HLA-DQ7 beta(1) and beta(2) derived peptides as immunomodulators.

Modulation of protein-protein interactions involved in the immune system by using small molecular mimics of the contact interfaces may lead to the blockage of the autoimmune response and the development of drugs for immunotherapy. The nonpolymorphic beta-regions, exposed to the microenvironment, of the modeled HLA-DQ7, which is genetically linked to autoimmune diseases, were determined. Peptides 132-141 and 58-67, located at the beta(1) and beta(2) domains of HLA-DQ7, respectively, were tested for their involvement in the interactions with CD4(+) T lymphocytes. Linear, cyclic, and dimeric analogs that mimic the exposed surfaces of HLA-DQ7 were designed and synthesized. Their immunosuppressory activities, found in the secondary, humoral immune response to sheep erythrocytes (SRBC) in mice in vitro, ranged from 11% to 53%. The significance of the total charge of the peptides, the pattern of the hydrogen bonding, and the presence of secondary structure were investigated in relation to the immunomodulatory effect of the peptides. Two dimeric analogs of the HLA-DQ7 58-67 fragment, consisting of the two monomers covalently linked by a polyethylene glycol (PEG) spacer, able to mimic the superdimers, were also synthesized and studied. As the 58-67 segment is located at the beta(1) region of HLA-DQ7, close to the major histocompatibility complex (MHC) groove, one may assume that the 58-67 peptide could accommodate the association between T-cell receptor (TCR) and human leukocyte antigen (HLA) by activating a co-stimulatory molecule of the TCR/HLA interaction. This hypothesis is supported by the confocal laser image of the fluorescein-labeled 58-67 peptide and by the fact that it is an immunostimulator at low concentration.

Inhibition of the alloimmune response through the generation of regulatory T cells by a MHC class II-derived peptide.

We have previously shown that HLA-DQA1, a peptide derived from a highly conserved region of MHC class II, prevents alloreactive T cell priming and effector function in vivo, although underlying mechanisms are obscure. In this study, we demonstrate that 28% of mice treated with HLA-DQA1 combined with low-dose rapamycin achieved permanent engraftment of fully MHC-disparate islet allografts and significantly prolonged survival in the remaining animals (log rank, p < 0.001). Immunohistologic examination of the grafts from HLA-DQA1/rapamycin-treated animals revealed up-regulated expression of TGF-ss and FoxP3. In vivo administration of blocking anti-TGF-ss or depleting anti-CD25 mAb augmented T cell alloimmunity and prevented the long-term engraft induced by HLA-DQA1. In vitro experiments further showed that HLA-DQA1 induced differentiation of CD4(+) T cells into CD4(+)CD25(+)FoxP3(+) regulatory T cells. Together, these data provide the first demonstration that HLA-DQA1, a MHC class II-derived peptide, can prolong allograft survival via a TGF-beta and regulatory T cell-dependent mechanisms.

MHC Class II-mediated apoptosis by a nonpolymorphic MHC Class II peptide proceeds by activation of protein kinase C.

It was demonstrated previously that a peptide derived from a conserved region of MHC class II, HLA-DQA1, inhibits proliferation of allogeneic T cells in vitro. Administration of HLA-DQA1 in conjunction with allogeneic cells at the time of priming or at the time of rechallenge prevented the development of the delayed type hypersensitivity response in vivo. The immunomodulatory effects of HLA-DQA1 were associated with the induction of apoptosis in B cells, macrophages, and dendritic cells via a caspase-independent pathway. This study investigated the binding site and mechanism that mediates cell death induced by HLA-DQA1. It was demonstrated that HLA-DQA1 binds to MHC class II on the cell surface, causing MHC class II signaling, initiation of protein kinase C signaling, and mitochondrial membrane depolarization with resultant apoptosis. The data indicate that HLA-DQA1 binds to MHC class II outside the groove, in a manner similar to superantigen. These results suggest that HLA-DQA1 is a novel immunotherapy that may provide an effective means of targeting professional antigen-presenting cells, in particular B cells.

Differential effects of human papillomavirus DNA types on p53 tumor-suppressor gene apoptosis in sperm.

OBJECTIVE: Sperm DNA undergoes apoptotic fragmentation when exposed to HPV DNA. Details of the specific gene regions targeted by HPV in sperm are lacking. The objective of this study was to determine the integrity of exons 5 and 8 of the p53 gene in sperm exposed to HPV DNA. METHODS: Washed sperm were exposed to either HLA-DQA1 (control) or HPV type 6b/11, 16, 18, 31, or 33 DNA fragments for 24 h at 37 degrees C. The integrity of sperm p53 exons 5 and 8 was assessed using a novel DNA disc chip assay based on comparative genomic hybridization. RESULTS: Fragmentation of exon 5 occurred after exposure to HPV DNA type 18. In contrast, only exon 8 was affected by HPV type 16. HPV DNA from type 31 or 33 was without effect on the p53 exons. Sperm motility but not hyperactivation was reduced in all HPV groups. CONCLUSION: The data suggest that different HPV types preferentially degrade different exons of important genes. Decreased motility but not hyperactivation in HPV-exposed sperm suggests retention of some fertilizing capacity and the possibility of transmitting virus-destabilized genes through fertilization.

