Inhibition of allorecognition by a human class II MHC–derived peptide through the induction of apoptosis

Abstract

MethodsClass II MHC peptides. We synthesized four 15-to 16-mer peptides, two derived from human class II MHC: HLA-DQA1 (residues 62-77 of the α chain of DQA*0101) and HLA-DQB1 (corresponding residues of the β chain of DQB1*0501) chain, and two derived from rat class II MHC: RT1.D u α (residues 61-75) and RT1.B u α (residues 62-78). The peptides were synthesized by Chiron Mimotopes (Victoria, Australia) using an automated peptide synthesizer. Peptides were purified by reverse-phase HPLC and shown to be >95% homogenous by analytical reverse HPLC and mass spectroscopy. RT1.D u β2 (residues 20-44), a Wistar-Furth (WF) class II MHC peptide, was synthesized in the Protein/Nucleic Acid Laboratory, Brigham and Women's Hospital, Department of Medicine (7,8). Before use, the peptides were dissolved in sterile PBS at a concentration of 1 mg/ml. Peptide sequences are shown in Table 1.Animals. Male Lewis (LEW), Wistar-Furth (WF), and Brown Norway (BN) rats, 8-12 weeks old, were obtained from Harlan-Sprague-Dawley (Indianapolis, Indiana, USA). Adult male CBL/6j and DBA mice, 4-6 weeks old, were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). Rat mixed lymphocyte response. Cervical lymph nodes were har- Received for publication November 6, 1998, and accepted in revised form January 26, 1999. Inhibition of allorecognition by a human class II MHC-derived peptide through the induction of apoptosisThe interaction of the T-cell receptor with the major histocomatibility complex (MHC)-peptide complex is central to T-cell activation. Variation in the nature of the peptide bound within the groove of the MHC molecule may result in an altered T-cell response. Because some naturally processed peptides bound within the groove of the class II MHC molecule are derived from the MHC molecules themselves, we studied the inhibitory effects of synthetic class II MHC peptides on alloimmune responses in vitro. Three peptides derived from a highly conserved region of the class II MHC α chains inhibited the rat mixed lymphocyte response (MLR) in a dose-dependent manner, with the human HLA-DQA1 peptide also inhibiting the human and mouse MLR. No effect was seen on mitogen-induced T-cell proliferation. HLA-DQA1 inhibited cytolytic T lymphocyte (CTL) generation in a dose-response fashion, with no reduction in preformed CTL killing, suggesting that the inhibitory effect is targeted at CD4 + T-cell function. Cell-cycle analysis by flow cytometry showed that restimulation of primed T cells in the presence of HLA-DQA1 resulted in increased apoptosis, whereas unstimulated cells were not affected. These data demonstrate that synthetic peptides derived from highly conserved regions of the class II MHC α chain can alter CD4 + T-lymphocyte alloimmune responses in vitro, and this effect is mediated by the induction of apoptosis in activated T cells. vested from naive LEW, WF, and BN rats, and the lymphocytes were isolated as described previously (9). The cells were then washed twice and resuspended in RPMI-1640 medium (BioWhittaker Inc., Walkersvi...

A Novel Mechanism for the Immunomodulatory Functions of Class II MHC–Derived Peptides

“…We next examined whether inhibition of proliferation of murine lymphocytes caused by HLA-DQA1 was mediated by apoptosis, as shown in previous studies with rat lymphocytes (15). BALB/c lymphocytes primed to RT1.D␤ 2 by immunization were stimulated in the presence or absence of either HLA-DQA1 or HLADQB1, and apoptosis determined at 24 h. Cells incubated in medium alone served as negative control.…”

“…Peptides derived from conserved regions of class II MHC were synthesized by Global Peptide Services (Fort Collins, CO) using an automated peptide synthesizer, purified by reverse phase HPLC, and shown to be greater than 95% homogenous by analytical reverse HPLC and mass spectroscopy (15). The immunogenic peptide RT1.D␤ 2 (20 -44), a Wistar Furth (WF) class II MHC peptide, was synthesized by Macromolecular Resources (Fort Collins, CO), purified by reverse phase HPLC, and shown to be greater than 80% homogenous by analytical reverse HPLC and mass spectroscopy (16).…”

“…Cells incubated in medium alone served as negative control. The induction of apoptosis was assessed by cell cycle analysis after propidium iodide uptake as described previously (15). In addition, apoptosis was demonstrated by staining for annexin V-PE in combination with the viability dye 7-AAD.…”

“…We have previously demonstrated that HLA-DQA1 (62-77) inhibits T cell proliferation in an allele and species nonspecific manner (15). To further study the mechanism of action of this immunomodulatory peptide, we used an antigen-specific mouse model in which BALB/c T cells were primed by immunization to the Wistar Furth rat MHC class II peptide, RT1.D␤ 2 (20 -44).…”

“…Our initial investigations focused on the influence of a group of peptides derived from a highly conserved region of the ␣ chain (residues 62 to 77) of three class II MHC molecules (15). All three peptides inhibited the rat MLR independent of responder or stimulator MHC.…”