Human CD8+ T Cell Memory
                                     Generation in Puumala Hantavirus
The Journal of Immunology

Human CD8 T Cell Memory Generation in Puumala
Hantavirus Infection Occurs after the Acute Ph...
The Journal of Immunology                                                                                                 ...
1990                                                              CD8 T CELL RESPONSE IN ACUTE PUUMALA VIRUS INFECTION

The Journal of Immunology                                                                                                 ...
1992                                                           CD8 T CELL RESPONSE IN ACUTE PUUMALA VIRUS INFECTION

The Journal of Immunology                                                                                                 ...
1994                                                                          CD8 T CELL RESPONSE IN ACUTE PUUMALA VIRUS I...
The Journal of Immunology                                                                                                 ...
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  1. 1. Human CD8+ T Cell Memory Generation in Puumala Hantavirus Infection Occurs after the Acute Phase This information is current as of October 19, 2010 and Is Associated with Boosting of EBV-Specific CD8+ Memory T Cells Tamara Tuuminen, Eliisa Kekäläinen, Satu Mäkelä, Ilpo Ala-Houhala, Francis A. Ennis, Klaus Hedman, Jukka Mustonen, Antti Vaheri and T. Petteri Arstila Downloaded from on October 19, 2010 J. Immunol. 2007;179;1988-1995 References This article cites 46 articles, 15 of which can be accessed free at: Subscriptions Information about subscribing to The Journal of Immunology is online at Permissions Submit copyright permission requests at Email Alerts Receive free email alerts when new articles cite this article. Sign up at The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright ©2007 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
  2. 2. The Journal of Immunology 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 Cells1 Tamara Tuuminen,2* Eliisa Kekalainen,2† Satu Makela,¶ Ilpo Ala-Houhala,¶ Francis A. Ennis,‡ ¨ ¨ ¨ ¨ Klaus Hedman,* Jukka Mustonen, Antti Vaheri,* and T. Petteri Arstila3† §¶ The induction and maintenance of T cell memory is incompletely understood, especially in humans. We have studied the T cell response and the generation of memory during acute infection by the Puumala virus (PUUV), a hantavirus endemic to Europe. It causes a self-limiting infection with no viral persistence, manifesting as hemorrhagic fever with renal syndrome. HLA tetramer staining of PBMC showed that the CD8 T cell response peaked at the onset of the clinical disease and decreased within the next 3 wk. Expression of activation markers on the tetramer-positive T cells was also highest during the acute phase, suggesting that the peak population consisted largely of effector cells. Despite the presence of tetramer-positive T cells expressing cytoplasmic Downloaded from on October 19, 2010 IFN- , PUUV-specific cells producing IFN- in vitro were rare during the acute phase. Their frequency, as well as the expression of IL-7R mRNA and surface protein, increased during a follow-up period of 6 wk and probably reflected the induction of memory T cells. Simultaneously with the PUUV-specific response, we also noted in seven of nine patients an increase in EBV- specific T cells and the transient presence of EBV DNA in three patients, indicative of viral reactivation. Our results show that in a natural human infection CD8 memory T cells are rare during the peak response, gradually emerging during the first weeks of convalescence. They also suggest that the boosting of unrelated memory T cells may be a common occurrence in human viral infections, which may have significant implications for the homeostasis of the memory T cell compartment. The Journal of Immunology, 2007, 179: 1988 –1995. I mmunological memory rests on a population of long-lived T ena such as immunological memory should be verified before ex- and B lymphocytes, which are both quantitatively and qual- trapolating them to humans. It is quite likely that the maintenance itatively different from naive cells (for review, see Refs. 1 of T cell memory in mice over periods of months may have and 2). Despite the theoretical and practical importance of memory different requirements than the often decades-long immunity T cells, many aspects of their development and survival are inad- encountered in humans. Studies in murine models indicate that equately understood especially in humans, in whom individual dif- the memory phenotype is gained gradually following clearance ferences in the genetic background and disease history pose addi- of the pathogen, with measurable changes occurring in the T tional problems of interpretation. The longer lifespan, outbred cell population for up to 40 days postinfection (3). This sug- nature, and varied environmental exposures of humans make it gests that the first weeks after the acute phase of an infection, obvious that results obtained in the mouse on long-term phenom- when the declining Ag load, the disappearance of most effector cells, and