Critical role of endogenous Mtv in acute lethal graft-versus-host disease

Little is known about the etiology of the graft-versus-host disease (GVHD) occuring after transplantation of lymphoid cells incompatible for minor histocompatibility antigens (mHAg). Here, the potential role of host endogenous mouse mammary tumor virus (Mtv)-encoded superantigens (SAg) in the development of lethal GVHD was investigated. In a combination of H-2^d compatible mice, the presence of Mtv-7 and, to a lesser extent, of Mtv-1, -6, -13 in the host genome, highly increases the rate and severity of GVHD. Kinetic analyses of TCR Vβ gene expression in recipient mice consistently indicate a dramatic but transient infiltration of GVHD target organs by Mtv-SAg-specific T cells. This suggests that SAg encoded by endogenous Mtv, by activating large T cell subpopulations, would help the response to mHAg and thus play a critical role in the initiation or aggravation of GVHD.     

T cell receptor Vβ repertoire in mice lacking endogenous mouse mammary tumor provirus

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) Vβ segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, ?7, ?8 and ?9) and 38CH (Mtv^?) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, ?7, ?8 or ?9. The TcR Vβ chain (TcR Vβ) usage in these mice was analyzed using monoclonal antibodies specific for TcR Vβ2,Vβ3, Vβ4,β5,Vβ6,Vβ7,Vβ8,Vβ11,Vβ12 and Vβ14 segments. Both Mtv-8⁺ mice and Mtv-9⁺ mice deleted TcR Vβ5⁺ and Vβ11⁺ T cells. Moreover, we also observed the deletion of TcR Vβ12⁺ cells by Mtv-8 and Mtv-9 products. Mtv-6⁺ and Mtv-7⁺ animals deleted TcR Vβ3⁺ and Vβ35⁺ cells, and TcR Vβ6⁺,Vβ7⁺ and Vβ8.1⁺ cells, respectively. Unexpectedly, TcR Vβ8.2⁺ cells were also deleted in some backcross mice expressing Mtv-7. TcR Vβ8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn → Asp) in position 19 on the TcR Vβ8.2 fragment. Reactivities of BALB.D2 TcR Vβ8.2 and 38CH TcR Vβ8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR Vβ2⁺, Vβ4⁺ and Vβ8⁺ CD4⁺ T cell subsets in Mtv-8⁺ and Mtv-9⁺ mice, and TcR Vβ4⁺ CD4⁺ T cells in Mtv-6⁺ and Mtv-7⁺ mice, when compared with the T cell repertoire of Mtv^? mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.     

The structural basis of MHC control of collagen-induced arthritis; binding of the immunodominant type II collagen 256 – 270 glycopeptide to H-2Aq and H-2Ap molecules

The A^q major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen-induced arthritis (CIA), whereas the closely related H-2A^p molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H-2A^q and H-2A^p molecules (referred to as A^q and A^p) to bind and present collagen type II (CII)-derived glycosylated and non-glycosylated peptides. T cell clones specific for the immunodominant CII 256 – 270 peptide and restricted to both A^q and A^p molecules were identified. When these clones were incubated with CII protein and either A^q- or A^p-expressing antigen-presenting cells (APC), only A^q-expressing APC were able to induce stimulation. With the use of A_β transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC- or non-MHC genes. Peptide binding studies were performed using affinity-purified MHC class II molecules. The CII 256 – 270 peptide bound with lower affinity to the A^p molecule than to the A^q molecule. Using a set of alanine-substituted CII 256 – 270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the A^q and A^p molecule, i. e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslotionally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256 – 270 peptide did not change the anchor positions used for binding to the A^q or A^p molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the A^q molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256 – 270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II-associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.     

Endogenous retroviral superantigen presentation by B cells induces the development of type 1 CD4+ T helper lymphocytes

The endogenous retroviral superantigen, minor lymphocyte stimulating antigen (Mls 1^a, encoded by Mtv-7), when presented by highly purified B cells induced the development of a highly polarized population of T helper (Th)1 cells from naive peripheral CD4⁺ T cells in vitro. Immobilized anti-Vβb6⁺ antibodies similarly generated highly polarized, largely Vβ6⁺, Th 1 populations in vitro. In the presence of exogenous interleukin-4, both stimuli were capable of generating Th 2, rather than Th 1 populations. Mls 1^a presentation by B cells in vivo led to the development of an equally polarized Th 1 population. Using monoclonal antibodies against interferon-γ and transforming growth factor-β it was demonstrated that maximal Th 1 development with either stimulus in vitro was dependent on the endogenous production of these two cytokines. Thus, our results demonstrate that the retroviral encoded superantigen, Mls 1^a, drives the development of Th 1 cells both in vitro and in vivo, and they suggest that B cell presentation does not, in itself, lead to the generation of Th 2 cells.     

