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Antigen-Specific CD8⁺ T Cells Mediate a Peptide-Induced Fatal Syndrome¹

Aaron J. Johnson^*,, Yanice Mendez-Fernandez^*, Ann M. Moyer^*, Cari R. Sloma^*, Istvan Pirko, Matthew S. Block^*, Moses Rodriguez^*, and Larry R. Pease^2,*,

Departments of^* Immunology, Neurology, and Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Peptide immunotherapy both activates and suppresses the T cell response against known peptide Ags. Although pretreatment with VP2_121–130 peptide inhibits the development of antiviral CTL specific for the immunodominant D^b:VP2_121–130 epitope expressed during acute Theiler’s murine encephalomyelitis virus infection, i.v. injection of this same peptide or MHC tetramers containing the peptide during an ongoing antiviral CTL response results in a peptide-induced fatal syndrome (PIFS) within 48 h. Susceptibility to PIFS is dependent on peptide-specific CD8⁺ T cells, varies among inbred strains of mice, and is not mediated by traditionally defined mechanisms of shock. Analyses using bone marrow chimeras and mutant mice demonstrate that susceptibility to PIFS is determined by the genotype of bone marrow-derived cells and requires the expression of perforin. Animals responding to peptide treatment with PIFS develop classical stress responses in the brain. These findings raise important considerations for the development of peptide therapies for active diseases to modify immune responses involving expanded populations of T cells. In summary, treatment with peptides or MHC-tetramers during a peptide-specific immune response can result in a fatal shock-like syndrome. Susceptibility to the syndrome is genetically determined, is mediated by CD8⁺ T cells, and requires expression of perforin. These findings raise concerns about the use of peptides and MHC tetramers in therapeutic schemes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The discovery that cell-mediated immunity focuses on peptide Ags led to the development of strategies for peptide-based immunotherapy. Peptides derived from allergens have been used to suppress CD4⁺ T cells important for promoting hypersensitivity reactions (1, 2, 3). Similarly, peptide treatments that inhibit T cells specific for graft-derived peptides presented by self MHC class I and class II molecules provides a basis for prolonging graft survival (4, 5). Whereas peptides have been used successfully to inhibit immune responses, attempts to activate T cells with peptide treatments after the disease process is established have not been as successful. Peptides administered in adjuvant stimulate a stronger CTL response toward some tumor Ags (6, 7, 8, 9, 10, 11, 12, 13). In other cases, peptide treatment induced unresponsiveness of tumor-specific CTL (14, 15, 16, 17). The physiological basis of these different outcomes is unknown. The preponderance of evidence indicates that it is more difficult to promote immunity toward known peptide:MHC epitopes than to inhibit peptide-specific responses.

We have sought to inhibit peptide-specific T cells in the Theiler’s murine encephalomyelitis virus (TMEV)³ model of multiple sclerosis. TMEV is a murine picornavirus that causes chronic inflammation, demyelination, and neurologic deficits in susceptible, but not resistant, strains of mice (18, 19). Resistant H-2^b haplotype mice develop a robust CTL response in the CNS against a peptide (VP2_122–130, FHAGSLLVFM), derived from a TMEV capsid protein by 7 days postinfection (20, 21). Using peptide:MHC tetramers, we demonstrated in H-2^b haplotype mice that 35–70% of CD8⁺ T cells express TCRs specific for the D^b:VP2_121–130 epitope (22).

We have demonstrated that i.v. injection of VP2_121–130 peptide before TMEV infection inhibits the appearance of D^b:VP2_121–130 epitope-specific CTL in the brain on day 7 and markedly protects the animals from ensuing paralytic disease (23, 24). Strategies that block peptide-specific CD8⁺ T cells before infection provide the immune system the opportunity to adapt by developing alternative responses to the Ag. Therefore, we sought to inhibit the response after infection and during active disease in an attempt to prevent the recruitment of alternative responses against the virus. Surprisingly, administration of VP2_121–130 peptide during an ongoing antiviral CTL response had dramatically adverse effects. In C57BL/6 mice with an expanded D^b:VP2_121–130 epitope-specific CTL response, the administration of VP2_121–130 peptide induced a fatal syndrome. Remarkably, even administration of MHC tetramers bearing the peptide in an attempt to image an ongoing immune response elicited the adverse response. This peptide-induced fatal syndrome (PIFS) is mediated by CD8⁺ T cells specific for the inciting peptide and is dependent on the expression of perforin. Furthermore, PIFS is not mediated by pathways previously defined in systemic shock. Susceptibility differs among mouse strains, is determined by the genotype of bone marrow-derived cells, and is inherited in a complex genetic fashion. The finding that peptide therapy or attempts to monitor Ag-specific responses can induce dramatically adverse effects provides important considerations for the development of schemes using peptides to suppress or monitor active immune responses that contain populations of expanded T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Virus infection and mice