the effect of prosurvival cytokines shape the emerging memory population, are crucial in determining the characteris- Haartman Institute, *Department of Virology and †Department of Immunology, Uni- versity of Helsinki, Finland; ‡Center for Infectious Disease and Vaccine Research, tics of T cell memory. University of Massachusetts Medical School, Worcester, Massachusetts 01655; Little is known of memory T cell differentiation in humans dur- § Medical School, University of Tampere and ¶Department of Internal Medicine, ing the important postinfection period. Based on the differential Tampere University Hospital, Tampere, Finland; and HUSLAB, Helsinki Uni- versity Hospital, Helsinki, Finland behavior and tissue homing, the memory T cells have been divided Received for publication May 11, 2006. Accepted for publication May 17, 2007. into two main subsets: the CCR7 central memory T cells found The costs of publication of this article were defrayed in part by the payment of page predominantly in the lymphoid tissues and the CCR7 effector charges. This article must therefore be hereby marked advertisement in accordance memory T cells found in peripheral nonlymphoid tissues (4). In with 18 U.S.C. Section 1734 solely to indicate this fact. this scenario, it remains unclear whether one of the memory 1 This work was supported by grants from the Academy of Finland, the Sigrid Juselius ´ subsets is a precursor for the other or whether the subsets de- Foundation, the Finnish Technology Advancement Fund, the Finnish Cancer Re- search Foundation, the Helsinki University Central Hospital Research and Education velop independently. Others, however, have argued that instead Fund, and the Medical Research Fund of Tampere University Hospital, European of two distinct memory phenotypes, the human CD8 T cell Commission Contracts QLK2-CT-1999-01119 and GLK2-CT-2002-01358, and Na- tional Institutes of Health/National Institute of Allergy and Infectious Diseases Grant memory acquisition is a linear process, with the Ag-experi- U19 AI057319. enced cells gaining gradually a more differentiated phenotype 2 T.T. and E.K. contributed equally to this work. and simultaneously approaching proliferative senescence (5). 3 Address correspondence and reprint requests to Dr. T. Petteri Arstila, Haartman How far along this scheme of differentiation the cells proceed Institute, Department of Immunology, PB21, University of Helsinki, Finland. E-mail seems to depend on the type of infection. Thus, hepatitis C or address: influenza-specific memory T cells have an early memory phe- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 notype, retaining partly the expression of naive cell markers
  3. 3. The Journal of Immunology 1989 such as CD27, CD28, and CCR7. In infections by EBV or HIV, Table I. The highest values of the laboratory findings measured during CD28 and CCR7 are largely down-regulated, whereas CMV hospital care infection seems to induce the most highly differentiated late- stage memory T cells (5). Days after the Laboratory Finding Median (Range) Onset of Fever One problem in reconciling these different views of human CD8 T cell memory may be the nature of the pathogens studied. Maximum plasma creatinine 445 (79 –1285) 6–8 In the case of the influenza virus it is unlikely that what is studied ( mol/L) truly represents a primary infection, given the repeated reinfections Maximum plasma 152 (30 –269) 3–7 C reactive protein (mg/L) by this pathogen. The other pathogens mentioned establish a Maximum blood leukocyte 12.9 (8.5–26.8) 4–8 chronic, persistent infection, so it is not possible to study T cell count (109/L) memory generation during virus clearance. Moreover, some of these viruses, most obviously HIV and EBV that infect CD4 T cells and B cells, respectively, have profound pathological effects predispose to an imbalance of T cell repertoire and immuno- on the immune system, further confounding the interpretation of T logical senescence. cell dynamics following the acute phase. Attempts to circumvent these problems have involved experiments with live or killed vac- Materials and Methods cines, making it possible to control the kinetics of the response. Patients However, it is unclear whether a vaccine exposure, which by def- Patients (16 altogether) presenting with acute PUUV infection at the Tam- inition should be harmless, reliably reproduces all the immunolog- pere University Hospital (Tampere, Finland) were partially HLA-typed for ical phenomena associated with an infection caused by a virulent HLA-A2 and B8 haplotypes with Abs purchased from One Lambda. Nine patients with either or both haplotypes and positive PUUV serology at any pathogen. The relatively short half-life of T cell memory reported point of the acute disease (one woman, eight men, age 36 –72 years, mean in some vaccine studies may thus reflect qualitatively or quantita- 58 years) were enrolled. PBMC, sera, and plasma were collected daily Downloaded from on October 19, 2010 tively insufficient effector activation. In support of this, the pattern during hospitalization and after discharge on two follow-up visits, roughly of T cell expansion and memory generation after vaccination is 3 and 6 wk after