A viral epitope that mimics a self antigen can accelerate but not initiate autoimmune diabetes (original) (raw)
Challenge of unprimed, naive animals with viruses that express lower-avidity mimic ligands of β-cell epitopes fails to induce autoimmune diabetes. Infection of RIP-LCMV-NP H-2b transgenic mice, which express the LCMV strain Armstrong (LCMV-Arm) NP protein (LCMV-NP) in β cells, with LCMV-Arm leads to T1D 2–4 months after infection in more than 95% of mice. By 10–14 days after LCMV infection, virus is cleared from infected mice (29). In order to identify the T cell epitopes of the viral NP molecule responsible for induction of diabetes, we infected transgenic mice with LCMV-Arm that contains LCMV-NP396 and LCMV-NP205 cytotoxic T cell epitopes or with an LCMV-NP396 cytotoxic T lymphocyte (CTL) escape variant (LCMV-Arm escape variant; LCMV-Arm-Var) that contains a single amino acid substitution of phenylalanine to leucine at position 403 (substitution underlined: FQPQNGQFI to FQPQNGQLI; Table 1) (33). While infection with LCMV-Arm caused diabetes with the expected kinetics, infection with LCMV-Arm-Var, which lacked the LCMV-NP396 epitope but still expressed the normal LCMV-NP205 epitope (33), did not (Figure 1A). Thus, the LCMV-NP396 epitope that is recognized with comparatively high avidity (34) was essential for initiation of T1D in RIP-NP H-2b mice.
Molecular mimicry can accelerate but not easily initiate autoimmune diabetes. (A) Molecular mimicry is insufficient to prime naive autoreactive CD8 T cells and cause autoimmune diabetes. RIP-LCMV-NP mice were infected with 105 PFU LCMV-Arm (open circles), LCMV-Arm-Var (filled triangles), or PV alone (filled circles). (B) Primed autoreactive cells can become activated via molecular mimicry and accelerate disease. RIP-LCMV-NP mice were infected with either 105 PFU LCMV-Arm (open circles, filled circles) or 105 PFU PV (open triangles, filled triangles) on day 0 and, as indicated, received a secondary inoculation (2nd inf.) with PV (open triangles, filled circles) 28 days after the priming LCMV infection. As a comparison, the incidence data to RIP-LCMV-NP mice infected with LCMV alone are displayed (open circles). For both studies, blood glucose values were determined at weekly intervals. Mice with blood glucose levels above 300 mg/dl were considered diabetic. It is evident from these studies that secondary infection but not primary infection with PV can accelerate T1D development. Statistical analysis was done using the log rank test. Note that the diabetes onset curves for the groups LCMV alone versus LCMV-PV are significantly different (P = 0.0066).
CD8 T cell epitopes of LCMV-NP and PV-NP
To assess the contribution of the LCMV-NP205 CD8 T cell epitope for induction of diabetes, we utilized Pichinde virus (PV), another member of the arenavirus family, which contains the cross-reactive H-2Kb–restricted epitope PV-NP205, YTVKFPNM, that shares six of eight amino acids with the LCMV-NP205 epitope, YTVKYPNL (substitutions underlined; Table 1) (27). Although binding of the LCMV-NP205 and PV-NP205 epitopes to H-2Kb was similar (despite differences in the MHC anchoring residues at positions 209 and 212), they exhibited differential antigenic properties. PV infection elicited more CD8 T cells to the PV-NP205 than to the LCMV-NP205 epitope in earlier studies (27), indicating that true cross-reactivity of the interaction of the NP205-peptide presented by MHC class I in conjunction with TCR was operational rather than reduced binding affinity of the mimic peptide to MHC. Despite the presence of this cross-reactivity with the LCMV-NP transgene, infection of transgenic mice with PV failed to induce diabetes (Figure 1A). This lack of disease induction was likely associated with the approximately 100-fold lower avidity of PV-NP205 compared with LCMV-NP396 in cytotoxicity assays (27), resulting in a failure to induce a robust primary CD8 T cells response to the lower-avidity PV-NP205 after LCMV infection. Collectively, these data indicate that upon infection of RIP-LCMV-NP mice with LCMV-Arm, induction of diabetes is dependent on the higher-avidity H-2Db–restricted LCMV-NP396 epitope and not the lower-avidity H-2Kb–restricted LCMV-NP205 epitope. Based on this study (Figure 1A) and previous studies (35), we conclude that naturally occurring viral mimics recognized with comparatively lower avidity are unable to activate a sufficient number of naive autoreactive lymphocytes to cause clinical diabetes, even if the infecting virus is tropic to the pancreas and induces local inflammation of the target organ (36, 37).
