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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Hepatology. 2010 Sep;52(3):1068–1077. doi: 10.1002/hep.23745

Direct, help-independent priming of CD8+ T cells by AAV-transduced hepatocytes

Sherry A Wuensch 1, Jessica Spahn 1,*, Ian N Crispe 1,*
PMCID: PMC2932746  NIHMSID: NIHMS207626  PMID: 20607836

Summary

Both HBV and HCV frequently establish chronic infection, raising the question whether T cells are poorly primed in the liver. To determine the role of different cell types in the activation of CD8+ T cells against hepatocellular antigens, we used an Adeno-Associated Virus to deliver ovalbumin to hepatocytes. In contrast to CD8+ T cells, CD4+ T cells were not activated. The CD8+ T cells were activated even in the absence of endogenous CD4+ T cells; however in the liver these cells were high in PD-1 and low in CD127. Chimera experiments revealed that these CD8+ T cells were activated on a solid tissue cell.

Conclusion

Priming of CD8+ T cells directly on non-hematopoietic cells, in the absence of CD4+ T cell help, results in suboptimal T cell activation. This could explain the impaired function of CD8+ T cells seen in chronic liver infection.

Keywords: AAV, CTL, direct priming, help-independence

Most people infected with HCV progress to chronic infection. This is partly due to an inadequate CD8+ T cell response that lacks breadth, intensity and CD4+ T cell help (1-3). The CD8+ T cells generated in response to HCV often display an “exhausted” phenotype expressing high levels of PD-1 and low levels of CD127(4). Inadequate immunity is also seen in HBV, and against the liver stage of the malaria parasite. The common factor in these diseases is infection of hepatocytes, bringing up the idea that the liver environment is contributing to the development of a defective immune response. This may be due to the liver's constant exposure to endotoxin, raising the threshold for immune activation (5, 6).

Multiple liver cell types may present antigens. In the mouse, the liver contains plasmacytoid and myeloid Dendritic Cells (DC), as well as more unusual DC subsets (7) and Kupffer cells. In addition the liver sinusoidal endothelial cells (LSEC) and the hepatic stellate cells (HSC), both have credentials as antigen-presenting cells (APC) (8-10). Hepatocytes also present antigens (11-13). This profusion of potential APC raises the issue of which are actually important in priming immune responses against hepatocellular antigens. To clarify these issues, we used an Adeno-Associated Virus (AAV2)-based gene therapy vector (AAV2-ova) delivered by direct injection into the liver. This vector was expressed exclusively in the liver, based on RT-PCR analysis of multiple tissues, and exclusively in hepatocytes, based on immunohistochemistry (14).

Here we examine the priming of CD8+ T cells against this AAV vector. Previous work suggested that AAV vectors did not generate cross-primed immunity that could engage transduced hepatocytes (15), and that AAV could induce tolerance in CD4+ T cells (16) However, we found that CD8+ T cells were effectively primed against the same antigen. Activation of these OT-1 cells was CD4+ T cell help-independent, and was independent of bone marrow-derived APC. The implication is that hepatocellular antigens are excluded from bone marrow-derived “professional” APC, including DC and macrophages, but can nevertheless engage CD8+ T cells.

Results

Activation of CD8+ but not CD4+ T cells

To test the capacity of the AAV2-ova vector to activate CD4+ T cells in vivo, mice received an intra-hepatic injection of either AAV2-ova, or a control vector AAV2-gfp. After 3 weeks, mice were given CFSE-labeled OT-II transgenic CD4+ T cells, specific for the ISQAVHAAHAEINEAG peptide (ova 323-339). These T cells did not respond, similar to OT-II T cells infused into mice that had been given the antigen-negative AAV2-gfp control vector (Figure 1A, two upper left panels). However, the OT-II cells were competent to proliferate in vivo, revealed by their response to peptide-pulsed splenocytes (marked “pep” in Figure 1); after this treatment, we observed divided OT-II T cells in the liver (17), spleen (SPL) and PLN (6). To detect T cell activation we also measured the expression of the lymph node homing receptor, CD62L (Figure 1B). Non-dividing OT-II T cells maintained high expression of CD62L, while responding T cells expressed less. These results were confirmed using D0.11.10 transgenic CD4+ T cells, which recognize the ISQAVHAAHAEINEAG peptide in the context of the I-Ad molecule in BALB/c mice (Figure 1C,D).