DQ 65-79, a peptide derived from HLA class II, induces I kappa B expression.

A synthetic peptide corresponding to residues 65-79 of the alpha helix of the alpha-chain of the class II HLA molecule DQA03011 (DQ 65-79) inhibits the proliferation of human T lymphocytes in an allele nonrestricted manner. By using microarray technology, we found that expression of 29 genes was increased or decreased in a human CTL cell line after treatment with DQ 65-79. This study focuses on one of these genes, IkappaB-alpha, whose expression is increased by DQ 65-79. IkappaB proteins, including IkappaB-alpha and IkappaB-beta, are increased in T cells treated with DQ 65-79. Nuclear translocation of the NF-kappaB subunits p65 and p50 is decreased in T cells after treatment with DQ 65-79, while elevated levels of p65 and p50 are present in cytosol. DQ 65-79 inhibits the degradation of IkappaB-alpha mRNA and inhibits the activity of IkappaB kinase. These findings indicate that the DQ 65-79 peptide increases the level of IkappaB proteins, thereby preventing nuclear translocation of the transcription factor, NF-kappaB, and inhibiting T cell proliferation.

Proliferating cell nuclear antigen as the cell cycle sensor for an HLA-derived peptide blocking T cell proliferation.

Synthetic peptides corresponding to structural regions of HLA molecules are novel immunosuppressive agents. A peptide corresponding to residues 65-79 of the alpha-chain of HLA-DQA03011 (DQ65-79) blocks cell cycle progression from early G1 to the G1 restriction point, which inhibits cyclin-dependent kinase-2 activity and phosphorylation of the retinoblastoma protein. A yeast two-hybrid screen identified proliferating cell nuclear Ag (PCNA) as a cellular ligand for this peptide, whose interaction with PCNA was further confirmed by in vitro biochemistry. Electron microscopy demonstrates that the DQ65-79 peptide enters the cell and colocalizes with PCNA in the T cell nucleus in vivo. Binding of the DQ65-79 peptide to PCNA did not block polymerase delta (pol delta)-dependent DNA replication in vitro. These findings support a key role for PCNA as a sensor of cell cycle progression and reveal an unanticipated function for conserved regions of HLA molecules.

Inhibition of cell cycle progression by a synthetic peptide corresponding to residues 65-79 of an HLA class II sequence: functional similarities but mechanistic differences with the immunosuppressive drug rapamycin.

A synthetic peptide corresponding to a region of the alpha1 alpha-helix of DQA03011 (DQ 65-79) inhibits the proliferation of human PBL and T cells in an allele-nonspecific manner. It blocks proliferation stimulated by anti-CD3 mAb, PHA-P, and alloantigen, but not by PMA and ionomycin. Substitution of each amino acid with serine shows that residues 66, 68, 69, 71-73, and 75-79 are critical for function. Inhibition of proliferation is long lasting and is not reversible with exogenous IL-2. The peptide can be added 24 to 48 h after stimulation and still block proliferation. The DQ 65-79 peptide does not affect expression of IL-2 or IL-2R; however, IL-2-stimulated proliferation is inhibited. Cell cycle progression is blocked at the G1/S transition, and the activity of cdk2 (cyclin-dependent kinase 2) kinase is impaired by the continued presence of p27. Although these results suggest a mechanism similar to that of rapamycin, the peptide inhibition is not reversed with FK-506, which indicates a distinct mechanism.

Further investigations on thymopentin-like fragments of HLA-DQ and their analogs.

Recently we showed that the fragments of HLA-DQ with the Thr-Pro-Gln-Arg-Gly-Asp-Val-Tyr-Thr and Gln-Arg-Gly-Asp-Val-Tyr-Thr sequences strongly suppress the immune response, while their shorter analogs, Arg-Gly-Asp-Val, Arg-Gly-Asp-Val-Tyr and Gln-Arg-Gly-Asp-Val-Tyr, show very weak stimulatory activity with respect to humoral immunological response. The fragments contain the sequence which is very similar to thymopentin (pentapeptide Arg-Lys-Asp-Val-Tyr, an active fragment (32-36) of thymopoietin, an immune system activator produced in thymi), and at the same time contains the Arg-Gly-Asp (RGD) sequence, known as an inhibitor of adhesion processes. In the present study we found that a hexapeptide: Arg-Gly-Asp-Val-Tyr-Thr is the smallest size fragment of HLA-DQ having both cellular and humoral immunosuppressive activity. We also found that linear and cyclic fragments of HLA-DQ do not affect cell line production of various cytokines, what suggests that the mechanism of interactions of these peptides with the immunological system is different as compared with most other known immunosuppressors.