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Keywords:

  • CD8+ T cells;
  • costimulatory molecules;
  • transgenic models;
  • virology

Abstract

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

The importance of costimulation on CD4+ T cells has been well documented. However, primary CTLs against many infections including influenza can be generated in the absence of CD4+ T-cell help. The role of costimulation under such “helpless” circumstances is not fully elucidated. Here, we investigated such a role for CD28 using CTLA4Ig transgenic (Tg) mice. To ensure valid comparison across the genotypes, we showed that all mice had similar naïve precursor frequencies and similar peak viral loads. In the absence of help, viral clearance was significantly reduced in CTLA4Ig Tg mice compared with WT mice. CD44+BrdU+influenza-specific CD8+ T cells were diminished in CTLA4Ig Tg mice at days 5 and 8 postinfection. Adoptive transfer of ovalbumin-specific transgenic CD8+ T cells (OT-I)-I cells into WT or CTLA4Ig Tg mice revealed that loss of CD28 costimulation resulted in impairment in OT-I cell division. As shown previously, neither viral clearance nor the generation of influenza-specific CD8+ T cells was affected by the absence of CD4+ T cells alone. In contrast, both were markedly impaired by CD28 blockade of “helpless” CD8+ T cells. We suggest that direct CD28 costimulation of CD8+ T cells is more critical in their priming during primary influenza infection than previously appreciated.


Introduction

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

CTL induction is important for the clearance of many viral infections including influenza [1]. CD4+ T-cell help potentiates CTL induction by licensed dendritic cells (DCs) [2-5] and is critical for the generation of CD8+ T-cell memory [6-9]. This licensing is mainly mediated by CD40L on CD4+ T cells, although other co-stimulatory moleculesmay play a role [10]. There may also be some contribution of cytokines released by CD4+ T cells that directly influence CD8+ T-cell activation [10-12]. Yet another way CD4+ T-cell help may work is in the protection of APCs from killing by effector CTLs [13]. However, it should be noted that those hallmark papers on helper-mediated licensing were performed with noninfectious antigens [2-4] and CD4+ T-cell help is not essential for the induction of primary CTLs to several viruses including influenza [6, 14-17], lymphocytic choriomeningitis virus (LCMV) [18, 19], and vaccinia [20]. We have previously reported that viruses that can upregulate CD40L on DCs can activate “helpless” CTLs [21] and others have reported that type I IFN can do likewise during LCMV infection [22]. The fact that influenza can elicit CTLs without help led us to explore further the role of costimulation in “helpless” CTL induction and viral clearance.

Unlike influenza, certain viruses such as LCMV can elicit strong CTL responses without the need for costimulation [23-25]. Highly virulent strains of vaccinia (unlike attenuated strains) elicit CD28-independent responses [26]. Previous studies on the effect of costimulation on CD8+ T-cell responses have mainly been performed in the presence of CD4+ T cells, for example for anti-LCMV [24, 27-29] and for anti-influenza [30] responses. In these previous studies dissecting the requirement for CD8+ T-cell responses, the contribution of CD4+ T-cell help and costimulation were not addressed separately. We opined that the induction of “helpless” primary anti-influenza CTLs is contributed by the direct interaction between costimulatory molecules on DCs and CD8+ T cells. Therefore, we sought to determine the role of costimulation in mice depleted of CD4+ T cells. CD28 is a pivotal costimulation pathway, although there are many other costimulatory molecules including CD40L, CD27, CD134, CD137, and CD278 [31, 32]. We compared mice that were deficient in CD4+ T cells alone with mice that were deficient in CD4+ T cells and CD28 costimulation. We measured the clearance of influenza from the lung and the frequency of antigen-specific CD8+ T-cell responses against a peptide of influenza polymerase acidic protein (PA224–233). Our findings reveal that unlike immunization with noninfectious antigens, direct CD28 costimulation of CD8+ T cells was more important than help in eliciting primary CD8+ T-cell responses for influenza. These findings may have implications for attempts at inducing CTLs in HIV patients with reduced CD4+ T cells. Also, with the advent of costimulation, blockade being clinically used in transplantation and autoimmunity [33-35], our findings suggest that there should be vigilance on infection rates for patients undergoing such treatments.