the onset of symptoms. The group of healthy controls was often highly variable (6). recruited from students and staff with negative PUUV serology and con- sisted of six men, mean age 31 years. All patients gave written, informed Puumala virus (PUUV),4 a zoonotic pathogen carried by ro- consent and the study was approved by the ethics committee of the Tam- dents, is a member of Hantaviridae and is endemic in large areas pere University Hospital. of Europe (7–9). It causes a self-limiting and relatively mild form of hemorrhagic fever with renal syndrome termed nephropathia IFN- ELISPOT epidemica (NE). The disease usually follows a characteristic PBMC were harvested with Ficoll-Paque (Amersham Biosciences) gradi- course with a sudden onset of high fever in the absence of respi- ent centrifugation and cryopreserved in 90% heat-inactivated FCS and 10% ratory symptoms accompanied by headache, hemodynamic DMSO (Sigma-Aldrich) until use. ELISPOT was performed in duplicate using the ELISpotPRO human IFN- kit (Mabtech) according to the man- changes, and acute renal failure that sometimes necessitates he- ufacturer’s instructions. Control Ags were tested in single wells. As a modialysis treatment. During the acute phase, viral RNA can be viability control the cells were stimulated with phytohemagglutinin detected in the blood and urine of many patients, but only until 9 (Sigma-Aldrich) (7 g/ml) and heat-killed (30 min at 65°C) Candida days from the onset of the symptoms (10, 11). Reinfection by albicans (2.5 g/ml). Interassay stability was monitored by testing mul- tiple aliquots of a single buffy coat isolation of human PBMC (Finnish PUUV has not been reported, yet Abs have been detected for Red Cross Blood Service, Helsinki, Finland) simultaneously with the decades after the infection (12). We have previously shown that patient samples. PUUV-specific CD8 memory T cells also persist for more than The antigenic PUUV peptides from the nucleocapsid (N) protein a decade after the acute infection in the absence of viral per- (N204 –212 is HLA-A2 restricted; N243–51 and N173– 81 are HLA-B8 re- stricted) have been previously described (13). They were synthesized by sistence or reinfection (13). This, combined with the readily the Peptide and Protein Laboratory of the Haartman Institute, University of diagnosable acute syndrome, makes PUUV infection an excel- Helsinki (Helsinki, Finland) with standard methods. The HLA-A2- and lent model for analysis of the generation of human T cell mem- HLA-B8-restricted control peptides were from the BMLF-1 (GLCTL- ory during an infection with a virulent pathogen. Hantaviral VAML) and EBNA-3 (FLRGRAYGL) proteins of EBV and the Env infections may also serve as a model of immunopathology (9). (KLTPLCVTL) and Gag (GEIYKRWII) proteins of HIV, respectively. EBV peptides were used as a positive control and HIV peptides as a neg- PUUV-infected patients with the HLA-B8-DR3 haplotype have ative control and both were purchased, together with the HLA-A2-re- a more severe course of NE (14, 15), and in hantavirus pulmo- stricted influenza matrix epitope peptide GILGFVFTL, from ProImmune. nary syndrome the number of Sin Nombre virus-specific CD8 All peptides were also tested, with no detectable spot formation, in the T cells correlates with disease severity (16). These data suggest ELISPOT assay plates without cells. that the T cell response in the acute phase may contribute to the HLA tetramer analysis and flow cytometry pathogenesis of the disease. In this report, we characterize the dynamics of the Ag-specific Customized, PE-labeled HLA-A2 tetramers with the PUUV N204 –212 pep- tide were purchased from Beckman Coulter and ProImmune, and the PE- CD8 T cell population in patients during primary PUUV infec- labeled HLA-A2 tetramers with the EBV epitope GLCTLVAML were tion and the immediate recovery period. Our results show that from ProImmune. To minimize nonspecific background, the cells were also following the contraction of the effector population, memory T stained with anti-CD3 and anti-CD8 mAbs (BD Biosciences) and gated to cells appear during the first weeks after the clinical disease the CD3 population before analysis. mAbs to CD122 and Ki-67 were followed by an attrition of unknown kinetics. Our data also purchased from BD Biosciences, and the mAbs to HLA-DR, CD127, and IFN- were from eBioscience. Intracellular staining with anti-IFN- or reveal an instance of simultaneous boosting of unrelated anti- Ki-67 mAb was done using a fixation and permeabilization kit purchased viral T cell memory, a phenomenon that may be of importance from eBioscience according to the manufacturer’s instructions. For the to the maintenance of immunological memory but may also testing of apoptosis susceptibility, the cells were cultured overnight in RPMI 1640 and supplements with anti-CD3 mAb immobilized to the bot- tom of the culture well. The detection of apoptotic cells was done using the Annexin-V:FITC apoptosis detection kit from BD Biosciences. Flow cy- 4 Abbreviations used in this paper: PUUV, Puumala virus; N, nucleocapsid protein; tometry samples were analyzed by using the FACScan or FACSAria in- NE, nephropathia epidemica. strument (BD Biosciences).
