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The Journal of Immunology, 1998, 160: 2706-2714.
Copyright © 1998 by The American Association of Immunologists

Nucleic Acid Vaccine-Induced Immune Responses Require CD28 Costimulation and Are Regulated by CTLA41

James H. Horspool*, Peter J. Perrin{dagger}, Juliana B. Woodcock*, Josephine H. Cox{ddagger}, Christopher L. King§, Carl H. June*,¶, David M. Harlan*,¶, Daniel C. St. Louis|| and Kelvin P. Lee2,*,¶

* Immune Cell Biology Program, Naval Medical Research Institute, Bethesda, MD 20889; {dagger} Allergy and Immunology Section, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104; {ddagger} SRA Technologies, Rockville, MD 20850; § Division of Tropical Medicine, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106; Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; and || Military Medical Consortium for Applied Retroviral Research, Rockville, MD 20850 ...............


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunization with plasmids expressing specific genes (DNA or nucleic acid vaccination (NAV)) elicits robust humoral and cell-mediated immune responses. The mechanisms involved in T cell activation by NAV are incompletely characterized. We have examined the costimulatory requirements of NAV. CD28-deficient mice did not mount Ab or CTL responses following i.m. immunization with eukaryotic expression plasmids encoding the bacterial gene ß-galactosidase (ßgal). Because these mice retained their ability to up-regulate the CTLA4 receptor (a negative regulator of T cell activation), we examined CTLA4’s role in the response of wild-type BALB/c mice to NAV. Intact anti-CTLA4 mAb but not Fab fragments suppressed the primary humoral response to pCIA/ßgal without affecting recall responses, indicating CTLA4 activation inhibited Ab production but not T cell priming. Blockade of the ligands for CD28 and CTLA4, CD80 (B7-1) and CD86 (B7-2), revealed distinct and nonoverlapping function. Blockade of CD80 at initial immunization completely abrogated primary and secondary Ab responses, whereas blockade of CD86 suppressed primary but not secondary responses. Simultaneous blockade of CD80 + CD86 was less effective at suppressing Ab responses than either alone. Enhancement of costimulation via coinjection of B7-expressing plasmids augmented CTL responses but not Ab responses, and without evidence of Th1 to Th2 skewing. These findings suggest complex and distinct roles for CD28, CTLA4, CD80, and CD86 in T cell costimulation following nucleic acid vaccination.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Robust immune responses can be generated following vaccination solely with eukaryotic expression plasmids encoding an "Ag" gene (nucleic acid vaccination, or NAV3). Seminal studies have demonstrated that these plasmids can be delivered i.m. (1), intradermally (2), i.v., or mucosally (3). Long-lived cellular, humoral, and protective immune responses have been generated against a variety of Ags in a wide range of hosts (reviewed in Refs. 4 and 5).

Following i.m. injection, myocytes take up and express the plasmid (6), but cellular and molecular components involved in subsequent Ag presentation to and activation of T cells remain largely uncharacterized (7). Myocytes inducibly express MHC class II but not the costimulatory ligands characteristic of professional APC (8, 9). Bone marrow-derived APC are essential in generating CTL responses (10, 11, 12, 13), possibly by picking up myocyte-produced Ag as secreted extracellular protein (4), by direct cell to cell transfer (11), or following CTL-mediated myocyte destruction (4, 10, 14). Direct plasmid uptake and Ag presentation by resident dendritic cells without a role for myocytes has also been proposed (15, 16, 17). The requirement for immunostimulatory CpG plasmid sequences and differential generation of Th1 or Th2 responses depending on the site of injection (muscle vs skin) also suggests that several different APC are involved (18, 19, 20, 21).

As with the cellular components, the molecular APC:T cell interactions required for the generation of responses to NAV are largely undefined. Following protein immunization, T cell activation requires delivery of both a primary signal via TCR binding to MHC/nominal Ag and a second signal via a costimulatory receptor. Although several accessory molecules can deliver costimulation, CD28 appears to be the most important (22). Responses to protein immunization are abrogated by blocking CD28 receptor binding to its ligand(s), CD80 (B7-1) and CD86 (B7-2) with the chimeric fusion protein CTLA4Ig (23). Similarly, CD28-deficient mice have diminished Ab responses to vesicular stomatitis virus infection (24) and cardiac myosin (25). A central role of CD28 in CTL generation has also been shown (26, 27). Conversely, activation of the second B7-binding receptor, CTLA4, appears to inhibit CD28-mediated T cell activation (28, 29, 30). Distinct and sometimes conflicting roles of CD80- and CD86-mediated costimulation during Ag presentation by APC have been described for both humoral and cellular responses (31, 32, 33, 34, 35, 36, 37).

