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Keywords:

  • Antigen amount;
  • CD8+ T cell;
  • Chronic viral infection;
  • Dysfunction;
  • Immunopathology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Chronic viral infections lead to CD8+ T-cell exhaustion, characterized by impaired cytokine secretion and loss of proliferative capacity. While viral load and T-cell dysfunction correlate, it is currently unclear whether the quality of a cell type presenting antigen determines the degree of T-cell exhaustion or if the overall amount of antigen recognized by T cells promotes exhaustion. We found that chronic lymphocytic chorio-meningitis virus infection led to decreased CD8+ T-cell exhaustion in DC-MHC class I (MHCI) mice, in which CD8+ T cells can only recognize antigen on DCs. However, this increase in CD8+ T-cell function came at the expense of fatal immunopathology. Additional antigen recognition on nonhematopoietic cells in DC-MHCI mice promoted T-cell exhaustion and avoidance of immunopathology. Likewise, increased numbers of antigen-expressing hematopoietic cells, as well as a selective elevation of the number of DCs as the only cell type presenting antigen in DC-MHCI mice, resulted in compromised T-cell function. These results favor a scenario in which the overall amount of antigen exposure, rather than the type of cell engaging with virus-specific CD8+ T cells, is responsible for their functional exhaustion. Furthermore, exhaustion of virus-specific CD8+ T cells leads to avoidance of life-threatening immunopathology.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

During highly replicative chronic viral infections such as HIV-1, hepatitis B or hepatitis C virus infection in humans, or lymphocytic choriomeningitis virus (LCMV) in mice, virus-specific CD8+ T cells initially expand and acquire effector functions early after infection but then gradually lose these functions [1-8] in a hier-archical manner: first the ability to produce IL-2 and to proliferate, then TNF-α secretion and last IFN-γ production become impaired [9-12]. This T-cell dysfunction, also termed CD8+ T-cell exhaustion, is well studied after infection of mice with a high dose of LCMV clone 13 or LCMV-Docile [13]. Infection of mice with a low dose of the same virus strains results in an acute resolved infection [13].

Besides a role for immunoregulatory cytokines such as IL-10 [14, 15], IL-6 [16], or TGF-β [17], a role for inhibitory receptor expression (reviewed in [18]) and perhaps regulatory T cells [19] as well as the availability of T-cell help [10, 20] and IL-21 [21-23], the overall duration and amount of antigen exposure seems to be a critical parameter driving CD8+ T-cell exhaustion. Recent work has highlighted a negative correlation between viral load and CTL function in chronically infected humans and mice [24-27]. Even though these studies indicate that high overall antigen levels correlate with T-cell exhaustion, it was never directly shown whether alterations of antigen levels can substantially influence T-cell function independently of the cell type presenting the antigen in vivo. Furthermore, as T-cell exhaustion and viral persistence mutually depend on each other and since alterations in one of the parameters affects the other one, it is often difficult to assign causal relationships, particularly in humans.

Here, we made use of Tg(CD11c-β2m) × Tg(K14-β2m) × β2m−/− (DC-MHCI, where DC is defined as dendritic cell; MHCI is defined as MHC class I) mice to assess the role of antigen presenting cells versus antigen load in induction of CD8+ T-cell exhaustion upon chronic LCMV infection. DC-MHCI mice are β2m−/− mice that transgenically express β2m in DCs, keratinocytes, and thymic cortical epithelial cells, ensuring positive selection of CD8+ T cells in the thymus [28]. Thus, DC-MHCI mice can mount normal endogenous CD8+ T-cell responses, but peripheral CD8+ T cells can only recognize their cognate antigens selectively on DCs and keratinocytes, the latter not being known as a target for LCMV in vivo. As priming of CD8+ T cells is strictly dependent on the presence of DCs during LCMV infection [29], MHCI-DC mice are a suitable system to study the impact of selective antigen presentation by only one cell type on the size and function of the LCMV-specific CD8+ T-cell response. A second advantage of DC-MHCI mice is that the extent of the innate response to LCMV is expected to be similar in wild-type and DC-MHCI mice as virus titers are similar during chronic LCMV infection.

Selective antigen presentation on DCs during chronic LCMV infection resulted in LCMV-specific CD8+ T-cell responses of higher magnitude and polyfunctionality. However, this superior CD8+ T-cell function came at the expense of fatal CD8+ T-cell dependent immunopathology. Increasing the antigen exposure of CD8+ T cells by expanding antigen recognition to all cells of nonhematopoietic origin promoted exhaustion and at the same time inhibited immunopathology. Moreover, elevation of antigen-expressing hematopoietic cells or only increasing DC numbers in DC-MHCI mice also resulted in decreased LCMV-specific CD8+ T-cell immunity. These findings indicate a direct role of antigen load rather than the type of antigen presenting cells in driving CD8+ T-cell exhaustion which in itself represents an important means of the host to avoid life-threatening immunopathology.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Antiviral CD8+ T cells cannot control LCMV after acute infection of DC-MHCI mice

Based on the requirement of DCs for priming of CD8+ T-cell responses during LCMV infection [29], we aimed to investigate how a monoclonal antiviral CD8+ T-cell population is affected when antigen recognition is restricted to one cell type, namely DCs, during acute LCMV infection. Therefore, naive Ly5.1+ TCR transgenic P14 CD8+ T cells specific for the LCMV-derived epitope gp33 were adoptively transferred to DC-MHCI and C57BL/6 mice. Recipients were infected with 200 pfu of LCMV-Docile 1 day later. Acute infection of DC-MHCI mice led to lower numbers of TCR transgenic CD8+ T cells (Fig. 1A and C) and to lower numbers of degranulating (Fig. 1B and D), IFN-γ (Fig. 1B and E), TNF-α (Fig. 1F) or IL-2 (Fig. 1G) producing antiviral CD8+ T cells than in lungs of infection-matched C57BL/6 mice on day 8.5 post infection. Similar trends were observed when splenocytes were analyzed (Fig. 1H–M). Due to the absence of MHCI expression on the majority of infected cells, and due to LCMV control via direct CD8+ T-cell mediated cytotoxicity [30], LCMV titers were significantly higher in DC-MHCI mice compared with those of C57BL/6 mice in all organs analyzed at day 8.5 post infection (Fig. 1N–P).

