Longitudinal Analysis of the Human T Cell Response during Acute Hantavirus Infection

Therese Lindgren; Clas Ahlm; Nahla Mohamed; Magnus Evander; Hans-Gustaf Ljunggren; Niklas K Björkström

doi:10.1128/JVI.05548-11

. 2011 Oct;85(19):10252–10260. doi: 10.1128/JVI.05548-11

Longitudinal Analysis of the Human T Cell Response during Acute Hantavirus Infection ^{^▿}^†

Therese Lindgren ¹, Clas Ahlm ¹, Nahla Mohamed ¹, Magnus Evander ², Hans-Gustaf Ljunggren ³, Niklas K Björkström ^3,^*

PMCID: PMC3196424 PMID: 21795350

Abstract

Longitudinal studies of T cell immune responses during viral infections in humans are essential for our understanding of how effector T cell responses develop, clear infection, and provide long-lasting immunity. Here, following an outbreak of a Puumala hantavirus infection in the human population, we longitudinally analyzed the primary CD8 T cell response in infected individuals from the first onset of clinical symptoms until viral clearance. A vigorous CD8 T cell response was observed early following the onset of clinical symptoms, determined by the presence of high numbers of Ki67⁺CD38⁺HLA-DR⁺ effector CD8 T cells. This response encompassed up to 50% of total blood CD8 T cells, and it subsequently contracted in parallel with a decrease in viral load. Expression levels of perforin and granzyme B were high throughout the initial T cell response and likewise normalized following viral clearance. When monitoring regulatory components, no induction of regulatory CD4 or CD8 T cells was observed in the patients during the infection. However, CD8 as well as CD4 T cells exhibited a distinct expression profile of inhibitory PD-1 and CTLA-4 molecules. The present results provide insight into the development of the T cell response in humans, from the very onset of clinical symptoms following a viral infection to resolution of the disease.

INTRODUCTION

Hantavirus infection in humans causes hemorrhagic fever with renal syndrome (HFRS), a disease characterized by severe vascular symptoms and sporadic mortalities (39). Hantaviruses have been documented to infect endothelial cells, causing a viremia that typically clears within the first 2 weeks after symptom debut (10, 29). Previous studies have described high frequencies of hantavirus-specific memory CD8 T cells in previously infected individuals (21, 38). These findings, together with the absence of evidence for hantavirus persistence or symptomatic reinfection in humans, have suggested a role for CD8 T cells in the generation of protective long-lasting immunity. However, the primary antiviral CD8 T cell response, which is likely responsible for viral clearance and T cell memory formation, is not well characterized in this or similar human diseases.

Much of our present knowledge of CD8 T cell responses to acute viral infections originates from experimental model systems in which mice have been infected with viruses such as lymphocytic choriomeningitis virus (LCMV) or vaccinia virus (VV) (6, 11, 25). In these model systems, infection induces rapid expansion and activation of antigen-specific cells and the emergence of large numbers of activated effector CD8 T cells (6, 25). The expanding population of effector cells is highly susceptible to apoptosis, and the response culminates soon after viral clearance. At that point, the virus-specific CD8 T cell population contracts, leaving behind long-lived memory cells (25). Human antiviral CD8 T cell responses have been analyzed primarily in settings of chronic infection, such as hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus 1 (HIV-1), and Epstein-Barr virus (EBV) infections (19, 22, 33, 37). In many respects, these disease settings differ from those of the murine models classically used to study viral infection; the latter often lead to rapid T cell-dependent clearance of viral infection. Recently, however, effector CD8 T cell responses were analyzed longitudinally in human recipients of vaccines containing live yellow fever and smallpox viruses (1, 23). The results have provided insights into the generation of human functional CD8 T cell immunity from the time of vaccination.

A Puumala hantavirus outbreak in northern Sweden (5, 28) has now enabled us to longitudinally study the antiviral T cell response in a setting of a natural viral infection. Briefly, clinical samples were prospectively collected, and the emerging effector CD8 T cell responses were documented in 15 patients from their first presentation at the emergency unit with acute symptoms, during the entire disease period, and until the viral infection resolved. Furthermore, we characterized inhibitory immunoregulatory components of the response that may fulfill the purpose of balancing the activated effector CD8 T cells. The results of the study are presented here.

MATERIALS AND METHODS

Study design and patient material.

A prospective study design was used. Peripheral blood was collected from 15 patients with acute hantavirus infection. Patients included in the study met the following inclusion criteria: (i) verified diagnosis of acute hantavirus infection via immunofluorescence test for hantavirus-reactive IgM and IgG antibodies from sera (10); (ii) access to a first sample drawn during presentation at the emergency clinic, i.e., at around day 6 of clinical symptoms; and (iii) access to two sequential samples of peripheral blood during the acute phase and one follow-up sample during the convalescent phase (average, day 60). Written and oral informed consent was obtained from all included patients. The study was approved by the regional ethics committee of Umeå University (approval number 04-113 M). Controls were 15 uninfected blood donors that had been matched with the infected patients with respect to age and sex. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood collected in CPT tubes (BD Biosciences) and cryopreserved in liquid nitrogen in 90% human albumin (Octo-Pharm), 10% dimethyl sulfoxide (DMSO) (WAK-Chemie Medical), and 50 IU/ml heparin (LEO Pharma) for later analysis. Standard procedures, including lymphocyte counts, were used to obtain clinical data.

Antibodies for flow cytometry.

Commercially available monoclonal antibodies (MAbs) against the following proteins were used: CD3, CD4, CD8, CD14, CD25, CD28, CD38, CD45RA, CD127, perforin, granzyme B, HLA-DR, Ki67, CTLA-4, PD-1, CCR7, and FoxP3. For detection of biotinylated MAbs, streptavidin Qdot 605 or streptavidin Pacific Blue was used.

Flow cytometry.

Cell surface staining of PBMC was performed as previously described (4). For intracellular staining of Ki67, CTLA-4, perforin, and granzyme B, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences). For intracellular staining of FoxP3, cells were permeabilized with FoxP3 fixation/permeabilization concentrate and Dilutent (eBioscience). A LIVE/DEAD Fixable Aqua dead cell stain kit (Invitrogen) was used to exclude dead cells from the analyses. Polystyrene single-stained beads (BD Biosciences) were used for compensation purposes. Samples were acquired on a CyAN ADP 9-color flow cytometer (Beckman Coulter) with a 25-mW 405-nm laser, a 20-mW 488-nm laser, and a 25-mW 635-nm laser. Software-based compensation was performed using the compensation platform in FlowJo software version 8 (Treestar).