Results

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

Impaired viral clearance under CD28 blockade in the absence of CD4+ T cells

To investigate the role of costimulation in CTL induction under “helpless” circumstances, WT and CTLA4Ig Tg mice were depleted of CD4+ T cells and infected intranasally (i.n.) with the same dose of influenza virus. However, as the mice had different genetic modifications, we wanted to check that the virus replicated to a similar extent in the different strains of mice, that is, the antigen load would be equivalent among the different strains. To do this, we chose a time point when virus had grown enough to be easily and accurately measured but before any clearing effect had been educed by the adaptive immune system [36]. We therefore harvested the lungs at day 5 postinfection and found that the two groups of infected mice had similar viral loads (Fig. 1). Thus, this establishes that any differences in CTL response magnitude are not due to differences in antigen load.

image

Figure 1. Viral load in lungs of infected mice at day 5. To deplete CD4+ T cells, all mice were treated with 0.5 mg GK1.5 Ab 1 day before and one day after intranasal infection with 104.5 pfu of Mem71 virus. Lungs were harvested at day 5 postinfection and viral titres were determined by plaque assay using Madin Darby canine kidney (MDCK) cells. Data are shown as mean + SEM of n = 12, 7, and 9 for WT, CTLA4Ig Tg, and naïve mice, respectively. Data were pooled from two independent experiments. The detection limit of the virus assay is 1.8 log.

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Administration of GK1.5 Ab led to >99% depletion of CD4+ T cells. We had previously shown that such treated mice were protected against allograft rejection and the IgG Ab responses to OVA and keyhole limpet hemocyanin [37, 38] were abrogated.

As virus begins to be cleared by CTLs at around day 8 after initial infection in WT [6, 36], we decided to compare viral clearance at day 8 postinfection in the different groups of mice. We observed that, in the absence of CD4+ T cells, the CTLA4Ig Tg mice had significantly higher viral titer as compared with WT mice (mean ± SEM: 2.72 ± 0.35 and 1.85 ± 0.04 log10pfu/lungs, respectively; p < 0.005) (Fig. 2); note that the limit of our detection of virus was 1.8 log10pfu/lungs. WT mice with or without CD4+ T cells cleared the virus similarly (1.8 versus 1.85 log10pfu/lungs) as has been shown previously by many researchers [6, 14, 16]. Our data therefore suggest that during acute infection, CD28 costimulation of CD8+ T cells is critical for the efficient clearance of influenza virus infection.

image

Figure 2. Viral load in the lungs of mice at day 8. To deplete CD4+ T cells, all mice were treated with 0.5 mg GK1.5 Ab 1 day before and one day after intranasal infection with 104.5 pfu of Mem71 virus. Lungs were harvested at day 8 postinfection and viral titres were determined by plaque assay using MDCK cells. Data are shown as mean + SEM of n = 14, 7, and 9 for WT, CTLA4Ig Tg, and naïve mice, respectively. Data were pooled from two independent experiments. Statistical analysis was performed using Mann–Whitney U test. The detection limit of the virus assay is 1.8 log.

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Ab titres in “helpless” CTLA4Ig Tg mice were similar to “helpless” WT mice

To determine if the loss of costimulation had an effect on the generation of influenza-specific antibodies, ELISA titers of IgM and IgG specific for purified whole Mem71 virus were measured in sera taken from mice at day 8 postinfection, the time of viral clearance in WT mice. As expected, the loss of CD4+ T cells resulted in the decrease of IgM and total IgG titres in WT mice; for IgM, the geometric mean titres (GMT) of Ab titers in nontreated WT and GK1.5-treated WT mice were 4.1 ± 0.2 and 3.1 ± 0.1, respectively; for IgG, the GMT of Ab titres in nontreated WT and GK1.5-treated WT mice were 3.6 ± 0.1 and 2.0 ± 0.2, respectively (Supporting Information Fig. 1). In CTLA4Ig tg mice, GMT of IgM titre (4.1 ± 0.1) were similar to WT but the IgG titre was decreased (3.2 ± 0.1). Additional loss of CD4+ T cells in CTLA4Ig tg mice resulted in further reduction in both IgM (3.0 ± 0.1) and IgG (1.7 ± 0.1) Ab titres as compared to CTLA4Ig tg mice with intact CD4+ T cells. However, there was no difference in both IgM and IgG Ab titres between CD4-deficient WT and CD4-deficient CTLA4Ig tg mice. Therefore, the difference in the ability to clear viral infection between these two groups of mice was unlikely to be due to production of antibodies.