A lower-avidity viral mimic ligand of a β-cell CD8 T cell epitope can significantly accelerate an ongoing autoimmune process and the development of clinical disease. We next asked whether the lower-avidity LCMV-NP205 epitope could influence the diabetic outcome of mice in the prediabetic stage in which autoreactive processes were already established. We addressed this question through the use of sequential heterologous infections of transgenic mice with LCMV-Arm and PV separated by a 4-week interval. As described earlier (27), immunity to PV exhibits clear cross-reactivity of the PV epitope PV-NP205 to the LCMV-NP epitope LCMV-NP205. Indeed, as shown in Figure 1B, PV infection administered 1 month after the initial autoimmunity-initiating LCMV infection considerably accelerated T1D in RIP-LCMV-NP mice. Importantly, the PV infection had to occur at a time when islet destruction was already ongoing (initiated by LCMV infection 4 weeks earlier), as the reverse scenario (when PV was given first followed by secondary PV or LCMV infection) did not accelerate T1D (Figure 1B). This observation shows that PV can enhance LCMV-induced T1D in RIP-LCMV-NP mice when given after but not before the initiation of islet destruction by LCMV-NP396–specific CTLs.
To define the precise role of cross-reactive CD8 T cells that specifically react to the NP205 epitope of LCMV and PV (LCMV/PV-NP205–specific cross-reactive CD8 T cells) in these different infection scenarios, we quantified their numbers and functional activity after primary and secondary PV and LCMV infection, respectively. Figure 2 shows the frequencies of IFN-γ–producing CD8 T cells in response to the LCMV H-2Db–restricted LCMV-NP396 and dominant LCMV-glycoprotein 33 (LCMV-GP33) peptides, the LCMV cross-reactive subdominant H-2Kb–restricted LCMV-NP205 peptide, and the PV dominant PV-NP38 peptide (internal PV control) after primary LCMV or PV infection and after secondary infection with LCMV or PV. As expected, primary and secondary PV infection expanded the dominant PV-NP38–specific population (Figure 2B). Furthermore, as described earlier (29), infection of RIP-LCMV-NP mice with LCMV alone resulted in high numbers of LCMV-GP33–specific CD8 T cells but much lower numbers of LCMV-NP396–specific CD8 T cells, as this mouse line expresses LCMV-NP in the thymus as well as in the pancreas, resulting in thymic negative selection of a significant proportion of NP-specific CD8 T cells. The slower development of disease in RIP-LCMV-NP mice than in RIP-LCMV-GP mice (38) can be attributed to this fact. Important for our investigation here is that secondary PV infection in LCMV-infected RIP-LCMV-NP mice strongly and selectively expanded the LCMV-NP205–specific CD8 T cell population but none of the other LCMV-specific populations (LCMV-GP33 and LCMV-NP396). This finding indicates that LCMV/PV-NP205–specific cross-reactive CD8 T cells are alone not sufficient to cause T1D in RIP-LCMV-NP mice but are important for the acceleration of disease observed after secondary PV infection. Most likely they must be present in islets together with LCMV-NP396–specific CD8 T cells in order to cause disease, as diabetes never developed after single PV infection (Figure 1A). The selective expansion of LCMV/PV-NP205–specific cross-reactive CD8 T cell populations is in concordance with observations of Brehm et al. (27) and strengthens the hypothesis that PV-NP205–specific CD8 T cells play a role in the acceleration of diabetes in mice with preclinical diabetes. Challenge of mice immune to LCMV (LCMV-immune mice) with a secondary LCMV infection did not result in a long-lasting expansion of either LCMV-NP396–specific or LCMV-NP205–specific cross-reactive CD8 T cell populations (Figure 2B). This stands well in agreement with our earlier findings that sequential infection with LCMV-Arm has no influence on the incidence and kinetics of diabetes in RIP-LCMV-NP mice (39).