Quantitative RT-PCR for hantavirus.

RNA from patient plasma (or serum when plasma was not available) was extracted using a QIAamp viral RNA kit (Qiagen) followed by cDNA synthesis with a GoScript reverse transcriptase (RT) kit (Promega) according to the manufacturer's instructions. The real-time RT-PCR was performed as previously described, using ABI Prism 7900HT sequence detection system 2.0 (Applied Biosystems) (10). Numbers of viral RNA copies per milliliter were determined twice for every time point, and the mean of the results of the two separate experiments was used.

Statistical analysis.

Unless otherwise indicated in the figure legends, the following statistical tests were used. For multiple group comparisons of matched groups, two-way repeated tests (by two-way analysis of variance [ANOVA]) were used with Bonferroni posttests. For single comparisons of matched groups, two-tailed paired Student t tests were performed. For single comparisons of unrelated groups, two-tailed unpaired Student t tests were performed. Statistical analyses were performed using GraphPad software version 5.0.

RESULTS

Identification of responding CD8 T cells during acute hantavirus infection in humans.

Fifteen patients with acute hantavirus infection, sampled longitudinally during the acute (average, days 6 and 10) and the convalescent (average, day 60) stages of disease, and 15 uninfected controls were included in the study. First, total lymphocyte counts and numbers of CD8 as well as CD4 T cells were determined. Minor changes were observed in total lymphocyte counts as well as CD8 and CD4 T cell numbers during the infection (see Fig. S1 in the supplemental material). Early after infection, CD8 T cells were increased in numbers in the patients compared to CD4 T cell numbers. At day 60 after infection, these numbers had started to normalize to the levels observed in uninfected individuals (see Fig. S1 in the supplemental material). We subsequently evaluated the expression of Ki67, CD38, and HLA-DR on CD8 T cells. A substantial number of CD8 T cells expressed Ki67, CD38, and HLA-DR during the acute phase of infection but not at day 60 after symptom debut or in the uninfected controls (Fig. 1 a). On average, 54%, 51%, and 30% of total CD8 T cells expressed Ki67, CD38, and HLA-DR, respectively, at day 6 after symptom debut (Fig. 1b). Next, when evaluating the peak of the response, we found that 13 of 15 patients had higher levels of Ki67⁺, CD38⁺, or HLA-DR⁺ cells at day 6 than at day 10. Analysis of coexpression patterns of Ki67, CD38, and HLA-DR revealed that levels of triple-positive CD8 T cells peaked at day 6 with an average of 26% total CD8 T cells. In some patients, however, effector Ki67⁺CD38⁺HLA-DR⁺ CD8 T cells represented up to 50% of total CD8 T cells. The size of the Ki67⁺CD38⁺HLA-DR⁺ CD8 T cell subset decreased by day 10, and the subset was virtually undetectable at day 60 (Fig. 1c). In absolute numbers, Ki67⁺CD38⁺HLA-DR⁺ CD8 T cells represented 240 blood cells/μl at day 6 but only 70 cells/μl at day 10 (Fig. 1d). To compare the kinetics of responding CD8 T cells to viral burden, we quantified the viral load in all patients at multiple time points during the acute phase. The viral load peaked 5.4 days after symptom debut with, on average, 107,000 viral copies/ml (Fig. 1e and data not shown). From day 7 after symptom debut, the viral load declined in all patients; at day 10, a majority of the patients had no detectable virus, and no virus was found in infected patients from day 16 after symptom debut and thereafter (Fig. 1e and data not shown). Interestingly, the decline observed in effector CD8 T cells coincided with the decline in viral load and clearance of the virus from the infected patients.

Fig. 1. — Identification of effector CD8 T cells after hantavirus infection. (a) Expression of Ki67, CD38, and HLA-DR on CD8 T cells from one representative hantavirus-infected patient at days 6, 10, and 60 after symptom debut and from one representative uninfected control. (b) Frequency of Ki67, CD38, and HLA-DR expression on CD8 T cells summarized for hantavirus-infected patients and uninfected controls (n = 15 for all groups; ***, P < 0.0001) (box plots show median, 25th and 75th percentiles, and minimum and maximum). UC, uninfected controls. (c) Frequency of Ki67, CD38, and HLA-DR coexpression on CD8 T cells as determined with Boolean gating summarized for hantavirus-infected patients and uninfected controls (n = 15 for all groups; ***, P < 0.001) (bars represent standard errors of the means [SEM]). (d) Absolute numbers of Ki67⁺CD38⁺HLA-DR⁺ CD8 T cells from patients with hantavirus infection measured over time (n = 15; bars represent SEM). (e) Viral load and frequency of Ki67⁺CD38⁺HLA-DR⁺ CD8 T cells measured over time during acute and convalescent phases shown for three representative hantavirus-infected patients.

When extending our analysis to CD4 T cells, we did not detect Ki67⁺CD38⁺HLA-DR⁺ cells early after symptom debut in any of the infected patients. However, both an increase in Ki67-positive cells and a trend toward higher CD38 expression were evident, although no differences were apparent in levels of HLA-DR expression in comparisons of the acute with the convalescent phase of infection and the uninfected controls (see Fig. S2A and S2B in the supplemental material).

These data demonstrate that the primary effector CD8 T cell response occurs rapidly and vigorously, encompassing (at the peak of response) up to 50% of all CD8 T cells. Thereafter, the response decreases, paralleled by a decline in viral load and clearance of viremia in the infected patients.

Responding CD8 T cells display an effector phenotype.