Figure 1.

Figure 1

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The AAV2-ova vector induced proliferation in OT-1 CD8+ T cells, but not OT-II or DO.11.10 CD4+ T cells. Figure 1A and 1C show CFSE staining of OT-II and OT-1 T cells and for DO.11.10 T cells respectively in mice transduced with AAV2-ova. There was no cell division of OT-II or DO.11.10 cells in response to AAV2-ova (marked OVA), nor in mice given the control vector (marked GFP), but these cells were competent to respond in vivo to peptide-pulsed spleen cells (marked pep). In contrast, OT-1 cells proliferated efficiently to AAV2-ova (marked OVA). Figure 1B and 1D show that non-dividing cells remained mostly CD62L-high, while dividing cells became CD62L-low. Histograms represent an individual animal from three independent experiments for a total 5-7 mice per group.

To confirm the expression of the antigen, mice that were given the AAV2-ova vector were infused with OT-1 T cells, a CD8+ T cell population specific for the SIINFEKL peptide (ova 257-264). These T cells divided and down-regulated CD62L (Figure 1B, right panels), verifying that the antigen was expressed. We conclude that AAV-2-ova vector stimulated CD8+ but not CD4+ T cell responses.

The CD8+ T cell response is CD4+ independent

Two different TCR transgenic CD4+ T cells failed to respond to AAV-OVA (Figure 1). To test whether endogenous CD4+ T cells were helping the CD8+ cells, MHC class II deficient mice, lacking CD4+ T cells, were given AAV2-ova vector and then OT-1 T cells. Figure 2A shows the response of OT-1 T cells, measured using CFSE, at day 3 (D3), day 5 (D5) and week 8 (W8) after adoptive transfer (shaded profiles). In the liver (17), OT-1 cells divided as early as day 3, and almost all of the cells were CFSE-low by day 5; these cells were also present at week 8. In control mice given the AAV2-gfp vector (non-shaded profiles), there was very little OT-1 T cell division at days 3 and 5, although the cells were subject to some loss of CFSE staining by week 8. Strikingly, there was no difference in the division of OT-1 T cells between normal B6 mice and MHC class II deficient mice. In the spleen (SPL) and the PLN there was essentially no cell division in any of the mice at day 3, but divided cells appeared in these tissues on day 5, as previously reported (14), and again there was no difference between normal and MHC class II deficient mice.

Figure 2.

Figure 2

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MHC class II-restricted CD4+ T cell help is irrelevant to the OT-1 response to AAV in vivo. A. The dilution of CFSE in OT-1 T cells in mice given AAV2-ova (shaded profiles in the liver (LIV), spleen (SPL) and PLN was identical in normal B6 and MHC class II deficient (MHC II-/-) mice. This was true at day 3 (D3), day 5 (D5) and week 8 (W8) after adoptive transfer of the T cells. B. The antigen-driven increase in OT-1 T cells numbers is shown by the difference between mice given AAV2-ova (solid bars) and those given AAV2-gfp (cross-hatched). This measure of responses peaked in the liver (left panel), spleen (middle panel) on day 5. Neither in these organs, nor in PLN (right panel), was there a significant difference between normal B6 and MHC class II deficient mice. In panels B, differences between groups were compared using Student's t test. Differences marked with symbols are significant (p<0.05). Plots represent means ± SEM; data represent two independent experiments with 6-8 mice per group.

