HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lim, D.-G. Articles by Hafler, D. A. Search for Related Content PubMed PubMed Citation Articles by Lim, D.-G. Articles by Hafler, D. A.

Examination of CD8⁺ T Cell Function in Humans Using MHC Class I Tetramers: Similar Cytotoxicity but Variable Proliferation and Cytokine Production Among Different Clonal CD8⁺ T Cells Specific to a Single Viral Epitope

Dong-Gyun Lim^1,*, Katarzyna Bieganowska Bourcier^1,*, Gordon J. Freeman and David A. Hafler^2,*

^* Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, and Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Following infection by human T cell lymphotrophic virus-I (HTLV-I), high frequencies of polyclonal Tax11–19-reactive CD8⁺ T cells can be detected in the peripheral blood. To investigate whether there are differences in the effector functions of these cells, we generated a panel of Tax11–19-reactive T cell clones by single cell sorting of HLA-A2/Tax11–19 tetramer binding CD8⁺ T cells followed by repeated stimulation with PHA and IL-2. Examination of the TCRs revealed 17 different T cell clones with unique clonal origins. Nine representative CD8⁺ T cell clones showed a similar cytotoxic dose-response activity against Ag-pulsed target cells, even though they express different TCRs. This cytotoxic effector function was not influenced by the engagement of either CD28 or CD2 costimulatory molecules. In contrast to the cytotoxic activity, qualitatively different degrees of proliferative response and cytokine secretion were observed among T cell clones of different clonal origin. The induction of proliferation and cytokine secretion required the engagement of costimulatory molecules, particularly CD2-LFA-3 interaction. These results indicate that functionally diverse, polyclonal CTL populations can be activated specific to a single immunodominant viral epitope; they can manifest virtually identical cytotoxic effector function but have marked differences in proliferation and cytokine secretion.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Viral infections can induce an intense activation and expansion of CD8⁺ T cells. It was recently established that a high proportion of activated CD8⁺ T cells are specific for these viral Ags (1, 2, 3). CD8⁺ CTLs recognize viral peptide fragments complexed with class I MHC molecules on the surface of virus-infected cells. Depending on the infected virus and MHC haplotype of the host, CTL responses are focused on a few immunodominant viral peptides. The clonality of CTLs specific to a single immunodominant viral epitope has been extensively studied in different viral infections by the determination of TCR fine specificity and TCR- and -chain usage. Most studies demonstrated the polyclonality of CTL responses to a single viral epitope (4, 5, 6, 7), whereas highly restricted oligoclonal CTL responses directed against a few viral epitopes were observed (8, 9, 10). However, the relevant functional role of these polyclonal CTLs on the immune response, particularly in humans, has not been well explored. Here, we examined this issue using the Tax peptide of human T cell lymphotropic virus type I (HTLV-I)³ as a model viral epitope.

HTLV-I is a human retrovirus and can cause human adult T cell leukemia/lymphoma (11) and a slowly progressive demyelinating neurologic disease, HTLV-I-associated myelopathy/tropical spastic paraparesis (12, 13). Tax11–19 was defined as the immunodominant CTL epitope in HTLV-I-infected patients expressing the HLA-A2 allele (14). CTLs recognizing this viral epitope are expanded in vivo and occupy a high proportion of total peripheral blood as well as cerebrospinal fluid CD8⁺ T cells (15, 16). Previous analysis of TCR repertoire of these cells revealed an oligoclonal expansion of a few founder T cells in each individual patient (17, 18). Our previous ex vivo study using Tax11–19/HLA-A2 tetramer has also demonstrated high frequencies of circulating Tax11–19-reactive CD8⁺ T cells with several different but one dominant TCR -chain usage (19).

The current study was performed to determine whether CTLs having different clonal origins could perform the same effector functions following the cognate recognition of MHC-peptide ligand. Single cell cloning and expansion of cloned cells is an inevitable step for the functional analysis of individual clones. We took advantage of MHC-peptide tetramer staining for the relatively unbiased selection of Tax11–19-reactive CTLs and directly sorted these as single cells followed by expansion with PHA stimulation. From this approach, we could obtain a diverse panel of Tax11–19-reactive CTL clones. Our data demonstrate that all Tax11–19-reactive CD8⁺ T cell clones exhibited virtually identical dose responses for cytotoxic effector function, though they had different clonal origins and TCR usage. In contrast, proliferative responses and cytokine secretion were qualitatively different depending on the individual clones and costimulatory signals.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peptide and cell line

Tax11–19 peptide (LLFGYPVYV) was synthesized (Quality Controlled Biochemicals, Hopkinton, MA) and was >98.9% pure as determined by HPLC.