Generation of “helpless” antigen-specific CD8+ T-cell responses is impaired in the absence of costimulation

We wanted to determine the requirement for costimulation for generating influenza-specific CTL responses in WT and CTLA4Ig Tg mice, in the absence of CD4+ T cells. To ensure that the different groups of mice had equivalent naïve precursor frequencies, the number of naïve PA-specific CD8+ T cells in uninfected mice was determined after tetramer enrichment [39, 40]. As we have previously shown that the number of PA-specific CD8+ T cells (both naïve precursor and activated) was higher than NP-specific CD8+ T cells from day 0 to day 8 postinfection [39], we decided to focus on PA-specific CD8+ T cells in our study. We found that the number of PA-specific naïve precursors was similar between the different groups of mice (Fig. 3A). Unfortunately, our preliminary studies had shown that CD80/86 KO mice had a lower starting precursor frequency (half of WT); therefore, we did not use these mice for these comparative in vivo studies. We also reasoned that CD28 KO mice might have a similar reduction in the frequency of influenza-specific precursors, thereby seriously jeopardizing any interpretation of subsequent reactivity; therefore, CTLA4Ig mice, with a similar starting precursor frequency of influenza-specific CD8+ T cells to WT mice, were chosen for this study.

image

Figure 3. Number of PA-specific CD8+ T cells in infected mice. (A) The total number of naïve PA precursors was determined in uninfected littermates treated with 0.5 mg GK1.5 1 day before harvest. Data are shown as mean + SEM of n = 15 (WT) and n = 6 (CTLA4Ig Tg) pooled from two independent experiments. (B) To deplete CD4+ T cells, mice were treated with 0.5 mg GK1.5 Ab 1 day before and 1 day after intranasal infection with 104.5 pfu of Mem71 virus. Total LNs and spleen were harvested from infected mice at day 8 postinfection to determine the total number of PA-specific CD8+ T cells. Data are shown as mean + SEM of n = 8, 6, 14, 7, and 12 for WT, CTLA4Ig Tg, GK1.5-treated WT, GK1.5-treated CTLA4Ig Tg, and naïve mice respectively. Data were pooled from two independent experiments. ns = not significant; *p < 0.05, Mann–Whitney U test.

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At day 8 postinfection, as expected from previous reports [6, 14, 16], we found that CD4+ T-cell deficiency did not lead to a reduction in PA-specific CD8+ T cell numbers (17.5 ± 3.1 versus intact WT mice 13.0 ± 1.4; p = 0.52; Fig. 3B). CD4+ T-cell deficiency also had no effect on PA-specific CD8+ T cell numbers in CTLA4Ig tg mice (6.3 ± 1.5 versus intact CTLA4Ig tg mice 7.5 ± 1.2; p = 0.73). In contrast, in the absence of CD4+ T cells, we found that the number of PA-specific CD8+ T cells was threefold less for CTLA4Ig Tg mice when compared to WT mice (p < 0.05) (Fig. 3B). This showed that the optimal generation of PA-specific CD8+ T cells was dependent upon direct CD28 costimulation of CD8+ T cells. We then looked at the activation of PA-specific CD8+ T cells by staining for CD44. We found that CTLA4Ig Tg mice had more PA-specific CD8+ T cells that were CD44low than those found in WT mice (Fig. 4A), that is, fewer specific cells were activated during CD28 blockade. When we looked at the proliferation of activated PA-specific CD8+ T cells by CD44 staining and BrdU incorporation, the percentage of PA-specific CD8+ T cells that were CD44+BrdU+ in CTLA4Ig Tg mice was reduced when compared to WT mice (52.8 ± 8.2% and 70.3 ± 3.1% respectively; p < 0.05) (Fig. 4A and B). The total number of CD44+BrdU+ PA-specific CD8+ T cells was threefold less in CTLA4Ig Tg mice than in WT mice (3.9 ± 1.2 and 12.4 ± 2.2 × 103 cells, respectively; p < 0.005) (Fig. 4C). The most dramatic effect of CD28 blockade was in the lower numbers of activated (CD44+) CD8+ T cells and absolute numbers of proliferated cells rather than the percentage. This suggested that the main effect of CD28 was on initial activation.

