is known to enhance B-lymphocyte expression of the MHC class II molecule RT1.B, predominantly in BN rats. We demonstrate that, in contrast, expression of RT1.D was unmodified on these B cells, whereas both RT1.B and RT1.D were up-regulated on epithelial cells. Regulation of B-cell MHC class II isotype expression was further studied in vitro, using BN rat lymph node (LN) cells. Interleukin-4 (IL-4) strongly enhanced B-cell expression of RT1.B (2.8-fold), whereas RT1.D expression was only slightly, although significantly, modified (1.2-fold). B cells from Lewis rats showed a similar IL-4-induced enhancement of RT1.B expression (2.5-fold), whereas, in contrast, RT1.D expression was unmodified. Exposure of LN cells from BN rats to interferon-gamma induced a moderate increase of B-cell MHC class II expression, predominantly of RT1.B. Strong and rapid enhancement of B-cell RT1.D expression was observed after stimulation by phorbol 12-myristate 13-acetate and ionomycin. Rat IL-13 did not modify B-cell MHC class II expression; however, it induced typical morphological changes in peritoneal macrophages. These experiments demonstrate isotype-specific and strain-dependent regulation of MHC class II expression on rat B lymphocytes, which may be of pathophysiological relevance for the strain-dependent susceptibility for Th1- or Th2-mediated autoimmunity.     

Tolerance to Mlsa by clonal deletion of V beta 6+ T cells in bone marrow and thymus chimeras

To evaluate the rules of tolerance induction to Mlsa we investigated maturation of T cells in irradiation bone marrow chimeras and thymus transplanted mice expressing the strongly stimulatory Mlsa or the nonstimulatory Mlsb in presence or absence of the permissive MHC class II IE molecule. As shown previously, deletion of V beta 6+, Mlsa reactive T cells required the presence of Mlsa and IE products either on donor cells or on recipient tissue. Additional experiments revealed that tolerance was induced when Mlsa and IE were expressed by distinct cells of the chimera. Similar observations were made in chimeras reconstituted with bone marrow stem cells of two different strains of mice. The presented data confirm earlier evidence that tolerance is induced by cooperation of Mlsa-expressing cells and IE+ Mlsb antigen-presenting cells (APCs). Preliminary results suggest that APCs presenting Mlsa during negative selection of V beta 6+ cells in the thymus are radiosensitive and derived from lymphohemopoietic stem cells.     

Facial nerve transection causes expansion of myelin autoreactive T cells in regional lymph nodes and T cell homing to the facial nucleus.

Nervous tissue expression of immunological signal and recognition molecules, as well as lymphoid tissue immune responses after facial nerve trauma was studied in male rats of the Lewis and Brown Norway (BN) strains. In both rat strains nerve transection caused within four days the appearance of IFN-gamma-like immunoreactivity in the cytoplasm of axotomized motor neurons and an induction of MHC class I and II, and CD4 molecules on surrounding glial cells to a similar extent. T lymphocytes also infiltrated the facial nuclei ipsilateral to the axotomy in all animals. The number of autoreactive T cells in superficial cervical lymph nodes, which in response to whole myelin or peptides of myelin basic protein (MBP) secreted IFN-gamma increased markedly after axotomy. This response was more conspicuous in Lewis rats, which are susceptible to experimental allergic encephalomyelitis (EAE), than in BN rats, which are EAE resistant. A proportion of the axotomized Lewis rats also developed widespread perivascular infiltration of mononuclear cells in the CNS, reminiscent of EAE. Hypothetically, a strong expansion of myelin autoreactive IFN-gamma producing T cells secondary to nerve trauma may have immunopathological consequences in genetically predisposed individuals. It is also possible that myelin reactive T cells, whether recruited to the lesioned nerve, could have impact on macrophage function during Wallerian degeneration in the distal stump.     

Self-reactive anti-class II T helper type 2 cell lines derived from gold salt-injected rats trigger B cell polyclonal activation and transfer autoimmunity in CD8-depleted normal syngeneic recipients