Male and female C57BL/6J, C57BL/10J, 129S3/SvImJ, C57BL/6-Pfp^tm1Sdz, B6–129S-Tnf^tm1Gkl (TNF^–/–), C57BL/6-Tnfrsf1a^tm1Mak (TNFRI^–/–), C57BL/6-Tnfrsf1b^tm1Mwm (TNFRII^–/–), and C57BL/6-TgN(ACTbEGFP)1Osb (GFP) mice were obtained from The Jackson Laboratory. 129-Ifngr^tm1 (IFN-R^–/–) mice were gifts from M. Aguet (Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland). The C57BL/10 class II^–/– mice were generated by C. David (Mayo Foundation, Rochester, MN) using H-2AB⁰ (class II^–/– mice; gifts from C. Benoist and D. Mathis, Harvard University, Cambridge, MA). FVB/D^b transgenic mice were generated with a D^b genomic transgene in collaboration with C. David. Mice were given an intracerebral injection with 2 x 10⁶ PFU of Daniel’s strain of TMEV. Handling of all animals conformed to the National Institutes of Health and Mayo Clinic institutional guidelines and were approved by the Mayo Clinic Institutional Animal Care and Use Committee.

Preparation of H-2D^b and H-2K^b tetramers

H-2D^b tetramers were prepared as described (22, 25, 26). H-2K^b was inserted into the pET23 expression vector (Novagen), and K^b tetramers were assembled using the same procedures.

Flow-cytometric analysis

Brain-infiltrating lymphocytes were extracted from the brains of virus-infected mice. Brains were homogenized and lymphocytes were recovered using a step gradient of 30% Percoll mix. Percoll was diluted with serum-free RPMI 1640. In all FACS samples, erythrocytes were removed by lysis with ACK. Lymphocytes isolated from brain 7 days post-TMEV infection were stained with R-PE D^b/VP2_121–130, D^b/E7, or K^b/SIYR tetramer for 1 h, adding anti-CD8-FITC and anti-CD4-PerCP (BD Pharmingen) during the final 20 min. Samples were washed twice with FACS buffer (1% BSA and 2% sodium azide), resuspended in cold PBS, and fixed in 1% paraformaldehyde. Samples were analyzed on a BD Biosciences FACScan instrument.

Analysis of vascular leakage

Eight days post-TMEV infection, C57BL/6 mice were injected i.v. with VP2_121–130 or E7 peptide. The following day, both VP2_121–130- and E7 peptide-treated groups were injected i.v. with HRP (Sigma-Aldrich). After 1 h, the mice were killed and the heart, lungs, kidney, liver, and brain were frozen to chucks in OCT compound (Miles) at –80°C. Sections (20 µm) of each of these organs were mounted to slides. Dried, unfixed slides were developed in Hanker Yates substrate solution (Polysciences). H&E staining was performed on brain tissue from mice anesthetized i.p. with 10 mg of sodium pentobarbitol and perfused by intracardiac puncture with Trump’s fixative (phosphate-buffered 4% paraformaldehyde with 1.5% glutaraldehyde).

Peptides

VP2_121–130 (FHAGSLLVFM), E7 (RAHYNIVTF), SIYR (SIYRYYGL), and OVA (SIYNFEKL) peptides were synthesized in the Mayo Foundation Protein Core Facility. To inhibit the development of D^b:VP2_121–130 epitope-specific CD8⁺ T cells 7 days post-TMEV infection, 0.01 or 0.1 mg of VP2_121–130 was administered three times at 4-h intervals 1 day before TMEV infection. E7 peptide was administrated as a mock pretreatment. To initiate VP2_121–130 PIFS, 8 days post-TMEV infection, 0.1 mg of VP2_121–130 or E7 peptide was administered once or three times at 4-h intervals. Both regimens of VP2_121–130 peptide injection resulted in PIFS in 8-day infected mice. Animals were then monitored the following 72 h for morbidity.

Administration of OVA peptide (SIINFEKL) to reduce CD4⁺CD8⁺ double-positive thymocytes in OT-1 transgenic mice without fatal consequences has been documented (27). In these experiments, 0.1 mg of peptide was injected i.p. once a day for 3 days. Our approach used the same amount of peptide (0.1 mg), but the treatment was administered i.v. We have found that a single injection of peptide is sufficient to induce PIFS in these animals.

Analysis of SIYR peptide-induced shock in 2C TCR transgenic mice

2C TCR transgenic mice were administered one i.v. injection of 0.1 mg of SIYR peptide and then monitored for changes in behavior. To deplete mice of CD8⁺ T cells, 1.0 mg of Lyt 2.43 Ab was injected i.p., 1 day before administration of peptide (28, 29). The day following mAb injection, PBL were assayed for cells expressing CD8 that were specific for the K^b:SIYR epitope by staining with anti-CD8 Ab and K^b/SIYR tetramer as described under flow-cytometric analysis. Following evidence that depletion of CD8⁺ T cells had occurred, 0.1 mg of SIYR peptide was administered, and animals were observed for changes in behavior. CD4⁺ and CD8⁺ T cells were isolated using a MACS column (Miltenyi Biotec) 4 h after the administration of SIYR peptide or PBS. RNA was then isolated from these cells and analyzed on a mouse cytokine GEArray (Super Arrays) according to the manufacturer’s protocol to determine expression of cytokine RNA.