  4. 4. 1990 CD8 T CELL RESPONSE IN ACUTE PUUMALA VIRUS INFECTION Quantitative real-time PCR Total RNA was isolated by using the TriPure reagent (Roche Diagnostics), and first-strand cDNA was synthesized with oligo(dT) primer and avian myeloblastosis virus reverse transcriptase (Finnzymes). Quantitative PCR was done in duplicate with commercially available primer-probe sets for human IL7-R and IL-15R and the TaqMan master mix, all purchased from Applied Biosystems. The results were normalized against -actin mRNA levels in the samples, and relative quantitation was achieved by comparison with a standard dilution curve. Reactions were performed by using the ABI7700 instrument (Applied Biosystems). Determination of TNF- by ELISA Human TNF- was determined from patients’ sera collected during the hospital stay and after recovery. TNF- was also measured from superna- tants harvested from cell cultures stimulated with the various PUUV pep- tides. Analysis of cytokine levels was done by using a commercial enzyme immunoassay for human TNF- as instructed (Mabtech). EBV serology and detection IgG and IgM class Abs to EBV were determined with the Enzygnost anti- EBV IgG and IgM kits by using the Behring ELISA processor. The IgG avidity was determined as described (17). Real-time quantitative PCR for EBV DNA in plasma was performed as published (18). Results Downloaded from on October 19, 2010 Kinetics of the T cell response in acute PUUV infection The typical incubation period of PUUV infection is 2– 4 wk, and all of our nine patients were seropositive at presentation. The clin- ical picture was typical of NE in all patients. A summary of the main laboratory findings is shown in Table I, which also includes the periods at which the highest values were measured in different patients. Serum creatinine concentrations were elevated in all but one patient and three needed transient hemodialysis treatment. All patients had proteinuria and hematuria. Seven of nine patients had thrombocytopenia. One patient was hypotensive (blood pressure 95/60 mmHg), but none had a clinical shock. By the end of hos- FIGURE 1. Cytotoxic T cell response to an acute infection by PUUV. A, Kinetics of a HLA-A2 restricted, PUUV N204 –212-specific response in pitalization (8 –12 days after onset) the laboratory findings had three representative patients as measured by HLA tetramers. B, The fre- normalized. quency of circulating CD8 T cells during the acute phase and convales- The first blood sample, at presentation, was drawn 4 –7 days cence (p 0.05 between d1 and c2; Student’s two-tailed t test). C, Com- after clinical onset (fever). Analysis of the frequency of PUUV N parison of the frequency of HLA-A2/PUUV N204 –212 tetramer-reactive protein-specific CD8 T cells using HLA-A2 tetramers showed (circle) and IFN- producing PUUV N204 –12-specific (square) cells at that the patients had a strong T cell response (Fig. 1A), with up to different stages of the infection. d1, Presentation; c1, first control, on 19,800 N204 –212 specific cells per million PBMC (range 3,200 – average 3 wk after the onset, c2, second control, 6 wk after the onset. 19,800) or 1.3%–5.7% of all circulating CD8 T cells. At control In A only CD3 cells are shown, while the results in B and C are shown visits 3– 6 wk after the onset the response detectable with HLA-A2 as mean SD. tetramers had mostly subsided (Fig. 1A). At presentation, the proportion of CD8 T cells to all CD3 cells was also close to FIGURE 2. Frequency of PUUV- specific, IFN- -producing CTLs during acute PUUV infection and convalescence. Responses against HLA-A2-restricted N204 –212 (trian- gle) and HLA-B8 restricted N243–251 (diamond) and N173–181 (square) PUUV peptides were measured by us- ing an IFN- ELISPOT assay. Non- specific background was subtracted from the results, and only responses higher than twice the background were considered positive.
