A FUNCTIONAL VARIANT OF HLA-DR1 DELINEATED BY T-LYMPHOCYTE CLONE REACTIVITY, PROTEIN CONFORMATION, AND GENOMIC RESTRICTION SITES

This research characterized a serologically indistinguishable form of HLA-DR1 that: (1) cannot stimulate some DR1-restricted or specific T-lymphocyte clones; (2) displays an unusual electrophoretic pattern on two dimensional gels; and (3) is marked by a polymorphic restriction site of the alpha gene. Inefficient stimulation of some DR1-restricted clones was a property of DR1$\sp{+}$ cells that shared HLA-B14 on the same haplotype and/or were carriers of 21-hydroxylase (21-OH) deficiency. Nonclassical 21-OH deficiency frequently demonstrates genetic linkage with HLA-B14;DR1 haplotypes and associates with duplications of C4B and one 21-OH gene. Cells having both stimulatory (DR1$\sb{\rm n}$) and nonstimulatory (DR1$\sb{\rm x}$) parental haplotypes did not mediate proliferation of these clones. However, heterozygous DR1$\sb{\rm x}$, 2 and DR1$\sb{\rm x}$, 7 cells were efficient stimulators of DR2 and DR7 specific clones, respectively, suggesting that a trans acting factor may modify DR1 alleles or products to yield a dominant DR1$\sb{\rm x}$ phenotype. Incompetent stimulator populations did not secrete an intercellular soluble or contact dependent suppressor factor nor did they express interleukin-2 receptors competing for T-cell growth factors. Two dimensional gel analysis of anti-DR immunoprecipitates revealed, in addition to normal DR$\alpha$ and DR$\beta$ chains, a 50kD species from DR1$\sb{\rm x}$ but not from the majority of DR1$\sb{\rm n}$ or non-DR1 cells. The 50kD structure was stable under reducing conditions in SDS and urea, had antigenic homology with DR, and dissociated after boiling into 34kD and 28kD peptide chains apparently identical with DR$\alpha$ and DR$\beta$ as shown by limited digest peptide maps. N-linked glycosylation and sialation of DRgp50 appeared to be unchanged from normal DR$\alpha$ and DR$\beta$. Bg1II digestion and $DR\alpha$ probing of DR1$\sb{\rm x}$ genomic DNA revealed a 4.5kb fragment while DR1$\sb{\rm n}$ DNA yielded 3.8 and 0.76kb fragments; all restriction sites mapped to the 3$\sp\prime$ untranslated region of $DR\alpha$. Collectively, these data suggest that DRgp50 represents a novel combinatorial association between constitutive chains of DR that may interfere with or compete for normal T cell receptor recognition of DR1 as both an alloantigen and restricting element. Furthermore, extensive chromosomal abnormalities previously mapped to the class III region of B14;DR1 haplotypes may extend into the adjacent class II region with consequent intrusion on immune function. ^

STUDIES ON THE CLASS I GLYCOPROTEINS OF THE MURINE MAJOR HISTOCOMPATIBILITY COMPLEX (TRANSPLANTATION ANTIGEN, LYMPHOCYTE, CELL SURFACE MOLECULE)

A series of studies were undertaken to analyze and compare various aspects of murine class I glycoproteins. An initial area of investigation characterized the Qa-1 alloantigens using two-dimensional gel electrophoresis. Analysis of the products of the Qa-1('b), Qa-1('c) and Qa-1('d) alleles indicated that these were distinct molecules as determined by their lack of comigration upon comparative two-dimensional gel analysis. The importance of asparagine-linked glycosylation in the cell surface expression of class I molecules was also examined. These studies employed tunicamycin, an inhibitor of N-linked glycosylation. Tunicamycin treatment of activated T lymphocytes diminished the surface expression of Qa-1 to undetectable levels; the levels of other class I molecules exhibited little or no decrease. These results indicated that N-linked glycosylation has a differential importance in the cell surface expression of various class I molecules. The molecular weight diversity of class I molecules was also investigated. Molecular weight determination of both the fully glycosylated and unglycosylated forms of H-2 and Qa/Tla region encoded molecules established that there is a significant variation in the sizes of these forms of various class I molecules. The most significant difference ((TURN)9,000 daltons) exists between the unglycosylated forms of H-2K('b) and Qa-2, suggesting that the structural organization of these two molecules may be very different. A comparative two-dimensional gel analysis of various class I glycoproteins isolated from resting and activated T and B lymphocytes indicated that class I molecules expressed on activated T cells exhibited an isoelectrophoretic pattern that was distinct from the isoelectrophoretic pattern of class I molecules expessed on the other cell populations. This difference was attributed to a lower sialic acid content of the molecules expressed on activated T cells. Analysis of cell homogenates determined that activated T cells contained a higher level of endogenous neuraminidase activity than was detected in the other populations, suggesting that this may be the basis of the lower sialic acid content. The relationship of the Qa-4 and Qa-2 alloantigens was also examined. It was established that upon mitogen activation, the expression of Qa-4 was greatly decreased, whereas Qa-2 expression was not decreased. However, an anti-Qa-2 monoclonal antibody blocked the binding of an anti-Qa-4 monoclonal antibody to resting cells. These studies established that Qa-4 is a determinant restricted to resting cells, which is closely associated on the surface with the Qa-2 molecule. ^