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Figure 1. CD8+ T cells cannot clear LCMV and become exhausted after acute infection of DC-MHCI mice. One day after transfer of 104 Ly5.1+ T-cell receptor transgenic CD8+ T cells specific for the LCMV-derived epitope gp33 (P14), DC-MHCI, and C57BL/6 mice were infected with 200 pfu LCMV-Docile i.v.. (A, C, H) On day 8.5 post infection, the number of the transferred Ly5.1+ CD8+ T cells was determined in the lung and spleen by flow cytometry. Data are shown as (A, B) representative flow cytometry plots and (C–M) summary of all mice, each symbol representing an individual mouse and lines representing the mean. (B, C–M) The number of (B, D, I) degranulating, (B, E, K) IFN-γ-secreting, (F, L) TNF-α-producing, and (G, M) IL-2-secreting Ly5.1+ CD8+ T cells was measured after restimulation with the peptide epitope gp33. The virus titers in the (N) spleen, (O) liver, and (P) lung were determined on day 8.5 post infection. Each symbol represents an individual mouse, the lines indicate the mean and the dashed line the detection limit. All data shown are from one representative experiment out of two. Statistical significance was determined using unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Thus, in DC-MHCI mice, the inability of the CD8+ T cells to clear LCMV in MHCI cells leads to an increased presence of antigen for an extended period of time. The increased amount of antigen sensed by LCMV-specific CD8+ T cells selectively on DCs is associated with reduced numbers of LCMV-specific CD8+ T cells in DC-MHCI mice compared with C57BL/6 recipients which resolve the infection.

Antiviral CD8+ T cells are less exhausted during chronic LCMV infection in DC-MHCI mice

Next, we analyzed the consequences of DC-restricted antigen presentation on LCMV-specific CD8+ T-cell responses in the setting of a chronic LCMV infection. Notably, when DCs were the only cell type capable of presenting antigen, the transferred P14 TCR transgenic CD8+ T cells proliferated to higher numbers in the spleens compared with wild-type recipients in which P14 cells could recognize antigen on all infected nucleated cells (Fig. 2A and C), despite similar virus titers in various organs (Fig. 2G–J). Similarly, DC-MHCI mice exhibited higher numbers of degranulating (Fig. 2B and D) and IFN-γ (Fig. 2B and E) or TNF-α secreting (Fig. 2F) P14 CD8+ T cells than C57BL/6 mice. Moreover, P14 CD8+ T cells also exhibited reduced effector functions on a per-cell level in C57BL/6 mice (Supporting Information Fig. 1), indicating that the overall lower number of P14 CD8+ T cells with effector function was due both to lower numbers of P14 cells and due to increased cellular exhaustion in C57BL/6 mice compared with DC-MHCI mice.

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Figure 2. Antiviral CD8+ T cells are less exhausted in DC-MHCI mice during chronic LCMV infection. One day after transfer of 104 Ly5.1+ P14 T cells, DC-MHCI and C57BL/6 mice were infected with 106 pfu LCMV-Docile i.v. (A, C) On day 8 post infection, the number of the transferred Ly5.1+ CD8+ T cells was measured in the spleen. The data are shown as (A) representative flow cytometry plots and (C) summary of all mice, each symbol representing an individual mouse and lines representing the mean. (B, D–F) Splenocytes were restimulated with the epitope gp33 and the number of (B, D) degranulating, (B, E) IFN-γ-secreting and (F) TNF-α-producing Ly5.1+ CD8+ T cells was determined. Data are shown as (B) representative flow cytometry plots and (D–F) summary of all mice, each symbol representing an individual mouse and lines representing the mean. The virus titers in the (G) blood, (H) liver, (I) spleen, and (J) kidney were determined on day 8 post infection. Each symbol represents an individual mouse, the solid line indicates the mean value and the dashed line the detection limit. (K) On day 6 post infection, the number of the transferred Ly5.1+ CD8+ T cells was measured in the lung. The number of (L) degranulating, (M) IFN-γ-secreting, and (N) TNF-α-producing Ly5.1+ CD8+ T cells in the lung was determined after restimulation with the peptide epitope gp33. Each symbol represents an individual mouse and the solid line indicates the mean value. All data shown are from one experiment representative of three. Significance was determined by unpaired two-tailed Student's t-test. *p < 0.05; **p < 0.01.

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Furthermore, we determined the number and effector functions of antiviral CD8+ T cells in lungs and livers as peripheral organs. Similar to spleen, P14 TCR transgenic CD8+ T cells were present at higher numbers in the lung of DC-MHCI recipients than in wild-type mice (Fig. 2K; comparable data for liver, not shown). In addition, DC-MHCI mice exhibited higher numbers of degranulating (Fig. 2L), IFN-γ-secreting (Fig. 2M), and TNF-α-producing (Fig. 2N) CD8+ T cells in the lung and liver (not shown). Thus, selective antigen presentation on DCs induces a larger pool of cytokine-producing antiviral CD8+ T cells in the setting of a chronic viral infection, presumably due to reduced antigen exposure of the CD8+ T cells.

Next, we investigated if the reduced number of transgenic CD8+ T cells in C57BL/6 mice reflected increased apoptosis or decreased cell proliferation. BrdU incorporation was not higher in DC-MHCI mice than in C57BL/6 mice, indicating that the reduced number of P14 cells in C57BL/6 mice does not result from decreased proliferation (Supporting Information Fig. 2A and B). However, the percentage of early apoptotic (Annexin V+ 7AAD) cells was increased in C57BL/6 mice (Supporting Information Fig. 2C and D). The percentage of dead (Annexin V+ 7AAD+) cells was not different in C57BL/6 and DC-MHCI mice (data not shown), likely reflecting the quick removal of dead cells by phagocytes in vivo. Thus, the elevated numbers of P14 cells in DC-MHCI mice compared with those from C57BL/6 mice result from similar proliferation, but increased apoptosis in C57BL/6 mice.