  5. 5. The Journal of Immunology 1991 contraction phase of the acute response, suggesting that the in- crease of the IFN- -producing population may have corre- sponded to the emergence of a memory T cell population, peak- ing between the days 20 to 45 after the onset of the symptoms. Interestingly, the highest frequencies of IFN- -producing cells at this convalescence stage were roughly an order of magnitude higher than those of memory T cells found in individuals with old immunity (13). We have previously shown that patients with the HLA-B8-DR3 haplotype have a more severe course of PUUV infection (14, 15). FIGURE 3. Changes in the expression of IL-7R mRNA during PUUV This has been suggested to be due to exaggerated T cell responses infection. cDNA was synthesized from total RNA by using an oligo(dT) and subsequent immunopathology. In our patient cohort we ob- primer and IL-7R levels were measured by quantitative PCR. The results served no clear difference in the frequency of HLA-B8-restricted T were normalized against human -actin levels and relative quantitation was cells as compared with HLA-A2-restricted cells, but because only achieved by comparing the results with a standard dilution curve. The mean three of our patients had the B8 haplotype this result should be expression level of six healthy controls is indicated by a dotted line. The assessed with caution. However, the same conclusion was also values are shown in arbitrary units. drawn from an earlier analysis of the frequency of PUUV-specific memory T cells (13). 50% and decreased significantly at later sampling days to 35% (Fig. 1B). ELISPOT analysis of the frequency of T cells secret- ing IFN- in response to three different PUUV N peptides Expression of IL-7R and IL-15R Downloaded from on October 19, 2010 (N204 –212, N243–251, and N173–181), including the A2-restricted In addition to the Ag-specific signals, several cytokines have been tetramer epitope (Fig. 1C), produced different results. Measured shown in murine studies to be important for the generation of by the ELISPOT assay, the frequency of reactive T cells at CD8 T cell memory (22). Some of these, especially IL-7 and presentation was low, ranging from 300 to 50 cells/million IL-15, are produced constitutively so that the regulation of their PBMC. Thus, the increased CD8 T cell population at presenta- effects takes place on the receptor level. We analyzed the expres- tion consisted to a large degree of cells that can be visualized by sion of IL-7R (CD127) and IL-15R mRNA during the acute direct HLA tetramer staining but are not readily detectable by an phase and convalescence by using the average expression in six in vitro functional assay such as IFN- ELISPOT. healthy volunteers as a baseline. Analysis of PBMC by quantita- A dissociation between staining and functional assays has been tive PCR showed that the expression levels of IL-7R mRNA demonstrated in many experimental models and can be due to the were low in the acute phase but increased in most patients during susceptibility of effector cells to activation-induced cell death (19). the follow-up (Fig. 3). These data are in accordance with previous The ELISPOT assay also showed lower responses to the poly- reports that have shown that IL-7R is down-regulated during T clonal stimulator PHA during the acute phase than at later sam- cell activation but re-expressed at the transition to the memory pling days (not shown), perhaps because of the tendency of the phenotype (23). This provides further support for the view that the effector population to respond to stimulation by apoptosis. A sim- increase of IFN- -producing T cells during the follow-up corre- ilar transient decrease in PHA responses has also been observed in other acute viral infections (20, 21). To test the susceptibility of the sponded to the emergence of the specific memory population. IL- isolated cells to activation-induced apoptosis, we stimulated them 15R has been reported to be up-regulated in activated T cells and overnight in vitro with an immobilized anti-CD3 mAb and then maintained in memory T cells (24), but in our patients the expres- performed annexin-V staining to detect apoptotic cells. The results sion of IL-15R showed no apparent change and was comparable showed that cells isolated during the acute phase were more prone to that in noninfected controls (data not shown). to apoptosis than cells isolated during the convalescence, with We also measured the production of TNF- , reported to be high 16.3% (range 7.2–23.9%) and 7.3% (range 5.7–10.0%) annexin- during the acute phase of PUUV infection and suggested to be positive cells, respectively. important in pathogenesis (25). In our hands the concentrations of During follow-up the frequency of IFN- -producing T cells TNF- in the patient sera were all below the detection level of the started to increase, reaching in some subjects nearly 2,800 reactive kit (8 pg/ml). However, TNF- was detectable in supernatants col- cells per million PBMC (Fig. 2), while at the same time the fre- lected from the cultures of T cells stimulated with PUUV Ags in quency of both CD8 T cells and A2 tetramer-positive cells de- amounts proportional with the frequency of IFN- producing cells. creased and the results from the different methods of analysis drew This suggests that local production of TNF- may play a role in closer to each other (Fig. 1C). These changes thus reflected the the clinical disease. FIGURE 4. Activation status of PUUV-specific T cells detected by the HLA-A2 tetramer. The acute samples were drawn early during the hospital- ization and separated from the control samples by an average of 15 days. The frequency of cells expressing the indi- cated marker within the CD8 tetramer population during the acute phase and convalescence is shown, each line rep- resenting an individual patient.