CD8 T cell populations specific for the mimicking epitope PV-NP205 are significantly expanded after sequential infection with PV. (A and B) RIP-LCMV-NP and RIP-LCMV-NP × Kb(–) mice were infected with 105 PFU LCMV or PV. After 4 weeks, the mice received a secondary infection of either LCMV or PV. (A) Intracellular cytokine staining (ICCS) after stimulation with PV-NP205 is displayed for 1 representative mouse infected first with LCMV (LCMV alone) and then with PV (LCMV-PV) (mean frequencies are indicated in boxed areas). (B) The frequency of epitope-specific CD8 T cells in the blood was determined by ICCS for IFN-γ after stimulation with the indicated peptides (key) immediately before (upper panel) and 7 days after (lower panel) secondary infection. (C) Numbers of H-2Kb–restricted PV-NP205–specific lymphocytes after LCMV or PV infection, assessed by ICCS for IFN-γ. Splenocytes were harvested on day 35 from mice that received LCMV at day 0 (d0) and, for the PV group, PV at day 28 (d28). Means (± SEM) are displayed. (D) Lytic precursors after LCMV-NP396 versus PV-NP205 antigenic stimulation for 10 days. In addition to lytic activity, IFN-γ production was assessed in the supernatant of each well; wells with IFN-γ levels of more than 0.05 ng/ml by ELISA were counted as positive. IFN-γ production was on average 13 (± 3.5) ng/ml in LCMV-NP396–stimulated cultures and 7.1 (± 3.1) ng/ml in PV-NP205–stimulated cultures. This experiment was repeated three times and mean values (± SEM) are displayed. *P < 0.05.
We further investigated whether the expansion/activation of previously primed LCMV/PV-NP205–specific CD8 T cell populations by the mimic epitope expressed by PV enhanced the effector functions of these cells. Primary infection of C57BL/6 wild type or RIP-LCMV-NP mice with PV induced no detectable CD8 T cells response to whole LCMV-NP protein (Table 2). However, PV-challenged, LCMV-primed mice made clearly detectable recall responses to whole LCMV-NP, as evidenced by killing assays (Table 2), and to LCMV-NP205 as shown by intracellular cytokine staining for IFN-γ by flow cytometry (Figure 2C). In agreement with the results presented in Figure 2C is the substantially increased number of PV-NP205 CTL precursors in mice that received LCMV and then PV sequentially (Figure 2D). Thus, expansion of LCMV/PV-NP205 cross-reactive CD8 T cell populations with lytic activity (31) and IFN-γ production occurs after PV infection only in LCMV-immune mice.
Cytotoxic T cells specific for LCMV-NP are found in LCMV-immune but not naive mice after PV infection
Sequential infection with LCMV and PV results in accumulation of PV-NP205–specific CD8 T cells in islets. Histological examination of the pancreas at week 3 after secondary infection of LCMV-primed RIP-LCMV-NP mice with PV revealed increased lymphocyte infiltration and destruction of the islets of Langerhans (Figure 3A, lower left panel). There was profound difference in islet infiltration by CD8 T cells in these mice versus mice that had only been administered a single LCMV infection (Figure 3A, upper left panel).
Sequential infection with LCMV and PV results in accumulation of PV-NP205–specific CD8 T cells in the islets of Langerhans. (A) RIP-NP mice were infected with 105 PFU LCMV. After 4 weeks, one group of mice received a secondary infection of PV (105 PFU, i.p.). Left panels, pancreata were harvested at week 3 after secondary infection and 6-μm tissue sections were stained for cellular infiltration with a monoclonal antibody against CD8. Sections were counterstained with hematoxylin. Right panels, pancreata were harvested at day 5 after secondary infection and 6-μm tissue sections were cut and were stained for CD8 T cells with rhodamine X–conjugated anti-CD8 (red) and for PV-NP205–specific CD8 T cells with allophycocyanin-conjugated H-2Kb–PV-NP205 tetramers (green). Note that only after sequential infection with LCMV followed by PV are PV-NP205–specific CD8 T lymphocytes (yellow) found in the islets of Langerhans. Original magnification, ×20. (B) Expansion of PV-NP205–specific CD8 T cell populations in blood and pancreatic lymph nodes after secondary PV infection. Upper panels, flow cytometry of PV-NP205–specific CD8 T cells in the blood of LCMV-immune mice that did or did not receive secondary infection with PV, as detected by H-2Kb–PV-NP205 tetramers; mean frequencies are indicated in boxed areas. Lower panel, frequencies of PV-NP205–specific CD8 T cells were determined by flow cytometry using H-2Kb–PV-NP205 tetramer staining and by ICCS for IFN-γ expression after 5 hours of in vitro stimulation with PV-NP205 peptide.
