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. ^

Molecular analysis of the human $\gamma$-chain T cell receptor genes

In our studies we have focused on the issue of variability and diversity of the $\gamma$ (or $\delta)$ chain T cell receptor (TCR) genes by studying cDNA transcripts in peripheral blood mononuclear cells or $\gamma\delta$ TCR+ T cell clones. The significance of these studies lies in the better understanding of the molecular biology of the $\gamma\delta$ T cell receptor as well as in answering the question whether certain molecular forms predominate in $\gamma\delta$ T cells exhibiting specific immunologic functions. We establish that certain $\gamma$-chain TCR genes exhibit particular patterns of rearrangements in cDNA transcripts in normal individuals. V$\gamma$I subgroup were shown to preferentially rearrange to J$\gamma$2C$\gamma$2 gene segments. These preferential VJC rearrangements, may have implications regarding the potential for diversity and polymorphism of the $\gamma$-chain TCR gene. In addition, the preferential association of V$\gamma$I genes with J$\gamma$2C$\gamma$2, which encode a non-disulfide-linked $\gamma\delta$ TCR, suggests that $\gamma$ chains utilizing V$\gamma$I are predominantly expressed as non-disulfide-linked $\gamma\delta$ TCR heterodimers. The implications of this type of expression remain to be determined. We identified two alternative splicing events of the $\gamma$-chain TCR genes occurring in high frequency in all the normal individuals examined. These events may suggest additional mechanisms of regulation and control as well as diversification of $\gamma\delta$ TCR gene expression. The question whether particular forms of $\gamma$ or $\delta$-chain TCR genes are involved in HLA Class I recognition by specific $\gamma\delta$ cytotoxic T cell clones was addressed. Our results indicated that the T cell clones expressed identical $\gamma$ but distinct $\delta$-chains suggesting that the specificity for recognition of HLA-A2 or HLA-A3 may be conferred by the $\delta$-chain TCR. The issue of the degree of diversity and polymorphism of the $\delta$-chain TCR genes in a patient with a primary immunodeficiency (Omenn's syndrome) was addressed. A limited pattern of rearrangements in peripheral blood transcripts was found, suggesting that a limited $\gamma\delta$ TCR repertoire may be expressed in this particular primary immunodeficiency syndrome. Overall, our findings suggest that $\delta$-chain TCR genes exhibit the potential for significant diversity and that there are certain preferential patterns of expression that may be associated with particular immunologic functions. ^

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.