EBV-transformed B cell line expressing HLA-A*0201 (KS.B) was generated by incubation of PBMCs in the presence of supernatant from EBV-producing cell line B95.8 and 1 µg/ml cyclosporin A. This lymphoblastoid cell line was cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS and used as APCs or target cells.

Transfected cell lines

cDNAs for HLA-A*0201 and human ₂-microglobulin in pCMV were generously provided by Geoffrey Davis (Repligen, Cambridge, MA) and were linearized with ApaLI and XmnI, respectively. A cDNA for CD58 was generously provided by Brian Seed (Massachusetts General Hospital, Boston, MA), subcloned into pAXEF, and linearized with KpnI. Human B7-1 in pLEN was linearized with PvuI. Chinese hamster ovary (CHO) cells were transfected with HLA-A*0201, human ₂-microglobulin, and SV2-Neo, or together with LFA-3 or B7-1 by electroporation and selected in media containing 400 µg/ml G418. Transfected CHO cells were stained with FITC-conjugated anti-human HLA class I/₂-microglobulin-specific mAb (W6/32; Sigma, St. Louis, MO) and PE-conjugated anti-LFA-3 mAb (L306.4; Becton Dickinson, San Jose, CA) or PE-conjugated anti-B7-1 mAb (L307; Becton Dickinson), sorted twice, and single cell cloned.

Generation of human CD8⁺ T cell clones reactive to Tax11–19

PBMCs from peripheral blood of an HLA-A*0201-expressing patient with typical HTLV-I-associated myelopathy were isolated by Ficoll-Hypaque density gradient centrifugation. PBMCs were stained with FITC-conjugated anti-CD8 mAb and PE-conjugated HLA-A2/Tax11–19 tetramer (kindly provided by Beckman-Coulter, Miami, FL) as previously described (19). CD8⁺/Tax11–19 tetramer-positive and CD8⁺/Tax11–19 tetramer-negative T cells were directly sorted into single wells of 96-well round-bottom microplates by use of a fluorescence cell sorter (Vantage; Becton Dickinson) and stimulated with 2 µg/ml of PHA (Murex, Dartford, England) in the presence of allogeneic irradiated (5000 rad) mononuclear cells in complete medium (RPMI 1640 supplemented with 10% human serum (BioWhittaker, Walkersville, MD), 4 mM glutamine, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10 mM HEPES). Two days later, an equal volume of complete medium containing 10% T-Stim (Collaborative Biomedicine Products, Bedford, MA) was added as a source of IL-2, and cultures were maintained for 2 wk with a change of half volume of media with fresh complete media containing 10% T-Stim every 3 days. Afterward, each T cell clone was maintained by weekly restimulation with PHA and irradiated mononuclear cells.

Abs and flow cytometry

Indirect immunofluorescence staining was used for the determination of TCR V-chain usage as previously described (19). Anti-TCR V2, 3, 5.1, 5.2, 5.3, 6.1, 8, 9, 11, 12, 13.1, 13.6, 14, 16, 17, 18, 20, 21.3, 22, 23 mAb (Immunotech, Marseille, France) and FITC-conjugated anti-mouse IgG F(ab')₂ (Biosource, Camarillo, CA) as secondary Ab were used. To analyze the expression of cell surface molecules, cloned T cells were stained for 30 min on ice with FITC-conjugated anti-CD8 (clone SFCI21Thy2D3), anti-CD2 (clone SFCI3Pt2H9), anti-CD28 (clone CD28.2), or PE-conjugated anti-TCR (clone BMA031) purchased from Coulter-Immunotech. Transfected CHO cells were directly stained with PE-conjugated anti-CD58 (clone AICD58; Immunotech) or PE-conjugated anti-CD80 (clone MAB104; Immunotech) and indirectly stained with anti-HLA-A2 (BB7.2; American Type Culture Collection, Manassas, VA) followed by FITC-conjugated F(ab')₂ goat anti-mouse IgG (115-096-062; Jackson ImmunoResearch, West Grove, PA). Stained cells were fixed in 1% paraformaldehyde (Sigma) and analyzed by flow cytometry on a FACScan equipped with CellQuest software (Becton Dickinson).

