Ling Tian

Synergistic mechanisms by which sirolimus and cyclosporin inhibit rat heart and kidney allograft rejection

The studies presented herein examined the mechanism(s) whereby sirolimus (SRL) and cyclosporin (CsA) act synergistically to block allograft rejection. Combination index (CI=1 reflects additive, CI<1 antagonistic, and CI<1 synergistic, effects) analysis documented potent synergism between SRL and CsA to block allograft rejection. Combinations of the two drugs produced synergistic prolongation of heart (CI=0.001–0.2) or kidney (CI=0.03–0.5) allograft survival at SRL/CsA ratios ranging from 1:12.5 to 1:200. Pharmacokinetic analysis of the individual drugs showed that CsA does not affect the blood levels of SRL, and SRL mildly increases the levels of CsA in SRL/CsA‐treated rats. Quantitative polymerase chain reaction analysis was used to document that both subtherapeutic (1.0 mg/kg) and therapeutic (2.0 or 4.0 mg/kg) CsA doses inhibited the expression of interferon‐gamma (IFN‐γ) (P<0.03) and IL‐2 (P<0.003) mRNA produced by T helper (Th) 1 cells, as well as IL‐10 (P<0.001), but not IL‐4 (NS) mRNA produced by Th2 cells. Contrariwise, all tested SRL doses (0.02, 0.04 or 0.08 mg/kg) did not affect cytokine mRNA expression. However, heart allografts from rat recipients treated with synergistic SRL/CsA doses displayed reduced levels of IFN‐γ (P<0.01), IL‐2 (P<0.001) and IL‐10 (P<0.001) mRNA. Thus, because subtherapeutic doses of CsA reduce Th1/Th2 activity, thereby facilitating the inhibition of signal transduction by low does of SRL, the two agents act synergistically to inhibit allograft rejection.

Cytokine mRNA expression in tolerant heart allografts after immunosuppression with cyclosporine, sirolimus or brequinar

We sought to examine the impact of the preferential activation of Th2 cells on the induction and maintenance of a tolerant state in heart allograft rat recipients treated with a short course of cyclosporine (CsA), sirolimus (SRL) or brequinar (BQR). A quantitative polymerase chain reaction (PCR) method was used to measure the levels of cytokine mRNAs, namely interferon (IFN)-gamma and interleukin (IL)-2 in T helper 1 (Th1) cells and IL-4, IL-5 and IL-10 in Th2 cells. Our main findings were that on day 5 postgrafting allografts from untreated recipients had increased levels of IFN-gamma (216 +/- 119 fg), IL-2 (449 +/- 75 fg), IL-4 (6.2 +/- 1.3 fg), IL-5 (34.8 +/- 9.3 fg) and IL-10 (1554 +/- 184 fg) mRNAs compared with normal hearts. CsA reduced the levels of IFN-gamma, IL-2, IL-5 and IL-10, but not IL-4, mRNAs. SRL did not affect the expression of cytokine mRNAs. BQR decreased the levels of IFN-gamma, IL-2 and IL-10, but not IL-5 or IL-4 mRNAs. Compared with grafts from untreated recipients, those from CsA- or BQR-treated tolerant hosts (day 100) displayed undetectable IL-2 mRNA levels, and reduced levels of IFN-gamma, IL-4 and IL-10 mRNAs. In fact, the patterns of cytokine mRNA expression in grafts from CsA- and BQR-treated tolerant hosts were similar to those of normal hearts. Grafts from SRL-treated tolerant hosts merely showed slightly increased Th2 cell activity. In conclusion the selective activation of Th2 cells is not absolutely required for induction or maintenance of tolerance.

Nucleotide sequences of three distinct clones coding for rat heavy chain class I major histocompatibility antigens

Poly(A){sup +} RNAs were isolated from ConconavalinA stimulated splenocytes of BUF (RT1.A{sup b}), PVG (RT1.A{sup c}), or PVG.1U (RT1.A{sup u}) rats, respectively, using a Micro-Fast Track kit. After reverse transcription with a synthetic oligo-d(T) primer (5{sup {prime}}-CAT GAT CGA ATT CAC GCG TCT AGA TTT TTT TTT TTT TTT TTT TTT TTT TVN-3{sup {prime}}, V = A+G+C, N = A+T+G+C; Genosys, Woodland, TX), 1.6 kilobase products, which encode the entire MHC class I protein and the 3{sup {prime}} non-translated region including the poly-A tail, were amplified by polymerase chain reaction (PCR) using two synthetic oligonucleotide primers (Genosys). The upstream primer (5{sup {prime}}-GTC CGG GWT CTC AGA TGG GG C-3{sup {prime}}, W = A+T) was designed based upon the published rat class I sequences of eight genes: RT1.1{sup a} M31018; rat LW2 gene X70066; RT1.1{sup 1}, L26224 X79719; RT1.A{sup u} X82669, and RT1.Aw3 L40363, RT1.E{sup u} L40365, RT1.C{sup 1} L40362. The downstream primer (5{sup {prime}}) ATG ATC GAA TTC ACG CGT CTA GA-3{sup {prime}} was the portion of the oligo-d(T) primer used for reverse transcription. The purified PCR products were inserted into pCR II cloning vectors (Invitrogen). Automated sequencing of plasmid cDNAs from the positive clones obtained from three repeated PCRmorexa0» amplifications identified by restriction enzyme mapping were reproducible. Comparison between new sequences of the heavy chain class I genes and those available in GenBank. 7 refs., 1 fig.«xa0less