Although coinjection of CD80- and CD86-expressing plasmids augments CTL responses (38, 39, 40, 41), whether NAV actually requires CD28-mediated costimulation (as does protein immunization) is unclear. We have examined the costimulatory requirements of NAV, focusing primarily on Ab responses given the absolute necessity for Ag processing, APC:T cell interaction, and CD4+ help. We find a central role for CD28-mediated costimulation for immune responses to i.m. DNA immunization. In addition, we find distinct roles for CTLA4, CD80, and CD86 in the generation of these responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids

The bacterial gene encoding ß-galactosidase (ßgal) (Clontech, Palo Alto, CA) was subcloned into the eukaryotic expression plasmid pCIA (derived from pcDNAI and pcDNA/amp (Invitrogen, San Diego, CA; CMV promoter). Expression of ßgal was confirmed by enzymatic histochemistry of transfected COS7 cells (42, 43). The murine B7 coexpression plasmids pCIA/mB7-1 and pCIA/mB7-2 were derived by subcloning CD80 or CD86 cDNA (gift of Dr. G. Gray, Genetics Institute, Cambridge, MA) into the expression vector pRSV (Invitrogen). The RSV promoter-B7 cDNA-SV40 intron-poly(A) cassette was then subcloned 880 bp 3' of the pCIA CMV promoter. Expression was confirmed by flow cytometric analysis of transfected COS7 cells.

DNA purification

Plasmids were purified by alkaline lysis and two sequential high-speed centrifugations over cesium chloride gradients (44). Endotoxin was removed using the Detoxigel kit (Pierce, Rockford, IL) per the manufacturer’s instructions and the DNA resuspended at 1 mg/ml in sterile normal saline. Endotoxin levels were less than 0.03 pg/µg DNA.

Antibodies

Abs used were: anti-murine B7-1 mAb 16-10A1 (gift of Dr. H. Reiser, Dana-Farber Cancer Institute, Boston, MA), anti-murine B7-2 mAb GL-1 (gift of Dr. R. Hodes, National Institute of Aging, National Institutes of Health, Bethesda, MD), anti-mouse CTLA4 mAb UC10-4F10-11 (gift of Dr. J. Bluestone, University of Chicago, Chicago, IL), anti-murine CD28 mAb PV-1.1 (45), and anti-murine CD3 (145-2C11 (46)). Hamster IgG was used as a control for 16-10A1, PV1.1, and UC10-4F10-11. A rat IgG2a Ab was used as a control for GL-1. For FACS analysis, phycoerthrin (PE)-conjugated Armenian hamster anti-CTLA4 (UC10-410-11; PharMingen, San Diego CA; IgG), FITC-conjugated Armenian hamster anti-CD3 (145-2C11; PharMingen; IgG), FITC or PE-conjugated Armenian hamster anti-trinitrophenol (clone UC8-4B3; PharMingen; an IgG isotype control), were used. Fab fragments were made and tested as previously described (33).

Immunizations

The experiments were conducted according to institutional guidelines (67). Female BALB/cByJ mice (The Jackson Laboratory, Bar Harbor, ME), CD28 knockout mice, and wild-type littermate controls (H2d (24)), 5 to 10 wk old, were utilized. Groups of three to six mice were injected i.m. in the tibialis anterior with 100 µg of DNA in 100 µL of normal saline with a 30-gauge needle. This immunization protocol routinely gives a 75% response rate and typically similar titers in the responders. Mice assayed for CTL activity were boosted with 100 µg of DNA 2 wk before performance of the assay. In blocking studies, 50 µg of Ab was injected i.p. 1 h before and 48 h after the first immunization.