Magnetic resonance imaging (MRI) with superparamagnetic tetramer

Seven-day TMEV-infected C57BL/6 mice were injected i.v. with superparamagnetic D^b:VP2_121–130 tetramer or D^b:E7 tetramer 1 day before imaging with MRI. Superparamagnetic tetramer was assembled by conjugating 10 µl of superparamagnetic streptavidin (Miltenyi Biotec) to 0.025 mg of biotinylated D^b:VP2_121–130 monomer for 30 min before injection. For live-animal MRI, mice were anesthetized by inhalational isoflurane anesthesia using a calibrated evaporator. The gas flow was maintained at 1.5 v/v% during the entire imaging session. A Bruker Avance 300 nuclear magnetic resonance spectroscope was used to obtain in vivo images of the mouse brain (Bruker Biospin). The field strength was 7 T; the apparatus was equipped with a mini-imaging birdcage resonator coil suitable for imaging small rodents (38-mm bore diameter; vertical bore). After obtaining localizer images, a T2-weighted fast spin-echo volume acquisition sequence was used to obtain data from the entire cranial volume (repetition time, 1500 ms; echo time, 60 ms; rapid acquisition relaxation enhancement factor, 16; field of view, 3 cm³; matrix size, 128 x 128 x 128; resolution, 234 µm; total acquisition time, 26 min). Two-dimensional slice data were reconstructed from the three-dimensional datasets using Bruker’s proprietary software, ParaVision.

Adoptive transfer of GFP splenocytes into C57BL/6 perforin^–/– mice

C57BL/6 perforin^–/– mice were irradiated with 400 rad. One day following irradiation, 10⁸ GFP-expressing splenocytes were i.v. injected into the irradiated C57BL/6 perforin^–/– mice. One day following GFP splenocyte transfer, mice were intracranially infected with TMEV. Eight days following TMEV infection, animals were administered 0.1 mg of VP2_121–130 or E7 peptide. One day following peptide treatment, brains were excised, fixed in 4% paraformaldehyde, and cut with a vibratome. The sections were adhered to slides and the presence of GFP-expressing cells in both E7 and VP2_121–130 peptide-treated groups was observed by fluorescent microscopy to confirm successful adoptive transfer.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
TMEV-infected mice develop anti-TMEV responses dominated by a single peptide epitope

CNS-infiltrating lymphocytes were isolated from the brains of several strains of H-2^b haplotype mice 7 days postinfection with TMEV. Approximately 35–70% of CD8⁺ T cells isolated from brains of TMEV-infected C57BL/6, 129, TNF-^–/–, and TNFRII^–/– mice stained positive for both CD8 Ab and D^b:VP2_121–130 tetramer (Fig. 1, A-D), but not with a control tetramer bearing an irrelevant peptide derived from human papilloma virus (E). Also, as previously described, TMEV-infected IFN-R^–/–, class II^–/–, TNFRI^–/–, and perforin^–/– mice have a similar percentage of D^b:VP2_121–130 epitope-specific CD8⁺ T cells in their brains (22). CD4⁺ T cells did not express TCR specific for D^b:VP2_121–130 in these mice. As expected, few CNS-infiltrating lymphocytes were detected in the brains of uninfected C57BL/6 mice (22). These observations demonstrate that, at the peak of virus infection, many CD8⁺ T cells at the site of infection have TCRs specific for the VP2_121–130 peptide presented in the context of D^b.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Clonal dominance of brain infiltrating lymphocytes isolated from the brains of 7-day TMEV-infected mice. A–D, Lymphocytes isolated from the brains of C57BL/6 (A), 129 (B), TNF-–/– (C), and TNFRII–/– (D) were stained with CD8-specific Ab and Db/VP2121–130 tetramer and analyzed using a FACScan instrument. E, C57BL/6 brain-infiltrating lymphocytes were stained with Ab specific for CD8 and with Db/E7 tetramer.

The development of D^b:VP2_121–130 epitope-specific CTL during acute TMEV infection of C57BL/6 mice can be inhibited by administering VP2_121–130 peptide to naive animals 1 day before virus challenge (23, 24). Three i.v. injections of 0.01 mg of VP2_121–130 in PBS reduced D^b:VP2_121–130 epitope-specific CD8⁺ T cells infiltrating the brain from 70 to 18% (Fig. 2A). Complete removal of D^b:VP2_121–130 epitope dominance was achieved with three injections of 0.1 mg of VP2_121–130 peptide (Fig. 2B), but not with irrelevant D^b binding E7 peptide (C).