Mls-1 and Mls-2 superantigens do not control susceptibility to collagen-induced arthritis in HI and HII mice.

The HI mouse line is sensitive to collagen-induced arthritis (CIA), whereas HII is refractory, although both express the H-2q permissive haplotype. The two lines also share the same T-cell receptor (TcR) gene haplotypes for alpha and beta chains. The distribution of mouse mammary tumour viruses (MMTV), which encode endogenous superantigens (SAg) such as minor leucocyte-stimulating antigens (Mls) known to modulate the available TcR-V beta repertoire, was investigated in the two lines. Mls-1 is present in HI-susceptible mice, while Mls-2 and Mls-2-like SAg are absent in both lines. This suggests that Mls antigens play no significant role in the resistance to CIA. Moreover, HI and HII exhibit close V beta gene usage as assessed by fluorescence staining with 11 V beta-specific monoclonal antibodies (mAb). These results indicate that mechanisms other than clonal deletion based on V beta expression and induced by SAg are involved in the resistance of H-2q-positive mice to experimental arthritis. Yet, a slightly reduced level of V beta 5+ T cells is observed in HII animals which might correlate with the presence of Mtv-6 and Mtv-9 proviruses.     

The phenotype of H-2M-deficient mice is dependent on the MHC class II molecules expressed

For a broader view of the role of H-2M as an accessory molecule in antigen presentation, we investigated the degree to which different MHC class II isotypes and alleles depend on H-2M to function in vivo. We generated H-2M-deficient animals expressing E^{k / b} or A^k molecules in addition to the A^b molecules already present in the mutant strain, and compared the ability of the different MHC class II molecules to present antigen at the cell surface for recognition by T cells, and contribute to positive selection of CD4⁺ T cells in the thymus. Biochemical analyses were performed to assess MHC class II maturation, and to determine the peptide content of the molecules. In the absence of H-2M, E^{k / b} molecules containd a more heterogeneous set of class II-associated invariant chain peptides (CLIP) than A^b did, which, unlike A^b -CLIP complexes, were not SDS-stable. Unlike A^b molecules, both E^{k / b} and A^k efficiently presented exogenously added peptides to T cells in the absence of H-2M. In addition, epitopes from some proteins, especially those known to be invariant chain independent, were presented by A^k molecules in the mutant animals. To our surprise, expression of E^{k / b} overcame the positive selection defect observed in H-2M-deficient mice expressing A^b alone. In contrast, A^k expression did not augment positive selection of CD4⁺ T cells in the mutant animals. Some of these findings in vivo contrast significantly with findings from in vitro studies on murine MHC class II molecules in human DM-deficient cell lines.     

Vβ11+ T-lymphocyte expansion by toxic shock syndrome toxin-1 differs in mice bearing H-2q versus H-2b haplotypes

We have recently demonstrated that toxic shock syndrome toxin-1 (TSST-1) expanded Vβ11⁺ T lymphocytes contribute to Staphylococcus aureus arthritis and sepsis-induced mortality. Interestingly, Vβ11⁺ T-cell mediated joint pathology varies in different mouse strains. In this study, we characterized the in vitro pattern of Vβ11⁺ T-cell expansion by TSST-1 in mice with various genetic backgrounds. Mice expressing major histocompatibility complex (MHC) class II I-E molecules did not expand Vβ11⁺ T cells upon stimulation with TSST-1. Using B10 congeneic I-E negative mouse strains, we found that the TSST-1-expanded Vβ11⁺ T cells in B10Q (H-2^q) and B10M (H-2^f) mice but not in B10B (H-2^b) mice. Antigen-presenting cells (APC) from B10Q mice, L cells and lymphoma cell line transfected with a q gene did not restore the deficient Vβ11⁺ T-cell expansion by TSST-1 in purified T cells from B10B mice. In contrast, I-A^b APC were able to stimulate Vβ11⁺ T cells from H-2^q mice. Furthermore, Vβ11⁺ T cells in H-2^b mice did expand when exposed to staphylococcal enterotoxin A (SEA). These findings suggest that the T-cell repertoire, skewed by clonal deletion and inactivation of self-reactive T cells, accounts for the different magnitude of Vβ11⁺ T-cell expansion among the different mouse strains.     