ELISA

Serum was collected at the times indicated, pooled, and analyzed for anti-ßgal IgG Ab concentration by standard ELISA methodology. Immulon 2 (Dynatech Laboratories, Chantilly, VA) flat-bottom plate wells were coated with 0.5 µg of ßgal protein (Sigma, St. Louis, MO) diluted in PBS and incubated for 12 to 15 h at 37°C in a humidity chamber. The plates were then washed with PBS-Tween 20 (0.1%). Test sera serially diluted twofold (starting at 1:20) in borate-buffered saline (0.5% Tween-20 and 0.5% BSA (Sigma) or control anti-ßgal Ab (Sigma or Organon Technika Corporation, Durham, NC), were then applied to the plate. The plates were incubated (37°C) for 2 h and washed as above. Alkaline phosphatase-labeled second Abs (Organon Technika) were then added to the plate. The 2-h incubation was repeated. After the second wash step, phosphate substrate (Sigma) diluted in diethanolamine buffer (Aldrich Chemical, Milwaukee, WI) was applied to the plates, incubated (37°C) for 20 min, and stop solution (2 M NaOH) added. OD readings were taken at 450 nm. The concentration of ßgal-specific Ab was calculated from the control mAb standard curve and is expressed as arbitrary units. Experiments standardized to the Sigma mouse anti-ßgal mAb (Figs. 1Go, 3B, and 5) typically gave a 10-fold higher relative Ab concentration than those standardized to the Organon Technika polyclonal rabbit anti-ßgal Ab (Figs. 3GoA and 4) for the same OD450nm. Vaccination-specific Ab concentrations were obtained by subtracting out the vector-alone Ab concentration.



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  		FIGURE 1. Absent Ab and CTL responses of CD28-deficient mice to NAV. A, Ab response. CD28-deficient mice (CD28-/-) or wild-type (CD28+/+) littermate controls (three per group) were immunized i.m at time 0, 2, and 4 wk (arrows) with 100 µg of pCIA (to determine background) or 100 µg of pCIA/ßgal plasmid (CD28+/+ ({square}), CD28-/- ({blacktriangledown})). Anti-ßgal IgG Ab concentrations were assayed per Material and Methods. Vaccination-specific Ab concentrations were obtained by subtracting out the vector-alone Ab concentration. In this experiment, the background vector-alone Ab concentration was 6226 at wk 0 and 6413 U at wk 10 in wild-type mice, and 2166 at wk 0 and 3740 U at wk 10 in the CD28-deficient mice. Results are representative of two separate experiments. B, CTL response. CD28-/- or CD28+/+ mice were immunized i.m. at time 0, 2, 4, and 15 wk with 100 µg of pCIA (CD28+/+ (•), CD28-/-({blacktriangledown})), or 100 µg of pCIA/ßgal plasmid (CD28+/+ ({square}), CD28-/- ({triangledown})) and killed at 17 wk. CTL assays were performed per Material and Methods. Data are represented as the % specific lysis of individual mice at the E:T ratios indicated.

 


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  		FIGURE 3. CTLA4 activation inhibits primary but not secondary Ab responses to NAV. A, Anti-ßgal Ab responses to NAV following anti-CTLA4 mAb treatment. Groups of four BALB/c female mice were immunized with 100 µg/100 µl of pCIA/ßgal plasmid i.m. at time 0 (arrow), and given 50 µg of control hamster Ig (ßgal + control Ig, •), whole murine anti-CTLA mAb (UC10-4F10) (ßgal + {alpha}CTLA4, {triangledown}), or anti-CTLA Fab (ßgal + {alpha}CTLA4 Fab, {blacksquare}) i.p. at time -1 h and 48 h. A fourth group was immunized with 100 µg of vector alone to establish background. Mice were boosted at 10 wk with 100 µg of the original immunizing plasmid without Ab retreatment. Anti-ßgal IgG Ab concentrations were assayed per Materials and Methods. Vaccination-specific Ab concentrations were calculated as in Figure 1GoA. In this experiment, the background vector-alone Ab concentration was 126 at wk 0 and 107 U at wk 16. These data are representative of two independent experiments. B, Ab isotypes. Mice were immunized with ßgal and treated with control Ig

(•, inset) or whole anti-CTLA4 mAb ({triangledown}, inset) as above. Animals were boosted without Ab retreatment. ßgal-specific IgG or IgM responses were determined 4 wk after the primary immunization (week 4) and boost (week 12).