  6. 6. 1992 CD8 T CELL RESPONSE IN ACUTE PUUMALA VIRUS INFECTION FIGURE 5. Boosting of EBV-spe- cific memory T cells during acute PUUV infection. The frequency of EBV-specific T cells was measured us- ing an IFN- ELISPOT assay. The scale on the left indicates the number of cells producing IFN- in response to GLCTLVAML (open diamonds) and FLRGRAYGL (closed squares). In the case of a positive EBV PCR result the number of EBV genomes is indicated by the vertical bars and the scale on the right. The period when no EBV-spe- cific T cells or EBV DNA was detected is indicated by the horizontal bars. Expression of activation markers on PUUV-specific T cells follow-up to 165-1845 cells per million PBMC. To exclude the To verify the results described above and to further characterize possibility of an immunosuppressive effect during the acute phase, the composition of the PUUV-specific CD8 T cell population, we we also used HLA-A2 tetramers bound to the same EBV epitope used flow cytometry to analyze the activation status of the PUUV to directly measure the frequency of EBV-specific T cells in six tetramer-positive cells in seven HLA-A2 patients, comparing an patients. The results were in close agreement with the ELISPOT Downloaded from on October 19, 2010 early sample taken from each patient during hospitalization with data, with an average of 129 (range 0 –207) specific cells per mil- the first control sample. The average time between the two samples lion PBMC in the acute phase and 328 (range 107– 613) in the first was 15 days (range 9 –20). In contrast to the ELISPOT results control sample. obtained after in vitro stimulation, when analyzed directly ex vivo We then used the ELISPOT assay to test the response of 6 by intracellular cytokine staining the frequency of cells producing HLA-A2 patients to an immunodominant A2-restricted influenza IFN- was higher in the acute phase than during convalescence in matrix epitope in the acute phase and in the first control sample. A all seven patients (15.2% vs 5.1% on the average; Fig. 4). Simi- response was detected in five of the patients, but the frequency of larly, the frequency of cells expressing the activation markers responding cells was low ( 60 specific cells per million PBMC). HLA-DR and CD122 (the high-affinity IL-2R -chain) was higher An increase was observed in only one patient, from 15 specific in the acute phase in most of the patients. We also analyzed the cells in the acute phase to 60 specific cells per million PBMC in expression of the cell cycle-associated intracellular Ki-67 Ag in the control sample, and on the average there was no change. It is five patients and found that in four of them the frequency of Ki- thus unlikely that the increase in EBV-specific T cells would have 67 cells among the HLA-A2 tetramer-positive population de- been due to nonspecific, generalized memory T cell activation creased sharply during the transition to convalescence. Taken to- caused by cytokines or putative virus-induced lymphopenia. gether, these data show that the population detected by the Serological analysis showed that all nine patients had pre-exist- tetramers in the acute phase consists of highly activated cells un- ing immunity to EBV and were thus carriers of a latent infection. detectable by the IFN- ELISPOT assay. To determine whether the EBV-specific T cell response was ac- Because the bulk analysis of IL-7R mRNA levels indicated an companied by viral reactivation, we used quantitative PCR to mea- increase during convalescence, we next analyzed IL-7R surface sure the number of EBV DNA copies in the patients’ plasma. In expression on the tetramer-positive cells (Fig. 4). In six of seven three patients EBV DNA was detected during the acute phase of patients the frequency of IL-7R cells increased during the fol- the PUUV infection but not during follow-up. Strikingly, two of low-up, consistent with the emergence of a memory population. the three patients with a transient viremia had no measurable EBV- Finally, we measured the mean fluorescence intensity of tetramer specific T cell response while only one of the seven patients with staining to determine the amount of surface TCR expressed by the an EBV-specific T cell response had detectable EBV DNA, sug- tetramer-positive cells. T cell activation is associated with rapid gesting a link between the T cell response and the suppression of TCR down-modulation, whereas on memory T cells the levels are EBV reactivation. Thus, all nine patients had either a measurable restored (reviewed in Ref. 26). When compared with the samples EBV-specific T cell response or transient EBV viremia. It is also taken during the acute phase, the level of surface TCR increased noteworthy that in five of the seven patients with an EBV-specific during convalescence in all seven patients, on average by 80% T cell response the levels of specific CD8 T cells remained higher (range 15–258%). at the last control sampling than at the beginning of the study. These results suggest that the PUUV-associated EBV reactivation EBV-specific T cell response and viral reactivation and the resultant immune response also served to boost the EBV- specific T cell memory. For specificity control of our ELISPOT assay we used antigenic peptides of EBV and HIV. As expected, there was no detectable response to HIV peptides in our HIV-negative patients, whereas Discussion some reactivity to EBV peptides was observed in most patients. The requirements for the maintenance of T cell memory have been Unexpectedly, however, during the follow-up seven of our nine the object of a lengthy debate, with special attention given to the patients also showed a clear increase in the frequency of EBV- question of whether contact with the original or a cross-reactive reactive T cells. The EBV-specific response followed closely the Ag is needed. In one extreme, some experimental models have kinetics of the PUUV-specific response as measured by the ELIS- indicated that memory may be a form of chronic, low-grade stim- POT assay (Fig. 5). During the acute phase the responses were low ulation rather than a latent state of more or less resting T cells (27). (25–225 specific cells per million PBMC) but increased during the In other studies, MHC-deficient mice without any T cells were