Figure 2B shows the outcome of these experiments in terms of the numbers of OT-1 T cells in the liver (left panel of Figure 2B), spleen (center) and PLN (18) of normal B6 versus MHC class II deficient mice at day 3, day 5 and week 8 after adoptive transfer. In liver, there was statistically significant expansion of OT-1 T cells at all three time points, but no significant difference between B6 and MHC class II deficient mice. In spleen, we observed significant clonal expansion only on day 5, but not later. Again, there was no difference between normal B6 and MHC class II deficient mice. In PLN, we observed no clear effects on overall OT-1 T cell numbers on days 3 and 5, although there was a significant increase in the numbers of OT-1 T cells in MHC class II deficient mice on day 5, followed by a significant loss of OT-1 T cells in the AAV2-ova transduced mice at week 8. The overall conclusion is that MHC class II-restricted helper T cells did not influence the response of OT-1 CD8+ T cells to the AAV2-ova vector.

To determine whether the absence of MHC class II restricted CD4+ T cell help was modifying the immune response of CD8+ T cells in more subtle ways, we evaluated the expression of the lymph node homing receptors CD62L, the global activation marker CD44, the IL-7Rα (CD127), and the co-inhibitory molecule PD-1. In every case, OT-1 T cells “parked” in mice transduced with the AAV2-gfp control vector served as the control. The results are represented as the MFI in groups of at least 5 mice, and are shown in Figure 3A. In the liver (17), the presence of AAV-OVA caused the down-regulation of CD62L and up-regulation of CD44 at all time-points. The CD127 marker was down-regulated at day 3 and 5, but was restored by 8 weeks. The PD-1 marker was powerfully induced in the AAV-OVA mice from day 3 to week 8. None of these effects was modified in the absence of MHC class II.

Figure 3.

Figure 3

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The expression of key activation markers on OT-1 T cells, responding in the liver (LIV), spleen (SPL) and PLN of normal B6, or MHC class II deficient mice. Solid bars are data obtained from mice given the AAV2-ova vector; hatched bars are data from control mice given the AAV2-gfp vector. Two independent experiments with 6-8 mice per group at each time point. Data in A are the mean fluorescence index (MFI) +/- SEM of greater than five mice per group. Figures B (Day 5) and C (Week 8) represent the percentage of OT-1 cells expressing IFNμ in the presence or absence of SIINFEKL peptide. Black bars are data obtained from B6 mice given AAV2-ova vector and white bars from B6 mice given AAV2-gfp vector. Grey bars are data from MHC II -/- mice given AAV2-ova vector and hatched bars from MHC II -/- mice given AAV2-gfp vector.

To determine if this PD-1 high phenotype correlated with impaired function, we tested the ability of these cells to produce IFN-γ. Figure 3B shows OT-1 cells in WT vs. MHC II deficient mice on Day 5 (B) and Week 8 (C). On Day 5, OT-1 cells in both WT and MHC II deficient hosts were capable of making IFN-γ in the presence of antigen. However, by Week 8, these cells made less IFN-γ than those without antigen. Thus the high expression of PD-1 correlated with loss of function.

In the spleen (SPL), the down-regulation of CD62L was clear-cut only at week 8, while increased CD44 was seen at day 5 and week 8. These data are consistent with our previous demonstration that the anti-AAV immune response starts in the liver, rather than in lymph nodes (14). The down-regulation of CD127 expression on OT-1 T cells in the spleen was not seen on day 3, but was present at day 5 and week 8. PD-1 was up-regulated in OT-1 T cells on day 5 and week 8, but the level of PD-1 expression was at least ten-fold less than on the OT-1 T cells in the liver; the PD-1 MFI data are shown on the same scale to emphasize this difference. None of these effects were different between normal B6 mice and MHC class II deficient mice. Effects on OT-1 T cells in the PLN were smaller, but there was up-regulation of CD44 and PD-1 expression on day 5 and at week 8. Again, there was no effect of MHC class II-restricted help on any of these phenotypic changes. These effects on CD8+ T cell surface phenotype in B6 versus MHC class II deficient mice agree with Figure 2, and support the conclusion that CD4+ T helper cells are not involved in the CD8+ T cell response to AAV2-ova transduced liver cells.