View larger version (37K): [in this window] [in a new window]   FIGURE 2. Abrogation of brain-infiltrating lymphocytes in 7-day TMEV-infected C57BL/6 mice by pretreatment with VP2121–130 peptide. Lymphocytes isolated from mice pretreated with 0.01 mg of VP2121–130 peptide (A), 0.1 mg of VP2121–130 peptide (B), or 0.1 mg of E7 peptide (C) were stained with Ab to CD8 and Db/VP2121–130 tetramer and analyzed by FACS.

Administration of VP2_121–130 peptide at 8 days post-TMEV infection induces a fatal syndrome in C57BL/6 and IFN-R^–/– mice

In an attempt to inhibit the effects of virus-specific CD8⁺ T cells at the peak of CTL activity on mice susceptible to virus-induced paralytic disease (23), VP2_121–130 peptide was administered i.v. to C57BL/6 mice and IFN-R^–/– mice 8 days postinfection. Rather than inhibiting Ag-specific function, severely adverse reactions were observed in both strains of mice. By 24 h after peptide treatment, a change in overall activity was observed among the mice, because they lost coordination, became emaciated, and ceased grooming. Ninety-seven percent (28 of 29) of the animals became critically ill or died within 48 h following peptide treatment (Table I). We refer to this moribund state as PIFS. Death resulting from PIFS in TMEV-infected mice was Ag specific, because infected animals administered the irrelevant D^b binding E7 peptide remained asymptomatic (Table I).

Administration of VP2_121–130 peptide 8 days post-TMEV infection does not increase TMEV virus in IFN-R^–/–, C57BL/6, and 129 mice undergoing VP2_121–130 PIFS

The mechanism underlying the pathology of PIFS was not evident. We considered the possibility that a rapid and overwhelming rise in virus load might result from depletion of effective CTL by peptide treatment. To address this possibility, we prepared whole-brain RNA from IFN-R^–/– mice 1 day after the VP2_121–130 or E7 peptide treatment. Using probes complementary to TMEV RNA (VP2 capsid region) and to endogenous mouse GAPDH, we determined the ratio of virus to mouse RNA in individual animals. The ratio of virus to mouse GAPDH RNA present was not significantly different among the VP2_121–130 (1.1 ± 0.04) and E7 (1.3 ± 0.8) peptide with a trend toward a lower virus load in VP2_121–130 peptide-treated animals. In addition to the Northern blots, real-time RT-PCR was used to detect virus RNA load in C57BL/6 and 129 animals 1 day after VP2 and E7 peptide treatment. Consistent with IFN-R^–/– mice, neither of these strains showed evidence of an increased virus load following VP2_121–130 PIFS. These measurements ruled out the possibility that PIFS is the result of increased virus load.

PIFS is mediated by Ag-specific CD8⁺ T cells

We next considered the possibility that PIFS is mediated by activation of T cells by peptide treatment. To determine whether antiviral CD8⁺ T cells specific for the D^b:VP2_121–130 epitope were mediating PIFS observed in infected C57BL/6 and IFN-R^–/– mice, the development of these T cells was inhibited by pretreating the mice with VP2_121–130 peptide 1 day before TMEV infection. Pretreatment with VP2_121–130 peptide results in a complete loss of D^b:VP2_121–130 epitope-specific CD8⁺ T cells in the brain at 7 days post-TMEV infection (Fig. 2B). Elimination of this specific set of CD8⁺ T cells prevented PIFS at day 8 post-TMEV infection (Table II). This result, observed in both C57BL/6 and IFN-R^–/– mice, supports the hypothesis that VP2_121–130 PIFS was dependant on the presence of a CD8⁺ response specific for the D^b:VP2_121–130 epitope in the CNS.

View this table: [in this window] [in a new window]   Table II. PIFS does not occur in C57BL/6 and IFN-R–/– mice depleted of Db:VP2121–130 epitope-specific CD8+ T cells

PIFS is not class II dependent

Because rapid deterioration of the mice observed after VP2_121–130 peptide treatment resembled symptoms observed in class II-restricted toxic shock, we addressed the possibility that CD4⁺ T cells were somehow involved in mediating PIFS. Class II^–/–H-2 AB° mice cannot present the VP2_121–130 peptide or any other peptide in the context of class II molecules and have greatly reduced numbers of CD4⁺ T cells, but still have high numbers of CNS-infiltrating CD8⁺ T cells specific for D^b:VP2_121–130 (22). Administration of the VP2_121–130 peptide 8 days after TMEV infection resulted in PIFS in five of seven mice (Table I), demonstrating that the mechanisms mediating VP2_121–130 PIFS are not dependent on class II presentation of peptide.

