Clifford V. Harding, 111 Department of Pathology Medicine, Washington St.University Louis School of
1865
Antigen electroporation and class I MHC processing inhibitors
Eur. J. Immunol. 1992. 22: 1865-1869
Electroporation of exogenous antigen into the cytosol for antigen processing and class I major histocompatibility complex (MHC) presentation: weak base amines and hypothermia (18OC) inhibit the class I MHC processing pathway* While endogenous antigens are presented by class I major histocompatibility complex (MHC) molecules, exogenous antigens generally require a means for penetration into the cytosol for processing prior to class I MHC presentation.We have optimized conditions for electroporation as a means to experimentally introduce exogenous antigens into the cytosol, providing a system with a number of advantages for dissecting the class I MHC processing pathway. Presentation was assessed by the response of class I or class I1 MHC-restricted T hybridoma cells. Essentially instantaneous antigen delivery by electroporation facilitated kinetic analysis of the class I pathway and investigation of the effects of various inhibitors or hypothermic conditions on class I h4HC antigen processing. This pathway was inhibited by weak base amines (e.g. chloroquine and NH&l), cycloheximide, and hypothermia (18 “C, which inhibits certain intracellular vesicular processing pathways). The electroporation technique provides a simple, consistent approach for rapid cytosolic antigen delivery for analysis of class I MHC processing.
1 Introduction
tration or synthesis. In most cases, the delivery of antigen cannot be accomplished within a narrow kinetic window.
T lymphocytes are activated when clonotypic T cell receptors recognize antigenic peptides bound to MHC molecules. Class I1 MHC (MHC-11) molecules primarily present peptides derived from “exogenous” antigens, internalized by the antigen-presenting cell from the external milieu. Class I MHC (MHC-I) molecules present peptides derived from “endogenous” antigens that are synthesized within the antigen-presenting cell. Thus, many studies of MHC-I processing have investigated the processing of “endogenous” viral antigens after viral infection [ l , 21. In addition, several other systems exist to introduce exogenous antigens into the class I MHC processing pathway, for subsequent presentation by MHC-I molecules [3-71. These approaches entail penetration of antigenic materials from vesicular endocytic compartments into the cytosol. This penetration is achieved by various means, including osmotic [3] or enzymatic [4] disruption of vesicular membranes, encapsulation of antigen within fusogenic acid-sensitive liposomes [5, 61, or virus penetration [7]. All of the above methods require prior exposure of the antigen-presenting cells to antigen for varying periods to allow for its uptake, pene-
[I 102781
*
This work was supported by an American Cancer Society Junior Faculty Research Award and a Pfizer Scholar Award.
Correspondence: Clifford V. Harding, Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, 1-JSA . ~
Abbreviations: MHC-I: Class I MHC MHC-11: Class I1 MHC EK: Endoplasmic reticulum
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
This report examines the advantages of electroporation as a simple means for delivering antigenic proteins into the cytosol. Electroporation involves the permeabilization of cells by application of a pulsed high-intensity electric field (reviewed in [S, 91). Oscillating (radio frequency) electric fields may also be used [ 101. Pulsed electric fields introduce transient pores in the plasma membrane of cells [9, 111, which allows the introduction of large macromolecules such as DNA [12] or proteins such as antibody molecules [13]. Rapid-freezing electron microscopy of erythrocytes has revealed the formation of 20-120-nm pores within 3 ms of the application of a radio frequency electric field [14], and resealing was observed within 10-20 s (in other systems resealing may be slower). Thus, electroporation allows for extremely rapid introduction of molecules into the cytosol (essentially instantaneous for many applications).Viability of cells may be affected by electroporation [8], but this can be minimized by optimization of electroporation parameters. Viability of up to 80%-90% may be achieved with 90% of the cells sufficiently permeabilized to allow the penetration of antibodies into the cytosol [13]. In fact, under appropriate conditions, intracellular vesicular organelles may remain largely intact and functional even with transient plasma membrane permeabilization [151. We have performed several experiments on the class I processing of antigen introduced into the cytosol by electroporation that would be difficult using other established methods. These include a kinetic analysis of MHC-I processing and the effects Of hypothermia and various inhibitors present over strictly defined periods relative to the introdkction of antigen. -Electropdration had only a minor effect on cell viability and the processing of exogenous antigens for MHC-11-restricted presentation.