These effects of the OT-1 T cell phenotype could be summarized as follows: while other markers fluctuated in a similar way in both help-intact and help-deficient mice in all of the organs sampled, the expression of PD-1 was dramatically different. Its expression was very high on OT-1 T cells in the liver; however this expression was not influenced by the presence or absence of CD4+ T cell help.

High PD-1 due to priming in the liver

Figure 3 shows that high PD-1 expression is unique to OT-1 cells in the liver. This could be due to the liver environment causing all liver CD8+ T cells to become PD-1 high, or alternatively by intra-hepatic priming. We investigated this by comparing host CD8+ T cells in the liver to OT-1 cells. As shown in Figure 4, the majority of host CD8+ T cells in the liver are PD-1 negative however, there is a population of PD-1 positive cells. These PD-1 positive cells are also CD62L low indicating that they are recently activated CD8+ T cells. We have previously shown that activated CD8+ T cells are trapped in the liver in a TLR4 dependent manner(19).However, we cannot assume that the host liver PD-1 high cells were trapped thus.

Figure 4.

Figure 4

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The expression of CD62L and PD-1 on OT-1 T cells (filled grey histogram) or host CD8+ T cells (black line) at Day 3, Day 5, or Week 8 in Liver or lymph nodes.

To test the hypothesis that activation in the liver upregulates PD-1 on CD8+ T cells, we compared OT-1 cells activated by AAV-OVA in the liver and OT-1 cells activated in primary lymphoid tissues by SIINFEKL-pulsed DCs. Figure 5 shows that PD-1 expression is an indication of activation, as this molecule is expressed on OT-1 cells both in the liver of AAV-OVA transduced mice, and in liver and lymphoid organs of DC-SIINFEKL stimulated mice. Further, OT-1 cells activated by DC SIINFEKL in all organs, and OT-1 cells activated by AAV-OVA in the liver, expressed a significantly higher level of PD-1 than OT-1 cells taken from untreated mice. However, expression of PD-1 was significantly higher on OT-1 cells in the liver of mice stimulated with AAV-OVA, compared both with OT-1 cells from unstimulated mice, and with those in any organ of mice stimulated with DC-SIINFEKL. This shows that while PD-1 expression follows activation, the generation of PD-1hi cells is unique to cells primed in the liver. We can conclude that high PD-1 expression is not simply due to activated CD8+ T cells migrating to liver.

Figure 5.

Figure 5

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PD-1 expression in OT-1 cells from either untreated, AAVOVA- or SIINFEKL pulsed DC- treated mice. Histograms of PD-1 expression on OT-1 cells (filled grey) or host CD8+ T cells (black line) in liver, spleen or lymph node. Mean Fluorescence Intensity of PD-1 on untreated, AAVOVA stimulated and DC SIINFEKL stimulated OT-1 and CD8+ T cells.

Antigen presentation by non-bone marrow-derived cells

Cross-presentation depends on the transfer of antigen from an antigen-expressing cell to a distinct APC, and can lead either to cross-priming or to cross-tolerance (20-22). The APC are generally MHC class I+ II+ bone marrow-derived cells, such as macrophages or DC. To test the participation of such cells in the OT-1 T cell response to AAV2-ova, we used bm8 mice. These mice harbor several mutations in the Kb MHC class I molecule, which prevent the presentation of the SIINFEKL peptide (23). We created radiation bone marrow chimeras in which bm8 bone marrow was used to reconstitute lethally irradiated B6 mice, or vice versa. Since a subset of bone marrow-derived Kupffer cells is resistant to depletion by radiation alone, mice were additionally treated with clodronate liposomes after the bone marrow transplant. This treatment effectively depletes both subsets of Kupffer cells (24).