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Figure 4. Activation and proliferation of PA-specific CD8+ T cells at day 8 postinfection. To deplete CD4+ T cells, all mice were treated with 0.5 mg GK1.5 Ab 1 day before and 1 day after intranasal infection with 104.5 pfu of Mem71 virus. Mice were given BrdU i.p. on day 6 postinfection, together with BrdU in drinking water on day 6 and day 7 postinfection. Total LNs and spleen were harvested at day 8 postinfection. (A) Activ-ation and proliferation of PA-specific CD8+ T cells were determined by staining for CD44 and BrdU, respectively. (B) The percentage and (C) absolute number of PA-specific CD8+ T cells was determined by counting the entire tetramer-specific population. Data are shown as mean + SEM of n = 14, 7, and 9 for WT, CTLA4Ig Tg, and naïve mice, respectively and were pooled from two independent experiments. Statistical analysis was performed using Mann–Whitney U test.

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CD28 is critical for early activation

To explore further how CD28 costimulation affects the early development of antigen-specific CD8+ T cells responses, we looked at an earlier time point viz. 5 days postinfection, even though the numbers were expected to be low [39]. We found that the total number of PA-specific CD8+ T cells in CTLA4Ig Tg mice was significantly lower than those in WT mice (Mean ± SEM: 62 ± 3 and 134 ± 15 cells respectively; p < 0.005). Although the percentage of PA-specific CD8+ T cells that were CD44+BrdU+ in CTLA4Ig Tg mice was not significantly less than in WT mice (13 ± 4% and 18 ± 3% respectively; p = 0.38), the absolute numbers of CD44+BrdU+ PA-specific CD8+ T cells in CTLA4Ig Tg mice were significantly reduced (8.0 ± 2.4 and 25.3 ± 3.8 cells respectively; p < 0.005) (Supporting Information Fig. 2).

Even though the difference was statistically significant, the above numbers of endogenous PA-specific CD8+ T cells at this early time point were so low that the results warranted confirmation in some other way. Therefore, we took advantage of the X31-OVA influenza virus that expresses the OVA epitope in the neuraminidase stalk and adoptively transferred CellTrace Violet-labeled OT-I cells to accurately map the contribution of costimulation to amplification of antigen-specific T cells. We infected WT or CTLA4Ig Tg mice with X31-OVA before adoptively transferring CellTrace-labeled OT-I into infected mice. The OT-I cells were harvested 5 days posttransfer and proliferation profiles were analyzed. We found that the numbers of OT-I cells that proliferated were lower for the CTLA4Ig Tg mice than for WT in both spleen (Fig. 5A) and LN (Fig. 5B). Therefore, the results obtained with PA-specific CD8+ T cells and OT-I cells indicate that CD28 costimulation plays a critical role in the early activation and expansion of CD8+ T-cell responses in vivo.

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Figure 5. Proliferation of OT-I cells in mice infected with X31-OVA. WT and CTLA4Ig Tg mice depleted of CD4+ T cells were infected with X31-OVA. CellTrace-labeled OT-I cells were adoptively transferred into the mice 1 day after infection. Spleen and mediastinal LNs were harvested 5 days after transfer of OT-I cells. The absolute number of OT-I cells at each division was determined for (A) spleen and (B) mediastinal LN using counting beads. Data are shown as mean + SEM of WT (n = 5), CTLA4Ig Tg (n = 5), and naïve mice (n = 2) and are representative of two experiments.

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CD80/86 on DC are important for the activation of “helpless” CD8+ T cells in vitro

Next, we sought to determine which costimulatory molecule was important for which cell type viz. CD8+ T cells versus DCs. As a model of activation of naïve CD8+ T cells, we used an in vitro MLR between CD8+ T cells and DCs. We found that CD8+ T cells isolated from CD80/86 KO mice were able to proliferate as well as WT (Fig. 6A). However, proliferation of BALB/c CD8+ T cells was reduced threefold if incubated with CD80/86 KO DCs compared with that incubated with WT DCs (p < 0.0005, Fig. 6B). Therefore, the loss of CD80/86 on DCs but not on CD8+ T cells is critical for the priming CD8+ T cells.

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Figure 6. MLR using CD8+ T cells or DCs from various mice. (A) CD8+ T cells from WT or CD80/86 KO mice were incubated with DCs from BALB/c mice and allogeneic proliferation was measured 4 days later by thymidine incorporation. (B) Conversely, DCs from WT B6 mice or CD80/86 KO mice were incubated with CD8+ T cells from BALB/c mice and allogeneic proliferation was measured 6 days later by 3H-thymidine incorporation. These incubation times were previously determined to be optimal. Data are shown as mean + SEM of n = 5 and 10 replicates for CD8+ T cells and DCs respectively and are representative of two independent experiments. Statistical analysis was performed with Mann–Whitney U test.