 
For isotype analysis, plates were coated with ßgal as above. Plates were washed five times in PBS-Tween 20 (0.1%). Plates were then incubated (37°C) in BSA Diluent/Blocking Solution (KPL, Gaithersburg, MD) for 1 h. After the plates were washed, serum from vaccinated mice diluted in BSA Diluent/Blocking Solution was applied to the plate. A rabbit anti-ßgal Ab (Organon Technika) standard was also added to each plate. The plates were incubated (37°C) for 3 h, washed, and the blocking step was repeated. Horseradish peroxidase-labeled isotype-specific Abs (Southern Biotechnology Associates, Birmingham, AL), diluted 1:1000 in BSA Diluent/Blocking Solution, were then applied to the plates. The plates were incubated (37°C) for 1 h. After washing, ABTS Peroxidase Substrate (KPL) diluted in Peroxidase Solution B (KPL) was added to each plate. The plates were incubated at room temperature for 5 min and Peroxidase Stop Solution (KPL) was added. OD were read at 410 nm.

CTL assay

CTL assays were performed as previously described (47). Splenic mononuclear cells were stimulated for 5 days with the synthetic ßgal peptide TPHPARIGL spanning the H2d epitope (Peptide Technologies, Washington, DC). 51Cr-labeled P815 cells pulsed with 1 µg/ml of peptide or no peptide were used as targets at E:T ratios of 100:1, 30:1, 10:1, and 3:1.


The percentage of specific lysis of P815 pulsed with no peptide was always <10%. Experimental values represent the mean of triplicate cpm values.

T cell activation and flow cytometric analysis

Splenocytes were harvested from CD28-deficient or wild-type littermate controls and 4 x 106 cells/ml were cultured in 24-well plates (Costar, Cambridge, MA) in complete media containing 1 µg/ml of anti-CD3 mAb (145-2C11). Cells were harvested at 18, 24, 48, and 72 h, washed twice with PBS plus 3% FBS (staining buffer, SB), resuspended in 50 µl of SB containing 10 µg of goat IgG (Sigma), and incubated for 10 min on ice to block nonspecific binding. Without washing, directly conjugated anti-surface marker mAb (1 µg/106 cells) was added and the samples incubated for an additional 30 min on ice in the dark. Samples were washed three times with SB, fixed in 2.5% paraformaldehyde, and analyzed by flow cytometry. Data were acquired and analyzed using a FACScan instrument and CellQuest software, version 3.1 (Becton Dickinson, Mountain View, CA). CTLA4 expression by CD3+ cells in the lymphocyte gate was determined using a CD3-FITC vs side-scatter gate.

Northern blot analysis

Splenocytes from CD28-deficient or wild-type littermate controls were enriched by nylon wool depletion, resuspended at 5 x 104 cells/well, stimulated with 1 µg/ml of anti-CD3 mAb (2C11), and cultured at 37°C in humidified 5% CO2 air for the times indicated. RNA was extracted from the harvested cells with RNAzol (Cinna Biotecs, Friendswood, TX) per the manufacturer’s instructions. The RNA concentration of each time point was determined by ethidium bromide visualization and equalized by serial dilution. Equal amounts of each RNA sample were separated by electrophoresis through formaldehyde agarose gel, transferred to a nylon membrane, probed with 32P-labeled (random hexamer priming) murine CTLA4 exon 2 cDNA fragment, and analyzed on a Phosphorimager 445 (Molecular Dynamics, Sunnyvale, CA). The blot was then reprobed with a ß-actin cDNA fragment to confirm mRNA integrity.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD28 is required to generate Ab and CTL responses following NAV

CD28-deficient mice have impaired Ab responses to virus and protein Ag, impaired Ig isotype switching, but intact CTL responses to lymphocytic choriomeningitis virus (LCMV) infection (24, 25). To assess CD28’s role in NAV, we i.m. immunized CD28-deficient mice (H-2d) and wild-type littermate controls with ßgal-expressing plasmids. In contrast to the wild-type controls, the CD28-deficient mice failed to mount ßgal-specific Ab responses (Fig. 1GoA) despite multiple immunizations. Similarly, CD28-deficient mice failed to generate ßgal-specific CTL responses (Fig. 1GoB).