It is conceivable, but unlikely, that C57BL/6 and DC-MHCI mice already exhibit differences during the CD8+ T-cell priming phase. To exclude that potential priming differences were leading to the numeric and functional differences observed at day 8 post infection, we analyzed early CFSE dilution profiles of transferred P14 CD8+ T cells. On day 1.5 post infection, CFSE dilution within P14 cells was similar in C57BL/6 and DC-MHCI mice (data not shown). Similarly, the number of P14 cells was not different in C57BL/6 and DC-MHCI mice on day 3.5 post infection (data not shown). These results indicate that priming is not affected by the absence of MHCI on all cell types except DCs in DC-MHCI mice, showing that the smaller pool of P14 cells in C57BL/6 mice at later time points reflects exhaustion rather than priming defects.

DC-MHCI mice succumb to chronic LCMV infection

Unexpectedly, we observed that DC-MHCI mice developed pathology including symptoms such as hunchback, ruffled fur, ataxia, and apathy after transfer of P14 CD8+ T cells in combination with either low- or high-dose LCMV infection (Fig. 3A and B). The velocity of disease development was consistent within experiments but varied slightly from experiment to experiment. This was likely due to slight variations in the number of transferred transgenic CD8+ T cells and due to slight variances of the viral inoculum. To quantitatively determine disease onset and progression, we longitudinally weighed the mice and measured their body core temperature. The observed pathology was not accompanied by drastic weight loss (data not shown), but by a drop in body core temperature concurrent with the emergence of disease symptoms (Fig. 3C). To analyze if the observed pathology reflected an immunopathology dependent on CD8+ T cells, we transferred different numbers of P14 CD8+ T cells into DC-MHCI and C57BL/6 mice and infected them with a high dose of LCMV-Docile. Indeed, the induction of pathology proved to be dependent on the number of transferred antiviral CD8+ T cells (Fig. 3C). However, C57BL/6 also developed pathology after transfer of 5 × 104 or more P14 CD8+ T cells (Fig. 3D). Nevertheless, the disease induction after transfer of a similar number of P14 CD8+ T cells was always quicker in DC-MHCI than in wild-type mice, likely reflecting the expansion of the TCR transgenic CD8+ T cells to higher numbers of functional effector cells in DC-MHCI mice (Fig. 2A, C and K). Thus, we conclude that downregulation of the T-cell response by means of T-cell exhaustion represents an important means of the host to avoid life-threatening immunopathology and that such downregulation is faster and more pronounced when CD8+ T cells are exposed to higher antigen levels.

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Figure 3. DC-MHCI mice succumb to chronic LCMV infection. One day after transfer of 104 P14 T cells, DC-MHCI and C57BL/6 mice were i.v. infected with (A) 200 or (B) 106 pfu LCMV-Docile. (A, B) The percentage of healthy mice over time is shown. The data depicted are summaries of all mice from (A) three or (B) eight independent experiments each performed with three to ten mice per group. (C, D) The indicated numbers of T-cell receptor transgenic CD8+ T cells were transferred and body core temperature was measured as indicator of disease development in (C) DC-MHCI and (D) C57BL/6 mice. Means + SEM of three to four mice per group are depicted. (E–K) Mice received 104 MACS-purified P14 CD8+ T cells 1 day prior to infection with 106 pfu LCMV-Docile. As indicator of liver pathology, the levels of the liver transaminases (E) ALT and (F) AST were measured in the serum of C57BL/6 and DC-MHCI mice at the time point when DC-MHCI mice showed disease symptoms. Mean + SD of three mice are depicted. (G) Tissue penetration of Evan's Blue into the indicated tissues was measured at the time point when DC-MHCI mice showed disease symptoms. Each symbol represents one mouse and data shown are pooled from two experiments with three to five mice per group. (H–J) The levels of (H) IFN-γ, (I) TNF-α, and (J) IL-6 were measured in the serum by cytometric bead array at the time point of disease symptoms in DC-MHCI mice. Each symbol represents one mouse and data are representative of three independent experiments. (K) DC-MHCI mice were left untreated or i.p. injected with neutralizing antibodies against TNF-α and/or IFN-γ and/or IL-6R on days −1, 1, 3, and 5 post infection. Untreated C57BL/6 mice served as negative controls. The body temperature was measured as indicator of disease. Mean + SEM of three to four mice per group are depicted. One representative of two experiments is shown. Significance was determined by unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significant.

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LCMV-induced immunopathology is often associated with a CD8+ T-cell mediated liver damage, reflected by elevated levels of the liver transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST) [31]. As DC-MHCI mice do not express MHCI on hepatocytes, killing of hepatocytes would not be expected. To formally exclude this, ALT and AST were measured and proved not to be elevated in DC-MHCI mice compared with those of C57BL/6 mice, but were rather reduced in the former (Fig. 3E and F).

In contrast to liver transaminase levels, vascular permeability was significantly elevated in kidneys (2-fold), livers (2-fold), and lungs (3.8-fold), but not in brains of DC-MHCI mice compared with those of wild-type mice (Fig. 3G). The increased vascular permeability was most striking in the lung. To investigate if CD4+ T cells might be responsible for the observed pathology in DC-MHCI mice, the number of IFN-γ- and/or TNF-α-producing CD4+ T cells was compared in DC-MHCI and C57BL/6 mice. However, the number of cytokine-producing CD4+ T cells was not elevated in DC-MHCI mice (data not shown). Although elevated concentrations of serum cytokines (IFN-γ, TNF-α, IL-6) could be observed in DC-MHCI mice compared with C57BL/6 mice (Fig. 3H–J), neutralization of different combinations of IFN-γ, TNF-α, and/or IL-6 neither led to decreased vascular permeability (data not shown) nor to delayed or alleviated disease symptoms as assessed by body core temperature, but rather to a trend toward accelerated disease progression when IFN-γ was neutralized (Fig. 3K).