Major histocompatibility complex class I presentation of exogenously acquired minor alloantigens initiates skin allograft rejection

Major histocompatibility complex (MHC) class I molecules present peptides from endogenous proteins. However, in some cases class I-restricted peptides can also derive from exogenous antigens. This MHC class I exogenous presentation could be involved in minor histocompatibility antigen (mHAg)-disparate allograft rejection when donor alloantigens are not expressed in graft antigen-presenting cells (APC) that initiate the rejection mechanism. Here we addressed this question by using a skin graft experimental model where donors (H-2^b or H-2^d Tgβ-gal mice) expressed the mHAg like β-galactosidase (β-gal) in keratinocytes but not in Langerhans' cells (LC) which have an APC function. Rejection of Tgβ-gal skin by a β-gal-specific CD8 cytotoxic T lymphocyte (CTL) effector mechanism should require presentation by donor and/or recipient LC of MHC class I-restricted peptides of exogenous β-gal shed by keratinocytes. Indeed, our results showed that 1) H-2^b Tgβ-gal skin was rejected by H-2^bxs and H-2^bxd recipients; 2) rejection was mediated by β-gal-specific CD8⁺ CTL effectors; and 3) H-2^bxd mice having rejected H-2^b Tgβ-gal skin generated β-gal-specific CTL restricted by H-2^b and H-2^d class I molecules and rejected subsequently grafted H-2^d Tgβ-gal skin in an accelerated fashion, demonstrating that recipient LC have presented exogenous β-gal-derived MHC class I epitopes. These results lead to the conclusion that MHC class I exogenous presentation of donor mHAg can initiate allograft rejection.     

T CELL REPERTOIRE SELECTION BY MOUSE MAMMARY TUMOUR VIRUSES

Mouse mammary tumour viruses (Mtv) are B-type retroviruses. These can be exogenous, transmitted via maternal milk, or endogenous, as proviral integrations into the mouse genome, transmitted vertically in a Mendelian fashion. A number of different sites of integration of endogenous Mtvs have been reported in various inbred mouse strains. An open reading frame (ORF), within the long terminal repeat (LTR) of Mtv, encodes a type 2 integral membrane glycoprotein. The ORF products are expressed in association with MHC class II molecules at the cell surface and have an affinity for certain T cell receptor (TCR) Vβ chains such that CD4⁺8⁺ TCR⁺ double positive thymocytes expressing these Vβ chains undergo programmed cell death in mice carrying the appropriate endogenous or exogenous Mtvs. This constitutes a measurable part of negative repertoire selection of the T cell repertoire. Some positive selection of the T cell repertoire also appears to be TCR Vβ-specific, although the involvement of polymorphic ligands other than MHC molecules is not apparent. This minireview summarizes the published work on the TCR Vβ specificity and chromosomal localization of the various mouse mammary tumour proviral integrations leading to negative selection, and discusses the nature of TCR Vβ-specific positive selection.     

PREDNOD,a prediction server for peptide binding to the H-2g7 haplotype of the non-obese diabetic mouse

The non-obese diabetic (NOD) mouse is a widely used animal model for study of autoimmune diseases, in particular human type 1 diabetes mellitus (T1DM). Identification of the subset of peptides that bind MHC molecules comprising the H-2^g7 haplotype of NOD mouse and thereby representing potential NOD T-cell epitopes is important for research into the pathogenesis and immunotherapy of T1DM. The H-2^g7 haplotype comprises the MHC class-I molecules K^d and D^b and a single class-II molecule I-A^g7. We have developed a prediction system, PRED^NOD, for accurate identification of peptides that bind the MHC molecules constituting the H-2^g7 haplotype. PRED^NOD is accessible at http://antigen.i2r.a-star.edu.sg/Ag7.     

Chronic deletion, escape from deletion and activation of mouse mammary tumor virus superantigen-reactive T cells in C57BL/10 mice.

