Testing for adjuvant activity of cell-sized beads, foreign proteins, and primary cells. (A) Similar to Fig. 1A except that OVA beads were injected with GL261 cells (10⁶), 25 μM latex beads (cell size, 2 × 10⁶), or 10 μM of latex beads coated with FBS (2 × 10⁶; 10⁶ beads with FBS passively absorbed and the other 10⁶ beads with FBS covalently conjugated). GL261 cells cultured in serum-free media gave nearly identical results as cells cultured with FBS (not shown). (B) Similar to Fig. 1A except 2 μg OVA/BioMag beads was injected with or without 10⁵ C57BL/6 splenocytes. Syngeneic splenocytes also enhanced CTL responses to gp120 beads in BALB/c mice (not shown). E:T ratio, effector-to-target cell ratio.

UV treatment leads to higher adjuvant activity. (A) BALB/c 3T3 cells were UV-irradiated for 5 min, incubated at 37°C for 5 h, and then subjected to two rounds of freeze thawing. The indicated number of cell equivalents were mixed with 0.5 μg gp120/latex beads and injected into BALB/c mice. CTL priming was evaluated as described in Fig. 1C. (B) Identical to A except that the 3T3 culture was covered with plastic lids during the UV treatment. No response was seen in mice injected with antigen beads without cells. E:T ratio, effector-to-target cell ratio.

The cytosol contains adjuvant activity. (A) EL4 cells were disrupted by nitrogen cavitation and fractionated by differential centrifugation. Cytosolic and nuclear fractions from 5 × 10⁴ EL4 cells were mixed with 2 μg OVA/latex beads, injected into mice, and assayed as described in Fig. 1A. Fractionated GL261 cells and 3T3 cells (in BALB/c mice) gave similar results. (B) 3T3 cells were UV-irradiated as described in Fig. 4A and then were fractionated as in A. Cytosolic fraction in quantities equivalent to 10⁵ and 10⁴ initial 3T3 cells was mixed with 0.5 μg of gp120/latex beads and injected into mice. An assay similar to the one described in Fig. 1C was performed. (C) Identical to B except that 3T3 cells were covered with plastic lids during the UV treatment. E:T ratio, effector-to-target cell ratio.

Syngeneic cells provide a potent adjuvant effect for CTL induction. (A) OVA/BioMag beads (5 μg) were injected s.c. into the hind flanks of C57BL/6 mice with either 10⁵, 10⁴, or 10³ mitomycin C-treated GL261 cells, or without cells in 100 μl of PBS. Seven days later, splenocytes were harvested and stimulated with OVA-transfected EL4 cells (EG7). CTL activity was measured against EG7 cells 5 days later in a ⁵¹Cr release assay. Control mice immunized with beads alone had no detectable CTL. Killing of control EL4 cells in this assay as well as all of the following assays was minimal. (B) Similar to A except that 10⁵ EL4 cells were used to replace GL261 cells. (C) Similar to A except that the antigen was gp120/latex beads (0.1 μg), the mice were BALB/c mice, and the coinjected cells were 5 × 10⁵ syngeneic 3T3 cells. Mice were killed 2 weeks after the injection and splenocytes were stimulated with and assayed for killing against gp120-transfected 3T3 cells (15–12). Killing against a control cell line 18neo (control transfected 3T3) in this assay as well as all of the following assays was minimal. E:T ratio, effector-to-target cell ratio.

The adjuvant effect is not mediated by secreted factors and does not require continued protein synthesis. (A) 10⁶ GL261 cells were untreated (open squares), subjected to two rounds of freeze thawing (closed circles), or treated with 8 μM emetine (closed diamonds; leading to complete cell death overnight) and then injected into C57BL/6 with 5 μg OVA//BioMag; or OVA/BioMag beads without cells were injected (open triangles). CTL priming was evaluated as described in Fig. 1A. (B) Emetine completely inhibits de novo protein synthesis. A20 cells (see Fig. 5B) were either untreated or treated with 8 μM or 0.02% azide (control), and cultured with radio-labeled methionine and cystine. Trichloroacetic acid precipitation assay of ³⁵S-labeled cells was performed as described in Materials and Methods. E:T ratio, effector-to-target cell ratio.

Apoptotic cells provide a stronger adjuvant effect. (A) A20 cells were treated at 37°C with 10 ng/ml Fas ligand (Alexis, San Diego) followed by an anti-Fas ligand antibody and incubated for 4 h. A portion of the cells was used for flow cytometry (see E) and the remainder was frozen and thawed; the indicated number of cell equivalents were coinjected with gp120/latex beads into BALB/c mice. The priming of anti-gp120 CTLs was determined as described in Fig. 1C. (B) Same as A except cells were treated with emetine (20 μM). (C) Same as A except cells were treated with oligomycin (25 μM) for at least 7 h. (D) Same as A except cells were untreated. (E) Treated and untreated cells were stained with Annexin V and propidium iodine (PI) and analyzed by flow cytometry. All Fas ligand-treated cells were Annexin V positive with a 30 min longer incubation (at the time of harvesting for injection, data not shown). 3T3 cells treated with UV as in Fig. 4A also become Annexin V+, PI- (data not shown). E:T ratio, effector-to-target cell ratio.

The adjuvant effect is systemic and long lasting: 10⁶ emetine-treated GL261 cells were injected either 10 days (-10 days) before, 2 days (-2 days) before, at the same time, or 2 days after the injection of 5 μg BioMag/OVA beads. The OVA beads and emetine-treated cells were either injected into the same flank (ipsilateral) or the opposite flank (contralateral). CTL priming was evaluated 9 days after injections as described in Fig. 1A. E:T ratio, effector-to-target cell ratio.