Cytotoxicity assay

Cytotoxicity was measured by a standard ⁵¹Cr-release assay. Target cells (2 x 10⁶) were labeled with 200 µCi of ⁵¹Cr (Amersham, Arlington Heights, IL) for 1 h, washed twice, and pulsed with peptide (concentrations indicated in result) for 2 h. ⁵¹Cr-labeled target cells (1 x 10⁴/well) were incubated with effector T cells at 10:1 E:T ratio in a final volume of 200 µl in 96-well U-bottom plates. Spontaneous release was assessed by incubating target cells in the absence of effector cells, and maximal release was determined by incubating target cells in the presence of 1% Triton X-100. After 4 h of incubation, 50 µl of supernatant was spotted on filtermat paper (Printed filtermat B 1205-404; LKB-Wallac, Gaithersburg, MD) and assayed for ⁵¹Cr-release in a 1205 Betaplate Liquid Scintillation Counter (LKB-Wallac). Percent specific lysis was calculated as 100 x (cpm of sample - cpm of spontaneous release)/(cpm of maximum release - cpm of spontaneous release).

Proliferation assay

Proliferation was assessed by [³H]thymidine incorporation assay. As APCs, KS.B, and transfected CHO cells were irradiated with 5000 rad or treated with mitomycin C (100 µg/ml for 2 h), respectively, pulsed with the indicated concentration of peptide for 2 h at 37°C, and washed twice in complete medium to remove free peptide. APCs (2 x 10⁴) and CD8⁺ T cell clone (10⁵) were added to each well of 96-well round-bottom plates to a final volume of 200 µl and cultured for 72 h including pulse with [³H]thymidine (1 µCi/well) for the last 18 h. Cells were harvested on a Tomtec cell harvester (Tomtec, Orange, CT), and incorporation of [³H]thymidine was determined using liquid scintillation counter (1205 Betaplate counter; LKB-Wallac).

Cytokine assay

Cloned CD8⁺ T cells were stimulated with KS.B or transfected CHO cells prepulsed with Tax11–19 peptide, as described in the proliferation assay. After 48 h of incubation, supernatants were collected and tested for the presence of cytokines. IFN- and IL-4 were quantified by capture ELISAs as previously described (20). Cytokine concentrations were calculated from a standard curve. The detection limits were 100 pg/ml for IFN- and 10 pg/ml for IL-4.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation and functional specificity of Tax11–19-reactive CD8⁺ T cell clones

Previous investigations using HLA-A2/Tax11–19 tetramer have demonstrated a high frequency of polyclonal Tax11–19-reactive T cells in the peripheral blood of patients with HTLV-I-associated myelopathy (16, 19). PBMCs isolated from peripheral blood of an HLA-A*0201-expressing patient with HTLV-I-associated myelopathy were stained with a PE-labeled HLA-A2/Tax11–19 tetramer and an FITC-labeled anti-CD8 mAb. As shown in Fig. 1A, the peripheral blood displayed a high proportion of CD8⁺ T cells labeled with Tax11–19 tetramer (13.8%).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Cellular origin and specificity of CD8+ T cell clones. A, PBMCs from HTLV-I-infected, HLA-A*0201-positive patient were stained with FITC-conjugated anti-CD8 and PE-conjugated HLA-A*0201/Tax11–19 tetramer. B, CD8+/Tax11–19 tetramer-positive and CD8+/Tax11–19 tetramer-negative T cells were directly single cell sorted and expanded with PHA stimulation. Tax11–19-specific proliferative activity of established clones from each cell population was assessed using HLA-A*0201/LFA-3 expressing CHO cells as APCs. Each dot represents a single clone. Tetramer-positive clones, n = 35; tetramer-negative clones, n = 22.

Heterogeneous clonal origin but similar cytotoxic effector function of T cell clones

The clonal origin of T cell clones was assessed by determination of TCR V-chain usage (Table I) and sequencing the CDR3 region of the TCR - and -chains (K. D. Bourcier, D.-G. Lim, Y.-H. Ding, K. J. Smith, K. Wucherpfennig, and D. A. Hafler, manuscript in preparation). We could identify 17 different clonal populations from 25 tetramer binding CD8⁺ T cell clones tested. To determine whether there is any difference in functional reactivity among these T cell clones with different clonal origins, we examined cytotoxic activity of nine representative CD8⁺ T cell clones to the target cells pulsed with different doses of Tax11–19 peptide. Interestingly, all nine different CD8⁺ T cell clones showed very similar peptide-Ag dose-response cytotoxic activity with 0.05 µM of peptide concentration inducing half-maximal response of cytotoxic activity (Fig. 2).