Though C57BL/10 mice express the mouse mammary tumor virus superantigens (sag) encoded by Mtv-8 and Mtv-9, it has been thought that these sag do not bind to the MHC class II molecule H2-Ab and consequently do not affect the T cell repertoire. However, we show that cells bearing TCR Vbeta chains specific for Mtv-8 and -9 sag are chronically deleted in C57BL/10 mice. Thymocytes and peripheral T cells escaping deletion by Mtv sag display a small reduction in the level of cell surface CD4. T cells escaping thymic deletion respond variably to endogenous Mtv sag with some, but not all, reactive populations appearing overrepresented in the activated/memory subset. The data suggest that in normal mice fine modulation of coreceptor expression levels may be a common way by which thymocytes escape elimination, that systems utilizing potentially Mtv sag-reactive TCR on a C57BL background may be inappropriate for the measurement of the affinity of TCR/MHC/peptide interactions required in thymic selection, and that detection of the activity of human sag may be aided by analysis of CD4 levels and activation markers on T cells in conjunction with studies of the frequency of cells bearing specific TCRVbeta chains.     

CD68-expressing cells can prime T cells and initiate autoimmune arthritis in the absence of reactive oxygen species

It is widely believed that DC, but not macrophages, prime naïve T cells in vivo. Here, we investigated the ability of CD68‐expressing cells (commonly defined as macrophages) in priming autoreactive T cells and initiating collagen‐induced arthritis (CIA) in the mouse. For this purpose, a transgenic mouse was developed (MBQ mouse) where macrophages exclusively expressed the MHC class II H2‐A^q (A^q) on an H2‐A^p (A^p) background. A^q, but not A^p expression mediates susceptibility to CIA through presentation of type II collagen (CII) to T cells. CIA severity is enhanced by a mutation in the Ncf1 gene, impairing reactive oxygen species (ROS) production by the phagocyte NADPH oxidase (NOX2) complex. Expression of functional Ncf1 on macrophages was previously shown to protect from severe CIA. To study the effect of ROS on macrophage‐mediated priming of T cells, the Ncf1 mutation was introduced in the MBQ mouse. Upon CII immunization, Ncf1‐mutated MBQ mice, but not Ncf1 wild‐type MBQ mice nor Ncf1‐mutated A^p mice, activated autoreactive T cells and developed CIA. These findings demonstrate for the first time that macrophages can initiate arthritis and that the process is negatively regulated by ROS produced via the NOX2 complex.     

HIERARCHY OF SPEA PRESENTATION TO T CELLS BY MOUSE MHC HAPLOTYPES

The ability of splenic antigen-presenting cells (APC) from nine independent mouse major histocompatibility complex (MHC) haplotypes to present recombinant streptococcal exotoxin A (rSPEA) to heterogeneous T cells and mouse T cell clones were compared using proliferation assays. We report that there is marked variation between MHC haplotypes, which can be ranked as follows: H2^z > H2^s = H2^f = H2^p = H2^r = H2^k > H2^d > H2^b = H2^q. In some haplotypes both A and E molecules bind rSPEA with low affinity. In addition, we show that presentation is preferentially by E molecules in haplotypes where A and E are co-expressed, but that A alleles also bind and present rSPEA, based on inhibition of responses by anti-E and anti-A mAbs. Furthermore, using strains of mice which fail to express E, we demonstrate that A^s and A^f present rSPEA with high efficiency, whereas A^q and A^b are the least efficient of all haplotypes. The results suggest that there is significant variation in the ability of different alleles of both E and A molecules to bind and present rSPEA.     

Mouse intestinal epithelial cells express the self superantigen Mis1

In previous studies, we demonstrated that intestinal epithelial cells of the mouse small intestine could present exogenous antigen to specific CD4⁺ T cell hybridomas. We now report on the abililty of normal enterocytes to present the self superantigen M1sl^a. Enterocytes from M1sl^a but not from M1sl^b strains stimulated interleukin-2 production through aVβ6⁺ T cell hybridoma specific for M1sl^a determinants. Antibody inhibition experiments showed that enterocytes presented M is determinants via a major histocompatibility complex class II-dependent mechanism. Furthermore, the ability of enterocytes to activate Vβ6⁺ M1sl^a-specific T cells was inhibited by monoclonal antibodies against the Orf protein encoded by an Mtv-7 provirus which is associated with M1sl^a expression. These findings provide evidence for the first time that M is determinants are expressed on normal enterocytes and support the theory of a possible role of these cells in extrathymic selection of T cell receptor Vβ repertoire of intraepithelial T lymphocytes.     