Of note, mice that were not transferred with TCR transgenic CD8+ T cells also developed a certain degree of pathology. This pathology was not life threatening, but a decrease of the body core temperature was apparent (Supporting Information Fig. 3A). As expected these mice showed elevated numbers of endogenous CD8+ T cells specific for the LCMV-derived epitopes gp33 and np396 (Supporting Information Fig. 3B and C).

MHCI expression on nonhematopoietic cells promotes T-cell exhaustion and guards from immunopathology

To analyze the role of hematopoietic versus nonhematopoietic cells for the induction of CD8+ T-cell exhaustion and the CD8+ T-cell mediated disease, bone marrow chimeric mice were generated. DC-MHCI or wild-type mice expressing the congenic marker Thy1.1 were lethally irradiated and reconstituted with bone marrow derived from DC-MHCI or Thy1.1+ mice. The presence of MHCI on DCs allowed for efficient priming of antiviral CD8+ T cells by DCs in all of these chimeras.

Interestingly, mice that were MHC-I deficient on nonhematopoietic cells succumbed to high-dose LCMV infection on day 6 p.i., while mice that expressed MHCI on nonhematopoietic cells stayed healthy (Fig. 4A). Consistent with the absence of disease symptoms, mice expressing MHCI on nonhematopoietic cells showed lower frequencies and absolute numbers of IFN-γ-, TNF-α-, and IL-2-producing P14 CD8+ T cells in lungs (Fig. 4B–G) and livers (Supporting Information Fig. 4). These results indicate that antigen recognition on nonhematopoietic cells in addition to DCs promoted T-cell exhaustion and hence avoidance of immunopathology. It is well known that a large variety of nonhematopoietic cells is infected by LCMV in vivo, including hepatocytes [32], fibroblastic reticular cells [33] and endothelial cells [32], which are all likely to contribute to the enhanced induction CD8+ T-cell exhaustion in DC-MHCI [RIGHTWARDS ARROW] Thy1.1 chimeras. The finding that the presence of antigen presentation on nonhematopoietic cells promotes CD8+ T-cell exhaustion was supported by experiments with bone marrow chimeras that express the P14 restricting H-2Db molecule on hematopoietic and/or nonhematopoietic cells or not at all. H-2Db or Thy1.1 mice were lethally irradiated and reconstituted with Thy1.1 or H-2Db bone marrow. To avoid differences in priming of P14 cells in these chimeras, memory P14 cells that had been primed in C57BL/6 hosts with an acute infection of LCMV-WE (Supporting Information Fig. 5A) were used for adoptive transfer into the chimeras. Consistent with the results in the DC-MHCI chimeras, CD8+ T-cell exhaustion was least pronounced when antigen presentation was absent on nonhematopoietic cells (Supporting Information Fig. 5B–D).

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Figure 4. MHC class I expression on nonhematopoietic cells induces T-cell exhaustion and protects from disease. DC-MHCI and wild-type mice expressing the congenic marker Thy1.1 were lethally irradiated and reconstituted with DC-MHCI or Thy1.1 bone marrow. One day after transfer of 104 Ly5.1+ P14 cells, the bone marrow chimeras were infected with 106 pfu LCMV-Docile i.v. (A) The health status of the mice was monitored twice daily. (B–G) On day 6 post infection, the percentage and number of (B, E) IFN-γ+, (C, F) TNF-α+, and (D, G) IL-2+ transgenic CD8+ T cells in the lung was determined after stimulation with the peptide epitope gp33. Each symbol represents an individual mouse, the line indicates the mean. Data shown are representative of two experiments performed. Significance was determined by unpaired two-tailed Student's t-test. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Antigen recognition on bone marrow derived cells can also induce T-cell exhaustion

As the data above indicate that nonhematopoietic cells induce a stronger degree of T-cell dysfunction, we next addressed whether this was mediated by a particular “exhaustion-inducing” qualitative feature of nonhematopoietic cells or whether this effect could be explained by the fact that a higher number of nonhematopoietic cells than hematopoietic cells is infected by LCMV and that therefore the CD8+ T cells sense far more antigen on nonhematopoietic cells than on hematopoietic cells. To address this, we generated a set of bone marrow chimeric mice that harbor varying amounts of LCMV gp33-expressing bone marrow derived cells. MHCI-DC mice were lethally irradiated and reconstituted with MHCI-DC bone marrow mixed with varying ratios of H8 bone marrow that transgenically expresses the glycoprotein gp33–41 epitope of LCMV in all nucleated cells [34]. To assure similar CD8+ T-cell priming in all experimental groups, P14 CD8+ T cells were primed in DC-MHCI mice and transferred to infection matched bone marrow chimeras on day 3 post infection (Fig. 5A). Analysis of splenocytes and lung lymphocytes showed an inverse correlation of the percentage of hematopoietic cells expressing antigen and the percentage and number of P14 CD8+ T cells on day 6 post infection (Fig. 5B, E, H, and I). Similarly, the number of degranulating and IFN-γ-producing Ly5.1+ CD8+ T cells decreased in a dose-dependent fashion with increasing frequencies of antigen-expressing hematopoietic cells (Fig. 5C, D, F, and G). The reconstitution efficacy was determined via the frequency of CD19+ cells expressing MHCI in the lung (Fig. 5J).

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Figure 5. Elevated antigen recognition on bone marrow derived cells induces T-cell exhaustion. (A) A schematic representation of the experimental design: DC-MHCI recipients were lethally irradiated and reconstituted with DC-MHCI bone marrow or DC-MHCI bone marrow mixed with 1, 10, or 50% of H8 bone marrow. The chimeras were infected with 106 pfu LCMV-Docile i.v. On day 3 post infection, they were transferred with 105 CD8+ T cells derived from infection matched DC-MHCI mice that had received 106 P14 T cells prior to infection. (B–G) On day 6 post infection, the frequency and number of (B, E) Ly5.1+ CD8+ T cells was assessed and the percentage and numbers (C, F) of degranulating and (D, G) IFN-γ-secreting Ly5.1+ CD8+ T cells in the lungs were analyzed after stimulation with the peptide epitope gp33. (H, I) The percentage (H) and number (I) of Ly5.1+ CD8+ T cells in the spleens are shown. (J) The percentage of MHC class I-expressing CD19+ cells was determined in the lung to assess the reconstitution efficacy. Each symbol represents an individual mouse and the lines represent the mean; data shown are pooled from two independent experiments. Significance was determined by unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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These results indicate that it is not a particular “exhaustion-inducing” qualitative feature of nonhematopoietic cells that promotes CD8+ T-cell exhaustion but that the degree of CD8+ T-cell dysfunction rather depends on the availability of antigen in the respective cell subsets.