View this table: [in this window] [in a new window]   Table I. TCR V chain usage and the expression level of cell surface molecules of T cell clones

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Dose-response cytotoxic activity of CD8+ T cell clones. KS.B expressing HLA-A*0201 was pulsed for 2 h at different peptide concentrations and used as target in a 51Cr release assay. E:T ratio was 10:1 and spontaneous release of 51Cr was <20% of total release. The lysis of peptide nonpulsed cells was <5%.

Previously, it has been shown that the level of MHC/peptide tetramer binding to T cells is directly correlated with the TCR affinity for MHC/peptide (21). The above observation that all the CD8⁺ T cell clones showed the identical dose-response cytotoxic activity even though they have different TCR usage might come from the preselection of CD8⁺ T cells having a similar TCR affinity to MHC-Tax11–19 ligand, because all these clones originated from the cells stained strongly with tetramer (Fig. 1). To address this possibility, we generated additional Tax11–19-reactive CD8⁺ T cell clones from the same subject, gating with different levels of tetramer-staining. In this second single cell-sorting and expanding experiment, the gate for the tetramer-high staining populations was set at the similar position as the first sorting gate (Fig. 1A) and the gate for the tetramer-low staining populations at a position very close to the negative population (Fig. 3A). Surprisingly, even CD8⁺ T cell clones that originated from T cells with a very weak tetramer-staining profile, if they were reactive to the Ag, exhibited similar Ag-dose response cytotoxic activity in comparison with tetramer-high staining T cell clones. The mean peptide concentration inducing half maximal lysis was 0.042 ± 0.004 µM in tetramer-high clones and 0.040 ± 0.007 µM in tetramer-low clones (Fig. 3, B and C).

View larger version (32K): [in this window] [in a new window]   FIGURE 3. The intensity of tetramer staining does not correlate with dose-response cytotoxic activity of Ag-reactive T cell clones. A, PBMCs from the same patient as in Fig. 1 were stained with PE-conjugated HLA-A*0201/Tax11–19 tetramer. B, Depending on the tetramer staining, tetramer-positive T cells were separated into tetramer high and low staining cells, and then directly single cell sorted and expanded as in Fig. 1. Dose-response cytotoxic activity of established clones were examined by 51Cr release assay using KS.B as target cells. E:T ratio was 10:1. Each line represents the cytotoxic activity of each clone. C, The peptide concentrations required for half maximal lysis were calculated from the peptide dose-response curve displayed in B, and plotted as dots for individual T cell clones. Horizontal bars are mean values of the peptide concentration required for half maximal lysis in each groups. Tetramer low (TL) group includes T cell clones showing Ag-reactivity (n = 11).

We examined whether T cell clones of different clonal origins exhibited similar dose responses to antigenic stimulation by measuring other functional T cell properties. T cell clones stimulated with HLA-A*0201 expressing KS.B prepulsed with different doses of Tax11–19 were examined for proliferative responses and secretion of cytokines (IFN- and IL-4). In contrast to the cytotoxic activity, markedly different magnitudes of proliferation and, especially, variable amounts of cytokine secretion were observed among different T cell clones, even though they expressed similar level of TCR on their cell surfaces (Table I). For example, TP59 and TP35 required 5–10 times more Tax11–19 peptide for inducing half maximal proliferation with Tax11–19 peptide stimulation (Fig. 4). All the T cell clones secreted variable amounts of IFN- in a similar Ag dose-responsive fashion. However, IL-4 secretion profile was qualitatively different depending on the individual T cell clone. Two clones (TP41 and TP60) secreted relatively high amounts of IL-4, whereas two other clones (TP35 and TP59) did not secrete IL-4 above the detection level, implying Tc1 type T cells.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Proliferative and cytokine-producing activity of T cell clones in response to Tax11–19. KS.B irradiated and pulsed with different doses of Tax11–19 was used as the APC. Proliferative activity was assessed by [3H]thymidine incorporation. IFN- and IL-4 were measured from culture supernatants by capture ELISA after 48 h of incubation and detection limits were 100 and 10 pg/ml for IFN- and IL-4, respectively.