Brown Norway (BN) rats given gold salts develop an autoimmune syndrome with an immune complex-type glomerulonephritis in the context of a polyclonal B cell activation that was suspected to be due to the emergence of anti-self major histocompatibility complex (MHC) class II T cells. In the present study, six anti-self MHC class II T cell lines have been derived from six gold salttreated rats by repeated stimulations with normal syngeneic MHC class II-bearing cells. The T cell lines proliferated in the presence of self MHC class II-positive B cell-enriched or B cell-depleted cells and the proliferation was inhibited by preincubating stimulator cells with an anti-IA monoclonal antibody. The T cell lines produced interleukin (IL)-4 only or IL-4 and some interferon (IFN)-γ and could, therefore, be considered as T helper type 2 (Th2) and Th0 cells, respectively. They triggered normal syngeneic B cells to produce in vitro IgE, anti-DNA, anti-laminin and anti-2,4-6-trinitrophenol antibodies through, at least in part, cognate interactions. More interestingly, these lines when transferred into normal BN rats induced an autoimmune syndrome similar to or even more severe than the one observed in the active gold model, provided the recipients were CD8 depleted. These manifestations included a dramatic increase in serum IgE concentration and the production of anti-DNA and anti-laminin antibodies. In addition, all recipients displayed an autoimmune glomerulonephritis due to anti-laminin antibodies, granular IgG deposits in the interstitium, in the vessel walls and along the tubular basement membranes and a severe tubulointerstitial nephritis with marked mononuclear cell infiltration. An anti-ovalbumin T cell line that produced IL-4 and low amounts of IFN-γ was used as a control and did not induce autoimmunity. These results demonstrate for the first time the ability of autoreactive Th2 as well as Th0 cell lines to induce antibody-mediated autoimmunity. They also show that CD8⁺ cells play a crucial role in the control of such autoreactive cells. Finally, this work suggests that Th2 cells could initiate cell-mediated reactions either directly or indirectly.     

Instillation of allogeneic lung antigen-presenting cells deficient in expression of major histocompatibility complex class I or II antigens have differential effects on local cellular and humoral immunity and on pathology in recipient murine lungs

Recognition of allogeneic major histocompatibility complex (MHC) molecules expressed on donor lung antigen-presenting cells (APCs) by host T lymphocytes is believed to stimulate lung allograft rejection. However, the specific roles of donor MHC molecules in the rejection response is unknown. We report a murine model in which instilling allogeneic lung APCs into recipient lungs induces pathology analogous to acute rejection, and the production of interferon (IFN)-gamma, immunoglobulin (Ig) G2a, and alloantibodies in recipient lungs. Using allogeneic lung APCs (C57BL/6, I-a(b), H-2(b)) deficient in MHC class I, II, or both for instillation into lungs of BALB/c mice (I-a(d), H-2(d)), the purpose of the current study was to determine the specific roles of donor MHC molecules in stimulating local alloimmune responses. The data show that MHC class I or II on donor APCs induced IFN-gamma and IgG2a synthesis locally, though less than that induced by wild-type cells. Both MHC class I and II were required to induce alloantibody production. Instillation of wild-type or class I- or class II-deficient APCs induced comparable pathologic lesions in recipient lungs, and more severe than that induced by MHC-deficient cells. These data show that donor MHC class I and II molecules have differential effects in the stimulation of local alloimmune responses.     

Is tumor expression of the major histocompatibility complex antigen required for T cell immune surveillance?

Tumor expression of major histocompatibility complex antigen (MHC) class I and class II is not essential for the induction of memory T cells. However, induction of MHC class I-restricted effector cytotoxic T cells (CTL) appears dependent on MHC class I expression on tumors. Moreover, the effector function of tumor-specific CTL requires direct recognition of the tumor. In contrast, both the inductive and the effector phases of MHC class II-restricted T cells are independent of MHC class II expression on tumors.     

移植心Th细胞免疫偏离与MHC Ⅱ类抗原表达的关系

目的:探讨Th细胞免疫偏离与移植心MHC Ⅱ类抗原表达的关系。方法:建立大鼠心脏移植模型,以同系移植和无移植动物作为对照,采用逆转录PCR技术测定移植心Ⅰ、Ⅱ类细胞因子IL-2、IL-4 mRNA水平变化,用免疫组化技术和单克隆抗体测定移植心MHC Ⅱ类抗原表达。结果:IL-2 mRNA水平和MHC Ⅱ类抗原表达随着移植心急性免疫排斥病变发展而显著增加(P＜0.01), IL-4 mRNA水平则显著降低(P＜0.01),急性免疫排斥发展到一定阶段Ⅰ、Ⅱ类细胞因子水平出现偏离时MHC Ⅱ类抗原由低表达变为高表达。结论:移植心脏急性免疫排斥过程中,Th细胞免疫偏离与MHC Ⅱ类抗原表达变化有相关性,并参与促进移植心脏MHC Ⅱ类抗原的高表达。     

Negative and positive selection of immature thymocytes: timing and the role of the ligand for alpha beta T cell receptor

Recent experiments show that CD4+8+ thymocytes represent the critical stage in T cell development at which the specificity of randomly generated ab T cell receptors is screened. These cells are deleted when the receptor binds to the MHC molecule plus specific peptide presented by bone marrow derived cells but are rescued from cell death and induced to mature if the receptor binds to the MHC molecule on thymic epithelium in the absence of the specific peptide. Different tolerogens delete CD4+8+ thymocytes earlier or later during their lifespan and negative selection can occur prior to positive selection. The specificity of the ab T cell receptor for either class I or class II thymic MHC molecules determines the CD4-8+ and CD4+8- phenotype of mature T cells.     

