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Figure S1: pMHCI tetramers in solution are prevented from re-binding as they are captured by the anti-HLA A2 antibody BB7.2. A ; 1.5x106 003 CTL were washed and resuspended in 100l ice cold azide buffer 0.1% NaN3 0.5% FCS in PBS ; and stained with PE-conjugated HLA A2SLYNTVATL tetramer for 20 min on ice. CTL were washed twice in ice cold azide buffer, resupended in 100l azide buffer and an excess of anti-HLA A2 antibody clone BB7.2 ; added at 100g ml. The sample was maintained at room temperature and analyzed on a FACSCalibur flow cytometer at 0, 2, 5, 8, and 20 min time points to demonstrate that tetramer levels decayed from the cell surface at the rate previously determined data not shown ; . After analyzing the level of tetramer staining at 2 hr, the sample was split into 2 separate aliquots. A ; One sample was washed in ice cold azide buffer and either resuspended with no BB7.2 antibody or with BB7.2 antibody at 100g ml. There is a time delay in the decay seen between 3-4 hr due to the washing step, but the normal rate of decay is resumed in the presence of BB7.2 antibody. If no BB7.2 antibody is added, then decay does not continue as tetramer rebinding from solution is no longer blocked by the antibody. B ; The remaining sample was split again and either maintained under the original decay conditions 100g ml BB7.2 ; or in the presence of an additional 200g ml BB7.2. The addition of further BB7.2 did not significantly increase the rate of decay suggesting that BB7.2 is present in excess when used at 100 g ml in our decay experiments. Figure S2: pMHCI tetramers do not decay significantly from cell surface TCR in the absence of the blocking antibody.1.5x106 868 CTL were resuspended in 100l azide buffer 0.1% NaN3 0.5% FCS in PBS ; and stained with HLA A2 Q115E-SLYNTVATL, HLA A2-SLYNTVATL, HLA A2 A245V-SLYNTVATL or HLA DT227 8KA-SLYNTVATL tetramers conjugated to PE, and 10l 7AAD, BD Biosciences for 20 mins on ice. The concentration of tetramer used was previously determined by titration to give a starting MFI of 200. Cells were then washed in ice cold azide buffer, resuspended in azide buffer without blocking antibody placed at room temperature and analyzed at the time points shown using a FACSCalibur flow cytometer. Similar results were obtained with other CTL data not shown ; . Figure S3. The pMHCI CD8 interaction enables recognition of natural weak agonist peptides and contributes to T cell cross-reactivity in SLYNTVATL-specific CTL. The HIV-1 p17 Gag epitope mutates in response to CTL attack to produce variant antigens that escape from recognition 31 ; . Some of these variants act as weak agonists of 003 and 868 CTL 31 ; and provide a natural system in which to test TCR cross-reactivity. A&B show the lysis of wildtype HLA A2 A ; or DT227 8KA CD8-null B ; targets pulsed with 1 M SLYNTVATL or the natural variant peptide SLHNTVATL that acts a s a weak agonist of these cells. C&D show the dynamic range of antigen concentrations eliciting IFN production by both of these peptides in the context of wildtype HLA A2 C ; or DT227 8KA HLA A2 mutant molecules D ; . E ; 105 C1R cells expressing HLA-A2 wildtype or DT227 8KA HLA A2 were incubated in triplicate with 250 HLA-A2 Gag-specific 868 CTL counted by tetramer staining ; and 100 nM SLYNTVATL or the natural variant SLFNTVATL, which acts as a weak agonist for these cells, in a 3 h IFN ELISpot assay as described in the experimental procedures. Both CD8-mediated prolongation of the TCR pMHCI interaction and cytoplasmic recruitment of p56lck are likely to play a role in recognition of weak agoinist peptides. It is not possible to determine which effect is dominant from these experiments.

The formulary beginning on the next page provides coverage information about some of the drugs covered by Sierra Nevada Spectrum. If you have trouble finding your drug in the list, turn to the Index that begins on page 38. The first column of the chart lists the drug name. Brand-name drugs are capitalized e.g., BENICAR ; and generic drugs are listed in lower-case italics e.g., amoxicillin ; . The information in the Requirements Limits column tells you if Sierra Nevada Spectrum has any special requirements for coverage of your drug. If there is an "X" in one of these columns, it means the marked requirement limit applies to the corresponding drug. If an "X" is in the: QL column, it means that Quantity Limits apply, PA column, it means that Prior Authorization requirements apply, and ST column, it means that step therapy requirements apply Table 5: number of samples in hsf- , hsf-7 and hsf-4 per digit class in nist sd19 and benzphetamine.

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