The efficient activation of T cells can be achieved by receiving two signals, one from TCR and the other from costimulatory molecules. The engagement of adhesion/costimulatory molecules is known to have a significant impact on the effector functions of T cells (22, 23). To determine whether the dissociation of effector functions observed in different T cell clones was due to differential influence by costimulatory molecules, we examined the effector functions of this panel of T cell clones after stimulation with Ag-pulsed transfected CHO cells expressing HLA-A*0201 alone or in combination with B7-1 or LFA-3 (Fig. 5A). As expected, cytotoxic activity of T cell clones was not augmented by the costimulatory molecules, and similar dose-response cytotoxic activity could be observed among different T cell clones against target CHO cells expressing HLA-A2 alone (Fig. 5B). In contrast to the cytotoxic activity, proliferation and cytokine secretion were highly dependent on the costimulatory signal. Ag-presenting CHO cells expressing HLA-A*0201 alone did not efficiently induce proliferative or cytokine-producing effector functions from T cell clones, and B7-1 only marginally enhanced these effector functions even in T cell clones expressing CD28 (Table I). In marked contrast, LFA-3 significantly augmented both proliferative responses and cytokine secretion of all the T cell clones. Perhaps of greater interest, engagement of CD2 by LFA-3 induced the secretion of IL-4 in a subset of Tax11–19-reactive T cell clones. However, qualitatively and quantitatively different functional activities of individual T cell clones observed using the EBV-transformed B cell line as APC were generally conserved in response to antigenic stimulation together with defined strong costimulation by LFA-3. These data suggest that the different proliferative and cytokine-producing effector functions observed in different T cell clones were not due to differential influence by two major costimulatory molecules such as B7-1 and LFA-3.

View larger version (49K): [in this window] [in a new window]   FIGURE 5. Requirement of the engagement of costimulatory molecules for the functional activity of CD8+ T cell clones. A, CHO cells transfected with HLA-A*0201 and human 2-microglobulin or together with LFA-3 or B7-1 were stained with PE-conjugated anti-CD58, PE-conjugated anti-CD80, or anti-HLA-A2 followed by FITC-conjugated goat anti-mouse IgG. B, CD8+ T cell clones were cultured with the various CHO transfectants pulsed with different doses of Tax11–19. Cytotoxicity was assessed by the 51Cr-release assay using 10:1 E:T ratio. Proliferation was determined by [3H]thymidine incorporation after a 72-h incubation including the last 18 h of [3H]thymidine pulse. Supernatants were collected after 48 h of culture and analyzed for IFN- and IL-4 by capture ELISA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

To study the effector function of individual T cells, their clonal expansion is a necessary step. In previous studies, repeated antigenic stimulation of T cells and limiting dilution methods have been used to obtain clonal populations of Ag-specific T cells. However, during this process, a few T cell clones tend to dominate and minor populations of Ag reactive T cells might be lost from the repertoire pool of Ag-specific T cells. This might be due to activation-induced cell death or poor proliferative capacity following repeated stimulation with Ag. In addition, depending on the in vitro stimulation protocols used to generate CTL clones, a biased T cell repertoire might be generated (6). To minimize these problems, we adapted a novel tetramer staining method for the selection of Ag-specific CD8⁺ T cells, followed by direct single cell sorting and expansion with PHA stimulation. The effectiveness of this approach was revealed in two aspects. First, all T cell clones generated from the tetramer binding CD8⁺ T cells specifically recognized the Ag, whereas all T cell clones generated from the tetramer staining negative CD8⁺ T cells did not. More importantly, the repertoire of Tax11–19-reactive CD8⁺ T cell clones was more diverse than that obtained from conventional methods by others (17, 18).

Previous studies using a limited number of CD8⁺ T cell clones generated after repeated antigenic stimulation suggested that all T cell clones have similar cytotoxic effector functions against target cells expressing cognate viral Ag (6, 24). Our data support these previous observations with a more comprehensive analysis of TCR repertoires. As expected, each representative CD8⁺ T cell clone expressing a different TCR exhibited its own fine specificity of cytotoxic responses to the antigenic peptide analogues (K. D. Bourcier, D.-G. Lim, Y.-H. Ding, K. J. Smith, K. Wucherpfennig, and D. A. Hafler, manuscript in preparation). There are several possibilities to explain why the Tax11–19-reactive T cell clones exhibited the same dose-response in respect to cytotoxic effector function. First, MHC/peptide tetramers might only detect T cells with a high affinity TCR. However, several experimental data argue against this possibility. It was shown that CD8⁺ T cells depleted of tetramer staining cells could not lyse the Ag-pulsed target cells (25). Moreover, all tetramer-negative T cells cloned from blood did not show any proliferative or cytotoxic activity with Tax11–19 stimulation (Fig. 1, and data not shown). A second possibility is that T cells expressing TCRs with affinities above a certain threshold to the cognate antigenic peptide are activated and expanded in vivo, whereas T cells expressing TCRs with affinities below this threshold could not be efficiently expanded, making them undetectable in our testing pool of repertoire.