From models of infection in mice and studies of CD8 T cells during chronic human viral infections in humans, phenotypic profiles have been established for naïve, effector, and memory CD8 T cell subsets (2, 3, 33, 43, 44). To ensure that the responding Ki67⁺CD38⁺HLA-DR⁺ CD8 T cells identified here represented effector CD8 T cells, we evaluated the expression patterns of CCR7, CD45RA, CD28, CD127, perforin, and granzyme B on responding (Ki67⁺) and nonresponding (Ki67⁻) CD8 T cells from patients and uninfected controls. Responding CD8 T cells, present in the patients only at days 6 and 10 after symptom debut (Fig. 1b), consistently exhibited low levels of CCR7, CD45RA, and CD127 (Fig. 2a). Additionally, the costimulatory molecule CD28 was expressed on approximately 50% of the Ki67⁺ CD8 T cells at days 6 and 10. When the expression levels of CCR7, CD45RA, CD127, and CD28 were analyzed simultaneously, using a Boolean gating strategy, more than 70% of the Ki67⁺ CD8 T cells were CCR7⁻CD45RA⁻CD127⁻CD28^+/− at day 6 after symptom debut (Fig. 2b). This phenotype was distinct from that of Ki67⁻ CD8 T cells at day 6 after infection as well as from the phenotype of CD8 T cells from uninfected controls (Fig. 2b). Furthermore, the frequency of perforin- and granzyme B-expressing cells was high in total CD8 T cells during the acute phase and then normalized at day 60 (Fig. 2c). When the responding CD8 T cells were specifically evaluated, >90% coexpressed perforin and granzyme B at day 6 after symptom debut and at a significantly higher level than nonresponding CD8 T cells (Fig. 2a and d). Similarly, CD4 T cells tended toward higher levels of effector molecules early after symptom debut (see Fig. S2C in the supplemental material). Collectively, these data strengthen the notion that Ki67⁺CD38⁺HLA-DR⁺ CD8 T cells are effector CD8 T cells responding to the viral infection.

Fig. 2. — Phenotype of effector CD8 T cells after hantavirus infection. (a) Expression of CCR7, CD45RA, CD28, CD127, granzyme B, and perforin on Ki67⁺ and Ki67⁻ CD8 T cells from one representative patient with hantavirus infection at day 6 after symptom debut. (b) Frequency of CCR7, CD28, CD45RA, and CD127 coexpression as determined with Boolean gating summarized for Ki67⁺ and Ki67⁻ CD8 T cells from patients with hantavirus infection at day 6 following symptom debut as well as uninfected controls (n = 15 for all groups; ***, P < 0.001) (bars represent SEM). (c) Frequency of perforin and granzyme B expression on CD8 T cells summarized for hantavirus-infected patients and uninfected controls (n = 15 for all groups; ***, P < 0.0001, **, P = 0.0010). UC, uninfected controls. (d) Frequency of perforin and granzyme B double-positive Ki67⁻ and Ki67⁺ CD8 T cells from hantavirus-infected patients at day 6 after symptom debut (n = 15 for both groups; ***, P < 0.0001). Box plots (in panels c and d) show median, 25th and 75th percentiles, and minimum and maximum.

No expansion of regulatory CD4 T cells in acute hantavirus infection.

Thus far, development of the effector T cell response during acute hantavirus infection was the focus of our analysis. To pinpoint mechanisms that could balance the effector T cell response, we assessed regulatory T cells and the effect of inhibitory receptors on responding effector T cells in the infected patients. T cells with a regulatory phenotype were defined as FoxP3⁺ CD4 or CD8 T cells that expressed high levels of CD25 and lacked expression of CD127 (Fig. 3a). In the patients, percentages of FoxP3⁺ CD4 T cells remained stable throughout the infection and were comparable to those in uninfected controls (Fig. 3a and b). Furthermore, no significant differences in absolute numbers were evident in comparisons of patients with uninfected controls (Fig. 3c). When we analyzed CD8 T cells, no FoxP3-expressing cells were detected either in the patients or in the controls (data not shown). We then evaluated the level of ongoing proliferation within the FoxP3⁺ CD4 T cell subset. On average, 20% of the FoxP3⁺ CD4 T cells expressed Ki67 early after infection (Fig. 3d and e). However, no sign of preferential proliferation was observed within the FoxP3⁺ CD4 T cell subset, since their levels of Ki67 expression were similar to those in FoxP3⁻ CD4 T cells (Fig. 3e). Clearly, CD4 T cells with a regulatory phenotype (FoxP3⁺CD25^highCD127^low) are present in peripheral blood during acute hantavirus infection in the absence of any detectable increase in percentages of regulatory CD4 T cells or preferential proliferation within this subset.

Fig. 3. — Numbers of FoxP3⁺ CD4 T cells remain unchanged during acute hantavirus infection. (a) Expression of FoxP3, CD127, and CD25 on CD4 T cells from one representative patient with acute hantavirus infection at day 6 after symptom debut. (b) Frequency of FoxP3⁺ CD4 T cells summarized for hantavirus-infected patients and uninfected controls (n = 15 for all groups). UC, uninfected controls. (c) Absolute numbers of FoxP3⁺ CD4 T cells from hantavirus-infected patients measured over time (n = 15; bars represent SEM, dashed lines represent upper and lower SEM intervals for means of FoxP3⁺ CD4 T cell numbers summarized for 15 uninfected controls). n.s., not significant. (d) Expression of FoxP3 and Ki67 on CD4 T cells from one representative patient with acute hantavirus infection at day 6 and from one uninfected control. Percentages denote relative frequencies of Ki67⁺ cells within FoxP3⁺ and FoxP3⁻ CD4 T cells. (e) Frequency of Ki67⁺FoxP3⁺ and Ki67⁺FoxP3⁻CD4 T cells summarized for hantavirus-infected patients over time and for uninfected controls (n = 15; **, P = 0.0046). UC, uninfected controls. Box plots (in panels B and E) show median, 25th and 75th percentiles, and minimum and maximum.

Transient expression of inhibitory receptors on responding T cells during acute hantavirus infection.