FGL2 knockdown improves heart function through regulation of TLR9 signaling in the experimental autoimmune myocarditis rats

Fibrinogen-like protein 2 (FGL2) is an important immune regulator of both innate and adaptive response. It is present on the surface of macrophages and endothelial cells, and can be constitutively secreted by CD4⁺CD8⁺ T cells. Previous studies showed that FGL2 is a potential target for the treatment of experimental autoimmune myocarditis. However, the molecular mechanism of the roles of FGL2 in experimental autoimmune myocarditis is poorly understood. Here, we silenced FGL2 gene by using FGL2-RNAi lentivirus to reveal the heart function in experimental autoimmune myocarditis rats. We found that the cardiac myosin of pigs’ hearts induced Lewis rats to come into being as autoimmune myocarditis. TLR9 was upregulated in the heart of experimental autoimmune myocarditis rats. After primary immunization (21 day), the cardiac function of the myocarditis model group improved (P < 0.05). Significantly, the levels of INF-α and NF-κB in the FGL2-RNAi-treated group were lower compared to those in the myocarditis model (EAM) group (P < 0.05). Notably, the inflammation score correspondence with the protein and mRNA levels of TLR9 in myocardial tissues was markedly reduced compared to that in the EAM group (P < 0.05). These results support a role of FGL2 to alleviate inflammatory situation in the myocardium through regulation of the TLR9 signaling pathway in the experimental autoimmune myocarditis rats.     

Protein sorting within the mhc class II antigen-processing pathway

Major histocompatibility complex (MHC) class II molecules are required for the presentation of antigenic peptides that are derived predominantly from internalized proteins. The assembly of MHC class II/peptide complexes occurs within endosomal compartments of antigen-presenting cells (APCs). Therefore, for assembly to occur, MHC class II molecules, foreign proteins, and accessory molecules must be sorted to appropriate intracellular sites. My laboratory is trying to understand how proteins are sorted to various antigen-processing compartments as well as to conventional endosomal organelles. Using chimeric marker proteins and a variety of biochemical and genetic approaches, we are addressing the specificity of protein sorting and the mechanisms by which sorting signals are deciphered. By using a similar chimeric protein approach to target endogenous proteins to distinct compartments, we hope to address the role of processing events in each compartment in the generation of MHC class II ligands.     

Modulation of CD4 T cell function by soluble MHC II-peptide chimeras

Peptides antigens of 8 to 24 amino acid residues in length that are derived from processing of foreign proteins by antigen presenting cells (APC), and then presented to T cells in the context of major histocompatibility complex molecules (MHC) expressed by APC, are the only physiological ligands for T cell receptor (TCR). Co-ligation of TCR and CD4 co-receptor on T cells by MHC II-peptide complexes (signal 1) leads to various T cell functions depending on the nature of TCR and CD4 co-ligation, and whether costimulatory receptors (signal 2) such as CD28, CTLA-4, CD40L are involved in this interaction. Recently, the advance of genetic engineering led to the generation of a new class of antigen-specific ligands for TCR, i.e., soluble MHC class I, and MHC class II-peptide chimeras. In principle, these chimeric molecules consist of an antigenic peptide which is covalently linked to the amino terminus of α-chain in the case of MHC I, or β-chains in the case of MHC II molecules. Conceptually, such TCR/CD4 ligands shall provide the signal I to T cells. Since soluble MHC-peptide chimeras showed remarkable regulatory effects on peptide-specific T cells in vitro and in vivo, they may represent a new generation of immunospecific T cell modulators with potential therapeutic applicability in autoimmune and infectious diseases. This review is focused on the immunomodulatory effects of soluble, MHC class II-peptide chimeras, and discuss these effects in the context of the most accepted theories on T cell regulation.     

Augmentation of antitumor immune responses after adoptive transfer of bone marrow derived from donors immunized with tumor lysate-pulsed dendritic cells.