Previous direct ex vivo investigations observed a very limited and oligoclonal expansion of CTLs reactive to immunodominant viral epitopes with chronic infections and memory immune responses (19, 26, 27, 28). However, our data using this novel method to generate a population of T cell clones indicated that there exist a rather diverse repertoire of T cells specific to an immunodominat epitope, and that these T cells exert a similar cytotoxic effector function. This then raises the question as to what generates dominant T cell populations in vivo. Recent studies have suggested two possible explanations. Busch and Pamer (29) and Savage et al. (30) provided evidence for an increase in average affinity of TCR for MHC/peptide that drives the selection for the preferred TCR in vivo. Another explanation comes from the observation by Bousso et al. (31) in that the differences in precursor pools appear to be the major source of individual variability in Ag-selected repertoires. If we extend this finding to the dominance of certain repertoires, we can speculate that the frequency of specific precursors in the preimmune repertoire reflects the dominance of certain repertoires.

It is well known that CD4⁺ T cells require two signals for activation; one from the TCR by engagement with MHC-peptide ligand, and the other from the engagement of costimulatory molecules (32, 33). On the contrary, there is a controversy in the requirement of costimulatory molecules for the activation of CD8⁺ CTLs. Some reports indicated that activation and effector cytotoxic function of CTLs are dependent on the engagement of costimulatory molecules, such as B7, LFA-3, and ICAM-1, in addition to the TCR signal (34, 35). In contrast, some CTLs could exert their cytotoxic effector function without the interaction of costimulatory molecules (36). Our CD8⁺ T cell clones recognizing Tax11–19 peptide did not require the engagement of costimulatory molecules for the cytotoxic effector function. The discrepancy of the requirement of the costimulatory molecules for the cytotoxic effector function could be explained by the difference of TCR affinity to the antigenic ligands depending on both the type of Ag and corresponding T cells tested (37, 38). In the light of this interpretation, all of our CD8⁺ T cell clones might have TCRs with strong affinity sufficient to induce cytotoxic activity with engagement of TCR alone.

In contrast to cytotoxic function, proliferation and cytokine secretion were significantly dependent on a second signal in our panel of T cell clones. Previous reports indicated that B7-CD28 interactions play an important role in the optimal and sustained proliferation rather than augmentation of cytotoxicity and cytokine production (36). However, the panel of T cell clones do not express or express very low levels of CD28 molecule on their surface (Table I), which might explain the limited enhancement of proliferation in the B7-1-transfected CHO system as an APC. LFA-3-CD2 interaction has been shown to augment the secretion of IFN- from Ag-stimulated CD8⁺ T cells (35, 39). Our finding that LFA-3-transfected CHO cells greatly enhanced IFN- production is in agreement with previous reports. Our data extend these observations to show that the LFA-3-CD2 engagement is also critical for Tc2, IL-4 secretion. Interestingly, LFA-3-CD2 engagement induced strong proliferative responses in the CD8⁺ T cell clones, and the extent of proliferation was equivalent to those obtained from the presentation of Ag by an EBV-transformed B cell line, which expresses high level of all major costimulatory molecules, including B7, ICAM-1, LFA-3, etc. (data not shown). This finding suggests that a costimulatory signal coming from LFA-3-CD2 engagement plays a critical role in the induction of both cytokine production and proliferation from the memory type of CD28-negative/weak positive CD8⁺ T cells.

The exact role of CD2 in T cell activation has not yet been clearly defined. Initial studies suggested the signaling role of CD2 molecule on the basis of the observation that certain combinations of Abs to CD2 can trigger T cell activation and that these effects require an intact cytoplasmic domain (40, 41). However, the signaling role of CD2 remains controversial because there is no clear signaling event by a physiological interaction between CD2 on T cells with a ligand on APCs. Recent structural studies and a detailed observation of cell to cell conjugation has led to the proposal that CD2 plays a critical role for the formation of the immunological synapse involving the redistribution of cell surface molecules for efficient T cell activation (42). This mechanism might explain the facilitated proliferation and cytokine production of CD8⁺ T cell clones following stimulation with LFA-3-transfected CHO cells in our experiments.