The complete absence of Ab and CTL responses following i.m. DNA vaccination suggests a profound dependence on CD28-mediated costimulation. Alternatively, if CD28-deficient T cells can still up-regulate expression of CTLA4, this "unopposed" signaling may suppress the responses to NAV. Although CTLA4 was initially described to oppose CD28 signals, recent findings suggest that CTLA4 can inhibit T cell activation independent of CD28 (68). We examined whether anti-CD3 activation of CD28-deficient T cells induced expression of CTLA4. As can be seen by Northern analysis (Fig. 2GoA), unstimulated T cells from both CD28+/+ and CD28-/- mice (seen more distinctly on longer exposures) express low levels of CTLA4 message that is substantially up-regulated at 48 h by anti-CD3 stimulation. Unexpectedly, this up-regulation is more pronounced in the CD28-/- mice. By 72 h, a small but distinct up-regulation of CTLA4 surface receptor expression is detected by FACS (Fig. 2GoB), with this also being greater on the CD28-/- cells.



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  		FIGURE 2. CD28-deficient mice retain the ability to up-regulate CTLA4 following anti-CD3 mAb activation. A, mRNA expression. Murine splenocytes from wild-type littermates (CD28+/+) or CD28-deficient mice (CD28-/-) were enriched for T cells by nylon wool depletion and stimulated with anti-CD3 mAb (1 µg/ml). Cells were harvested and mRNA isolated at the times indicated. Equal amounts of total RNA were separated by formaldehyde-agarose gel and transferred to nylon membranes. Blots were serially hybridized with a probe specific for murine CTLA4 and then with ß-actin to demonstrate RNA integrity. B, FACS analysis of CTLA4 expression. Murine splenocytes from CD28-deficient mice (CD28-/-) or wild-type littermates (CD28+/+) were stimulated with anti-CD3 mAb (1 µg/ml) for 0, 18, 48, and 72 h. Cells were then stained with anti-CD3 FITC and anti-CTLA4-PE or control Ab. CTLA4 expression by T cells was determined using a CD3-FITC vs side-scatter gate. No CTLA4 expression was detected at 0, 18, or 48 h poststimulation.

 
CTLA4 activation inhibits primary Ab responses to NAV

The complete impairment of Ab and CTL responses to NAV in CD28-deficient mice might be due to an absolute requirement for CD28, increased CTLA4 regulation, or a combination of both. Because the absence of CD28 costimulation in the knockout mice may mask any evidence of a suppressive effect of CTLA4 activation, we next assessed the potential regulatory role of CTLA4 in DNA vaccination in wild-type BALB/c mice. These mice were immunized i.m. at time 0 and treated with anti-CTLA4 mAb UC10-4F10-11 (whole and Fab fragments) i.p. at T = -1 h and T = 48 h. Whole UC10-4F10-11 Ab appears to be a mixed agonist/antagonist, cross-linking/activating CTLA4 in vitro (28, 29) but blocking in vivo (48, 49). We found that intact anti-CTLA4 mAb suppressed primary Ab responses to i.m. immunization (Fig. 3GoA). In contrast, coadministration of anti-CTLA4 Fab (which are strictly blocking) had no effect on ßgal titers compared with ßgal immunization alone. The difference between whole and Fab Abs suggests that intact anti-CTLA mAb is activating/cross-linking CTLA4 in this system. Reboosting (without Ab) resulted in similar early kinetics (wk 10–12) but lower sustained Ab responses (wk 12–16) in animals treated with whole anti-CTLA4 mAb vs control Ig or Fab. The rapid kinetics and predominant IgG isotype following reboosting (Fig. 3GoB) indicate a secondary (vs primary) response in these mice.

Distinct and nonoverlapping requirements for CD80- and CD86-mediated costimulation

Different roles for CD28 and CTLA4 in DNA immunization suggest the same for their ligands, CD80 and CD86. To assess this we immunized BALB/c mice with ßgal plasmid and blocked ligands individually with whole anti-CD80 mAb (16-10A1), whole anti-CD86 mAb (GL-1), or anti-CD80 + anti-CD86 mAb (both whole 16-10A1 and GL-1 have been previously shown to block cell-mediated responses in vivo (33)) only at the primary immunization. Treatment with anti-CD80 mAb completely suppressed primary Ab responses following DNA immunization (Fig. 4Go). These mice also failed to respond to reboosting in the absence of further anti-CD80 mAb treatment. Anti-CD86 mAb-treated mice had suppressed primary anti-ßgal Ab concentrations (48–90% reduction vs control Ig-treated animals) and a brisk response to reboosting but with lower sustained Ab concentrations (40–56% reduction). The combination of anti-CD80 + anti-CD86 mAb was unexpectedly less effective than either alone in blocking responses to the primary immunization. These mice were capable of mounting a response to reboosting, higher than anti-CD80-treated but lower than anti-CD86 or control Ig-treated animals (60–75% reduction compared with control Ig). Similar responses were found with CTLA4Ig, which also blocks both CD80 and CD86 (data not shown).