Expression of inhibitory receptors is yet another mechanism to ensure T cell tolerance during steady-state conditions. These receptors also regulate effector responses to pathogens. In particular, expression of inhibitory receptors has been linked to T cell dysfunction in settings of chronic viral infection (3, 13–15, 31) but has not yet been thoroughly evaluated during the very early phase of acute viral infections in humans. To address this issue in the present setting, we studied expression of PD-1 and CTLA-4 on responding T cells during acute hantavirus infection. However, no PD-1 was detected on CD8 T cells from hantavirus-infected patients or from the uninfected controls tested here (Fig. 4 a and c). On the other hand, on average 40% of total CD8 T cells expressed CTLA-4 early after symptom debut (Fig. 4b and d). CTLA-4 expression rapidly declined during the course of infection, and at day 60, few if any CD8 T cells showed signs of expressing CTLA-4. CTLA-4 appeared primarily on responding (Ki67⁺) CD8 T cells, 60% of which expressed CTLA-4 early after symptom debut (Fig. 4e). We also extended our analysis to CD4 T cells. A substantial fraction of CD4 T cells expressed CTLA-4 early after symptom debut in the patients (Fig. 5 a and b). Additionally, and distinct from the CD8 T cell results, on average 15% of total CD4 T cells also expressed PD-1 at day 6 after symptom debut (Fig. 5a and b). More than 90% of the PD-1 expressing CD4 T cells coexpressed CTLA-4 (Fig. 5b). Expression of both CTLA-4 and PD-1 (assessed longitudinally) decreased rapidly, and in patients at the convalescent stage of disease as well as in the uninfected controls, few CD4 T cells coexpressed CTLA-4 and PD-1 (Fig. 5b and e). Furthermore, all PD-1⁺CTLA-4⁺ CD4 T cells showed signs of recent or ongoing proliferation early after symptom debut in the infected patients (Fig. 5c). Similarly, more than 50% of the responding CD4 T cells coexpressed inhibitory PD-1 and CTLA-4 (Fig. 5e). Taken together, these results indicate the existence of cell-intrinsic processes that actively balance effector T cell responses during acute viral infections. These data also reveal that CD4 and CD8 T cells might utilize different inhibitory receptors to modulate effector responses during an acute viral infection.

Fig. 4. — Proliferating CD8 T cells express inhibitory CTLA-4, but not PD-1, early after hantavirus infection. (a) Expression of Ki67 and PD-1 on CD8 T cells from one representative patient with acute hantavirus infection and from one uninfected control. (b) Expression of Ki67 and CTLA-4 on CD8 T cells from one representative patient with acute hantavirus infection and from one uninfected control. (c) Frequency of PD-1 expression on CD8 T cells summarized for hantavirus-infected patients and for uninfected controls (n = 15 for all groups). (d) Frequency of CTLA-4 expression on CD8 T cells summarized for hantavirus-infected patients and for uninfected controls (n = 15 for all groups; ***, P < 0.001). (e) Frequency of CTLA-4⁺Ki67⁻ and CTLA-4⁺Ki67⁺ CD8 T cells from hantavirus-infected patients at day 6 after symptom debut (n = 15 for both groups, ***, P < 0.0001). Box plots (in panels c, d, and e) show median, 25th and 75th percentiles, and minimum and maximum. UC (in panels c and d), uninfected controls.

Fig. 5. — CD4 T cells coexpress inhibitory CTLA-4 and PD-1 during acute hantavirus infection. (a) Expression of CTLA-4 and PD-1 on CD4 T cells from one representative patient with acute hantavirus infection and from one uninfected control. (b) Frequency of PD-1 and CTLA-4 expression on CD4 T cells summarized for hantavirus-infected patients and for uninfected controls (n = 15 for all groups; ***, P < 0.0001). UC, uninfected control. (c) Expression of Ki67 on CD4 T cells expressing different combinations of CTLA-4 and PD-1 from one representative patient with acute hantavirus infection at day 6 after symptom debut. (d) Frequency of PD-1 and CTLA-4 double-positive Ki67⁻ and Ki67⁺ CD4 T cells from hantavirus-infected patients at day 6 after symptom debut (n = 15 for all groups; ***, P < 0.0001). (e) Absolute numbers of PD-1⁺CTLA-4⁺CD4 T cells from patients with hantavirus infection measured over time (n = 15; bars represent SEM). Box plots (in panels b and d) show median, 25th and 75th percentiles, and minimum and maximum.

DISCUSSION

To explore the primary human CD8 T cell response to an acute viral infection, we followed a cohort of patients with acute hantavirus infection from the very first presentation at the emergency clinic, only days after symptom debut, throughout the acute phase of infection into the convalescent phase of disease. Our results show that a primary effector CD8 T cell response developed rapidly after virus infection. The response peaked within 2 weeks after symptoms began and, at that time, encompassed up to 50% of total blood CD8 T cells and culminated in parallel with a decrease in viral load and clearance of viremia in the patients. In addition, careful monitoring of regulatory components did not reveal any marked induction of regulatory CD4 or CD8 T cells in the patients during acute infection. Instead, CD4 and CD8 T cells exhibited a distinct pattern of expression of inhibitory PD-1 and CTLA-4 early after infection.

From experimental models using mice, it is known that the primary effector CD8 T cell response during an acute viral infection includes a rapid and massive expansion of activated CD8 T cells occurring during the first 2 weeks after infection (6, 11, 25). At the peak of this response, up to 80% of all splenic CD8 T cells are specific for viruses such as LCMV or VV, proliferate, and produce gamma interferon (IFN-γ). Recent studies of humans vaccinated with yellow fever virus and smallpox vaccines showed a similar type of response (1, 23). In those studies, effector CD8 T cell numbers peaked within 2 weeks after infection. The response encompassed up to 12.5% and 40% of CD8 T cells after yellow fever virus and smallpox vaccination, respectively. In terms of time and magnitude, the levels of effector CD8 T cells that were observed early after acute hantavirus infection in the present study match or even exceed those of earlier human vaccination studies (1, 23).

Host immunity toward hantaviruses has previously been investigated primarily in patients long after the period of primary infection. Virus-specific CD8 T cells were detected in Puumala hantavirus-infected individuals up to 15 years after primary infection at frequencies similar to, or exceeding, those of influenza virus- or EBV-specific memory CD8 T cells (38). A recent study characterized memory CD8 T cell responses after Andes hantavirus infection (21). There, Andes hantavirus-specific CD8 T cells were detected ex vivo at least 13 years after acute infection, and in some individuals, 16% of total peripheral blood CD8 T cells were specific for a single Andes hantavirus epitope (21). The Andes hantavirus-specific CD8 T cells exhibited an effector memory phenotype (CD27⁻CD28⁻CCR7⁻CD127⁻) with revertant CD45RA expression. Not surprisingly, this phenotype is distinct from the phenotype of responding effector CD8 T cells seen in the study reported here (CD28^+/−CD127⁻CCR7⁻CD45RA⁻). On the other hand, CD8 T cells responding to acute Puumala hantavirus infection display a phenotype similar to that seen with the early responding cells after smallpox vaccination in humans (CD27⁺CD127⁻CCR7⁻CD45RA⁻) (23). Taken together, these outcomes highlight the phenotypic characteristics of effector and memory phases of acute human viral infections.