Differences in proliferation and cytokine secretion observed among the different T cell clones could not be ascribed to the differential engagement of CD2-LFA-3, a strong costimulatory pathway in our CD8⁺ T cell clones. Nevertheless, the effects of other costimulatory molecules cannot be excluded. Specifically, in a few T cell clones, there were small differences in both the magnitude of cytokine secretion and peptide dose-response proliferation between EBV-transformed B cell line or HLA-A2 and LFA-3-transfected CHO cells as APCs. Further studies are necessary to determine whether other costimulatory signals are brought into the immunologic synapse changing the immune function of T cell clones.

In conclusion, heterogeneous CTLs specific to a single viral peptide can exert their cytolytic activity against target cells with almost identical dose responses despite different TCR usage. This cytolytic effector function is not dependent on the engagement of costimulatory molecules. However, proliferation and cytokine secretion, which are strongly dependent on the engagement of costimulatory molecules, particularly CD2-LFA-3 interaction, are not the same among different T cells specific to a single viral peptide.

	Footnotes

 
¹ D.-G.L. and K.B. contributed equally to this work.

² Address correspondence and reprint requests to Dr. David A. Hafler, Center for Neurologic Diseases, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Boston, MA 02115.

³ Abbreviations used in this paper: HTLV-I, human T cell lymphotrophic virus type I; CHO, Chinese hamster ovary.