We demonstrated previously that tumor lysate-pulsed dendritic cells (TP-DC) could mediate a specific and long-lasting antitumor immune response against a weakly immunogenic breast tumor during early lymphoid reconstitution. The purpose of this study was to examine the potential therapeutic efficacy of bone marrow transplants from TP-DC-vaccinated donors. In 2 aggressive metastatic models, bone marrow transplantation with donor bone marrow cells from TP-DC-immunized mice mediated a tumor-specific immune response in the recipient, and this caused regressions of preexisting tumor metastases. After vaccination with TP-DC, donors harbored increased numbers of both activated CD4+ and CD8+ T-cell populations in the bone marrow. Adoptive transfer of T cells purified from the bone marrow of TP-DC-vaccinated mice led to a reduction in preestablished lung metastases, whereas depletion of T cells from bone marrow abolished this effect. By using T cells derived from the bone marrow of TP-DC-vaccinated major histocompatibility complex class I and class II knockout mice, the effector cells required for the observed antitumor effect were determined to be major histocompatibility complex class I-restricted CD8+ T cells. Additionally, the tumor burden in TP-DC-immunized transplant recipients could be reduced further by repetitive TP-DC immunizations after bone marrow transplantation. Collectively, these results demonstrate an important therapeutic role of bone marrow from TP-DC-immunized donors and raise the potential for this approach in patients with advanced cancer.     

Absence of nonhematopoietic MHC class II expression protects mice from experimental autoimmune myocarditis

Experimental autoimmune myocarditis (EAM) is a CD4⁺ T‐cell‐mediated model of human inflammatory dilated cardiomyopathies. Heart‐specific CD4⁺ T‐cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation‐induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV?/? K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α‐myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV?/? K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4⁺ T cells and in expression of IFN‐γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN‐γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN‐γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis.     

Susceptibility and resistance to autoimmunity following neonatal injection of semi-allogeneic spleen cells in rats.

A model of neonatal allotolerance was developed in rats. Brown-Norway (BN) neonates injected with semi-allogeneic (BN x Lewis) F1 hybrid spleen cells express a long-lasting chimerism and exhibit polyclonal B cell activation demonstrated by hyperimmunoglobulinemia affecting mainly IgE and IgG1, anti-laminin and anti-DNA autoantibodies as well as glomerulonephritis and anti-hapten antibodies. These abnormalities are autoregulated although the chimerism persists. In contrast, Lewis (LEW) neonates injected with semi-allogeneic (BN x LEW) F1 hybrid spleen cells exhibit a very short-lasting chimerism and transient activation of B cells, as reflected by increased allo-class II antigen expression, but do not develop an autoimmune disease. The autoimmune syndrome observed in BN rats is similar to that reported in mice during host-versus-graft reaction. Similarities between the drug-induced models of autoimmunity and allogeneic reactions in BN rats are also striking. The susceptibility of BN rats and the resistance of LEW rats to these autoimmune diseases might respectively reflect the involvement of TH2-like or of TH1-like subsets.     

Functional divergence of antigen-specific T-lymphocyte responses in syngeneic graft-versus-host disease.

Administration of cyclosporine after autologous bone marrow transplantation elicits a T-lymphocyte autoaggression syndrome with remarkable similarity to graft-versus-host disease (GVHD). This syndrome, termed syngeneic GVHD (SGVHD), with both acute and chronic phases, is mediated by a restricted repertoire of autoreactive T cells that promiscuously recognize major histocompatibility complex (MHC) class II determinants in association with a peptide from the invariant chain (MHC class II-invariant chain peptide; CLIP). This study evaluated and compared antigen-specific autoreactive T cells during acute and chronic SGVHD ex vivo isolated with a soluble MHC class II immunoglobulin molecular construct. This approach allows for the direct assessment of the functional behavior of the effector T cells without potential modification by in vitro culture. Two major subsets were detected that had overlapping specificity recognizing the MHC class II-binding domain of CLIP but that were differentially dependent on the N- and C-terminal flanking domains of this peptide. The N- and C-terminal subsets were primarily associated with acute and chronic SGVHD, respectively. The cytokine profiles of the CLIP-reactive T cells, however, were most informative and closely correlated with the onset and progression of disease. Levels of type 1 cytokine, particularly interferon-gamma, messenger RNA (mRNA) production, assessed by quantitative polymerase chain reaction, were dominant during acute SGVHD, whereas chronic SGVHD was associated with type 2 cytokine mRNA production. Although there was a dramatic polarization with respect to cytokine production, only subtle changes in antigen specificity could be detected. Unexpectedly, the functional behavior within the antigen-specific effector cell populations is not fixed and seems to change as the disease progresses to the chronic phase. Concordant with the evolution of the effector T-cell response is a differential loss in B7.1 mRNA expression in the N-terminal CLIP-reactive T-cell subset that may reflect the regulation of this autoimmune response. Of additional interest, autoreactive T cells producing interleukin-10 mRNA were detected in both acute and chronic SGVHD, suggesting that this cytokine may play an important but perhaps paradoxical role in both the onset and progression of this experimental autoaggression syndrome.