TNF-^–/–, TNFRI^–/–, TNFRII^–/–, and TNFRI^–/–TNFRII^–/– double-knockout mice are susceptible to VP2_121–130 PIFS

We next addressed the possibility that the VP2_121–130 peptide could be instigating a form of CD8⁺ T cell-dependent shock mediated by TNFRs. Septic shock is mediated by an increase in cytokines, most particularly TNF- and IL-1 (31). The importance of TNF- signal transduction in the development of septic shock has been addressed in TNFRI^–/– mice (32). To evaluate whether peptide-treated mice were succumbing to toxic shock mediated by a similar mechanism, TNF-^–/–, TNFRI^–/–, TNFRII^–/–, and TNFRI^–/–TNFRII^–/– double-knockout mice infected with TMEV were administered VP2_121–130 peptide 8 days postinfection. Peptide treatment induced shock in all four TNF-deficient mouse lines (Table III), demonstrating that neither TNF- nor the TNFRI or TNFRII receptors are required for the syndrome to develop. Furthermore, direct measurement of TNF- in the serum of mice treated with peptide revealed no increase over background levels (data not shown). To determine whether PIFS was mediated by lymphotoxin (LT) receptors, as previously suggested in the lymphocytic choriomeningitis virus (LCMV) model (33), seven C57BL/6 mice were administered 100 µg of rLT-receptor fusion protein 2 h before induction of PIFS. All seven mice developed PIFS with kinetics identical with six mice pretreated with PBS, indicating that blockade of LT receptor had no effect on fatal shock. Together, these data indicate that PIFS is mediated by a mechanism different from other shock syndromes that are mediated by members of the TNF family of cytokines.

View this table: [in this window] [in a new window]   Table III. PIFS occurs in TMEV-infected TNF–/– and TNFR-deficient micea

We noted a lower fatality in TNF-^–/– mice undergoing VP2_121–130 PIFS (Table III). Therefore, we considered the importance of mouse genetic background on susceptibility to PIFS. Unlike TNFRI^–/– and TNFRII^–/– mice, TNF-^–/– mice were not completely inbred to have a full B6 genetic background and are a mixture of the B6 and 129 genotypes. To address the role of genetic variability, the resistance of the 129 and FVB strains of mice to PIFS were assessed. Both 129 and FVB-H-2D^b transgenic mice develop robust numbers of brain-infiltrating CD8⁺ T cells specific for the D^b:VP2_121–130 epitope 7 days after TMEV infection (Fig. 1B and Ref. 22). Despite developing a substantial population of VP2_121–130 peptide-specific CD8⁺ T cells in the CNS, FVB-H-2D^b, (FVB x B6)F₁, 129, and (129 x B6)F₁ mice were more resistant to VP2 PIFS when compared with B6 animals (Table IV). Resistance to PIFS was highest in mice with the 129 background. None of the eight 129 mice or six (129 x B6)F₁ mice succumbed to PIFS. Because the amount of D^b:VP2_121–130-specific CD8⁺ T cells recovered from the brains of 129 and C57BL/6 mice were not different, resistance to VP2_121–130 PIFS observed in 129 mice cannot be attributed to greater infiltration of Ag-specific T cells in the brains of susceptible B6 mice. To a lesser degree, mice with FVB background were also more resistant, with only three of nine D^b transgenic and two of seven (FVB x B6)F₁ animals developing PIFS (Table IV). This pattern of resistance demonstrates that host genotype contributes to susceptibility of PIFS. C57BL strains are much more susceptible than 129-derived lines, whereas FVB-derived strains have an intermediate phenotype.

It should be noted that some chromosomal segments could not be analyzed using this procedure because of the highly similar microsatellite sequences present in the analyzed mouse lines. We attempted to specifically look at the importance of genes mapping to the region of chromosome 10 that includes the perforin locus, but were unsuccessful because no polymorphisms in this region were discernable. Even a search for single-nucleotide differences within the amplified segments failed to identify differences in this region of chromosome 10.

The question of whether genetic differences defining susceptibility to PIFS influence the properties of the immune system or influence the differential susceptibility of vital tissues to immune mediators was addressed using bone marrow chimera experiments. Lethally irradiated, resistant 129 mice were reconstituted with bone marrow from susceptible C57BL/6-ly5.1 mice. Six weeks after chimerization, the animals were challenged with TMEV intracranially. On day 7 postinfection, the animals were treated with 100 µg of VP2_121–130 peptide i.v. and scored for PIFS over the next 2 days. In contrast to the completely resistant phenotype of nonchimeric 129 mice to PIFS, seven of eight C57BL/6-ly5.1 129 chimeric animals succumbed to PIFS (p < 0.001). Analysis of the brain-infiltrating lymphocytes by flow cytometry revealed that >95% of the T cells in the brains of the TMEV-infected chimeric mice were of donor (susceptible phenotype) origin. This finding is consistent with the hypothesis that the genetic differences distinguishing resistance and susceptibility in C57BL/6 and 129 mice is a property of bone marrow-derived cells and likely is attributable to differences in the immune response. The reciprocal experiment was also performed in which resistant 129 bone marrow was used to reconstitute C57BL/6-ly5.1 animals. However, in this case, chimerism was incomplete with only 40–60% of the brain-infiltrating lymphocytes being of donor origin. This group of chimeric mice was somewhat more resistant (four of eight animals succumbed to PIFS) in comparison to the uniformly susceptible C57BL/6-ly.1 and standard C57BL/6 mice. The incomplete protection of the partially chimeric animals can be attributed to the substantial numbers of host T cells present in the TMEV-infected brains. Again, these results, although not definitive, are consistent with the view that susceptibility to PIFS is a determinant associated with traits expressed by C57BL/6 T cells.