One limitation of the current study was the fact that we were only able to assess the effector CD8 T cell response in a non-antigen-specific way by combining three activation markers. This strategy raises the important question as to whether non-hantavirus-specific CD8 T cells might have been bystander activated by the inflammatory response associated with hantavirus infection and might thus have affected the interpretation of our results. Although this is a possibility, evidence from murine studies of LCMV infection and assessment of acute EBV infection in humans suggests that T cell receptor engagement is the principal driving force behind CD8 T cell activation (25, 33). On the other hand, chronic or latent viruses might still be reactive during an acute viral infection, providing an opportunity for T cell receptor-specific bystander activation. Indeed, as recently shown, acute Puumala hantavirus infection can reactivate EBV to boost EBV-specific memory T cell levels (35). However, this boost and the subsequent increase in EBV-specific CD8 T cell levels occurred late (on average, approximately day 20 or later after symptom debut in the infected patients). Relative to this issue, we recently studied the cytomegalovirus (CMV) status of patients with acute hantavirus infection (5). No evidence was found for CMV reactivation during acute hantavirus infection; i.e., the number of CMV-specific CD8 T cells remained unchanged during the acute phase of hantavirus infection (5). This is in line with findings from Miller and colleagues (23), who showed that EBV-, CMV-, and influenza virus-specific CD8 T cells did not contribute to the peak of the effector CD8 T cell response to smallpox vaccine. However, in similarity to results seen in a study of acute hantavirus infection (35), a low-grade EBV reactivation could be detected in the smallpox-vaccinated individuals 3 weeks after vaccination (23). Thus, it still remains a possibility that bystander-activated CD8 T cells contributed to the peak of the effector CD8 T cell response studied here. However, we find it less likely.

Regulatory T cells with immunosuppressive functions, which maintain the balance between protective and tissue-damaging effects of the immune response, have been extensively studied in chronic and persistent viral infections of humans (12, 16). In these settings, the common notion is that regulatory T cells mitigate immunopathology at the expense of decreasing the effectiveness of antigen-specific CD8 T cell responses (12, 16). Importantly, since regulatory T cells are known to become compartmentalized at the sites of viral infection and/or replication (9, 26, 32), the local effects are sometimes difficult to interpret from studies of human peripheral blood. However, hantavirus replication has been documented to occur in the vascular endothelium (29); thus, by studying T cells with a regulatory phenotype in peripheral blood, we investigated the cells from a relevant site of infection. In another form of viral hemorrhagic fever, that caused by acute dengue virus infection, patients exhibited increased numbers of regulatory CD4 T cells in peripheral blood within 1 to 10 days of the first symptoms compared to the numbers seen at later phases of the disease (20). Furthermore, in acute infection of humans by West Nile virus, a virus with the capacity to infect endothelial cells (40), numbers of regulatory CD4 T cells in peripheral blood were unchanged early after symptom development (assessed, on average, at day 15 after symptom debut) but increased 2-fold during the next 3 months (18). Since FoxP3 can be transiently expressed in nonregulatory activated CD4 T cells (41), in the current study, we used multiple phenotypic markers (FoxP3, CD25, and CD127) to ensure that we were studying T cells of a regulatory phenotype. Interestingly, no CD8 T cells with a regulatory phenotype were induced. In contrast, regulatory CD4 T cells were readily found and represented (on average) 5 to 10% of total CD4 T cells in the infected patients. Although the absolute numbers of CD4 T cells with a regulatory phenotype increased slightly over time, we could not detect a specific increase of these cells occurring within the first 2 months after symptom debut in the hantavirus-infected patients. Therefore, whereas regulatory CD4 T cell numbers increased during the acute and follow-up phases of West Nile and dengue virus infections (18, 20), FoxP3⁺CD25⁺CD127⁻ CD4 T cells in blood from hantavirus-infected patients remained stable throughout the course of infection. This finding is supported by a recent report assessing the frequency of CD25^highCD4⁺ cells in hantavirus-infected HFRS patients, in whom the acute phase of disease was instead associated with reduced numbers of circulating CD25^highCD4⁺ cells (45).

Inhibitory members of the CD28:B7 family play an important role in regulating T cell activation by balancing proinflammatory responses and dampening excessive immune activation (14, 15, 27). This is particularly evident for the PD-1-PD-L1/2 and the CTLA-4-CD80/86 pathways, since deletion of any of these molecules in mice by gene-targeting strategies causes spontaneous autoimmune diseases (14, 15, 27, 31). In humans, PD-1 and CTLA-4 have been studied primarily during chronic viral infections. It has been suggested that chronically infecting pathogens, such as HIV-1 and HCV, exploit these pathways for the purpose of attenuating T cell immunity (3, 13, 14, 31). We extended those studies here by detecting CTLA-4 expression on a substantial fraction of responding CD4 and CD8 T cells early after acute hantavirus infection. CTLA-4 expression rapidly decreased and could not be detected in the infected patients at day 60 after symptom debut or in the uninfected controls. Previous reports suggested that responding effector CD8 T cells upregulate PD-1 during acute HCV infection and after human smallpox vaccination (23, 36). Induced PD-1 expression, as an inhibitory regulator of effector T cells, might be of particular relevance during acute hantavirus infection, since endothelial cells, the main cellular targets for hantaviruses, are known to express the PD-1 ligand PD-L1 and respond with induced PD-L1 expression upon cellular stress (8, 24). Interestingly, and in contrast to acute HCV and smallpox vaccination in humans, responding CD8 T cells showed no evidence of PD-1 upregulation during acute hantavirus infection in our hands. However, up to 50% of responding CD4 T cells exhibited signs of PD-1 expression early after hantavirus infection and a great majority of those cells coexpressed inhibitory CTLA-4. These results suggest that responding CD8 and CD4 T cells might utilize different inhibitory pathways during acute viral infections to regulate the strength of the effector responses.