A critical component of the hypothesis that LCMV/PV-NP205–specific cross-reactive CD8 T cells participate in the acceleration of diabetes is the identification of these cells at the right time and place after secondary PV infection in relation to the pancreas and, more specifically, the target of the autoimmune response (the islets of Langerhans). Thus, we generated H-2Kb–PV-NP205 tetramers in attempt to perform in situ tetramer staining for PV-NP205–specific CD8 T cells in quick-frozen pancreas sections, similar to what we previously reported for LCMV-GP33–specific CD8 T cells in the CNS (40). Histological examination using specific immunohistochemical staining for CD8 T cells and selective H-2Kb–PV-NP205 tetramer staining on tissue sections for NP205-specific CD8 T cells revealed that PV-NP205 CD8 T cells were present exclusively in islets of mice initially primed with LCMV and secondarily infected with PV. Between 1 and 3 PV-NP205–specific CD8 T cells were found in about 50% of all islet sections analyzed. In contrast, none of the islet sections examined in mice infected with LCMV alone contained any PV-NP205–specific CD8 T cells (Figure 3A, upper right panel). Quantification of our data indicated that a frequency of 2–4% of all infiltrating CD8 T cells were PV-NP205 specific in sequentially infected mice, which reflected the frequencies displayed in Figure 3B, assessed by flow cytometry in peripheral lymphoid organs, and corroborated frequencies of islet-infiltrating GP33-specific CD8 T cells observed both in the RIP-LCMV-GP fast-onset diabetes model (GP not expressed in thymus) using H-2Db–LCMV-GP33 tetramers (D.B. McGavern, unpublished results) and in LCMV-mediated leptomeningitis (40).
The expansion of LCMV/PV-NP205–specific cross-reactive CD8 T cell populations was also demonstrated by flow cytometry after staining with H-2Kb–PV-NP205 tetramers (Figure 3B) and intracellular cytokine staining for IFN-γ (Figure 3B, lower panels) in the blood as well as in the pancreatic draining lymph nodes (PDLNs) after sequential infection of transgenic mice with LCMV and PV. In particular, in the PDLNs, H-2Kb–PV-NP205 tetramer+ CD8 T cell populations expanded to a frequency of 4% after secondary PV infection, compared with only 0.4% after a single LCMV infection (Figure 3B). Thus, if PV infection occurs after LCMV infection, a selective and considerable expansion of initially subdominant, lower-avidity LCMV/PV-NP205–specific cross-reactive CD8 T cell populations is seen in the target organ and PDLNs.
Activation of LCMV/PV-NP205–specific cross-reactive CD8 T cells through molecular mimicry is absolutely essential for acceleration of diabetes and does not occur in mice genetically deficient in H-2Kb and in mice that were tolerized to PV-NP205. Further evidence that LCMV/PV-NP205–specific cross-reactive CD8 T cells were responsible for the acceleration of T1D was obtained from experiments using RIP-LCMV-NP mice bred onto a H-2Kb–deficient [Kb(–)] background (41). Because these mice still present the LCMV-NP396 epitope, which is H-2Db restricted, diabetes still occurred with the expected slower kinetics after a single LCMV infection (data not shown). However, sequential infection of these mice with LCMV followed by PV did not result in accelerated disease, which occurred as expected in RIP-LCMV-NP × H-2Kb–sufficient [Kb(+)] littermates (Figure 4A). Overall, the incidence of T1D in RIP-LCMV-NP × Kb(+) littermates mice was lower and its kinetics slower than in the original RIP-LCMV-NP line, because background genes introduced by the SV129 embryonic stem cells from knockout lines are known to protect from T1D in RIP-LCMV mice (42). In contrast to RIP-LCMV-NP × Kb(+) littermates, RIP-LCMV-NP × Kb(–) mice did not have accelerated T1D after secondary PV infection. In parallel with the lack of acceleration of diabetes in the RIP-LCMV-NP × Kb(–) mice, secondary infection with PV did not result in expansion of LCMV/PV-NP205–specific CD8 T cell populations (Figure 2B, right column) or in enhanced islet infiltration (Figure 4B).