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Discussion

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

The generation of primary CD8+ T-cell responses to noninflammatory Ag (e.g. soluble proteins, cell-associated Ag) largely requires help from CD4+ T cells [41]. In contrast, certain microbial infections (but not all) can elicit CTL responses in the absence of help. These include intracellular bacteria like Listeria [42] and viruses like influenza, LCMV, ectromelia virus, vesicular stomatitis virus, and HIV [16, 27, 43-45]. Although direct activation of DC by the microbial infection would seem an attractive explanation [44, 46], many infections that activate DCs cannot bypass help. For example, herpes simplex virus activates DCs yet CTL induction is largely dependent on CD4+ T cells [21, 47]. We have recently shown that upregulation of CD40L by DCs can explain why influenza but not herpes simplex can bypass help for CTL induction [21]. Interestingly, in humans with HIV infection (whose help is defective), herpes simplex is among the list of common opportunistic infections, whereas susceptibility to influenza is not significantly increased [48-50].

The role of costimulation and DC licensing by CD4+ T cells is now well established. The effect of a lack of costimulation on CD8+ T-cell responses has often been reported [24, 30, 51-55], but most of these studies were performed in the presence of CD4+ T cells. Therefore, it is difficult to distinguish whether reduced CTL responses were due to a lack of costimulation for CD8+ or CD4+ T cells or both. For example, although Lumsden et al. [30] found that CD28 blockade led to reduced anti-influenza CD8+ T-cell responses, it was not evident whether CD28 engagement was required by the CD4+ T cells (which then in turn affected CD8+ T cells) or was directly required by the CD8+ T cells. There are far fewer studies on the role of costimulation on CD8+ T cells directly (i.e. in the absence of CD4+ T cells). Nevertheless, several studies including our own have shown that disruption of CD40L costimulation can still hamper induction of CD8+ T-cell responses in the absence of CD4+ T-cell help [21, 56-58].

It is important that all groups of mice have similar starting numbers of antigen-specific CD8+ T-cell precursors to ensure that any interpretation of subsequent reactivity is accurate. We found that CD80/86KO mice have half the number of PA-specific CD8+ T-cell precursors as compared with WT mice. CD28/B7 costimulation has been shown to be important for thymic development of T cells [59-61], which explains the decrease in PA-specific CD8+ T-cell precursors in the CD80/86KO mice. Immunoglobulin molecules, however, do not penetrate the thymus efficiently [62-64]. This would explain why CTLA4Ig might have little effect on thymic development and hence precursor frequency.

We found that in the absence of CD4+ T-cell help but intact costimulation pathways, strong CD8+ T-cell responses were elicited in response to influenza virus and these efficiently cleared influenza on day 8 postinfection. These results concur with previous studies in MHC class II-deficient animals [6, 15, 16] and in mice treated with depleting anti-CD4 Ab [14, 17]. Our data suggest that whereas lack of help had little effect, the lack of direct CD28 costimulation for CD8+ T cells resulted in low numbers of PA-specific CD8+ T cells and delayed viral clearance.

The early clearance of influenza (before day 10) is mainly mediated by CD8+ T cells [65]. This timing is also reflected in the lack of any difference in weight loss between B-cell deficient and wild-type mice until after day 9 of influenza infection [66]. Our results concur with previous findings by Lumsden et al. [30], where the loss of CD28 costimulation resulted in the decrease in IgG titres. However, in our studies, the presence or absence of CD4+ T-cell help did not alter the IgG titres in mice deficient in CD28 costimulation. Therefore, the difference in viral clearance between the costimulation-deficient helper-deficient mice and helper-deficient mice is unlikely to be a reflection of the anti-influenza antibodies induced in the mice. We believe that the early differences in levels of viral clearance demonstrated on day 8, postinfection were attributable to differences in CD8+ T-cell response magnitude within the different groups of infected mice.

Our findings revealed that loss of CD28 costimulation in “helpless” mice resulted in the inadequate priming and activation of influenza-specific CD8+ T cells. This was demonstrated by the early reduction in numbers of activated PA-specific CD8+ T cells and the inability of OT-I cells to expand efficiently. Whereas the absolute numbers of activated proliferated cells were dramatically reduced with CD28 blockade, the proportion was either unchanged or only slightly reduced. This suggested that once the cells were activated, they proceeded to proliferate as normal. CD28 is involved during the very early stages of T-cell activation, when its binding to the CD80 and CD86 molecules on DC during TCR engagement, leads to organization of the immunological synapse [67]. That CD28 acts early has been previously described at least in vitro [68, 69]. For example, CD28 costimulation can reduce the lag time for T cells to first divide from 67 to 52 h, but had little effect on subsequent division times [69]. Moreover, CD28 costimulation promotes the survival of T cells by upregulating Bcl-xl [70] and IL-2 [71]; indeed, CD28 was obligatory for IL-2 production by human CD8+ T cells [72]. The lack of CD28 costimulation also upregulates apoptotic molecules like Bim [73]. Therefore, the decrease in CD44+BrdU+ PA-specific CD8+ T cells observed at day 8 could be due to either reduced activation or increased death.