Hantavirus infection causes tissue damage in infected humans (39). The pathology is believed to stem in part from capillary leakage and to result in transient kidney and lung failure (34). Hantaviruses, per se, are believed to be noncytopathic (29). Instead, it has been suggested that an overly vigorous CD8 T cell response might trigger symptomatic disease in the infected individuals (29, 34). However, contradictory reports exist on this topic. A positive correlation between the severity of disease during acute Sin Nombre hantavirus infection and the magnitude of responding hantavirus-specific effector CD8 T cell numbers has been previously shown (17). In contrast, a robust hantavirus-specific CD8 T cell response was recently shown to protect against severe disease in patients with Hantaan hantavirus infection (42). In this regard, it is intriguing that the zoonotic hosts for hantaviruses can be persistently infected without experiencing immunopathology (7). This discontinuity might be explained by observations indicating that Norway rats infected with Seoul hantavirus and deer mice infected with Sin Nombre hantavirus exhibited increased numbers of regulatory CD4 T cells (7, 30). Indeed, in humans, regulatory CD4 T cells have previously been suggested to influence the severity of disease during acute viral infections with West Nile and dengue virus, both of which target endothelial cells (18, 20). Interestingly, we were not able to detect an increase in CD4 T cell numbers with a regulatory phenotype during the acute and convalescent phases of infection. Thus, in line with a recent hypothesis put forward by Schönrich and colleagues (29), our results suggest the possibility that the absence of host immunoregulatory mechanisms might contribute to the vigorous effector CD8 T cell response observed and, secondary to that response, the capillary leakage and clinical disease that result in hantavirus-infected individuals.

In conclusion, we here provide a characterization of the primary effector CD8 T cell response to an acute viral infection in humans. We show that the effector response is induced rapidly after the onset of symptoms, engages a large part of total CD8 T cells, decreases in parallel with the decline in viral load, and includes specific inhibitory immunoregulatory components that potentially modulate the effector response. Altogether, our results provide new insights into the development of the effector CD8 T cell response to acute viral infections as it develops and clears the virus in humans.

Supplementary Material

[Supplemental material]

supp_85_19_10252__index.html^{(1.2KB, html)}

ACKNOWLEDGMENTS

We thank patients and blood donors who have contributed clinical material to this study. We are also thankful to the staff at the Department of Infectious Diseases, Laboratory of Clinical Hematology and Blood Bank, Umeå University Hospital, for assistance with the collection of clinical material.

This work was supported by grants from the Swedish Foundation for Strategic Research, the Swedish Research Council, the Swedish Cancer Society, the Royal Swedish Academy of Sciences, the Cancer Society of Stockholm, the Karolinska Institutet, the Karolinska University Hospital, the Swedish Heart-Lung Foundation, the County of Västerbotten, the Medical Faculty of Umeå University, and the County Councils of Northern Sweden.

Footnotes

^†

Supplemental material for this article may be found at http://jvi.asm.org/.

^▿

Published ahead of print on 27 July 2011.