The response of OT-1 T cells to AAV2-ova in the liver in such chimeras is shown in Figure 6. The negative controls were B6>B6 chimeras transduced with antigen-negative AAV2-gfp vector, and the positive control was B6>B6 mice given AAV2-ova. All mice received an IV adoptive transfer of CFSE-labeled OT-1 T cells. Flow cytometric measures of the T cell response are shown in Figure 6A. In the negative controls, there was no dilution of CFSE, no down-regulation of CD62L, and no up-regulation of CD44. In the positive controls, the T cells proliferated, CFSE staining was lost, and CD62L was downregulated. The effectiveness of direct presentation was revealed by the dilution of CFSE and down-regulation of CD62L expression in bm8>B6 chimeras; conversely the lack of cross-presentation was shown by the lack of CFSE dilution, and lack of CD62L down-regulation in B6>bm8 chimeras. The cell surface expression of CD44 also indicated cell activation in bm8>B6 chimeras, but not in B6>bm8 chimeras. These data also are consistent with a complete lack of cross-presentation by bone marrow-derived cells.

Figure 6.

Figure 6

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A. Radiation bone marrow chimeras were prepared using either B6 or bm8 bone marrow to reconstitute either B6 or bm8 host mice. Control chimeras used B6 bone marrow in B6 hosts (marked B6>B6). Such mice were given AAV2-gfp control vector and CFSE-labeled OT-1 T cells; these cells did not divide, nor modify their expression of CD62L or CD44. In contrast, B6>B6 chimeras given AAV2-ova supported a strong OT-1 T cell response. Thus, these cells diluted CFSE expression, down-regulated CD62L, and up-regulated CD44. The CFSE, CD62L and CD44 data all show that OT-1 T cells were able to respond in bm8>B6 chimeras, but not B6>bm8 chimeras, excluding cross-presentation. B. Estimation of the OT-1 cell numbers in the liver, spleen and PLN of chimeric mice. The experimental groups and labeling conventions are as in Figure 2A. The data show that OT-1 T cells could undergo clonal expansion in B6>B6, and bm8>B6 but not B6>bm8 chimeras. C. The percentage of OT-1 T cells, like the estimate of absolute cell number, strongly supports the concept that bone marrow-derived APC are not involved in OT-1 T cell activation. In panels B and C, differences between groups were compared using Student's t test. Differences marked with symbols are significant (p<0.05). Results represent two independent experiments for a total of 5-7 mice per group.

Figure 6B shows the estimated numbers of OT-1 T cells in the liver (17), spleen (SPL) and PLN in each experimental group. In the liver and in the spleen, there was a significant increase in the number of OT-1 T cells in B6>B6 chimeras given AAV2-ova, compared to similar chimeras given AAV2-gfp. Critically, there was no increase in cell numbers in B6>bm8 chimeras, but there was in bm8>B6 chimeras. Statistical tests confirm the significance of the difference between B6>B6 positive controls and the B6>bm8 chimeras. While the differences in the PLN did not give a significant result, the data from livers and spleens confirm that direct priming, and not cross-priming, leads to the clonal expansion of OT-1 T cells in response to AAV2-ova. In Figure 6C, we show the percentage of lymphocytes that were OT-1 T cells in each tissue. These results support the same conclusion, but also reveal that OT-1 T cells were most frequent in the liver, as we have documented in previous studies (14). Taken together, this data-set argues that AAV2-ova antigens are presented directly to OT-1 CD8+ T cells, without the involvement of bone marrow-derived APC. The liver contains multiple populations of potential APC, including HSC and LSEC. Neither of these cell types is significantly replaced in bone marrow chimeras, so their contribution to CD8+ T cell activation could not be excluded by this approach. However, we previously published that antigen was expressed exclusively in hepatocytes and not in other cell types (14), suggesting that direct priming was probably on hepatocytes.

Discussion

In chronic HCV and HBV infections, virus specific CD8+ T cells are impaired and ineffective at eliminating the virus. This correlated with an “exhausted” phenotype of high PD-1 and low CD127 (4, 25-28). Impaired virus specific CD8+ T cells may explain why these infections become chronic. Here we documented liver resident CD8+ T cells with this “exhausted” phenotype in the response to AAV-transduced hepatocytes in mice.

