Cell-sized, supported artificial membranes (pseudocytes): response of precursor cytotoxic T lymphocytes to class I MHC proteins

Abstract

Novel cell-sized, supported artificial membranes bearing class I antigens have been prepared by a simple dialysis procedure and then used to study the requirements for antigen recognition by precursor cytotoxic T lymphocytes (CTL). The membranes were made by mixing lipid, H-2 antigen, and C18 alkylated 5 microns silica beads in deoxycholate, and dialyzing to remove the detergent. The H-2 antigen-bearing, cell-sized beads, termed pseudocytes (artificial cells), were able to simulate generation of secondary CTL responses with the same specificity as alloantigen-bearing spleen cells. Comparative analyses demonstrated that the size of an antigen-bearing structure, and thus its potential for multivalent interaction, was a critical determinant of effectiveness of antigen recognition, and showed that H-2 antigen was recognized as effectively on cell-sized beads as on allogeneic spleen cells. Generation of a response to antigen on the cell-sized beads was completely dependent on addition of lymphokines to the cultures. Thus, unlike liposomes, H-2 antigen on beads was not available to accessory cells for stimulation of Ia-dependent production of lymphokines by T helper cells. These results, as well as direct observations by microscopy, strongly indicate that antigen is recognized on the surface of the beads. Despite effective stimulation of secondary CTL responses, antigen on beads was completely inactive in stimulating a primary CTL response by naive spleen cells. The results of mixing experiments by using beads and alloantigen-bearing cells or plasma membrane vesicles indicate that the lack of a primary response may result from a requirement for a soluble factor(s) that is not needed for generation of secondary responses. The unique advantages of cell-size supported membranes for studying antigen recognition by T cells are discussed. The beads can be handled and used like antigen-bearing cells in functional assays, while possessing well-defined, readily varied, and easily quantitated composition.

Figure 1.

Incorporation of lipid and H-2 antigen onto beads by dialysis. H-2K^kx antigen (6 μg) and 10⁷ beads were mixed with varying amounts of cell lipid in DOC/TBS, and were dialyzed as described in Materials and Methods. Trace amounts of ³H-dipalmitoylphosphatidylcholine (PC) or ¹²⁵I-H-2K^k were added to monitor incorporation onto the beads. After dialysis the beads were washed twice in serum-containing medium and radioactivity associated with the bead pellet was determined. In the absence of added beads, no radioactivity is recovered as a pellet. (○), ¹²⁵I-H-2K^k; (●), ³H-PC.

Figure 2.

STEM of beads. The 5 μm beads were either untreated or coated by dialysis with lipid and H-2 antigen, and were allowed to settle on poly-L-lysine-treated coverslips for processing and visualization by STEM as described in Materials and Methods. Untreated beads, (A) 20,000× and (B) 50,000×; beads coated with 5 nmol cellular lipid and 6 μg H-2K^k antigen/10⁷ beads, (C) 20,000× and (D) 50,000×; and (E) bead coated with 5 nmol cellular lipid and 12 μg H-2^d and a lymphocyte from an H-2^d anti-H-2^k secondary culture at 5000×. The size bar represents 0.5 μm (A to D) or 5 μm (E).

Figure 3.

CTL stimulation by pseudocytes. Splenocytes from (A) CD₂F₁ (H-2^d) or (B) C57BL/6 (H-2^b) mice immunized 2 to 6 mo previously with RDM-4 (H-2^k) cells were cultured in the presence of Con A supernatant and alloantigen as described in Materials and Methods. After 5 days, effector CTL generation was assessed by measuring the release from ⁵¹Cr-labeled RDM-4 cells. Antigens used were: 10⁶ H-2K^k-bearing beads/culture (B(H-2), ○); 10⁶ adherent cell-depleted A/J (H-2K^k/D^d) irradiated splenocytes/culture (SC, ●); 10⁶ beads coated only with lipid (B(O), Δ), or no antigen (C, ×). Data are shown as percent specific ⁵¹Cr release at varying effector to target ratios.

Figure 4.