Elevation of the DC number in DC-MHCI mice leads to impaired T-cell effector functions

The previous experiments demonstrated that the amount of antigen presented to LCMV-specific CD8+ T cells inversely correlated with the number of antiviral CD8+ T cells and the number of antiviral CD8+ T cells that were able to exert effector functions. However, our data so far do not distinguish whether increased exhaustion in presence of more antigen is only due to the amount of antigen that is recognized by CD8+ T cells, or if certain cell types induce exhaustion more potently than others. We therefore decided to analyze the effects of different amounts of antigen presented on only one cell type in vivo.

We made use of the finding that subcutaneous injection of irradiated B16 melanoma cells expressing GM-CSF (B16-GM-CSF) boosts the generation of mainly myeloid DCs (CD11bhi CD8α) while injection of irradiated B16 melanoma cells expressing Flt3L (B16-Flt3L) induces elevated numbers of lymphoid (CD11b CD8α+) and myeloid DCs [35]. A confirmation that such treatment (Fig. 6A) induced elevated frequencies of the different DC populations in DC-MHCI mice at the time point of infection is depicted in Fig. 6 B–F. The overall frequency of DCs induced after B16-GM-CSF injection was higher compared with that after B16-Flt3L injection (Fig. 6C). DC-MHCI mice with elevated numbers of DCs as the only cell type able to present antigen were transferred with P14 CD8+ T cells and analyzed on day 6 after high-dose infection with LCMV-Docile. Elevation of the DC number — and therefore the number of antigen presenting cells that are all of the same cell type — was found to induce lower numbers of transgenic CD8+ T cells in lung (Fig. 6G), spleen (Supporting Information Fig. 6A) and liver (Supporting Information Fig. 6D). Similarly, the numbers of degranulating (Fig. 6H, Supporting Information Fig. 6B and E), IFN-γ (Fig. 6I, Supporting Information Fig. 6C and F), TNF-α (Fig. 6J and Supporting Information Fig. 6G), and IL-2 (Fig. 6K and Supporting Information Fig. 6H) producing CD8+ T cells were inversely proportional to the number of DCs. Thus, these results clearly show that it is the amount of antigen which drives T-cell exhaustion and that this is independent of the cell type presenting the antigen. Even DCs, representing professional APCs, are able to induce CD8+ T-cell dysfunction on their own in the setting of a chronic viral infection if present at high numbers.

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Figure 6. Elevation of dendritic cell numbers leads to impaired T-cell effector functions. (A) Design of the experiment: DC-MHCI mice were s.c. injected with 106 irradiated B16, B16-Flt3L, or B16-GM-CSF cells on days −9 and −1. One day prior to infection with 106 pfu LCMV-Docile, 104 MACS-purified P14 CD8+ T cells were transferred i.v. T-cell responses were analyzed on day 6 post infection. (B–F) Different DC subsets were analyzed in the spleen on day 0. (B) Plots are pregated on I-A/I-E+ cells. The percentage of (C) total DCs, (D) CD8+ DCs, (E) MHCII+ CD11chi CD11b cells, and (F) MHCII+ CD11chi CD11b+ cells was analyzed. (G–K) On day 6 post infection, the (G) expansion of Ly5.1+ T-cell receptor transgenic CD8+ T cells was analyzed and the number of (H) degranulating and (I) IFN-γ, (J) TNF-α, and (K) IL-2-secreting T-cell receptor transgenic CD8+ T cells was assessed in the lung after stimulation with the peptide epitope gp33. Each symbol represents an individual mouse and lines represent the mean; data shown are representative of three independent experiments. Significance was determined by unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

The question whether high amounts of antigen are the cause or the consequence of CD8+ T-cell exhaustion is widely discussed. We show here that provision of elevated amounts of antigen induced a higher degree of exhaustion in a causal relationship, supporting the notion that high amounts of antigen directly induce CD8+ T-cell exhaustion.

A negative correlation between viral load and CTL function was observed in several settings of chronic infection in humans and mice [24-27]. However, it was so far unclear whether the quality of a cell type presenting the antigen determines the degree of exhaustion or if solely the overall amount of antigen recognized by T cells promotes exhaustion. Our data obtained in DC-MHCI mice confirm previous publications showing that the CD8+ T-cell dysfunction observed during chronic viral infections is highly influenced by the amount of antigen that is recognized by responding CD8+ T cells. Infection of bone marrow chimeric mice in which antigen presentation is selectively absent or present on nonhematopoietic cells suggested that high levels of antigen on nonhematopoietic cells strongly promotes T-cell exhaustion. This is likely due to a high number of infected nonhematopoietic cells (with hepatocytes [36], fibroblastic reticular cells [33], lamina propria cells [37], glial cells and microglia being infected [38], and high expression of the LCMV receptor α-dystroglycan being present on stromal and epithelial cells [39] in many tissues, suggesting that these cells might also be infected) compared with hematopoietic cells (with mainly DCs [40, 41] and macrophages [42] being infected by LCMV). However, by selective provision of antigen presentation in hematopoietic cells, CD8+ T-cell exhaustion can also be promoted, indicating that the overall amount of antigen, independent of the cell type (hematopoietic versus nonhematopoietic), can drive CD8+ T-cell exhaustion.