Nucleotide sequences of three distinct cDNA clones coding for the rat class I heavy chain RT1n antigen

We isolated and sequenced three different RT1n cDNA clones that each encode the entire major histocompatibility complex (MHC) class I heavy chain protein, including the 39 untranslated region (UTR) and the poly-A tail. The mRNA was isolated from Concanavalin A-stimulated (Pharmacia, Piscataway, NJ) splenocytes of BN (RT1n) rats (Harlan Sprague-Dawley, Indianapolis, IN). The cDNA library was synthesized from BN mRNA using an reverse transcription (RT)-polymerase chain reaction (PCR)-based cloning method (Wang et al. 1996). Using previously described upstream and downstream primers in a PCR-based method (Wang et al. 1996) we isolated three RT1n clones. To assure the accuracy of each RT1n clone, we individually sequenced the cDNA samples from triplicate PCR reactions as well as from triplicate TA-cloning reactions. Multiple RT1n clones for each cDNA were then sequenced in both directions to eliminate any possibility of random PCR mutations. Although we performed sequencing of multiple clones, we cannot completely exclude the possibility of PCR artifact. As previously suggested (Joly et al. 1995), undesirable PCR reactions such as polymerase “jumping” between different sequences are possible, especially at high concentrations of templates. We found that our three RT1n sequences are similar to rat class I MHC sequences available in the GenBank, including two RT1n sequences (Joly et al. 1995; X90375, X90376; Fig. 1). Interestingly, our clones 4 and 12 are identical in the 39 UTR and at the 39 half of the coding region. Furthermore, the sequence of our clone 12 (U50448) is similar to the partial cDNA sequence of RT1n (X90375) with the exception of two nucleotides (GC) at positions 564 and 565, which differ from those (CG) presented in their sequence. This difference in our amino acid sequence produced Trp and Leu amino acids at positions 191 and 192, instead of Cys and Val amino acids as in the RT1n (X90375) sequence. Our three cDNAs were subcloned into pMAMneo (CloneTech, Palo Alto, CA), an inducible eukaryotic expression vector, and transfected into rat BUF (RT1b) hepatoma cells. Neomycin (Life Technologies; Grand Island, NY)-resistant transfectants (0.5 × 106) stimulated with 1 μg dexamethasome (DEX; Sigma, St. Louis, MO) for 6 h were tested for cell-membrane expression of class I MHC molecules by FACS analysis with an FITC-conjugated mouse anti-rat class I MHC-specific monoclonal antibody (OX18; Pharmingen, San Diago, CA). Both non-induced and DEXinduced normal BUF cells expressed almost identical baseline amounts of class I RT1.Ab molecules (Fig. 2 A, B). Similarly, DEX-stimulated control BUF cells that had been transfected with pMAMneo alone (without RT1n cDNA) expressed a similar number of class I MHC molecules as non-transfected controls (Fig. 2 C). In contrast, following DEX induction, BUF hepatoma cells transfected with any of three RT1n clones displayed increased expression of membrane-bound class I MHC molecules (Fig. 2 D, E, F). Thus, our results document that all three transfected RT1n genes may be expressed on the cell surface, when under control of a DEX-inducible promoter.

Nucleotide sequences of three distinct complementary DNA clones encoding rat class II major histocompatibility complex RT1.D beta-chain proteins.