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  		FIGURE 4. Differential effects of CD80 or CD86 blockade on the Ab response to NAV. Groups of four BALB/c female mice were immunized with 100 µg/100 µl of pCIA/ßgal plasmid i.m. at time 0 (arrow), and given 50 µg of control Ig (•), anti-murine CD80 mAb (16-10A1) ({triangledown}), anti-murine-CD86 (GL-1) ({blacksquare}), or 50 µg of each anti-CD80 + anti-CD86 mAb ({diamond}) i.p. at time -1 h and 48 h after the first immunization only. A fifth group was immunized with 100 µg of vector alone to establish background. Animals were boosted with the original immunizing plasmid without Ab treatment at 14 wk. Anti-ßgal Ab concentrations were assayed as above. Vaccination-specific Ab concentrations were calculated as in Figure 1GoA. In this experiment, the background vector-alone Ab concentration was 151.3 at wk 0 and 617.6 U at wk 20. These data are representative of two independent experiments.

 
Coimmunization with CD80 and CD86 cDNA enhances responses to NAV

Based on these blocking studies, the distinct requirements for CD80 and CD86 predicted that coimmunization with B7-expressing cDNAs would enhance immune responses to nucleic acid vaccination. To assess this, mice were immunized i.m. on days 0, 14, and 21 with ßgal plasmid alone, or ßgal plasmid mixed (1:1) with CD80- or CD86-expressing plasmids. Coimmunization enhanced ßgal-specific CTL responses (Fig. 5GoA), CD86 more potently than CD80. However, in these same mice neither CD80 nor CD86 coinjection affected Ab responses, either in rate or magnitude of response (Fig. 5GoB) or in the minimum dose of ßgal plasmid required to generate equivalent Ab response (data not shown). We also did not find skewing of IgG isotypes (from IgG2a to IgG1) that would indicate redirection from a Th1 to Th2 response caused by CD80 or CD86 coimmunization (Fig. 5GoC).



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  		FIGURE 5. Effect of CD80 and CD86 coimmunization on CTL and Ab responses to NAV. A, CTL responses. BALB/c female mice were immunized i.m. at time 0, 2, 4, and 32 wk with 100 µg of pCIA (vector (O)), 100 µg of pCIA/ßgal ({square}), 100 µg of pCIA/ßgal coinjected with 100 µg of pCIA/CD80 (in 100 µl of total volume) ({blacktriangledown}), or 100 µg of pCIA/ßgal coinjected with 100 µg of pCIA/CD86 (in 100 µl of total volume) ({blacksquare}) and killed for CTL assays at week 34. CTL assays were performed per Material and Methods. Data are representative of two independent experiments and are shown as the % specific lysis of individual mice at the E:T ratios indicated. B, Ab responses. BALB/c female mice (three per group) were immunized i.m. at time 0, 2, and 4 wk (arrows) with 100 µg of pCIA/ßgal (•), 100 µg of CIA/ßgal coinjected with 100 µg of pCIA/CD80 (in 100 µl of total volume) ({triangledown}), or 100 µg of pCIA/ßgal coinjected with 100 µg of pCIA/CD86 (in 100 µl of total volume) ({blacksquare}). Three additional groups were immunized with 100 µg of the appropriate vector alone (pCIA, pCIA/CD80, and pCIA/CD86) to establish background. Anti-ßgal Ab concentrations were assayed as above. Vaccination-specific Ab concentrations were calculated as in Figure 1GoA. In this experiment, the background vector-alone Ab concentration was 3840 at wk 0 and 666.7 U at wk 9 for pCIA, 6307 (wk 0) and 1367 U (wk 9) for pCIA/CD80, and 5440 (wk 0) and 2080 (wk 9) U for pCIA/CD86.C, Ab isotypes. Week 9 serum from animals immunized inB above was analyzed for ßgal-specific IgG isotypes perMaterials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The mechanisms for T cell activation and a central role of CD28-mediated costimulation have been well established for protein Ag challenge (22). In contrast, the molecular and cellular components of Ag presentation and T cell activation are much less well defined for nucleic acid vaccination. Additional complexity is suggested by the differing immune responses (Th1 vs Th2) elicited by immunization at different sites (skin vs muscle) (50). In i.m. DNA vaccination, a role for the transfected myocyte as APC (39), Ag "factory" (11), and/or CTL target (and Ag source (14)) has been suggested. Whether myocytes have any role has been questioned, as the requirement for bone marrow-derived APC (10, 12) but not for the injected muscle site (after 10 min) (17) suggests transfection of a professional APC (dendritic cell?) that mediates the immune response.