Although only primary responses are dealt with in our study, others have shown that CD28 on memory CD8+ T cells remained important. For influenza, adoptive transfer into CD80/86 KO mice showed that CD28 was important during memory responses [74]; this requirement was independent of CD4+ T cells. For certain other viruses like herpes simplex, vaccinia, and gammaherpesvirus-68, recall CD8+ T-cell responses were suboptimal in the absence of CD28 costimulation [74, 75].

In accordance to previous reports [14, 17], we found no difference in the primary anti-influenza CD8+ T-cell numbers nor viral clearance after CD4+ T-cell depletion. For noninfectious antigens, CD4+ help “licenses” DCs via CD40L to elicit primary CTLs [2, 3, 76] and to elicit CD8+ T-cell memory [9]. However, for certain infectious diseases that can prime “helpless” CTLs (like Listeria and influenza), CD4+ T-cell help remains important for the generation of memory [6, 7]. However, for the generation of primary CD8+ T-cell responses per se for influenza, our data (on viral clearance or numbers of PA-specific T cells being unimpaired after CD4+ depletion) and those previously published [6, 66] indicate that CD4+ T-cell help is unimportant. In support of this, we found that CTLA4Ig Tg mice that were depleted of CD4+ T cells had viral titers similar to CTLA4Ig mice with intact CD4+ T cells (2.92 ± 0.44 and 2.72 ± 0.35, respectively; p = 0.4) (As expected, WT mice completely cleared the virus by day 8 whether CD4+ T cells were present or not). We opine that for infectious pathogens that can “license” DCs without help [3, 21], optimal primary CTL induction requires costimulation through engagement of CD28 on CD8+ T cells, as supported by recent findings by Dolfi et al. [73] and this may be more important than other signals provided by CD4+ T cells. In addition, our in vitro experiments concur with recent findings [73] that expansion of CD8+ T cells is not due to the lack of CD80/86 on the CD8+ T cells themselves but on DCs instead. Recently, belatacept, a high affinity variant of CTLA4Ig, has just been approved by the Food & Drug Administration for kidney transplantation [33]. Even though most adults have already experienced most viral infections and as such the immune responses mounted would not be primary ones, it would be interesting to monitor whether the use of this drug will further increase susceptibility to influenza or other infections in transplant patients, with or without other immunosuppressants that reduce helper function.

Materials and methods

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

Mice

C57Bl/6 mice were used as wild-type control mice. CTLA4Ig Tg mice, which express the fusion protein CTLA4Ig under the rat insulin promoter [77], originally made in B6.C-H2-Kbm1 mice were backcrossed to C57Bl/6 so that they were homozygous for H-2Kb. The impact of CTLA4Ig transgenesis on immune responses has been previously demonstrated [77, 78]. Mice with disruption to CD80/86 gene (CD80/86 KO) [79] and BALB/c mice were used as allogeneic responders or stimulators in MLR experiments. All mice used were 6–12 weeks old and maintained in specific pathogen-free conditions at the Walter and Eliza Hall Institute. Experiments were performed according to the guidelines of the Institute's Animal Ethics Committee.

Influenza virus and intranasal infection

The type A strain of influenza virus used in this study was a genetic reassortant of A/Memphis/1/71 (H3N2) × A/Bellamy/42 (H1N1) referred to as Mem71 (H3N1) virus. Mem71 virus was used to study viral clearance and primary anti-influenza CTL response in WT and CTLA4Ig Tg mice. Recombinant influenza virus, A/HKx31-OVA (X31-OVA) virus expressing the OVA257–263 peptide, described previously by Jenkins et al. [80], was used to study OVA-specific responses of adoptively transferred OT-I cells in WT and CTLA4Ig Tg mice. All mice were treated i.p. with 0.5 mg of the anti-CD4 monoclonal Ab (mAb) GK1.5 1 day before and after infection. Penthrane-anesthetized mice were infected i.n. with 104.5 or 104.0 pfu of Mem71 or X31-OVA, respectively in a volume of 30 μL.