+
0014-2980192l0707-1865$3.50 ,2510
1866
Eur. J. Immunol. 1992. 22: 1865-1869
C.V. Harding
2 Materials and methods 2.1 Cell culture
Cells were manitained in a standard medium: DMEM (Gibco, St. Louis, MO) with 10% fetal calf serum M 2-mercaptoethanol, (Hyclone, Logan, UT), 5 x antibiotics and the following supplements: L-arginine HCI (116 mg/l), L-asparagine (36 mg/l), NaHC03 (2 g/l), sodium pyruvate (1 mM). M12.B6 cells (generous gift of Osami Kanagawa, Washington University, St. Louis, MO) were used for antigen presentation; they were created by fusing M12.C3 murine B lymphoma cells [16] with LPS-stimulated splenocytes (source of B lymphoblasts) from a C57BL/6 mouse. Accordingly, they express the H-2bantigens. DOBW cells [7] are specific for the OVA(323-339) peptide bound to either I-Ah or I-Ad. B3.1 T hybridoma cells [7] respond to OVA(258-276)-Kb.
revealed that the optimal responses were obtained with 0.3-3 mg/ml (Fig. 1B). Lower responses were observed with 30-100 pg/ml, indicating that relatively efficient cytosolic delivery and processing of OVA was achieved (although less efficient in dose-response terms than OVA processing by the MHC-I1 pathway). Electroporation under these conditions had only minor effects on cell viability and function (viability remained at 92 % by Trypan blue exclusion). In addition, the endocytosis, processing and MHC-11-restricted presentation of OVA was not significantly affected by electroporation (Fig. 2). 6000
-
-
+ 600 uF
m
I 0 0 0 UF
4000-
2.2 Electroporation and antigen processing studies
Electroporation was performed with a BTX Electro Cell Manipulator 600 (Biotechnologies and Experimental Research, San Diego, CA) using 2-mm gap cuvette chambers. Timing resistance was 10 Q , capacitance was 200-1000 pF, and voltage was 50-400 V. Electroporation was performed in serum-free RPMI 1640 (Gibco) with 5 x 106-40 x lo6 M12.B6 cells/ml in the presence of OVA at room temperature. The cells were immediately placed on ice. Various inhibitors were then added and the cells were transferred to 18"C or 37 "C. Finally, the cells were lightly fixed with 1% paraformaldehyde (Figs. 3-6) and washed extensively, preventing further processing. In other experiments (Figs. 1 and 2) the cells were electroporated and then plated without fixation in the continued presence of OVA (but usually at a lower concentration) with the T hybridoma cells.The extent of processing (i.e. the level of specific peptide-MHC complexes expressed) was determined by the response of B3.1 or DOBW T hybridoma cells. T cells (los) were plated with the M12.B6 cells (2 x 10s if fixed, 5 x 104 if viable) in 0.2 ml for 18-24 h. Both T hybridomas respond to antigen stimulation by the secretion of IL-2 (assayed in the supernatants by IL2-dependent CTLL cell proliferation and [3H]thymidine incorporation) [17].
0
.
%
.
I00
8
.
200
0
.
I
400
300
Voltage
[OVA] ( u g / m l )
Figure 1. Optimizationof electroporation for cytosolic delivery of OVA to achieve MHC-I processing and presentation. (A) M12.B6 cells were suspended at 2.5 x 107/ml in serum-free RPMI 1640 with 0.5 mg/ml OVA. Following electroporation,they were diluted and combined with B3.1 cells in standard medium to 1 x los M12.B6 celldm1 and 20 pg/ml OVA.The B3.1 response serves as a measure of MHC-I processing and presentation of OVA(258-276)Kb. (B) M12.B6 cells were electroporated at 600 pF and 250 V in the presence of various concentrations of OVA. MHC-I and MHC-I1 presentation were determined using B3.1 and DOBW cells, respectively.