The initial CD8+ T cell response to AAV-encoded hepatocellular antigen is at first sight surprising, because the immune response to liver antigens is associated with many tolerance phenomena. Most dramatically, orthotopic transplantation of the liver between inbred strains of mice frequently results in tolerance without the need for immunosuppression (29). This effect could result from early T cell apoptosis (30, 31), from the immunosuppressive effects of antigen presentation by LSEC (10), the milieu created by the synthesis of both cytokines and PGE2 prostaglandins by Kupffer cells (32), or the action of regulatory T cells (29).

The data reported here support a different model in which the CD8+ T cells respond to an AAV2 vector-encoded transgene expressed in hepatocytes, and are primed without CD4+ T cell help by direct presentation of antigen. The absence of cross-priming is consistent with several other features of the AAV vectors. First, cell death and the subsequent phagocytosis of apoptotic bodies is a route by which cellular antigen enters the cross-presentation pathway (33), but these vectors are non-cytopathic so this pathway of antigen presentation would not be favored. Second, while transduction of liver cells with Adenovirus resulted in robust synthesis of type 1 IFN, this type of response was not elicited by AAV vectors (34). The type 1 IFNs play diverse roles in induction of specific immunity, and one of these roles is the promotion of cross-presentation (35).

The evidence in favor of a direct-primed response raises the question of how the T cells could actually be engaged. While hepatocytes are separated from circulating T cells by an endothelial barrier, this endothelium is fenestrated and direct contact between T cells and underlying hepatocytes can occur (12). In our model, the hepatocytes are the only cells transduced with the AAV vector. Thus, both the ultrastructural evidence and the expression pattern of AAV for the model that hepatocytes are directly priming the CD8+ T cells.

Our data also support the interpretation that AAV2-ova did not induce a CD4+ T cell response, and that furthermore the CD8+ T cell response was not modified by the absence of CD4+ T cell help. In similar studies using D0.11.10 T cells, the lack of a readily detectable immune response was attributed either to the induction of T cell anergy (16), or to the differentiation of the T cells into classic Tr3 cells expressing CD25 and the transcription factor, FoxP3 (36). However even if such cells were generated, a local immune response occurred in their presence.

The CD8+ T cell response to AAV was associated with elevated expression of the co-inhibitory molecule, PD-1. The PD-1 molecule is associated with a senescent phenotype, characteristic of inactive T cells found in the liver during chronic infections, including LMCV and HCV (37, 38). The regulation of PD-1 expression on CD8+ T cells is not fully understood, but our data contribute to this question by showing that the antigen-specific induction of PD-1 on CD8+ T cells in the liver was not different in mice lacking MHC class II; therefore we do not see an essential role either for CD4+ T-helper cells, or for CD4+ T-reg cells, in the induction of PD-1. Synthesizing the results from studies of LCMV and HCV with our own, we would suggest the hypothesis that it is continued contact with antigen in the liver environment and importantly, direct priming on hepatocytes that renders CD8+ T cells both PD-1-high, and functionally incompetent. Gaining a better understanding of how CD8+ T cells are activated in the liver, and in particular in chronic viral hepatitis, will give us more insight into how to better generate effective therapies.

Materials and Methods

Mice

Male C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Gene-targeted male B6.129-H2-Ab1tm1GruN12 (MHC class II-/-) mice were purchased from Taconic Farms (Germantown, NY). The OT-1 mice were on either a CD45.1/CD90.2 or CD45.2/CD90.1 background. The OT-II transgenic mice were on the CD45.2 background. B6.C-H-2bm8 (bm8) mice were a gift from L. R. Pease (Mayo Clinic, Rochester, MN). Mice were SPF, were used between 8 and 12 weeks of age, and experiments were approved by our IACUC.

