Biochemical analyses demonstrate that the murine and human “immune rebuilding factor” is B2M. (A-B) Murine and human NK/DC coculture supernatants were fractionated by HIC+RP. One out of the 30 fractions (fraction 22) displayed the most robust biological effect with enhanced murine thymocyte, T-cell, B-cell, and DC counts in vivo and increased human thymocyte cellularity in vitro in the human TEC/thymocyte cultures (the in vivo increase in murine T cells, B cells, and DCs is not shown). (C-D) Two parallel NK/DC coculture experiments were performed, 1 with and 2 without ³⁵S-methionine, and supernatants were fractionated by HIC+RP chromatography. (C) Fractions from the radioactive experiment were subjected to SDS-PAGE and autoradiography. An 11 to 12 kDa MW protein was detected in the fractions that corresponded to those that exerted maximal immune rebuilding activity in the nonradioactive experiment. (D) Two-dimensional electrophoresis showed the 11 to 12 kDa MW protein had isoelectric points (pI) of 7.5 and 8 for murine and 6 to 7 for human samples. (E) The 14 top-ranking proteins identified by mass spectrometry that displayed a ≥1.5-fold increase in protein content in alloreactive NK/DC coculture supernatants as compared with nonalloreactive NK/DC coculture supernatants. Proteins were ranked according to presence of (1) 11 to 12 kDa MW, (2) pI of 7.5 and 8 for murine and 6 to 7 for human samples (on top of panel E, the combination of the above biochemical features is denoted as “MW-pI”), and (3) detection of the protein displaying the above biochemical features in all murine and human experiments. The only protein that possessed all of these features was B2M.