-""-- I
3 Results We studied M12.B6 as an antigen-processing cell line expressing H-2hmolecules, capable of presenting t o the two OVA-specific T hybridomas B3.1 (Kb restricted) and DOBW (I-A" restricted). Conditions for electroporation were optimized to introduce OVA into the cytosolic MHC-I antigen-processing pathway, with MHC-I presentation measured by the response of B3.1 cells.While a wide range of conditions sufficed to induce some class I processing, the best results were obtained at relatively low voltage (150 V) and increased capacitance (1000 pF; Fig. 1A). These conditions were used in most later studies. OVA entered the MHC-I processing pathway only with adequate electroporation; processing and presentation were not seen without electroporation or with
1865
Antigen electroporation and class I MHC processing inhibitors
Eur. J. Immunol. 1992. 22: 1865-1869
Electroporation of exogenous antigen into the cytosol for antigen processing and class I major histocompatibility complex (MHC) presentation: weak base amines and hypothermia (18OC) inhibit the class I MHC processing pathway* While endogenous antigens are presented by class I major histocompatibility complex (MHC) molecules, exogenous antigens generally require a means for penetration into the cytosol for processing prior to class I MHC presentation.We have optimized conditions for electroporation as a means to experimentally introduce exogenous antigens into the cytosol, providing a system with a number of advantages for dissecting the class I MHC processing pathway. Presentation was assessed by the response of class I or class I1 MHC-restricted T hybridoma cells. Essentially instantaneous antigen delivery by electroporation facilitated kinetic analysis of the class I pathway and investigation of the effects of various inhibitors or hypothermic conditions on class I h4HC antigen processing. This pathway was inhibited by weak base amines (e.g. chloroquine and NH&l), cycloheximide, and hypothermia (18 “C, which inhibits certain intracellular vesicular processing pathways). The electroporation technique provides a simple, consistent approach for rapid cytosolic antigen delivery for analysis of class I MHC processing.
1 Introduction
tration or synthesis. In most cases, the delivery of antigen cannot be accomplished within a narrow kinetic window.
T lymphocytes are activated when clonotypic T cell receptors recognize antigenic peptides bound to MHC molecules. Class I1 MHC (MHC-11) molecules primarily present peptides derived from “exogenous” antigens, internalized by the antigen-presenting cell from the external milieu. Class I MHC (MHC-I) molecules present peptides derived from “endogenous” antigens that are synthesized within the antigen-presenting cell. Thus, many studies of MHC-I processing have investigated the processing of “endogenous” viral antigens after viral infection [ l , 21. In addition, several other systems exist to introduce exogenous antigens into the class I MHC processing pathway, for subsequent presentation by MHC-I molecules [3-71. These approaches entail penetration of antigenic materials from vesicular endocytic compartments into the cytosol. This penetration is achieved by various means, including osmotic [3] or enzymatic [4] disruption of vesicular membranes, encapsulation of antigen within fusogenic acid-sensitive liposomes [5, 61, or virus penetration [7]. All of the above methods require prior exposure of the antigen-presenting cells to antigen for varying periods to allow for its uptake, pene-
[I 102781
*
This work was supported by an American Cancer Society Junior Faculty Research Award and a Pfizer Scholar Award.
Correspondence: Clifford V. Harding, Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, 1-JSA . ~
Abbreviations: MHC-I: Class I MHC MHC-11: Class I1 MHC EK: Endoplasmic reticulum
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
This report examines the advantages of electroporation as a simple means for delivering antigenic proteins into the cytosol. Electroporation involves the permeabilization of cells by application of a pulsed high-intensity electric field (reviewed in [S, 91). Oscillating (radio frequency) electric fields may also be used [ 101. Pulsed electric fields introduce transient pores in the plasma membrane of cells [9, 111, which allows the introduction of large macromolecules such as DNA [12] or proteins such as antibody molecules [13]. Rapid-freezing electron microscopy of erythrocytes has revealed the formation of 20-120-nm pores within 3 ms of the application of a radio frequency electric field [14], and resealing was observed within 10-20 s (in other systems resealing may be slower). Thus, electroporation allows for extremely rapid introduction of molecules into the cytosol (essentially instantaneous for many applications).Viability of cells may be affected by electroporation [8], but this can be minimized by optimization of electroporation parameters. Viability of up to 80%-90% may be achieved with 90% of the cells sufficiently permeabilized to allow the penetration of antibodies into the cytosol [13]. In fact, under appropriate conditions, intracellular vesicular organelles may remain largely intact and functional even with transient plasma membrane permeabilization [151. We have performed several experiments on the class I processing of antigen introduced into the cytosol by electroporation that would be difficult using other established methods. These include a kinetic analysis of MHC-I processing and the effects Of hypothermia and various inhibitors present over strictly defined periods relative to the introdkction of antigen. -Electropdration had only a minor effect on cell viability and the processing of exogenous antigens for MHC-11-restricted presentation.