Transcriptional regulation of the invariant chain associated with MHC class II molecules

The invariant chain associated with the major histocompatibility complex (MHC) class II molecules is a non-polymorphic glycoprotein implicated in antigen processing and class II molecule intracellular transport. Class II molecules and invariant chain (In) are expressed primarily by B lymphocytes and antigen-presenting cells such as macrophages and can be induced by interferon gamma (IFN-$\gamma$) in a variety of cell types such as endothelial cells, fibroblasts, and astrocytes. In this study the cis-acting sequences involved in the constitutive, tissue-specific, and IFN-$\gamma$ induced expression of the human In gene were investigated and nuclear proteins which specifically bound these sequences were identified.^ To define promoter sequences involved in the regulation of the human In gene, 790 bp 5$\sp\prime$ to the initiation of transcription were subcloned upstream of the gene encoding chloramphenicol acetyl transferase (CAT). Transfection of this construct into In expressing and non-expressing cell lines demonstrated that this 790 bp In promoter sequence conferred tissue specificity to the CAT gene. Deletion mutants were created in the promoter to identify sequences important for transcription. Three regulatory regions were identified $-$396 to $-$241, $-$241 to $-$216, and $-$216 to $-$165 bp 5$\sp\prime$ to the cap site. Transfection into a human glioblastoma cell line, U-373 MG, and treatment with IFN-$\gamma$, demonstrated that this 5$\sp\prime$ region is responsive to IFN-$\gamma$. An IFN-$\gamma$ response element was sublocalized to the region $-$120 to $-$61 bp. This region contains homology to the interferon-stimulated response element (ISRE) identified in other IFN responsive genes. IFN-$\gamma$ induces a sequence-specific DNA binding factor which binds to an oligonucleotide corresponding to $-$107 to $-$79 bp of the In promoter. This factor also binds to an oligonucleotide corresponding to $-$91 to $-$62 of the interferon-$\beta$ gene promoter, suggesting this factor may be member of the IRF-1/ISGF2, IRF-2, ICSBP family of ISRE binding proteins. A transcriptional enhancer was identified in the first intron of the In gene. This element, located in a 2.6 kb BamHI/PstI fragment, enhances the IFN-$\gamma$ response of the promoter in U-373 MG. The majority of the In enhancer activity was sublocalized to a 550 bp region $\sim$1.6 kb downstream of the In transcriptional start site. ^

Deviation of the alloimmune response toward tolerance by chimeric class I MHC molecules