  7. 7. The Journal of Immunology 1993 grafted with memory T cells and the survival of the transferred following its induction. The kinetics of this decline is not known, cells was monitored in the absence of any possible TCR-mediated but it does suggest that the maintenance of high levels of memory stimulus (28). Although the transferred T cells seemed to survive cells may require occasional boosting. Such boosting may come for months, their functional properties have been reported to de- from Ags persisting in the host or from reinfection, whether clin- cline in the absence of boosting (28, 29). Even in murine models, ical or subclinical. A further possibility is that in some cases an the need for antigenic boosting therefore remains an open question. immune response against one pathogen may also boost the mem- In humans, where genetic manipulations are impossible, it is very ory response against another. This might be due to heterologous difficult to obtain a reliable answer. Both anecdotal and experi- immunity induced by a cross-reaction, homologous immunity in- mental evidence suggest that human T cell memory can be main- duced by the reappearance of latent pathogens or microbial com- tained for years and even decades in the apparent absence of boost- ponents, or nonspecific lymphopenia-driven or cytokine-driven ing (13, 30 –32), but it is difficult to exclude all forms of antigenic boosting. Such bystander maintenance of T cell memory has been stimulus. Our data allow several putative conclusions to be drawn. demonstrated in several experimental settings in mice, but clear First, the human memory T cell population seems to develop dur- examples from human systems are scarce (35). Our data putatively ing a period of a few weeks after the clearance of the pathogen. provide such an example, by showing an increase of EBV-reactive Second, the resulting population is significantly larger than the T cells in seven of nine patients with an acute PUUV infection. memory population detectable several years later. Third, the im- Reactivation of latent EBV is a well-known phenomenon, but as mune system seems to take advantage of the acute infection to a systemic manifestation it is generally linked with situations of boost the memory population specific to an unrelated pathogen. clinical immunosuppression such as organ transplantations and In acute viral infections the specific T cell response typically AIDS (36). In the absence of immunosuppression the EBV cycle reaches its peak by 2 wk after the infection. In our patients, al- becomes productive in the salivary glands and oropharyngeal mu- though the exact time of infection cannot be established, the high- cosa, with occasional shedding into saliva contributing to viral est frequency of circulating CD8 T cells and PUUV-specific tet- transmission (37). In contrast, plasma drawn from healthy people Downloaded from on October 19, 2010 ramer-positive cells was observed at presentation, suggesting that very rarely contains EBV DNA (18, 38). Here, a systemic EBV the peak response is roughly concurrent with the appearance of reactivation or a T cell response suggestive of such reactivation clinical symptoms. A large fraction of these cells also expressed was observed during an acute infection in otherwise healthy people activation markers, and a clear decrease was observed during the in every one of the nine patients studied. During latency EBV follow-up. It is thus likely that the cells forming the peak response persists in B cells, so the most likely explanation of our finding is were largely effector cells and were not readily detectable by in that EBV gets reactivated within EBV-infected, PUUV-specific B vitro stimulation, probably because of their susceptibility to acti- cells participating in the acute immune response. It is also possible vation-induced cell death (19). Interestingly, despite the presence that the cytokines produced during the immune response induce of cells with cytoplasmic IFN- , during the acute phase the fre- viral reactivation in B cells not directly involved in the PUUV- quency of cells detectable by ELISPOT was very low or even specific response. In either case, it may be that the phenomenon is below the sensitivity of the method in every patient, and in all nine restricted to EBV and might also be a part of the viral survival patients the frequency increased during convalescence. Thus, de- strategy. However, increased expression of microbial constituents spite the strong response detectable by direct staining, the popu- or even the appearance of viable organisms may be a more com- lation responsible for the in vitro IFN- production induced by the mon feature of immune responses. In an earlier study on herpes- same epitope at later stages was absent at the peak of the acute virus-specific B cell