Bone marrow chimeras were made in the strain combinations: B6→B6, B6→bm8, bm8→ B6. Donor and host differed in CD45 allotype. Eight-week-old recipients were irradiated (10Gy) using an RS2000 x-ray irradiator (Rad Source Technologies, Coral Springs, FL). T-depleted bone marrow (12 × 106 cells) was injected i.v. within 6 h of irradiation. Thirty days later, chimeras were i.v. injected with 200μl of clodronate liposomes from Encapsula NanoSciences (Nashville, TN) to deplete radio-resistant Kupffer cells (24). Vectors were injected 2 wks later, after the repopulation of the liver with Kupffer cells derived exclusively from the donor bone marrow.

AAV vectors

Serotype 2 AAV vectors encoding either ova or eGFP under the control of the CMV promoter were obtained from the Columbus Children's Research Institute Viral Vector Core Facility (Columbus, OH) (39).

Intrahepatic vector delivery

Mice aged 8-12 weeks were anesthetized using Avertin, and the central lobe of the liver was exposed through a 2-cm ventral midline incision. Using a 29G insulin syringe, 60μl (7.2 × 1010 DNAse Resistant Particles diluted in PBS) was slowly injected directly into the liver. The peritoneal cavity was sutured with 4-0 Vicryl (Ethicon) and the skin closed with wound clips.

Adoptive transfers

Spleen and PLN cells from either OT-1, OT-II or D0.11.10 transgenic mice were RBC-depleted using Lympholyte-M (Cedarlane, Ontario, Canada). Miltenyi MACS kits were used to isolate CD8+ or CD4+ T cells. For in vivo proliferation, cells in PBS were stained with 4 μM CFSE for 10 min at 37°C and washed in PBS. Three to four weeks after vector injection, either 1 × 106 (Fig 1C, Fig 5) or 5 × 106 (other Figures) of T cells (>90% pure) were injected i.v. in the tail vein.

Isolation of DCs

DCs were enriched from the spleen using the technique of Livingstone (17), with modifications (19). Spleens from C57BL6 mice were digested in HBSS containing 2.4mg/ml Collagenase IV (Sigma Aldrich), and 1mg/ml DNase (Sigma Aldrich) at 37° for 30 minutes. Cells were resuspended in 60% Percoll and overlayed with 2ml HBSS +5% FBS. This gradient was spun at 650 × g for 20 minutes. DCs from the interface were allowed to attach for 90 minutes and non-adherent cells washed away. Adherent cells were incubated overnight with 1ng/ml GM-CSF and 1μM SIINFEKL peptide, and harvested the next day by gentle washing. 1×106 DCs were given i.v. with the OT-1 cells.

Leukocyte isolation

Intrahepatic lymphocytes were isolated as described previously (14).

Flow cytometric analysis

Cells in staining buffer (1% FBS in PBS) were first incubated with Fc-block (Pharmingen) for 5 min. Antibodies used were anti-CD62L (PE), anti-CD44 (PE and PeCy5), anti-CD8 (PCP, APC and PeCy7), and anti-CD4 (PCP and Pacific Blue) all from Pharmingen. Pacific Blue-conjugated anti-CD127, anti-PD-1 (PE), anti-CD45.1 (APC and PeCy7), anti-CD45.2 (Alexa Fluor 700), anti-CD62L (APC-Alexa 750) were from eBioscience. Data were acquired using FACSCalibur or LSRII flow cytometers (26), and analyzed using FlowJo (TreeStar) on a iMac. Live lymphocytes were gated based on FSC/SSC.

Statistical analysis

Data in the figures represent the mean ± standard error of the mean (SEM). Student's t-test was used to analyze the results where applicable, and probability values of p < 0.05 were considered significant.

Acknowledgments

This work was supported by the NIH (DK075274). We thank Dr K. Reed Clarke for the preparation of the AAV vectors.

Abbreviations

PLN

peripheral lymph nodes

Footnotes

Conflict of Interest

The authors have no conflicts of interests to declare.

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