Assay for infectious virus

Mice were sacrificed at 5 and 8 days postinfection. The lungs were collected in 5 mL of RPMI media containing penicillin and streptomycin. Lungs were sieved and after centrifugation at 1000g for 5 min, the supernatant was stored at –70oC. The titer of virus in the lungs of infected mice was determined by plaque assay [81].

ELISA

ELISA was performed using serum from infected mice as previously described [82]. Briefly, purified, inactivated whole Mem71 virus was disrupted for 10 min at room temperature in a 1/10 dilution of lysis buffer (0.5% Triton-X100, Sigma Aldrich, St. Louis, MO, USA)/0.6 M KCl (Merck, Darmstadt, Germany)/0.05 M Tris HCl pH 7.5 (Invitrogen, Carlsbad, CA, USA) and adjusted to 5 μg/mL in PBS. Fifty microliters of diluted antigen were added into wells of flat-bottom 96-well polyvinyl chloride microtiter plates and incubated overnight at room temperature. Antigen was removed and wells were blocked with PBS containing 10% BSA (Sigma Aldrich) for 1 h at room temperature. Blocking solution was removed and plates were washed in PBS containing 0.05% Tween 20. Various dilutions of serum from infected mice were added to the wells and incubated overnight at room temperature. The plates were washed with PBST. Horseradish peroxidase conjugated anti-mouse IgM or IgG (Southern Biotechnology Associates, Birmingham, AL, USA) was added and plates were incubated at room temperature for 1 h. Wells were developed using 0.1 mg/mL 3,3′,5,5′–tetramethylbenzidine (Sigma Aldrich) in 0.1 M sodium acetate pH 5.5 containing 0.006% hydrogen peroxide. The reaction was stopped using 0.5 M sulphuric acid and absorbance was read at 450 nm. The Ab titer was expressed as the reciprocal of the dilution of serum giving an OD 2× above background.

Tetramer enrichment for influenza-specific CD8+ T cells

Influenza-specific CD8+ T cells were enriched using the method recently described by La Gruta et al. [39]. Briefly, mice were killed at 0, 5, or 8 days postinfection. Single cell suspensions were prepared from a pool of all major lymph nodes and spleen. Cells were resuspended in Fc block (1% mouse serum, 1% rat serum, and 40 μg/mL 2.4G2, FcγIII/II mAb) with PE-conjugated tetrameric complexes of the H-2Db influenza PA224–233 (SSLENFRAYV) peptide [83] and incubated at room temperature for an hour in the dark. The cells were washed with cold sorter buffer (ethylenediaminetetraacetic acid (EDTA) Balanced Salt Solution with 2% heat-inactivated FCS) and resuspended in 400 μL of the same buffer. One hundred microliters of anti-PE magnetic beads (Miltenyi Biotech, Auburn, CA, USA) were added to the cell suspension and incubated on ice for 30 min. The cells were washed twice and resuspended in 3 mL of sorter buffer and passed over an LS magnetic column (Miltenyi Biotech). Initial effluent was passed over the column again, followed by 3 × 3 mL washes of the column with cold sorter buffer. The column was removed from the magnet and again washed with 5 mL of cold sorter buffer. The cells were then incubated on ice for 30 min with an Ab cocktail of anti-CD3e- PerCP-Cy5.5 (clone 145-2C11), anti-CD8-allophycocyanin-Cy7 (clone 53-6.7), anti-CD44-PE-Cy7 (clone IM7) or anti-CD62L-PE-Cy7 (clone MEL14), anti-CD4 (clone RM4-5)/CD45R (clone RA3-6B2)/F4/80 (clone BM8)-allophycocyanin, and additional CD11c (clone HL3)/CD11b (clone M1/70)-allophycocyanin when looking at naïve precursors. Cells were washed and kept on ice for further staining for BrdU incorporation.

BrdU incorporation

Total LN and spleen were harvested at either day 0, day 5, or day 8 postinfection. Mice were given 100 mg/kg of BrdU (Sigma Aldrich) i.p. 2 days before each harvest and 0.5 mg/mL of BrdU with 10 mM glucose in the drinking water over the 2-day period. Single cell suspensions were prepared, subjected to tetramer enrichment, and staining for surface markers was carried out as previously described. Intracellular staining for BrdU was then performed according to the protocol outlined in the BrdU Flow Kit (BD Biosciences. San Jose, CA, USA). Cells were stored in PBS supplemented with 2% heat-inactivated FCS and 0.02% azide (FACS buffer) before analysis by flow cytometry.