The finding that 129 mice are resistant to PIFS permitted the analysis of the fate of VP2_121–130-specific T cells following treatment of mice with peptide at the height of the inflammatory response in the brain. We noted an equivalent reduction (by 30%) in the number of T cells 1 day after peptide treatment in both 129 and C57BL/6 animals. Whereas C57BL/6 mice succumb to PIFS by day 2, 129 animals survive indefinitely. By 4 days after peptide treatment, the number of VP2_121–130-positive T cells in the 129 mice had returned to normal. We also examined the possibility that the specificity of responding CD8⁺ T cells infiltrating the brains of infected B6 and 129 mice might be qualitatively different. We first compared the avidity of T cells for the viral peptide VP2_121–131 presented in the context of the D^b class I molecule. D^b-tetramers bearing the VP2 peptide were bound to brain-infiltrating T cells isolated from 129 and C57BL/6-virus-infected animals. Disassociation of labeled tetramers was assessed over time following blocking of their ability to reassociate with unlabeled tetramer. No differences in disassociation rates were observed in our analysis of 129 and C57BL/6 virus-specific T cells (data not shown). We next measured the direct cytolytic activity of the brain-infiltrating lymphocytes isolated from the brains of 8-day TMEV-infected B6 and 129 mice 1 day after VP2 peptide treatment. CTL from both strains showed equivalent ability to lyse C57SV targets expressing D^b and the VP2 protein in a standard ⁵¹Cr release assay (data not shown). Therefore, we found no evidence of increased apoptotic activity, differences in receptor repertoires, or changes in cytolytic activity that might account for the relative susceptibility of C57BL/6 mice in comparison to PIFS-resistant 129 mice.

Administration of VP2_121–130 at 8 days post-TMEV infection results in blood vessel permeability and tissue abnormality in the brain

Autopsies were performed on TMEV-infected C57BL/6 mice 1 day after injection of E7 or VP2_121–130 peptide. Mice given VP2_121–130 but not E7 peptide were moribund when tissue was harvested. Using H&E staining of frozen sections, we observed no changes in the liver, kidney, spleen, lung, lymph node, thymus, or skeletal muscle of mice treated with VP2_121–130 peptide when compared with organs of E7-treated controls not undergoing PIFS (data not shown). We concluded that the peptide-induced changes did not result in direct systemic damage, a finding consistent with our previous studies showing that peptide-specific T cells are largely colocalized in the CNS of infected animals (22).

Because PIFS resembled septic shock, we explored whether blood vessel permeability could be associated with the syndrome. C57BL/6 mice were injected i.v. with VP2_121–130 8 days after TMEV infection to initiate PIFS. The following day, VP2_121–130- or E7-treated mice were injected i.v. with HRP. One hour later, brain tissue was harvested for histological analysis. In comparison to brain tissue from mice treated with irrelevant E7 peptide (Fig. 3A), brains of mice succumbing to PIFS revealed extensive diffusion of HRP throughout the brain (B). We conclude that an extensive loss of vascular integrity occurs in the brains of mice with PIFS. No vascular leakage of HRP was observed in the heart, kidneys, lungs, liver, and peritoneum of these mice (data not shown). Furthermore, H&E staining of brain sections revealed extravasation of RBC in animals undergoing PIFS, but not in E7-treated control animals. In particular, extravascular erythrocytes were observed in the hypothalamus, cortex, hippocampus, striatum, and corpus callosum from moribund mice (Fig. 3, C and D). These experiments demonstrate that i.v. VP2_121–130 peptide treatment induced blood vessel permeability in the brain.

View larger version (45K): [in this window] [in a new window]   FIGURE 3. Vessel permeability in the brains of C57BL/6 mice with VP2121–130 PIFS. Leakage of HRP into the cortex of 8-day TMEV-infected C57BL/6 mice i.v. with E7 peptide (A) or VP2121–130 peptide (B). Erythrocyte leakage into the hypothalamus of E7 peptide-treated animals (C) and VP2 peptide-treated animals (D) undergoing PIFS (scale bar, 100 µm). Arrows denote clusters of erythrocytes located outside of blood vessels. Also shown is T2-weighted brain image of 7-day TMEV-infected C57BL/6 administered DbE7-tetramer (E) or DbVP2-tetramer (F). Arrows denote brain abnormalities detected by MRI.