Regulation of T cell function by Mtv-7 gene products.

Mls-1, a superantigen encoded by the endogenous mouse mammary tumor virus Mtv-7 induces immunological tolerance through deletion of antigen-reactive T cells. A remarkable difference between this self-antigen and self-MHC antigens is that while the mouse establishes tolerance against self MHC antigens by the time of birth it does not begin to delete T cells specific for the self-superantigen until they had mounted an immuneresponse against it. An immune response occurs normally several days after birth and may be delayed experimentally for weeks before the deletion process ensues. However, for effective deletion of Mls-1 reactive T cells the mouse must be exposed to Mtv-7 positive lymphoid cells within hours after birth. In reviewing here data obtained in this and other laboratories regarding experimental induction of Mls-1 tolerance in neonatal mice we are trying to make a case for the involvement of Mtv-7 encoded antigens distinct from the superantigen. We propose that T cells reactive with non superantigenic Mtv-7 determinants pose a threat to the establishment of chimaerism between Mls-1- neonates and Mls-1+ inocula, as they may cause the rejection of Mls-1 superantigen bearing lymphocytes. Chimaerism is essential for the establishment of lasting Mls-1 tolerance.     

Normal T cell receptor Vβ usage in a primary immunodeficiency associated with HLA class II deficiency

The human Tcell receptor was studied using an anchored-polymerase chain reaction (A-PCR) and hybridization with Vβ-specific oligonucleotide probes, together with the few anti-Vβ monoclonal antibodies (mAb) available. After confirming the semiquantitative and reproducible nature of the A-PCR technique, we assessed the complete Vβ repertoire in sorted CD4⁺ and CD8⁺ lymphocyte populations from three normal donors. These experiments confirmed the absence of Vβ-restricted deletions in human peripheral cells, in contrast to several mouse strains. This feature makes it difficult to study negative selection in man, given the apparent absence of an endogenous superantigen corresponding to the Mis system in the mouse. To investigate human Vβ repertoire shaping, we studied VP usage in CD4+ and CD8⁺ T cells from children with an inherited immunodeficiency characterized by defective expression of human leukocyte antigen class II molecules. An initial study using anti-Vβ monoclonal antibodies failed to show significant abnormalities in Vβ usage. Four patients analyzed using the A-PCR method all had a polyclonal Vβ repertoire, suggesting normal positive selection and raising questions as to the importance of VP major histocompatibility complex (MHC) interactions and the role of thymic MHC density in shaping the Vβ repertoire.     

Poor loading of major histocompatibility complex class II molecules with endogenously synthesized short peptides in the absence of invariant chain

In normal antigen-presenting cells, newly synthesized major histocompatibility complex (MHC) class II molecules associate with the invariant chain (Ii) glycoprotein in the endoplasmic reticulum (ER). They are loaded with peptides only after proteolytic removal of the Ii in post-Golgi endocytic vesicles. Since the Ii inhibits peptide binding to MHC class II molecules, this association could protect MHC class II molecules from being loaded with endogenous peptides early after biosynthesis. If this were an important function of the Ii in vivo, MHC class II molecules synthesized in cells lacking the Ii should be loaded efficiently with short endogenous peptides in the ER; such peptides are known to be present there due to TAP-mediated import from the cytosol. To examine this possibility, we have studied peptide loading in HeLa transfectants expressing murine H-2A^k MHC class II molecules either alone or together with an excess of Ii. Endogenous peptides could readily be extracted from conformationally intact A^k αβ dimers of biosynthetically labeled Ii⁺ cells, whereas peptide loading was greatly (> 95%) diminished in the absence of Ii. Significant amounts of sodium dodecyl sulfate-(SDS) stable 55-kDa peptide: A^k complexes were only found in the Ii⁺ transfectants. In the absence of Ii, the MHC class II molecules instead formed stable complexes with long (20 and 50 kDa) polypeptides. Known A^k-binding peptides bound stably to A^k molecules on Ii^? cells, could be co-purified with them, and were resistant to release in SDS, suggesting that poor recovery of endogenous peptides was not due to decreased stability of A^k: peptide complexes in the absence of Ii. We conclude that protection of MHC class II molecules from endogenous short peptides does not appear to be a quantitatively important function of the Ii molecule, because peptide loading is inefficient in its absence.