What is known about T cell costimulation in NAV adds clarity and confusion in equal measure. Blockade with CTLA4Ig results in only a slight decrease in CTL activity (40). Conversely, coimmunization with plasmids expressing the CD28 ligand CD86 significantly augments CTL responses (38, 39, 40, 41). However, it is unclear how transfection of professional APC (which natively express CD86) would lead to this enhancement. CD86 coimmunization of bone marrow chimeric mice failed to convert host nonmarrow cells into APC but also failed to augment generation of CTL (13), indicating a (later?) requirement for CD86 expression on nonhematopoietic cells. None of these studies addresses whether CD28-mediated costimulation is actually necessary to generate immune responses to NAV.

We have found that CD28-mediated costimulation plays a critical role in both humoral and cell-mediated responses to i.m. DNA vaccination, suggesting involvement in both CD4+ and CD8+ T cell activation. The lack of response in multiply vaccinated CD28-deficient mice was more profound than when these mice are immunized with protein (typically 10–20% of wild-type anti-trinitrophenol-KLH-specific IgG titers (P.J.P., K.P.L., manuscript in preparation)) or challenged with LCMV (intact CTL responses (24)). Although NAV is supposed to mimic viral infection (and thus predict intact CTL response in the CD28 knockout mice), it has been shown that CTL responses to infection by nonreplicating/poorly replicating viruses (such as vesicular stomatitis virus) are much more dependent on CD28-B7-mediated costimulation and T cell help than are robustly replicating viruses such as LCMV (66). These findings are also consistent with the dependence on CD28-mediated costimulation of immune responses to minute amounts of Ag (51) coupled with the lack of adjuvant. NAV may also be particularly sensitive to the immunoregulatory effects of CTLA4 activation. CTLA4 receptor engagement (by CD80 and/or CD86 in vivo) restricts progression of T cell activation (28, 29, 30, 52) even in the absence of CD28 signaling (C.H.J., manuscript in press). We find that CD28-deficient T cells retain the ability to up-regulate CTLA4 mRNA and surface receptor expression following stimulation with 1 µg/ml of anti-CD3 mAb, similar to what has been reported for a 10-fold higher dose of anti-CD3 (28). Since CD28 enhances CTLA4 gene transcription and mRNA stability (53), the greater induction in the CD28-deficient mice (particularly mRNA) was unexpected. This may be due to up-regulation of CTLA4 by its own (unopposed) binding to B7 ligands, a developmental defect (as the CD28 and CTLA4 pathways appear to be linked), or differences in the cytokine milieu between CD28+/+ and CD28-/- mice (54).

We hypothesized that Ab blockade could establish a role for CTLA4 following i.m. DNA vaccination of wild-type mice, as loss/blockade of CTLA4 activation enhances cellular immune responses in vivo (48, 49, 55, 56). Intact anti-CTLA4 mAb UC10-4F10 has been reported to be cross-linking/activating in vitro (28, 29) but blocking in vivo (48, 49). We unexpectedly found that treatment with intact UC10-4F10 inhibited primary Ab responses to NAV by cross-linking/activating CTLA4, as Fab fragments were nonsuppressive. The different effect of UC10-4F10 between this and previous studies (using protein + CFA) may be the "strength" of the TCR/CD3 signal. In vitro studies suggest that CTLA4 down-regulation of T cell activation is most pronounced with suboptimal TCR/CD3 signaling (28), which may be the case in NAV. Even though primary Ab responses were suppressed, CTLA4 activation did not induce tolerance or block priming, as rechallenge elicited typical secondary responses based on kinetics and Ab isotypes. Evidence for T cell priming also raises the possibility that inhibition occurs at the level of B cells, which also express CTLA4 when activated (57).