REFERENCES

1. Akondy R. S., et al. 2009. The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response. J. Immunol. 183:7919–7930 [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Appay V., et al. 2002. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat. Med. 8:379–385 [DOI] [PubMed] [Google Scholar]
3. Barber D. L., et al. 2006. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439:682–687 [DOI] [PubMed] [Google Scholar]
4. Björkström N. K., et al. 2010. Analysis of the KIR repertoire in human NK cells by flow cytometry. Methods Mol. Biol. 612:353–364 [DOI] [PubMed] [Google Scholar]
5. Björkström N. K., et al. 2011. Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus. J. Exp. Med. 208:13–21 [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Butz E. A., Bevan M. J. 1998. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity 8:167–175 [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Easterbrook J. D., Zink M. C., Klein S. L. 2007. Regulatory T cells enhance persistence of the zoonotic pathogen Seoul virus in its reservoir host. Proc. Natl. Acad. Sci. U. S. A. 104:15502–15507 [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Eppihimer M. J., et al. 2002. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells. Microcirculation 9:133–145 [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Epple H. J., et al. 2006. Mucosal but not peripheral FOXP3+ regulatory T cells are highly increased in untreated HIV infection and normalize after suppressive HAART. Blood 108:3072–3078 [DOI] [PubMed] [Google Scholar]
10. Evander M., et al. 2007. Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome. J. Clin. Microbiol. 45:2491–2497 [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Harrington L. E., van der Most R., Whitton J. L., Ahmed R. 2002. Recombinant vaccinia virus-induced T-cell immunity: quantitation of the response to the virus vector and the foreign epitope. J. Virol. 76:3329–3337 [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Joosten S. A., Ottenhoff T. H. 2008. Human CD4 and CD8 regulatory T cells in infectious diseases and vaccination. Hum. Immunol. 69:760–770 [DOI] [PubMed] [Google Scholar]
13. Kaufmann D. E., et al. 2007. Upregulation of CTLA-4 by HIV-specific CD4+ T cells correlates with disease progression and defines a reversible immune dysfunction. Nat. Immunol. 8:1246–1254 [DOI] [PubMed] [Google Scholar]
14. Kaufmann D. E., Walker B. D. 2009. PD-1 and CTLA-4 inhibitory cosignaling pathways in HIV infection and the potential for therapeutic intervention. J. Immunol. 182:5891–5897 [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Keir M. E., Francisco L. M., Sharpe A. H. 2007. PD-1 and its ligands in T-cell immunity. Curr. Opin. Immunol. 19:309–314 [DOI] [PubMed] [Google Scholar]
16. Keynan Y., et al. 2008. The role of regulatory T cells in chronic and acute viral infections. Clin. Infect. Dis. 46:1046–1052 [DOI] [PubMed] [Google Scholar]
17. Kilpatrick E. D., et al. 2004. Role of specific CD8+ T cells in the severity of a fulminant zoonotic viral hemorrhagic fever, hantavirus pulmonary syndrome. J. Immunol. 172:3297–3304 [DOI] [PubMed] [Google Scholar]
18. Lanteri M. C., et al. 2009. Tregs control the development of symptomatic West Nile virus infection in humans and mice. J. Clin. Invest. 119:3266–3277 [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Lechner F., et al. 2000. Analysis of successful immune responses in persons infected with hepatitis C virus. J. Exp. Med. 191:1499–1512 [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Lühn K., et al. 2007. Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection. J. Exp. Med. 204:979–985 [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Manigold T., et al. 2010. Highly differentiated, resting gn-specific memory CD8 T cells persist years after infection by Andes hantavirus. PLoS Pathogens 6:e1000779. [DOI] [PMC free article] [PubMed] [Google Scholar]
22. McMichael A. J., Rowland-Jones S. L. 2001. Cellular immune responses to HIV. Nature 410:980–987 [DOI] [PubMed] [Google Scholar]
23. Miller J. D., et al. 2008. Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines. Immunity 28:710–722 [DOI] [PubMed] [Google Scholar]
24. Mueller S. N., et al. 2010. PD-L1 has distinct functions in hematopoietic and nonhematopoietic cells in regulating T cell responses during chronic infection in mice. J. Clin. Invest. 120:2508–2515 [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Murali-Krishna K., et al. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177–187 [DOI] [PubMed] [Google Scholar]
26. Nilsson J., et al. 2006. HIV-1-driven regulatory T-cell accumulation in lymphoid tissues is associated with disease progression in HIV/AIDS. Blood 108:3808–3817 [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Okazaki T., Honjo T. 2006. The PD-1-PD-L pathway in immunological tolerance. Trends Immunol. 27:195–201 [DOI] [PubMed] [Google Scholar]
28. Pettersson L., Boman J., Juto P., Evander M., Ahlm C. 2008. Outbreak of Puumala virus infection, Sweden. Emerg. Infect. Dis. 14:808–810 [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Schönrich G., et al. 2008. Hantavirus-induced immunity in rodent reservoirs and humans. Immunol. Rev. 225:163–189 [DOI] [PubMed] [Google Scholar]
30. Schountz T., et al. 2007. Regulatory T cell-like responses in deer mice persistently infected with Sin Nombre virus. Proc. Natl. Acad. Sci. U. S. A. 104:15496–15501 [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Sharpe A. H., Wherry E. J., Ahmed R., Freeman G. J. 2007. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat. Immunol. 8:239–245 [DOI] [PubMed] [Google Scholar]
32. Siegmund K., et al. 2005. Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo. Blood 106:3097–3104 [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Tan L. C., et al. 1999. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers. J. Immunol. 162:1827–1835 [PubMed] [Google Scholar]
34. Terajima M., Hayasaka D., Maeda K., Ennis F. A. 2007. Immunopathogenesis of hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome: do CD8+ T cells trigger capillary leakage in viral hemorrhagic fevers? Immunol. Lett. 113:117–120 [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Tuuminen T., et al. 2007. Human CD8+ T cell memory generation in Puumala hantavirus infection occurs after the acute phase and is associated with boosting of EBV-specific CD8+ memory T cells. J. Immunol. 179:1988–1995 [DOI] [PubMed] [Google Scholar]
36. Urbani S., et al. 2006. PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion. J. Virol. 80:11398–11403 [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Urbani S., et al. 2002. Virus-specific CD8+ lymphocytes share the same effector-memory phenotype but exhibit functional differences in acute hepatitis B and C. J. Virol. 76:12423–12434 [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Van Epps H. L., et al. 2002. Long-lived memory T lymphocyte responses after hantavirus infection. J. Exp. Med. 196:579–588 [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Vapalahti O., et al. 2003. Hantavirus infections in Europe. Lancet Infect. Dis. 3:653–661 [DOI] [PubMed] [Google Scholar]
40. Verma S., et al. 2009. West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: transmigration across the in vitro blood-brain barrier. Virology 385:425–433 [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Wang J., Ioan-Facsinay A., van der Voort E. I., Huizinga T. W., Toes R. E. 2007. Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells. Eur. J. Immunol. 37:129–138 [DOI] [PubMed] [Google Scholar]
42. Wang M., et al. 2009. Cellular immune response to Hantaan virus nucleocapsid protein in the acute phase of hemorrhagic fever with renal syndrome: correlation with disease severity. J. Infect. Dis. 199:188–195 [DOI] [PubMed] [Google Scholar]
43. Wherry E. J., Barber D. L., Kaech S. M., Blattman J. N., Ahmed R. 2004. Antigen-independent memory CD8 T cells do not develop during chronic viral infection. Proc. Natl. Acad. Sci. U. S. A. 101:16004–16009 [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Zajac A. J., et al. 1998. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188:2205–2213 [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Zhu L. Y., Chi L. J., Wang X., Zhou H. 2009. Reduced circulating CD4+CD25+ cell populations in haemorrhagic fever with renal syndrome. Clin. Exp. Immunol. 156:88–96 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental material]

supp_85_19_10252__index.html^{(1.2KB, html)}

supp_85_19_10252__FigS1FigS2Legend.doc^{(66.5KB, doc)}

supp_85_19_10252__FigS1.zip^{(952.8KB, zip)}

supp_85_19_10252__figS2.zip^{(814.9KB, zip)}

[B1] 1. Akondy R. S., et al. 2009. The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response. J. Immunol. 183:7919–7930 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2. Appay V., et al. 2002. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat. Med. 8:379–385 [DOI] [PubMed] [Google Scholar]

[B3] 3. Barber D. L., et al. 2006. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439:682–687 [DOI] [PubMed] [Google Scholar]

[B4] 4. Björkström N. K., et al. 2010. Analysis of the KIR repertoire in human NK cells by flow cytometry. Methods Mol. Biol. 612:353–364 [DOI] [PubMed] [Google Scholar]

[B5] 5. Björkström N. K., et al. 2011. Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus. J. Exp. Med. 208:13–21 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Butz E. A., Bevan M. J. 1998. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity 8:167–175 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7. Easterbrook J. D., Zink M. C., Klein S. L. 2007. Regulatory T cells enhance persistence of the zoonotic pathogen Seoul virus in its reservoir host. Proc. Natl. Acad. Sci. U. S. A. 104:15502–15507 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8. Eppihimer M. J., et al. 2002. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells. Microcirculation 9:133–145 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9. Epple H. J., et al. 2006. Mucosal but not peripheral FOXP3+ regulatory T cells are highly increased in untreated HIV infection and normalize after suppressive HAART. Blood 108:3072–3078 [DOI] [PubMed] [Google Scholar]

[B10] 10. Evander M., et al. 2007. Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome. J. Clin. Microbiol. 45:2491–2497 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11. Harrington L. E., van der Most R., Whitton J. L., Ahmed R. 2002. Recombinant vaccinia virus-induced T-cell immunity: quantitation of the response to the virus vector and the foreign epitope. J. Virol. 76:3329–3337 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Joosten S. A., Ottenhoff T. H. 2008. Human CD4 and CD8 regulatory T cells in infectious diseases and vaccination. Hum. Immunol. 69:760–770 [DOI] [PubMed] [Google Scholar]

[B13] 13. Kaufmann D. E., et al. 2007. Upregulation of CTLA-4 by HIV-specific CD4+ T cells correlates with disease progression and defines a reversible immune dysfunction. Nat. Immunol. 8:1246–1254 [DOI] [PubMed] [Google Scholar]