The necessity of high antigen levels for the induction of CD8+ T-cell exhaustion was suggested in several settings of chronic infection such as LCMV infection of mice [25, 43], repeated injection of antigen into mice [44], but also in humans a correlation between high antigen levels and T-cell exhaustion was observed after Mycobacterium tuberculosis [27] and HIV [24, 26] infection. However, these studies were not able to distinguish whether increased exhaustion in the presence of more antigen is only due to the amount of antigen that is recognized by CD8+ T cells, or if certain cell types induce exhaustion more potently than others. Similarly, our experiments using bone marrow chimeric mice that present antigen on either hematopoietic or nonhematopoietic cells, cannot answer the question if different cell types induce exhaustion with different effectivity on a per-cell basis. Our data from bone marrow chimeric mice do not exclude that exhaustion is differentially induced by different cell types, for instance by displaying different amounts of peptide-MHC complexes or by exhibiting varying expression levels of costimulatory molecules or ligands for inhibitory receptors on CD8+ T cells. Indeed, the expression of PD-L1 was shown to differently control T-cell responses when expressed on hematopoietic and nonhematopoietic cells [45], albeit this might again be partially influenced by varying amounts of antigen presented on hematopoietic versus nonhematopoietic cells. To formally address whether diverse cell types have differential capacities to induce CD8+ T-cell exhaustion in vivo, one would need a system with comparable amount of antigen presentation on equal numbers of distinct cell types, which seems technically impossible. However, by varying the number of DCs as only cell type on which antiviral CD8+ T cells can see their antigen, our data extend the finding that high amounts of antigen drive CD8+ T-cell exhaustion by showing that this can be achieved independently of the cell type presenting antigen and that even high amounts of antigen presented by professional antigen presenting cells can induce CD8+ T-cell exhaustion. Our data also confirm previous findings, showing that the persistence of high levels of antigen can induce CD8+ T-cell exhaustion in mice that do not express MHCI on nonhematopoietic cells [25]. One further advantage of using DC-MHCI mice (or bone marrow chimeras thereof) as opposed to bone marrow chimeras with overall absence of the restricting MHCI molecule on hematopoietic or nonhematopoietic cells is the fact that these mice retain MHCI expression on DCs that is essential for the priming of naive CD8+ T-cell responses [29]. Thus, in previous reports [25] using Db bone marrow chimeras, it could only be addressed whether selective absence of the restricting MHCI molecule on nonhematopoietic cells would influence the degree of CD8+ T-cell exhaustion and a role for antigen presentation on hematopoietic cells could not be addressed. We believe that our data constructively address the issue of the role of antigen presentation on hematopoietic cells by showing that antigen expression on various percentages of hematopoietic cells (Fig. 5) or selectively on DCs (Fig. 6) also impact on CD8+ T-cell exhaustion.

Also in case of CD4+ T cells, it was recently shown that prolonged high-level antigen expression on MHC class II induces CD4+ T-cell exhaustion with the level of antigen exposure influencing the kinetics of CD4+ T-cell dysfunction [46].

Unexpectedly, DC-MHCI mice developed disease symptoms after transfer of 104 TCR transgenic CD8+ T cells and low-dose LCMV infection, presumably due to their failure to control infection. In addition, DC-MHCI mice succumbed to infection after adoptive CD8+ T-cell transfer and high-dose LCMV infection using CD8+ T-cell numbers where wild-type recipients stayed healthy. However, also mice that did not receive TCR transgenic CD8+ T cells lost body core temperature, indicating the development of a transient pathology. The severity and velocity of development of disease symptoms correlated with the number of transgenic CD8+ T cells transferred and likely goes in hand with the diminished CD8+ T-cell exhaustion observed in DC-MHCI mice. As observed before [47, 48], also C57BL/6 mice developed disease symptoms with increasing numbers of transferred CD8+ T cells. As CD8+ T-cell exhaustion was described in various settings of chronic infections of mice, nonhuman primates, and humans, these findings support the concept that CD8+ T-cell exhaustion might be an evolutionarily conserved mechanism in face of persistent antigen exposure that is beneficial for the host in order to avoid T-cell induced immunopathology.

Appearance of disease symptoms was accompanied by elevated numbers of cytokine-producing antiviral CD8+ T cells, elevated levels of the cytokines IFN-γ, TNF-α, and IL-6 in the serum and increased vascular permeability in DC-MHCI mice compared with C57BL/6 mice. To date, it is unclear what exactly drives the observed immunopathology as in vivo neutralization of IFN-γ, TNF-α, and IL-6 neither had an impact on the elevated vascular permeability nor on the disease symptoms. However, it cannot be excluded that these cytokines elicit the observed elevated vascular permeability and immunopathology in concert with additional factors. Earlier studies suggested that LCMV-induced immunopathology is mainly driven by perforin- and Fas/FasL-mediated hepatocyte lysis reflected by elevated levels of the liver transaminases ALT and AST [31]. However, the absence of MHCI on hepatocytes and decreased ALT and AST levels in DC-MHCI mice favor additional undefined effector mechanisms contributing to LCMV-induced CD8+ T-cell mediated immunopathology. It was recently suggested in a model of LCMV infection of the CNS, where fatal immunopathology is also induced in a CD8+ T-cell dependent manner, that CD8+ T cells facilitated secondary recruitment of inflammatory monocytes and neutrophils via secretion of multiple myelomonocytic chemoattractants, thereby promoting vascular leakage and acute lethality [49]. Whether a comparable mechanism of CD8+ T-cell mediated pathology operates in LCMV-infected DC-MHCI mice remains to be addressed in detail. It is also possible that alveolar macrophages might be involved in pathology development as they are known to express high levels of CD11c and could be presenting antigen in DC-MHCI mice, contributing to local CD8+ T-cell activation and lung immunopathology. Finally, it is conceivable that extensive killing of antigen-presenting DCs might be involved in the observed pathology in chronically infected DC-MHCI mice, potentially leading to severe immunosuppression and inability of chronically infected mice to cope with other viral or bacterial infections. However, this hypothesis is rather unlikely as the number of DCs was not severely compromised in chronically infected DC-MHCI mice and since development of pathology was very acute which is unlikely to be caused by heterologous infections in the setting of a clean specific pathogen free facility.