Adoptive transfer of OT-I cells

Total LNs were harvested from 6–12 week old OT-I/Ly5.1 transgenic mice and single cell suspension prepared. OT-I cells were then enriched by magnetic bead depletion with rat anti-mouse mAb M1/70 (anti-MAC-1), Ter119 (anti-erythrocytes), F4/80 (anti-Mac-3), RB6-8C5 (anti-Gr-1), M5/114 (anti-MHC Class II), and GK1.5 (anti-CD4), followed by goat anti-rat IgG-coupled magnetic beads (BioMag, Qiagen, Hilden, Germany). The nonbead-bound fraction enriched CD8+ T cells were washed once and resuspended in PBS. The cells were then stained using the CellTrace Violet according to manufacturer's instructions (Invitrogen). Cells were washed and resuspended in PBS. OT-I cells were adoptively transferred by i.v. injection of 0.5 × 106 Vα2+CD8+ T cells into X31-OVA-infected, CD4+ T-cell depleted mice.

Analysis of OT-I cell proliferation

Spleen, mediastinal LNs, and lungs from X31-OVA infected mice were harvested at day 5 post-transfer of OT-I cells. OT-I CD8+ T cells were enriched from organs of experimental mice by magnetic depletion as described above. The enriched cells were incubated on ice for 30 min with an Ab cocktail of anti-Vα2-biotin (clone B20.1)/Streptavidin-FITC, anti-CD8-allophycocyanin-Cy7 (clone 53-6.7), and anti-CD45-1-allophycocyanin (clone A20). Cells were washed and resuspended in FACS buffer containing 1 μg/mL of propidium iodide (PI) to exclude dead cells during flow cytometry analysis. A total of 1 × 104 Calibrite allophycocyanin beads (BD Biosciences) were added to each sample prior to flow cytometry to allow quantification of OT-I cells.

MLR

Total LNs were harvested from 6–12 week old WT, CD80/86KO, or BALB/c mice and single cell suspensions were prepared. CD8+ T cells were enriched using anti-CD8 PE conjugated Ab (BD Bioscience) and anti-PE magnetic MACS beads (Miltenyi Biotech). DCs from spleens of WT, CD80/86KO Tg, or BALB/c mice were enriched using protocol described by Vremec et al. [84]. Briefly, spleens were digested for 20 min at room temperature with collagenase–DNAase and then were treated with ethylenediaminetetraacetic acid (EDTA). Light density cells were selected by centrifugation in a 1.077 g/cm3 Nycodenz medium (Rodelϕkka, Oslo, Norway). CD11c+ DCs were then isolated using anti-CD11c FITC-conjugated Ab (BD Bioscience) and anti-FITC magnetic MACS beads (Miltenyi Biotech). All enriched cells were washed and resuspended in RPMI supplemented with 10% heat-inactivated FCS and 0.05 mM 2-mercaptoethanol (Sigma Aldrich). A total of 1 × 105 CD8+ T cells were then cocultured with WT or CD80/86KO DCs at 1:1 ratio for 4 or 6 days. To determine blastogenesis/proliferation, tritiated thymidine was added 16 h before harvest.

Data analysis

Flow cytometric data were analyzed using FlowJo software (version 3.6.1; Tree Star Inc., Ashland, OR, USA). The nonparametric Mann–Whitney U test was used to determine statistical significance (Prism 4, GraphPad Software Inc, San Diego, CA, USA).

Acknowledgments

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

We thank Dr. Ian Barr and Chris Durrant from WHO Collaborative Centre for Reference and Research on Influenza, Melbourne, for help in virus culture. This work was supported by National Health & Medical Research Council of Australia Program (#516700) and Project grants (#575543, 637324), Juvenile Diabetes Research Foundation grants (#447718), Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIIS, Pfizer Senior Research Fellowship (S. J. T.), Sylvia and Charles Viertel Fellowship (G. T. B.), and DSO National Laboratories Scholarship, Singapore (S. G. K. S.).

The authors declare no financial or commercial conflict of interest.

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  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. References
  9. Supporting Information
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Abbreviations
NP

influenza virus nucleoprotein

PA

influenza virus acid polymerase

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
eji2290-sup-0001-s1.pdf1970KSupplementary Data

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