[B14] 14. Kaufmann D. E., Walker B. D. 2009. PD-1 and CTLA-4 inhibitory cosignaling pathways in HIV infection and the potential for therapeutic intervention. J. Immunol. 182:5891–5897 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Keir M. E., Francisco L. M., Sharpe A. H. 2007. PD-1 and its ligands in T-cell immunity. Curr. Opin. Immunol. 19:309–314 [DOI] [PubMed] [Google Scholar]

[B16] 16. Keynan Y., et al. 2008. The role of regulatory T cells in chronic and acute viral infections. Clin. Infect. Dis. 46:1046–1052 [DOI] [PubMed] [Google Scholar]

[B17] 17. Kilpatrick E. D., et al. 2004. Role of specific CD8+ T cells in the severity of a fulminant zoonotic viral hemorrhagic fever, hantavirus pulmonary syndrome. J. Immunol. 172:3297–3304 [DOI] [PubMed] [Google Scholar]

[B18] 18. Lanteri M. C., et al. 2009. Tregs control the development of symptomatic West Nile virus infection in humans and mice. J. Clin. Invest. 119:3266–3277 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Lechner F., et al. 2000. Analysis of successful immune responses in persons infected with hepatitis C virus. J. Exp. Med. 191:1499–1512 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20. Lühn K., et al. 2007. Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection. J. Exp. Med. 204:979–985 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21. Manigold T., et al. 2010. Highly differentiated, resting gn-specific memory CD8 T cells persist years after infection by Andes hantavirus. PLoS Pathogens 6:e1000779. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. McMichael A. J., Rowland-Jones S. L. 2001. Cellular immune responses to HIV. Nature 410:980–987 [DOI] [PubMed] [Google Scholar]

[B23] 23. Miller J. D., et al. 2008. Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines. Immunity 28:710–722 [DOI] [PubMed] [Google Scholar]

[B24] 24. Mueller S. N., et al. 2010. PD-L1 has distinct functions in hematopoietic and nonhematopoietic cells in regulating T cell responses during chronic infection in mice. J. Clin. Invest. 120:2508–2515 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25. Murali-Krishna K., et al. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177–187 [DOI] [PubMed] [Google Scholar]

[B26] 26. Nilsson J., et al. 2006. HIV-1-driven regulatory T-cell accumulation in lymphoid tissues is associated with disease progression in HIV/AIDS. Blood 108:3808–3817 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. Okazaki T., Honjo T. 2006. The PD-1-PD-L pathway in immunological tolerance. Trends Immunol. 27:195–201 [DOI] [PubMed] [Google Scholar]

[B28] 28. Pettersson L., Boman J., Juto P., Evander M., Ahlm C. 2008. Outbreak of Puumala virus infection, Sweden. Emerg. Infect. Dis. 14:808–810 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Schönrich G., et al. 2008. Hantavirus-induced immunity in rodent reservoirs and humans. Immunol. Rev. 225:163–189 [DOI] [PubMed] [Google Scholar]

[B30] 30. Schountz T., et al. 2007. Regulatory T cell-like responses in deer mice persistently infected with Sin Nombre virus. Proc. Natl. Acad. Sci. U. S. A. 104:15496–15501 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31. Sharpe A. H., Wherry E. J., Ahmed R., Freeman G. J. 2007. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat. Immunol. 8:239–245 [DOI] [PubMed] [Google Scholar]

[B32] 32. Siegmund K., et al. 2005. Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo. Blood 106:3097–3104 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Tan L. C., et al. 1999. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers. J. Immunol. 162:1827–1835 [PubMed] [Google Scholar]

[B34] 34. Terajima M., Hayasaka D., Maeda K., Ennis F. A. 2007. Immunopathogenesis of hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome: do CD8+ T cells trigger capillary leakage in viral hemorrhagic fevers? Immunol. Lett. 113:117–120 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35. Tuuminen T., et al. 2007. Human CD8+ T cell memory generation in Puumala hantavirus infection occurs after the acute phase and is associated with boosting of EBV-specific CD8+ memory T cells. J. Immunol. 179:1988–1995 [DOI] [PubMed] [Google Scholar]

[B36] 36. Urbani S., et al. 2006. PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion. J. Virol. 80:11398–11403 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37. Urbani S., et al. 2002. Virus-specific CD8+ lymphocytes share the same effector-memory phenotype but exhibit functional differences in acute hepatitis B and C. J. Virol. 76:12423–12434 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38. Van Epps H. L., et al. 2002. Long-lived memory T lymphocyte responses after hantavirus infection. J. Exp. Med. 196:579–588 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39. Vapalahti O., et al. 2003. Hantavirus infections in Europe. Lancet Infect. Dis. 3:653–661 [DOI] [PubMed] [Google Scholar]

[B40] 40. Verma S., et al. 2009. West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: transmigration across the in vitro blood-brain barrier. Virology 385:425–433 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B41] 41. Wang J., Ioan-Facsinay A., van der Voort E. I., Huizinga T. W., Toes R. E. 2007. Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells. Eur. J. Immunol. 37:129–138 [DOI] [PubMed] [Google Scholar]

[B42] 42. Wang M., et al. 2009. Cellular immune response to Hantaan virus nucleocapsid protein in the acute phase of hemorrhagic fever with renal syndrome: correlation with disease severity. J. Infect. Dis. 199:188–195 [DOI] [PubMed] [Google Scholar]

[B43] 43. Wherry E. J., Barber D. L., Kaech S. M., Blattman J. N., Ahmed R. 2004. Antigen-independent memory CD8 T cells do not develop during chronic viral infection. Proc. Natl. Acad. Sci. U. S. A. 101:16004–16009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B44] 44. Zajac A. J., et al. 1998. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188:2205–2213 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B45] 45. Zhu L. Y., Chi L. J., Wang X., Zhou H. 2009. Reduced circulating CD4+CD25+ cell populations in haemorrhagic fever with renal syndrome. Clin. Exp. Immunol. 156:88–96 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Longitudinal Analysis of the Human T Cell Response during Acute Hantavirus Infection ^{^▿}^†

Therese Lindgren

Clas Ahlm

Nahla Mohamed

Magnus Evander

Hans-Gustaf Ljunggren

Niklas K Björkström

Abstract

INTRODUCTION