H-2Kb–restricted, autoreactive, LCMV/PV-NP205–specific cross-reactive CD8 T cells mediate the acceleration of diabetes. (A and B) RIP-LCMV-NP or RIP-LCMV-NP × Kb(–) mice were infected with LCMV or PV. After 4 weeks, the mice received a secondary infection of PV. (A) Blood glucose of RIP-LCMV-NP, RIP-LCMV-NP × Kb(–), and RIP-LCMV-NP × Kb(+) littermates was measured in weekly intervals. The diabetes onset curves (blood glucose values > 300 mg/dl) for the groups [RIP-LCMV-NP × Kb(–) vs. RIP-LCMV-NP × Kb(+)] are significantly different (log rank test; P = 0.0167). (B) Pancreas sections from 3–4 mice per group at week 3 after secondary infection with PV were stained for cellular infiltration of CD8 T cells. Sections of 1 representative RIP-LCMV-NP × Kb(–) and RIP-LCMV-NP × Kb(+) mouse are shown. Original magnification, ×20. (C) Mice were tolerized to PV-NP205 by injection of 2 × 107 ECDI–PV-NP205–coupled autologous splenocytes (ECDI + NP205) or with 2 × 107 splenocytes treated with EDCI alone, 5 days before infection with 105 PFU LCMV. After 4 weeks, mice were infected with PV. Diabetes incidence (blood glucose values > 300 mg/dl) at week 4 after PV infection is displayed; numbers of mice analyzed per group are indicated in parentheses. (D and E) Groups of 3–4 mice were infected with LCMV. After 4 weeks, the mice received 100 μg of PV-NP205 peptide or an H-2Kb–restricted control peptide (OVA; SIINFEKL). In addition, mice received three injections of poly(I:C) (7.5 μg/g body mass) at the time of peptide injection and then at days 2 and 4 thereafter. Controls received PV-NP205 only or PV infection. (D) The frequency of blood LCMV/PV-NP205–specific cross-reactive CD8 T cells was assessed by flow cytometry using H-2Kb–PV-NP205 tetramers (day 7 after peptide injection). (E) Mean blood glucose values (± SEM) measured at week 2 after peptide and/or poly(I:C) injection is displayed.
Furthermore, RIP-LCMV-NP mice that were tolerized to the PV-NP205 epitope showed no acceleration of T1D after secondary infection with PV (Figure 4C). Tolerization to PV-NP205 was achieved by i.v. injection of PV-NP205–coated splenocytes that were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI) (43, 44). Mice that were treated with 2 × 107 syngeneic EDCI–PV-NP205 cross-linked splenocytes 5 days before primary LCMV infection did not display accelerated T1D after subsequent infection with PV 1 month after infection with LCMV (Figure 4C). In contrast, mice that received splenocytes that were cross-linked with EDCI in absence of PV-NP205 displayed accelerated T1D after secondary infection as usual (Figure 4C). These experiments are consistent with the necessity of H-2Kb–restricted NP205-specific T cells in the acceleration of T1D.
LCMV/PV-NP205–specific cross-reactive CD8 T cell populations expand and accelerate disease in LCMV-immune mice challenged with PV-NP205 peptide and polyinosinic-polycytidylic acid. Final evidence that LCMV/PV-NP205–specific cross-reactive CD8 T cells are responsible for the acceleration of T1D was obtained from experiments using RIP-LCMV-NP mice that were administered PV-NP205 peptide together with synthetic polyinosinic-polycytidylic acid [poly(I:C)], a “mimic” of double-stranded viral RNA (Figure 4, D and E). LCMV/PV-NP205–specific CD8 T cell populations were significantly expanded in LCMV-immune RIP-LCMV-NP mice that received 100 μg PV-NP205 peptide (i.p.) together with a single injection of 7.5 μg poly(I:C) per gram body mass (i.p.) at week 4 after LCMV infection (Figure 4D). The frequency of LCMV/PV-NP205–specific CD8 T cells was much higher than after administration of the irrelevant H-2Kb–binding OVA peptide SIINFEKL followed by poly(I:C) or PV-NP205 alone and even exceeded the frequency observed when LCMV-immune mice received a secondary infection with PV (Figure 4D). As expected, T1D was accelerated in mice treated with PV-NP205 plus poly(I:C) in a way similar to that in mice that received a secondary PV infection (Figure 4E). Mice that were administered OVA peptide (SIINFEKL) and poly(I:C) had hyperglycemia (mean blood glucose value of 225 mg/dl) but no diabetes (Figure 4E). Furthermore, treatment with PV-NP205 alone did not accelerate diabetes (Figure 4E). These data demonstrate that in the proper inflammatory environment, the NP205 epitope that confers molecular mimicry between LCMV and PV is sufficient to accelerate autoimmune diabetes and requires no further assistance by other LCMV and/or PV epitopes.