Using cDNA reverse transcribed from A293T-CIITA RNA, we amplified HLA-DRa, HLA-DRb1 and HLA-DRb5 sequences (GenBank accession nos. NM019111, NM002121, and NM002125, respectively), using primers containing forward restriction site XbaI and the reverse restriction site HindIII (Table S1 ). The haplotyping of HLA-DR isotypes expressed by A293T cells was determined by the Transplantation Immunology Lab, Albany Medical College. Primers were designed to include an intact Kozak sequence upstream of the translation start site. HLA-DRa and b5 plasmids were then used for site-directed mutagenesis using primers indicated (Table S1 ). Transient transfections of all plasmids, including: pDNA3.FLAG.CIITA8 [75] , CIITA-GTP2 and -GTP3 [56] and CIITA -L1035P [58] , p33-143 (coding for the p33 and p35 isoforms of invariant chain-a kind gift provided by Dr. Eric O. Long, NIAID), HLA-DMa and b (pMCFR-PAC and pDMb/MCFRkindly provided by Dr. Lisa K. Denzin, Sloan-Kettering Cancer Center), eGFP-Rab4B (kindly provided by Dr. Marci Scidmore, Cornell University), and HIV Gag-iGFP [77] (kindly provided by Dr. Benjamin Chen, Mount Siani School of Medicine) were performed using a 3:1 ratio Fugene HD Transfection Reagent to DNA in serum-free media as suggested by the manufacturer (Roche, Indianapolis, IN). Virus plasmid DNA provided half of total DNA used in transfection reactions.