As a correlation of the degree of CD8+ T-cell exhaustion with the viral load was observed in M. tuberculosis [27] and HIV [24, 26] infected humans, our findings likely have a strong relevance for treatment of chronically infected patients. It is currently widely discussed to treat chronically infected patients with regimes aimed at boosting the antiviral CD8+ T-cell response, for example, with antibodies blocking the interaction of PD-1 and PD-L1 [50] (in combination with antibodies directed against other inhibitory receptors [51, 52]). However, as shown here, high viral loads in combination with boosted CD8+ T-cell responses are at risk to induce immunopathology. Our data suggest that the occurrence (or absence) of potentially lethal pathology likely depends on the number of virus-specific CD8+ T cells that are reinvigorated by specific interventions. It should be considered to treat patients with antivirals in combination with immunosuppressive drugs to avoid immunopathology until the virus titers drop and to continue the treatment with antivirals in combination with immunostimulatory regimens to give the T cells a chance to clear the virus without inducing overt immunopathology.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Virus and viral peptides

LCMV-Docile was propagated on MDCK cells and LCMV-WE was propagated on L929 cells as described [53]. The viral peptide gp33–41 (gp33; KAVYNFATM) was purchased from NeoMPS (Strasbourg, France). Determination of virus titers was done as described before [53].

Antibodies

Antibodies used for flow cytometry were purchased from BD Pharmingen (Allschwil, Switzerland) and Biolegend (LucernaChem, Luzern, Switzerland). IFN-γ (clone XMG1.1 or clone R4–6A2), TNF-α (clone XT3.11), and IL-6R (clone 15A7) neutralizing antibodies were purchased from BioXCell, West Lebanon, NH, USA.

Mice

C57BL/6 (Janvier Elevage, Le Genest Staint Isle, France), congenic Thy1.1 mice, H-2Db−/− mice [54], DC-MHCI mice selectively expressing β2m under control of the K14 and CD11c promoters (CD11c-β2m+K14-β2m+β2m−/−) [28, 55], H8 transgenic mice ubiquitously expressing the LCMV-derived epitope gp33 [34], gp33-specific T-cell receptor transgenic P14 mice expressing the congenic marker Ly5.1 [56] were kept under specific pathogen-free conditions and were intravenously (i.v.) infected with 200 pfu (low dose) or 106 pfu (high dose) of LCMV strains Docile or WE.

For the generation of bone marrow chimeras, mice were irradiated with 950 rad and reconstituted with 5 × 106 bone marrow cells. Six to eight weeks after reconstitution they were tested for chimerism.

The body core temperature of mice was measured by rectal insertion of a metal measuring probe of a precision thermometer (Hugo Sachs Elektronik, March-Hugstetten, Germany).

When indicated mice were treated with 500 μg of anti-IFN-γ and/or 500 μg anti-TNF-α and/or 33 μg/g body weight anti-IL-6R [57] by i.p. administration on days −1, 1, 3, and 5 post infection.

When indicated mice were subcutaneously injected with 106 irradiated (3500 rad with 60Co source) B16, B16-Flt3L, or B16-GM-CSF cells [35] that were kindly provided by Nicolas Mach, Geneva, Switzerland.

For measurement of BrdU incorporation, 1 mg of BrdU (BD Biosciences, Allschwil, Switzerland) was administered i.p. on days 4 and 5 post infection.

Animal experiments were performed according to the regulations of the cantonal veterinary office (animal experimentation number 146/2008). All animals were used at 6–12 weeks of age.

Adoptive transfers

Splenic CD8+ T cells were isolated by immunomagnetic sorting according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany) and i.v. transferred into recipients 1 day prior to virus infection.

Tetramers, BrdU, Annexin V, 7AAD, stimulation of lymphocytes, and flow cytometry

Peptide/MHCI tetrameric complexes were generated as described [58]. Splenocyte suspensions were prepared by passing the spleens of perfused mice through a metal mesh using syringe plungers. For the preparation of mononuclear cells from lungs and livers, the organs were removed from perfused mice, cut into small pieces and digested twice for 20 min at 37°C in RPMI containing 2.4 mg/mL collagenase type I (Gibco, Invitrogen, Basel, Switzerland) and 0.2 mg/mL DNase I (Roche Diagnostics, Rotkreuz, Switzerland). Mononuclear cells were purified by gradient centrifugation over 30% Percoll. Lymphocytes were stimulated with 1 μg/mL gp33 peptide in the presence of Brefeldin A (10 μg/mL; Sigma, St. Louis, MO, USA) or Monensin A (2 μM; Sigma) for 5 h at 37°C. For assessment of degranulation, 1 μL anti-mouse CD107a was added during the stimulation. Cells were surface stained for 30 min at 4°C before the cells were fixed and permeabilized in 500 μL 2× FacsLyse (BD Biosciences) containing 0.05% Tween 20 (Sigma-Aldrich) for 10 min at room temperature. After a wash step, the intracellular staining of cytokines was performed for 30 min at room temperature in the dark. Cells were then washed and resuspended in PBS containing 1% paraformaldehyde (Sigma-Aldrich). BrdU incorporation was stained using the BrdU Flow Kit (BD Biosciences) according to the manufacturer's instructions. Annexin V (BD Biosciences) and 7AAD (BD Biosciences) were applied in Annexin V binding buffer (BD Biosciences) according to the manufacturer's instructions. For the determination of cell numbers, a defined number of Calibrite beads (BD Biosciences) was added. Multiparameter flow cytometric analysis was performed using a FACS LSRII flow cytometer (BD Biosciences) with FACSDiva software. Analysis was performed using FlowJo software (Tree Star, San Carlos, CA, USA).

Analysis of cytokine levels in sera

The serum concentrations of IFN-γ, TNF-α, and IL-6 were determined by cytometric bead array (BD Biosciences) according to the manufacturer's instructions.

Analysis of liver transaminase levels in sera

The levels of AST and ALT were measured by the clinical chemistry department of the University Hospital Zurich.