Class I major histocompatibility complex (MHC) molecules induce either accelerated rejection or prolonged survival of allografts, presumably because of the presence of immunogenic or tolerogenic epitopes, respectively. To explore the molecular basis of this phenomenon, three chimeric class I molecules were constructed by substituting the rat class I RT1.A$\sp{\rm a}$ sequences with the N-terminus of HLA-A2.1 (N$\sp{\rm HLA-A2.1}$-RT1.A$\sp{\rm a}$), the $\alpha\sb1$ helix (h) with $\rm\alpha\sb{1h}\sp{u}$ sequences ( ($\rm\alpha\sb{1h}\sp{u}$) -RT1.A$\sp{\rm a}$) or the entire $\alpha\sb2$ domain (d) with $\rm\alpha\sb{2d}\sp{u}$ sequences ( ($\rm\alpha\sb{2d}\sp{u}$) -RT1.A$\sp{\rm a}$). Wild type (WT) and chimeric cDNAs were sequenced prior to transfection into Buffalo (BUF; RT1$\sp{\rm b}$) hepatoma cells. Stable transfectants were injected subcutaneously (s.c.) into different hosts 7 days prior to challenge with a heart allograft. In BUF hosts, chimeric ($\rm\alpha\sb{1h}\sp{u}$) -RT1.A$\sp{\rm a}$ accelerated the rejection of Wistar Furth (WF; RT1$\sp{\rm u}$) heart allografts, but had no effect on the survival of ACI (RT1$\sp{\rm a}$) grafts. In contrast, the ($\rm\alpha\sb{2d}\sp{u}$) -RT1.A$\sp{\rm a}$ (containing $\rm\alpha\sb{1d}\sp{a}$ sequences) immunized BUF recipients toward RT1$\sp{\rm a}$ grafts. In WF hosts, WT-RT1.A$\sp{\rm a}$ was a potent immunogen and accelerated ACI graft rejection, N$\sp{\rm HLA-A2.1}$-RT1.A$\sp{\rm a}$ was less effective and ($\rm\alpha\sb{\rm 1h}\sp{u}\rbrack$-RT1.A$\sp{\rm a}$ was not immunogenic. Thus, dominant and subdominant epitopes inducing in vivo sensitization to cardiac allografts are present in the $\alpha\sb1$ helix and the N-terminus, respectively. The failure of ($\rm\alpha\sb{2d}\sp{u}$) -RT1.A$\sp{\rm a}$ transfectants (containing recipient-type $\alpha\sb{\rm 2d}$ sequences) to sensitize WF hosts toward ACI (RT1$\sp{\rm a}$) grafts, despite the presence of donor-type immunogenic $\alpha\sb{\rm 1d}\sp{\rm a}$, suggests that "self-$\alpha\sb2$" sequences displayed on chimeric antigens interfere with immunogenicity. The ($\rm\alpha\sb{1h}\sp{u}$) -RT1.A$\sp{\rm a}$ transfectants injected s.c. prolonged the survival of WF (RT1$\sp{\rm u}$) hearts in ACI (RT1$\sp{\rm a}$) recipients. Furthermore, intra-portal injection of extracts from ($\rm\alpha\sb{1h}\sp{u}$) -RT1.A$\sp{\rm a}$, but not WT-RT1.A$\sp{\rm a}$ or RT1.A$\sp{\rm u}$, in conjunction with a brief cyclosporine course rendered ACI hosts permanently and specifically tolerant to donor-type WF cardiac allografts. Thus, immunodominant allodeterminants are present in the $\alpha\sb1$, but not the $\alpha\sb2$, domain of rat class I MHC molecules. Furthermore, the $\rm\alpha\sb{1h}\sp{u}$ immunogenic epitopes trigger tolerogenic responses when flanked by host-type N-terminal$\sp{\rm a}$ and $\rm\alpha\sb{2d}\sp{a}$ sequences. ^

THE CYTOPLASMIC TAIL OF MHC CLASS I MOLECULES PLAYS A CRITICAL ROLE IN DENDRITIC CELL-INDUCED T CELL IMMUNITY

The presentation of MHC class I (MHC-I)/peptide complexes by dendritic cells (DCs) is critical for the maintenance of central tolerance to self and for the regulation of cytotoxic T lymphocytes (CTL)-mediated adaptive immune responses against pathogens and cancer cells. Interestingly, several findings have suggested that the cytoplasmic tail of MHC class I plays a functional role in the regulation of CTL immune responses. For example, our previous studies demonstrated that exon 7-deleted MHC-I molecules not only showed extended DC cell surface half-lives but also induced significantly increased CTL responses to viral challange invivo. Although exon 7-deleted variant of MHC-I does not occur naturally in humans, the animal studies prompted us to examine whether exon 7-deleted MHC-I molecules could generate augmented CTL responses in a therapeutic DC-based vaccine setting. To examine the stimulatory capacity of exon 7-deleted MHC-I molecules, we generated a lentivirus-mediated gene transfer system to induce the expression of different MHC-I cytoplasmic tail isoforms in both mouse and human DCs. These DCs were then used as vaccines in a melanoma mouse tumor model and in a human invitro co-culture system. In this thesis, we show that DCs expressing exon 7-deleted MHC-I molecules, stimulated remarkably higher levels of T-cell cytokine production and significantly increased the proliferation of meanoma-specific (Pmel-1) T cells compared with DCs expressing wild type MHC-I. We also demonstrate that, in combination with adoptive transfer of Pmel-1 T-cell, DCs expressing exon 7-deleted Db molecules induced greater anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival as compared to DCs expressing wild-type Db molecules. Moreover, we also observed that human DCs expressing exon 7-deleted HLA-A2 molecules showed similarly augmented CTL stimulatory ability. Mechanistic studies suggest that exon 7-deleted MHC-I molecules showed impaired lateral membrane movement and extended cell surface half-lives within the DC/T-cell interface, leading to increased spatial availability of MHC-I/peptide complexes for recognition by CD8+ T cells. Collectively, these results suggesr that targeting exon 7 within the cytoplasmic tail of MHC-I molecules in DC vaccines has the potential to enhance CD8+ T cell stimulatory capacity and improve clinical outcomes in patients with cancer or viral infections.