The polymerase chain reaction (PCR)-based method with two designed primers was used to obtain three complementary DNA (cDNA) clones encoding the b chain of rat RT1.D class II major histocompatibility complex (MHC) molecules. The total RNA was isolated using an RNA isolation kit (Ambion Inc, Austin, Tex.) from normal and/or interferon-g (IFN-g; Genzyme Diagnostics, Cambridge, Mass.)-stimulated splenocytes harvested from either ACI (RT1), Brown Norway (BN, RT1), or Wistar Furth (WF, RT1) inbred rats (Harlan Sprague-Dawley, Indianapolis, Ind.). The cDNAs were synthesized from the RNA samples using synthetic oligo-d(T) plus primer (Wang et al. 1996) and reverse transcriptase enzyme (Promega, Madison, Wis.) in a reverse transcription reaction. The 1.1 kilobase products (encoding the entire RT1.D bchain protein and the 3’ nontranslated region) were amplified by PCR using two synthetic oligonucleotide primers (Genosys, Woodland, Tex.). We designed the upstream primer (5b-TCT CCT CTC CTG CAG CAT3b) based upon the analysis of seven genes encoding rat, mouse, and cat class II MHC b chains (rat: RT1.D X53054, Syha-Jedelhauser and Reske 1990; mouse: H2E, M36939 M18579, H2-E, M36940, King et al. 1988; H2-E, M35677 M34123; H2-E, M35680 M34124, Begovich et al. 1990; and cat: DRB*0507, U51537, DRB*0506 U51536, Yuhki and O’Brian 1997). The downstream primer (5b-ATG ATC GAA TTC ACG CGT CTA GA-3b) was previously designed to include the portion of the oligo-d(T) plus primer sequence (Wang et al. 1996). The PCR products were inserted into PCR 2.1 TA-cloning vectors (Invitrogen, San Diego, Calif.) and transformated into XL1-blue competent cells (Stratagene, La Jolla, Calif.). The positive clones with class II MHC cDNA obtained from three or four separated PCR reactions were individually sequenced using an automated sequencer (ABI Prism 377 DNA Sequencer, Applied Biosystems, Foster City, Calif.). Three RT1.D b chains were sequenced in both directions using two internal primers, with reproducibility confirmed at least three times for each PCR reaction and at least six times for each rat strain. A comparison of RT1.D, RT1.D, and RT1.D b-chain genes with a published RT1.D b-chain gene (X53054) documented a high degree of similarity at both the nucleotide and amino acid sequence levels (Fig. 1). In particular, sequence analysis showed 93.6–98.7% of identical nucleotides and 92.3–100% of identical amino acids in the leader domain; 85.6–88.8% and 73.7–82.1% in the b1 domain; 96.4–97.7% and 96.1–98.1% in the b2 domain; and 96.6–97.4% and 97.4–100% in the transmembrane/ cytoplasmic (TM/CYTO) domain. Furthermore, comparison of four RT1.D b-chain protein sequences revealed two polymorphic amino acids in the leader domain, 17–25 in the b1 domain, two to four in the b2 domain, and one in the TM/CYTO domain. Thus, most of the polymorphism is localized in the rat RT1.D b1 domain.

Localization of tolerogenic epitopes in the α1 helical region of the rat class I major histocompatibility complex molecule

Abstract Induction of tolerance to organ allografts remains an elusive goal in transplant biology. Tolerance toward heart allografts was induced by preoperative intrathymic (IT) inoculation of extracted soluble histocompatibility antigens (HAg) in combination with a single dose of anti-T-cell antibodies.1 In contrast, peritransplant intravenous (iv) or intraportal administration of HAg combined with a short course of cyclosporine (CyA) prolonged allograft survival but did not induce transplantation tolerance.2 IV injection of peptides corresponding to amino acid (AA) residues 75–84 of the human HLA-B7 protein combined with a short course of CyA before and after grafting induced donor-specific tolerance to heart allograft in rats.3 The present experiments examined the localization of tolerogenic epitopes in rat class I major histocompatibility complex RTI.Au and RT1.A1 molecules.

Nucleotide sequences of rat cDNA clones coding heavy chain class I major histocompatibility complex proteins.

Abstract The membrane-bound class I major histocompatibility complex (MHC) dimer is composed of a heavy chain (45-kd) that is noncovalently associated with a β 2 -microglobulin ( β 2 -m; 12-kd) light chain. 1 The heavy chain of the class I MHC dimer contains the membranedistal polymorphic α 1 (90 amino acids; AA) and α 2 (92 AA) domains, each of which forms an a-helix and four anti-parallel β-pleated strands. The α 2 domain is attached to a membrane-proximal α 3 (92 AA) domain, which is less polymorphic, and followed by the conserved transmembrane (25 AA) and cytoplasmic (30 AA) domains. The α 1 and α 2 domains of the rat class I MHC RT1.A a molecule bear highly polymorphic sequences which are immunogenic and induce allograft rejection. Two peptides were derived from the helical regions of the rat class I MHC RT1A a sequence. One peptide was localized in the a, helical region (56–72 AA and 72–83 AA), and one was localized in the α 2 helical region (145–155 AA). Both peptides elicited the proliferative response of alloantigen-specific T cells. 2 Our studies showed that the α 1 helical region of the RT1.A 1 molecule contains a potent immunogenic epitope localized at AA positions 62, 63, 65, and 69. 3 To examine the localization of other immunogenic epitopes, we isolated and sequenced additional rat class I MHC cDNAs.