The diametric roles of CD28 and CTLA4 in NAV suggested complex interaction with their ligands CD80 and CD86. Although both CD80 and CD86 bind to CD28 and CTLA4 (albeit with different kinetics (58)), previous studies have demonstrated distinct and conflicting roles for CD80 and CD86 in both humoral (35, 37) and cellular (33, 34, 36, 59) immune responses. Furthermore, the kinetics of early CD86 and later CD80 expression on APC suggest different functions (60). We found that CD80 blockade in NAV completely abrogated Ab responses to both primary and rechallenge immunizations, suggesting induction of tolerance. It has been reported that CD80 costimulates weak peptides but CD86 does not (61). Our findings are consistent with a scarce Ag/low TCR-signaling model in which CD80 is the primary ligand for CD28 and CD86 for CTLA4. Blockade of CD80 would allow CD86 to deliver suboptimal costimulation via CD28 and a full negative signal through CTLA4, two components required for tolerance induction (62). Likewise, Ab blockade of CD86 inhibits early activation of CD28 but also the down-regulatory effects of CTLA without affecting later up-regulation of CD80 (following MHC II engagement (63)) and binding to CD28. Like NAV, anti-CD86 mAb blockade in protein immunization results in a 50% decrease in Ab responses (64).

Blockade with anti-CD80 + anti-CD86 mAb was unexpectedly less inhibitory in the primary response than either alone. Blockade with CTLA4Ig (which also binds CD80 and CD86) yielded similar results (data not shown), consistent with previous reports using CTLA4Ig in NAV (40) and Ab blockade in experimental allergic encephalomyelitis (33). Given the opposing roles of CD28 and CTLA4, it is not unexpected that blockade of both B7 ligands results in a neutral event. However, since the knockout mice indicate that CD28-mediated costimulation is required to generate immune responses to NAV, these findings suggest the existence of a ligand that is not blocked by these anti-B7 Abs.

Although CD80-mediated costimulation is critical for Ab responses following NAV, coimmunization with CD80-expressing plasmids does not enhance humoral responses. CD86 coimmunization also does not increase Ab responses and neither appear to skew T cell responses from Th1 to Th2, a role that has been variably postulated for CD28 (32, 65). This failure of costimulatory coinjection to enhance humoral responses is similar to what has been reported by other groups (39, 40, 41). The fact that coimmunization with CD80 modestly and CD86 substantially augments CTL responses suggests that the amount of costimulation is less rate limiting for the generation of CD4+/T cell help responses than it is for CD8+/CTL activity. Alternatively, CTL augmentation may be a two-step process with initial T cell activation by bone marrow APC and subsequent enhancement by CD86-expressing myocytes upon T cell recirculation to the injection site. Both of these models predict that enhancement of CD4+ help/Ab responses may not be possible through coimmunization with costimulatory molecules.

These findings also suggest costimulation involved in the generation of CTL differs from that in Ab responses. We have focused primarily on Ab responses to NAV as Ag processing, APC:T cell interaction, and CD4+ help are all essential. Direct recognition of Ag on target cells, which may be a component of CTL responses, is not a confounding factor. Studies examining the costimulatory requirements of CTL generation are currently underway.

Our findings suggest that CD28 costimulation/CTLA4 countercostimulation play similar roles in i.m. nucleic acid vaccination as in protein immunization, particularly in the generation of Ab responses/CD4+ help. Ag processing, presentation, and T cell activation in NAV appear to involve multiple steps and components. Additional studies will be needed to uncover further complexities in costimulatory requirements for different immune responses and vaccination sites.


   Acknowledgments
 
We thank Dr. Kari Irvine (NCI, National Institutes of Health, Bethesda, MD) for originally providing the ßgal peptide and CTL protocol, and Alfred Black (ICBP, NMRI, Bethesda, MD) for producing many of the Ab reagents used in this study.


   Footnotes
 
1 This work was supported by the Naval Medical Research and Development Command, Research Task no. 61305D.B998.ABH29.1H.1281. Views presented in this paper are those of the authors; no endorsement by the Department of Navy, Department of the Army, or the Department of Defense has been given or should be inferred. Back

2 Address correspondence and reprint requests to Dr. Kelvin P. Lee, Immune Cell Biology Program, Immune Suppression Branch, Bldg. 17, Room 214, Naval Medical Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5067. E-mail address: Back

3 Abbreviations used in this paper: NAV, nucleic acid vaccination; ßgal, ß-galactosidase; PE, phycoerthrin; SB, staining buffer; LCMV, lymphocytic choriomeningitis virus. Back

Received for publication August 29, 1997. Accepted for publication November 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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