responses during acute CMV infection, the response. This suggests that the circulating effector population emergence or increment of Abs suggested the reactivation of EBV contained very few fully differentiated memory cells, at least if and also human herpesvirus 6, and in one patient the varicella virus judged by the functional criteria that could be used to identify them also (39). More recent studies using quantitative PCR or gene ar- years after the primary infection (13). Consistent with this sce- rays have provided direct evidence of the propensity of several nario, the kinetics of IL-7R expression also followed closely the herpesviruses to become reactivated at the same time (40, 41). appearance of PUUV-specific IFN- producing cells, suggesting Also, during acute HIV infection CD8 T cells specific for EBV, that they represented the emergent memory population (23). Pre- CMV, and influenza start to express activation markers without viously, a study of acute lymphocytic choriomeningitis virus in- detectable viral reactivation or infection (42). Cells of the immune fection in mice showed that the gene expression pattern associated system, especially phagocytic APCs like the macrophages, are with T cell memory is obtained gradually (3), a finding in good hosts to many kinds of microbes (43). Participation in an immune agreement with our results in regard to acute infection of humans. response against another pathogen might well have an impact on Whether the survival of some effectors and their further develop- the presentation of Ags from pathogens encountered earlier, ment into memory cells is linked to TCR affinity as suggested by whether or not they remain alive. In this way the heterologous some experimental models (33, 34), or if it happens stochastically, boosting of resting memory T cells could be a general feature of T remains unclear. Our results in the human system with a natural cell responses, creating a kind of ripple effect that might help to infection by a virulent pathogen show that the generation of mem- preserve pre-existing T cell memory in the face of a massive re- ory takes place only after the peak effector response has subsided. sponse directed against the infecting pathogen. Thus, either the induction of memory T cells is independent of and However, the consequences of EBV reactivation might not be significantly slower than the induction of effector cells, or, more all beneficial. One facet of heterologous T cell activation is its likely, the PUUV-specific memory cell generation reflects an end- potential to trigger pathological responses that might be of impor- point of T cell activation (5). tance to the pathogenesis of NE. The severity of acute PUUV Another significant observation was that roughly a month after infection is highly variable and can be modulated by genetic fac- the acute phase the frequency of PUUV-reactive T cells was an tors such as the HLA type of the patient (14, 15). It is possible that order of magnitude higher than that observed several years after the simultaneous EBV reactivation may contribute to the symp- infection (13). Although this comparison was made between two toms, the more so because PUUV and EBV infections can cause different patient cohorts, the results are likely to indicate a signif- similar complications including CNS inflammation, myocarditis, icant decline in the memory T cell population during the years hepatitis, nephritis, and arthritis (8, 44). These complications
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Analysis of Puumala hantavirus genome in and partly stochastic events during the primary infection and might patients with nephropathia epidemica and rodent carriers from the sites of infec- thus have a highly variable impact on the clinical course of the tion. J. Med. Virol. 59: 397– 405. 12. Settergren, B., C. Ahlm, P. Juto, and B. Niklasson. 1991. Specific Puumala IgG PUUV infection. A clear example of such a detrimental viral in- virus half a century after haemorrhagic fever with renal syndrome. Lancet teraction was provided by a recent demonstration that immunity 338: 66. against an influenza-derived Ag can cross-react with hepatitis C 13. Van Epps, H. L., M. Terajima, J. Mustonen, T. P. Arstila, E. A. Corey, A. Vaheri, and F. A. Ennis. 2002. Long-lived memory T lymphocyte responses after han- virus structures. In a minority of patients with acute hepatitis C tavirus infection. J. Exp. Med. 196: 579 –588. virus infection, this pre-existing, influenza-induced T cell popula- 14. Mustonen, J., J. Partanen, M. Kanerva, K. Pietila, O. Vapalahti, A. Pasternack, tion comes to dominate the T cell response and is associated with and A. Vaheri. 1996. Genetic susceptibility to severe course of nephropathia epidemica caused by Puumala hantavirus. Kidney Int. 49: 217–221. a severe form of hepatitis (45). Similarly, the patient’s pre-existing 15. Makela, S., J. Mustonen, I. Ala-Houhala, M. Hurme, J. Partanen, O. Vapalahti, ¨ pattern of EBV-specific T cell memory might be partly responsible A. Vaheri, and A. Pasternack. 2002. Human leukocyte antigen-B8-DR3 is a more for the outcome of the acute PUUV infection. important risk factor for severe Puumala hantavirus infection than the tumor necrosis factor- (-308) G/A polymorphism. J. Infect. Dis. 186: 843– 846. More generally, the responses against latent infections may 16. Kilpatrick, E. D., M. Terajima, F. T. Koster, M. D. Catalina, J. Cruz, and eventually grow to such a magnitude as to outcompete and crowd F. A. 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