Our findings of tissue damage in the brain by MRI and gross histological analysis is further supported by the activation of glia following systemic treatment with antigenic peptide. Up-regulated expression of glial fibrillary acidic protein, a pattern associated with the response of astrocytes to immunologic and traumatic injury, indicates that systemic peptide treatment leads to a widespread stress response in brain cells (Fig. 4) (34, 35). Tissue damage in the brain is normally associated with the influx of macrophages and the activation of resident microglial cells. The widespread appearance of cells bearing the macrophage/microglia marker F4/80 in the brains of C57BL/6 mice with PIFS is a marker for the onset of these inflammatory changes associated with damage in the brain. In contrast, TMEV-infected 129 mice treated systemically with peptide showed very little and localized evidence of macrophage infiltration or microglial activation (compare top and bottom right panels) and essentially no stress response by astrocytes (compare top and bottom left panels). This is further evidence that there are strong strain-dependent injury responses in the brain to systemic peptide treatment of mice undergoing an active T cell response.

View larger version (102K): [in this window] [in a new window]   FIGURE 4. Activation of stress responses in the brain accompany PIFS. C57BL/6 (B6) and 129 strain animals were infected for 7 days intracranially with TMEV before systemic treatment with TMEV-derived VP2121–130 peptide or E7 control peptide. Normal C57BL/6 mice were used as a standard. Twenty-four hours after peptide treatment, sections of the striatum were analyzed by immunoperoxidase staining for the expression of the stress-induced activation markers glial fibrillary acidic protein (GFAP) in astrocytes and F4/80 in macrophage and microglial cells. Image magnification is x10.

C57BL/6 mice with genetic disruption of the perforin gene are resistant to VP2_121–130 PIFS, with zero of nine animals succumbing to the fatal syndrome (Table V). Being asymptomatic, we next addressed whether perforin was responsible for vessel permeability in the CNS. Following administration of VP2_121–130 peptide, C57BL/6 perforin^–/– mice were found to have blood vessel permeability in the CNS comparable to a C57BL/6 mouse undergoing VP2_121–130 PIFS (Fig. 5, B and D). C57BL/6 and C57BL/6 perforin^–/– receiving the E7 peptide did not have appreciable vessel permeability (Fig. 5, A and C). This experiment demonstrates that perforin plays an important role in the development of symptomatic VP2_121–130 PIFS, but is not responsible for CNS blood vessel permeability. Surprisingly, the extensive blood vessel permeability observed in perforin^–/– mice occurred without observable clinical consequence to the animal.

View larger version (97K): [in this window] [in a new window]   FIGURE 5. Perforin is not necessary for CNS blood vessel permeability following administration of VP2121–130 peptide 8 days post-TMEV infection. A, Normal autofluorescence of hippocampus in E7 control peptide-treated C57BL/6 mice. B, Autofluorescence of hippocampus from VP2121–130 peptide-treated C57BL/6 mice. Strong autofluorescent spots correspond to blood deposits. C and D are as A and B, except hippocampus slice is from C57BL/6 perforin–/– mice. C57BL/6 perforin–/– mice have CNS vessel permeability but are asymptomatic when administered both E7 peptide (C) and VP2121–130 peptide (D) 8 days post-TMEV infection. Adoptive transfer of perforin-competent GFP-expressing splenocytes into perforin–/– mice results in no vessel permeability when administered E7 peptide (E) and extensive blood vessel permeability when administered VP2121–130 peptide (F). The hippocampus is shown in A–F. Blood in the CNS is autofluorescent. Note the presence of adoptively transferred GFP-expressing lymphocytes in the hippocampus in E and F. Representative examples are shown from groups of three animals per group.

	Discussion

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These experiments demonstrate that i.v. administration of class I-restricted peptides or class I tetramers that contain these peptides can be fatal to individuals harboring expanded, peptide-specific CD8⁺ populations. There is one well-characterized mechanism by which a response can lead to rapid death. Pathogen-derived superantigens simultaneously bind both the receptor (TCR) V chain and class II molecules (36). This cross-linking of TCR with class II leads to the polyclonal activation of CD4⁺ Th cells followed by the systemic increase of cytokines. Shock results from a dramatic rise in the cytokines TNF-, TNF-, IL-1, IL-6, IL-8, and IL-10. These cytokines initiate a cascade of fever, increased cardiac output, increased blood clotting, organ failure, and death (31). Our analysis rules out these classical shock pathways. In this study, we address a second T cell-mediated syndrome that is initiated by peptide-specific CD8⁺ T cells. Although CD4⁺ T cell-mediated shock is becoming better understood, the possibility of CD8⁺ T cells mediating similar syndromes has gone almost completely unnoticed. One study describing the possibility of CD8⁺ T cell-mediated shock used Abs that initiate polyclonal activation and their secretion of IFN- and TNF-. These cytokines were protective in the host response to Trypanosoma cruzi. However, when these T. cruzi-infected mice were administered anti-CD3 Ab, these mice underwent septic shock. Pretreatment with anti-CD8 Abs before this anti-CD3 treatment prevented septic shock (37). These observations suggested that, in this disease model, septic shock might be mediated by CD8⁺ T cells.