+
0014-2980192l0707-1865$3.50 ,2510
1866
Eur. J. Immunol. 1992. 22: 1865-1869
C.V. Harding
2 Materials and methods 2.1 Cell culture
Cells were manitained in a standard medium: DMEM (Gibco, St. Louis, MO) with 10% fetal calf serum M 2-mercaptoethanol, (Hyclone, Logan, UT), 5 x antibiotics and the following supplements: L-arginine HCI (116 mg/l), L-asparagine (36 mg/l), NaHC03 (2 g/l), sodium pyruvate (1 mM). M12.B6 cells (generous gift of Osami Kanagawa, Washington University, St. Louis, MO) were used for antigen presentation; they were created by fusing M12.C3 murine B lymphoma cells [16] with LPS-stimulated splenocytes (source of B lymphoblasts) from a C57BL/6 mouse. Accordingly, they express the H-2bantigens. DOBW cells [7] are specific for the OVA(323-339) peptide bound to either I-Ah or I-Ad. B3.1 T hybridoma cells [7] respond to OVA(258-276)-Kb.
revealed that the optimal responses were obtained with 0.3-3 mg/ml (Fig. 1B). Lower responses were observed with 30-100 pg/ml, indicating that relatively efficient cytosolic delivery and processing of OVA was achieved (although less efficient in dose-response terms than OVA processing by the MHC-I1 pathway). Electroporation under these conditions had only minor effects on cell viability and function (viability remained at 92 % by Trypan blue exclusion). In addition, the endocytosis, processing and MHC-11-restricted presentation of OVA was not significantly affected by electroporation (Fig. 2). 6000
-
-
+ 600 uF
m
I 0 0 0 UF
4000-
2.2 Electroporation and antigen processing studies
Electroporation was performed with a BTX Electro Cell Manipulator 600 (Biotechnologies and Experimental Research, San Diego, CA) using 2-mm gap cuvette chambers. Timing resistance was 10 Q , capacitance was 200-1000 pF, and voltage was 50-400 V. Electroporation was performed in serum-free RPMI 1640 (Gibco) with 5 x 106-40 x lo6 M12.B6 cells/ml in the presence of OVA at room temperature. The cells were immediately placed on ice. Various inhibitors were then added and the cells were transferred to 18"C or 37 "C. Finally, the cells were lightly fixed with 1% paraformaldehyde (Figs. 3-6) and washed extensively, preventing further processing. In other experiments (Figs. 1 and 2) the cells were electroporated and then plated without fixation in the continued presence of OVA (but usually at a lower concentration) with the T hybridoma cells.The extent of processing (i.e. the level of specific peptide-MHC complexes expressed) was determined by the response of B3.1 or DOBW T hybridoma cells. T cells (los) were plated with the M12.B6 cells (2 x 10s if fixed, 5 x 104 if viable) in 0.2 ml for 18-24 h. Both T hybridomas respond to antigen stimulation by the secretion of IL-2 (assayed in the supernatants by IL2-dependent CTLL cell proliferation and [3H]thymidine incorporation) [17].
0
.
%
.
I00
8
.
200
0
.
I
400
300
Voltage
[OVA] ( u g / m l )
Figure 1. Optimizationof electroporation for cytosolic delivery of OVA to achieve MHC-I processing and presentation. (A) M12.B6 cells were suspended at 2.5 x 107/ml in serum-free RPMI 1640 with 0.5 mg/ml OVA. Following electroporation,they were diluted and combined with B3.1 cells in standard medium to 1 x los M12.B6 celldm1 and 20 pg/ml OVA.The B3.1 response serves as a measure of MHC-I processing and presentation of OVA(258-276)Kb. (B) M12.B6 cells were electroporated at 600 pF and 250 V in the presence of various concentrations of OVA. MHC-I and MHC-I1 presentation were determined using B3.1 and DOBW cells, respectively.
-""-- I
3 Results We studied M12.B6 as an antigen-processing cell line expressing H-2hmolecules, capable of presenting t o the two OVA-specific T hybridomas B3.1 (Kb restricted) and DOBW (I-A" restricted). Conditions for electroporation were optimized to introduce OVA into the cytosolic MHC-I antigen-processing pathway, with MHC-I presentation measured by the response of B3.1 cells.While a wide range of conditions sufficed to induce some class I processing, the best results were obtained at relatively low voltage (150 V) and increased capacitance (1000 pF; Fig. 1A). These conditions were used in most later studies. OVA entered the MHC-I processing pathway only with adequate electroporation; processing and presentation were not seen without electroporation or with