CTL stimulation by pseudocytes is specific. Splenocytes from AKR/J (H-2^k) mice immunized 2 to 6 mo previously with P815 cells (H-2^d), or from CD₂F₁ (H-2^d) mice immunized 2 to 6 mo previously with RDM-4 cells (H-2^k), were cultured in the presence of Con A supernatant and 10⁶ beads per culture. Beads bore either H-2K^k (○) or H-2^d (×) antigen. After 5 days the cultures were assayed for lytic activity on both ⁵¹Cr-labeled RDM-4 and P815 target cells. Data are expressed as percent specific ⁵¹Cr release at varying effector to target ratios.

Figure 5.

The effect of varying lipid composition on CTL stimulation by pseudocytes. Splenocytes from CD₂F₁ (H-2^d) mice immunized 2 to 6 mo previously with RDM-4 (H-2^k) cells were cultured with Con A supernatant and 10⁶ beads. Beads were prepared from starting mixtures containing 6 μg H-2K^k/10⁷ beads and either 5 nmol or 50 nmol cellular lipid/10⁷ beads or 50 nmol synthetic lipid (DMPC:cholesterol, 98:2)/10⁷ beads. H-2K^k incorporation was the same in each case. Cytotoxicity was assessed on day 5 by using ⁵¹Cr-labeled RDM-4 target cells, and the results are expressed as lytic units per 10⁶ cells.

Figure 6.

Phase-contrast and fluorescence photomicrographs of pseudocyte-cell conjugates. Splenocytes from CD₂F₁ (H-2^d) mice immunized 2 to 6 mo earlier with RDM-4 (H-2^k) cells were cultured for 5 days with H-2K^k-bearing beads and Con A supernatant. Cultures containing both cells and beads were split into aliquots and were incubated with either: (A and B) an irrelevant anti-NP idiotype mAb (R3-I) or (C and D) an anti-H-2K^k mAb (11-4.1), and then were stained with a fluoresceinated goat-anti-murine γ_2a antiserum as described in Materials and Methods. The samples were viewed at 400× under phase-contrast (A and C) or fluorescent (B and D) illumination. Beads (arrows) are 5 μm in diameter.

Figure 7.

CTL stimulation by liposomes, liposome-coated beads, and pseudocytes bearing H-2K^k. Liposomes (L, ×) were prepared by mixing lipid and H-2K^k antigen in DOC, followed by dialysis to remove the detergent. Some of these liposomes were incubated with untreated 5 μm beads; the beads were pelleted and washed to yield liposome-coated beads (L/B, ●). Pseudocytes (B, ○) were formed by mixing H-2K^k (6 μg H-2K^k/10⁷ beads), lipid and beads in DOC/TBS and dialyzing. Details of the preparations are given in Materials and Methods. Splenocytes from CD₂F₁ (H-2^d) mice immunized 2 to 6 mo previously with RDM-4 (H-2^k) cells were cultured with Con A supernatant and varying amounts of the different antigens (increasing numbers of beads or liposomes; expressed as final H-2K^k concentration in culture) and were assayed for lytic activity on day 5 by using ⁵¹Cr-labeled RDM-4 cells. Data are expressed as lytic units per 10⁶ cells.

Figure 8.

Comparison of CTL stimulation by allogeneic spleen cells and pseudocytes. Splenocytes from CD₂F₁ (H-2^d) mice immunized 2 to 6 mo previously with RDM-4 cells (H-2^k) were cultured with Con A supernatant and increasing numbers of H-2K^k-bearing beads (B, ○) or irradiated A/J (H-2K^k/D^d) splenocytes depleted of adherent cells (SC, ●). Cytotoxicity was assessed by using ⁵¹Cr-labeled RDM-4 target cells after 5 days, and data are shown as lytic units/10⁶ cells.

Figure 9.

CTL stimulation by pseudocytes is completely dependent upon the addition of lymphokines. Splenocytes from CD₂F₁ (H-2^d) mice immunized 2 to 6 mo previously with RDM-4 cells (H-2^k) were cultured with H-2K^k-bearing beads (B, ○) or liposomes (L, ×) in the presence(—) or absence (----) of added Con A supernatant. Beads were made by using 6 μg H-2K^k and 5 nmol lipid per 10⁷ beads, and liposomes by using 1 μg H-2K^k per 20 nmol lipid. Antigen dose was varied by varying the number of beads or liposomes added, and is expressed as the final concentration of antigen in the culture. Cytotoxicity was assayed by using ⁵¹Cr-labeled RDM-4 cells after 5 days, and data are reported as lytic units/10⁶ cells.