Quantification of vascular permeability by injection of Evan's Blue

Mice were i.v. injected with 200 μL of 0.5% Evan's Blue in PBS. After 20 min, the mice were sublethally anesthetized with a cocktail containing 2 mg ketamine, 0.4 mg xylacine, and 60 μg acepromacine in PBS. After perfusion of the mice, the analyzed organs were removed and the Evan's Blue residing in the organs was extracted in 1 mL of formamid (Sigma-Aldrich, Buchs, Switzerland) over night at 56°C. Absorption was measured at 620 nm and quantified in relation to an Evan's Blue standard.

Statistical analysis

Significance was determined by two-tailed Student's t-test using GraphPad Prism software (GraphPad software, La Jolla, CA, USA).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

We thank Nicolas Mach for kindly providing B16-Flt3L and B16-GM-CSF cells, the clinical chemistry department of the university hospital Zurich for measurement of liver transaminases, Petra Wolint, Nathalie Oetiker, and Franziska Wagen for excellent technical assistance and members of the Oxenius group for fruitful discussions. This work was supported by the ETH Zurich and the Swiss National Science Foundation (Grant No. 310030–129751 to AO).

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information
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Abbreviations
ALT

alanine aminotransferase

AST

aspartate aminotransferase

LCMV

lymphocytic choriomeningitis virus

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
eji2338-sup-0001-figures1.pdf963KFigure S1: percentages of P14 cells in DC-MHCI and C57BL/6 mice after high dose LCMV infection One day after transfer of 104 Ly5.1+ T cell receptor transgenic CD8+ T cells specific for the LCMV-derived epitope gp33 (P14), DC-MHCI and C57BL/6 mice were infected with 106 pfu LCMV-Docile i.v.. On day 8 post infection the percentage of the transferred Ly5.1+ CD8+ T cells was measured in the spleen (A). The percentage of degranulating (B), IFN-γ secreting (C) and TNF-α producing (D) Ly5.1+ CD8+ T cells was determined after restimulation with the epitope gp33. Each symbol represents an individual mouse. The line indicates the mean value. Data from one representative experiment of two is shown. Statistical significance was determined using unpaired two-tailed student's t-test ** P < 0.01, *** P < 0.001 Figure S2: selective antigen recognition on DCs leads to lower percentages of early apoptotic cells One day after transfer of 104 Ly5.1+ P14 CD8+ T cells DC-MHCI and C57BL/6 mice were infected with 106 pfu LCMV-Docile i.v. On days 4 and 5 the mice were treated i.p. with 1 mg of BrdU. The mice were analyzed on d5.5 when disease symptoms became apparent. Representative FACS plots of BrdU stainings of splenocytes are shown in (A). The cells are pregated on CD8+ T cells. A summary of all mice analyzed is depicted in (B). Representative FACS plots of 7AAD and Annexin V stained splenocytes are shown in (C). The cells are pregated on Ly5.1+ CD8+ T cells. A summary of all mice analyzed is depicted in (D). Figure S3: The endogenous CD8+ T cell response induces pathology The body core temperature of C57BL/6 and DC-MHCI mice was monitored at the indicated time points after i.v. infection with 106 pfu LCMV-Docile (A). A summary of 8 mice per group from two independent experiments is shown. The mean ± SEM is depicted. The number of endogenous gp33-Tetramer+ CD8+ T cells (B) and np396-Tetramer+ CD8+ T cells (C) was determined in the spleen on day 17 post infection with 106 pfu LCMV-Docile i.v. Figure S4: MHC-I expression on non-hematopoietic cells induces T cell exhaustion and protects from disease DC-MHCI and wild-type mice expressing the congenic marker Thy1.1 were lethally irradiated and reconstituted with DC-MHCI or Thy1.1 bone marrow. One day after transfer of 104 Ly5.1+ P14 cells, the bone marrow chimeras were infected with 106 pfu LCMV-Docile i.v. On day 6 post infection the percentage and number of IFN-γ+ (A and D), TNF-α+ (B and E) and IL-2+ (C and F) transgenic CD8+ T cells in the liver was determined after stimulation with the epitope gp33. Each symbol represents an individual mouse. The line indicates the mean. One representative of two experiments is depicted. Significance was determined by unpaired 2-tailed Student's t test. * P < 0.05; ** P < 0 .01, *** P < 0.001, **** P < 0.0001. Figure S5: Antigen recognition on non-hematopoietic cells induces CD8+ T cell exhaustion of memory CD8+ T cells (A) Experimental setup: H2Db−/− and wild-type mice expressing the congenic marker Thy1.1 were lethally irradiated and reconstituted with H2Db-/- or Thy1.1 bone marrow. 2 × 104 MACS-purified CD8+ T cells from a C57BL/6 mouse that received 105 Ly5.1+ P14 splenocytes and 200 pfu LCMV-WE more than 6 months earlier were transferred into the chimeras. One day after adoptive T cell transfer the chimera were infected with 106 pfu LCMV-Docile. On day 12 post infection splenocytes were analyzed for the expansion of the transferred T cell receptor transgenic CD8+ T cells (B). The number of degranulating (C) and IFN-γ producing (D) T cell receptor transgenic. Figure S6: Elevation of dendritic cell numbers leads to impaired T cell effector functions. DC-MHCI mice were s.c. injected with 106 irradiated B16, B16-Flt3L or B16-GM-CSF cells on days -9 and -1. One day after transfer of 104 T cell receptor transgenic CD8+ T cells specific for the LCMV-derived epitope gp33, the mice were infected with 106 pfu LCMV-Docile i.v. On day 6 post infection, lymphocytes isolated from the spleen and liver were used to analyze the number of Ly5.1+ T cell receptor transgenic CD8+ T cells (A and D), degranulating (B and E) or IFN-γ (C and F), TNF-α (G) and IL-2 (H) producing Ly5.1+ CD8+ T cells. One representative of three experiments is shown. Significance was determined by unpaired 2-tailed Student's t test. *P < 0.05.

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