The importance of CD8⁺ T cells in inducing shock was suggested in other studies involving LCMV and tumor peptide vaccination (33, 38, 39). In the LCMV model, administration of peptide activated peptide-specific memory cells, inducing damage in the spleen that was generally immunosuppressive (38). Shock induced with LCMV was mediated by lymphotoxin as recombinant soluble chimeric lymphotoxin -receptor/Ab (LT-R) blocked virus-induced pathology (33). In the tumor peptide vaccination model, CD8⁺ T cell-mediated shock was mediated by TNF because anti-TNF Abs neutralized this fatal condition (39). In this study, we demonstrate that a fatal syndrome can be induced by the treatment of virus-infected mice with soluble peptide. Mice succumbing to PIFS display a dramatic breakdown of vascular integrity in the brain. In our system, TNF-, TNFRI, and TNFRII are not required for the development of the PIFS. Furthermore, induction of PIFS also is not influenced by treating animals with soluble LT-R fusion protein, indicating that none of the previously defined cytokine pathways appears to play a dominant role in this syndrome. Another possibility is that CD8⁺ T cells secrete cytokines that directly or indirectly lead to vessel permeability. Vessel permeability caused by cytokines has been reported by those investigating vascular leak syndrome (VLS), a condition where vascular epithelial cells become damaged and permeable to vascular fluids (40). In particular, the administration of rIL-2, a commonly emitted CD8⁺ cytokine, can initiate murine VLS (40, 41, 42). Although VLS is a trait of VP2_121–130 PIFS, permeability of blood vessels in the brain alone does not lead to death. Perforin-deficient mice used in this study have extensive vessel permeability in the CNS comparable to C57BL/6 controls, but remain asymptomatic. Therefore, we hypothesize that perforin destabilizes cells in the CNS involved with vital functions. This is consistent with previous observations that perforin contributes to pathogenesis (28, 29, 43, 44, 45). Therefore, we may be triggering a CD8⁺ T cell-mediated process that occurs naturally following infection in the CNS.

The observation that CD8⁺ T cells can mediate a PIFS extends beyond the Theiler’s virus system. We have investigated whether peptide could induce shock in the 2C transgenic mouse. 2C transgenic mice express receptors that are reactive with the SIYR peptide presented in the context of K^b class I molecules on >90% of peripheral T cells. The effect of injecting SIYR peptide i.v. into healthy 2C TCR transgenic mice was similar to that observed upon VP2_121–130 peptide treatment of 8-day TMEV-infected C57BL/6, class II^–/–, and IFN-R^–/– mice. PIFS was not reproduced with an irrelevant K^b-binding OVA peptide, demonstrating the peptide specificity of the syndrome. As little as 0.001 mg of SIYR peptide administered three times i.v. could induce PIFS in 2C transgenic mice. Similar to the VP2_121–130 peptide-induced syndrome, SIYR PIFS was mediated by an expanded population of epitope-specific CD8⁺ T cells.

Peptide-specific CD8⁺ T cells are necessary, but not sufficient, to render animals susceptible to PIFS. Besides expression of perforin, PIFS is also influenced by the genetic makeup of the host. More specifically, we present evidence that resistance to PIFS is conferred through a complex interaction of multiple genes. The influence of mouse genotype can, therefore, explain how 129 mice containing populations of D^b:VP2_121–130 epitope-specific CD8⁺ T cells equivalent to C57BL/6 mice can remain resistant to VP2_121–130 PIFS. More work is needed to determine whether mouse genotype influences perforin function. Because it is likely that peptide-mediated suppression will continue to be a strategy to down-regulate subsets of Ag-specific T cells, we find these observations to be highly relevant to those interested in the development of peptide therapies.

	Acknowledgments

 
We thank John Altman of Emory University (Atlanta, GA) and Mark Davis of Stanford University (Stanford, CA) for contributing the D^b and human ₂m expression plasmids. We also thank Lieping Chen of Mayo for soluble LT-R. We also thank Mike Bell, Charles Howe, Kevin Pavelko, Loc Nguyen, and Yanice Mendez-Fernandez for technical assistance in the completion of this work.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was funded by National Institutes of Health Grants N524180 and N532129.

² Address correspondence and reprint requests to Dr. Larry R. Pease, Department of Immunology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905. E-mail address: pease.larry{at}mayo.edu

³ Abbreviations used in this paper: TMEV, Theiler’s murine encephalomyelitis virus; PIFS, peptide-induced fatal syndrome; MRI, magnetic resonance imaging; LCMV, lymphocytic choriomeningitis virus; LT, lymphotoxin ; VLS, vascular leak syndrome.

Received for publication November 17, 2004. Accepted for publication February 18, 2005.

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