Received for publication April 14, 2000. Accepted for publication September 1, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Butz, E. A., M. J. Bevan. 1998. Massive expansion of antigen-specific CD8⁺ T cells during an acute virus infection. Immunity 8:167.[Medline]
2. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. D. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177.[Medline]
3. Callan, M. F. C., L. Tan, N. Annels, G. S. Ogg, J. D. K. Wilson, C. A. O’Callaghan, N. Steven, A. J. McMichael, A. B. Rickinson. 1998. Direct visualization of antigen-specific CD8⁺ T cells during the primary immune response to Epstein-Barr Virus in vivo. J. Exp. Med. 187:1395.[Abstract/Free Full Text]
4. Horwitz, M. S., Y. Yanagi, M. B. A. Oldstone. 1994. T-cell receptors from virus-specific cytotoxic T lymphocytes recognizing a single immunodominant nine-amino-acid viral epitope show marked diversity. J. Virol. 68:352.[Abstract/Free Full Text]
5. Cose, S. C., J. M. Kelly, F. R. Carbone. 1995. Characterization of a diverse primary herpes simplex virus type 1 gB-specific cytotoxic T-cell response showing a preferential V bias. J. Virol. 69:5849.[Abstract]
6. Ishikawa, T., D. Kono, J. Chung, P. Fowler, A. Theofilopoulos, S. Kakumu, F. V. Chisari. 1998. Polyclonality and multispecificity of the CTL response to a single viral epitope. J. Immunol. 161:5842.[Abstract/Free Full Text]
7. Naumov, Y. N., K. T. Hogan, E. N. Naumova, J. T. Pagel, J. Gorski. 1998. A class I MHC-restricted recall response to a viral peptide is highly polyclonal despite stringent CDR3 selection: implications for establishing memory T cell repertoires in "real-world" conditions. J. Immunol. 160:2842.[Abstract/Free Full Text]
8. Moss, P. A., R. J. Moots, W. M. Rosenberg, S. J. Rowland-Jones, H. C. Bodmer, A. J. McMichael, J. I. Bell. 1991. Extensive conservation of and chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc. Natl. Acad. Sci. USA 88:8987.[Abstract/Free Full Text]
9. Lehner, P. J., E. C. Wang, P. A. Moss, S. Williams, K. Platt, S. M. Friedman, J. I. Bell, L. K. Borysiewicz. 1995. Human HLA-A0201-restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the V17 gene segment. J. Exp. Med. 181:79.[Abstract/Free Full Text]
10. Bowness, P., P. A. H. Moss, S. Rowland-Jones, J. I. Bell, A. J. McMichael. 1993. Conservation of T cell receptor usage by HLA B27-restricted influenza-specific cytotoxic T lymphocytes suggests a general pattern for antigen-specific major histocompatibility complex class I-restricted responses. Eur. J. Immunol. 23:1417.[Medline]
11. Uchiyama, T., J. Yodoi, K. Sagawa, K. Takatsuki, H. Uchino. 1977. Adult T-cell leukemia: clinical and hematologic features of 16 cases. Blood 50:481.[Free Full Text]
12. Gessain, A., F. Barin, J. C. Vernant, O. Gout, L. Maurs, A. Calender, G. de The. 1985. Antibodies to human T-lymphotrophic virus type-I in patients with tropical spastic paraparesis. Lancet 2:407.[Medline]
13. Osame, M., K. Usuku, S. Izumo, N. Ijichi, H. Amitani, A. Igata, M. Matsumoto, M. Tara. 1986. HTLV-1 associated myelopathy, a new clinical entity. Lancet 1:1031.[Medline]
14. Koenig, S., R. M. Woods, Y. A. Brewah, A. J. Newell, G. M. Jones, E. Boone, J. W. Adelsberger, M. W. Baseler, S. M. Robinson, S. Jacobson. 1993. Characterization of MHC class I restricted cytotoxic T cell responses to Tax in HTLV-1 infected patients with neurologic disease. J. Immunol. 156:3874.
15. Elovaara, I., S. Koenig, A. Y. Brewah, R. M. Woods, T. Lehky, S. Jacobson. 1993. High human T cell lymphotrophic virus type 1 (HTLV-1)-specific precursor cytotoxic T lymphocyte frequencies in patients with HTLV-1-associated neurological disease. J. Exp. Med. 177:1567.[Abstract/Free Full Text]
16. Greten, T. F., J. E. Slansky, R. Kubota, S. S. Soldan, E. M. Jaffee, T. P. Leist, D. M. Pardoll, S. Jacobson, J. P. Schneck. 1998. Direct visualization of antigen-specific T cells: HTLV-1 Tax11–19-specific CD8⁺ T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients. Proc. Natl. Acad. Sci. USA 95:7568.[Abstract/Free Full Text]
17. Elovaara, I., U. Utz, S. Smith, S. Jacobson. 1995. Limited T cell receptor usage by HTLV-1 tax-specific, HLA class I restricted cytotoxic T lymphocytes from patients with HTLV-1 associated neurological disease. J. Neuroimmunol. 63:47.[Medline]
18. Utz, U., D. Banks, S. Jacobson, W. E. Biddison. 1996. Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8⁺ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion. J. Virol. 70:843.[Abstract]
19. Bieganowska, K., P. Höllsberg, G. J. Buckle, D.-G. Lim, T. F. Greten, J. Schneck, J. D. Altman, S. Jacobson, S. L. Ledis, B. Hanchard, et al 1999. Direct analysis of viral-specific CD8⁺ T cells with soluble HLA-A2/Tax11–19 tetramer complexes in patients with human T cell lymphotropic virus-associated myelopathy. J. Immunol. 162:1765.[Abstract/Free Full Text]
20. Fukaura, H., S. C. Kent, M. J. Pietrusewicz, S. J. Khoury, H. L. Weiner, D. A. Hafler. 1996. Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J. Clin. Invest. 98:70.[Medline]
21. Crawford, F., H. Kozono, J. White, P. Marrack, J. Kappler. 1998. Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes. Immunity 8:675.[Medline]
22. Janeway, C. A., M. Golstein. 1993. Lymphocyte activation and effector functions: the role of cell surface molecules. Curr. Opin. Immunol. 5:313.[Medline]
23. Jenkins, M. K.. 1994. The ups and downs of T cell costimulation. Immunity 1:443.[Medline]
24. Kalams, S. A., R. P. Johnson, M. J. Dynan, K. E. Hartman, T. Harrer, E. Harrer, A. K. Trocha, W. A. Blattner, S. P. Buchbinder, B. D. Walker. 1996. T cell receptor usage and fine specificity of human immunodeficiency virus 1-specific cytotoxic T lymphocyte clones: analysis of quasispecies recognition reveals a dominant response directed against a minor in vivo variant. J. Exp. Med. 183:1669.[Abstract/Free Full Text]
25. Kuroda, M. J., J. E. Schmitz, D. H. Barouch, A. Craiu, T. M. Allen, A. Sette, D. I. Watkins, M. A. Forman, N. L. Letvin. 1998. Analysis of gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex. J. Exp. Med. 187:1373.[Abstract/Free Full Text]
26. Moss, P. A. H., R. J. Moots, W. M. C. Rosenberg, S. J. Rowland-Jones, H. C. Bodmer, A. J. McMichael, J. I. Bell. 1991. Extensive conservation of and chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc. Natl. Acad. Sci. USA 88:8987.
27. Sourdive, D. J. D., K. Murali-Krishna, J. D. Altman, A. J. Zajac, J. K. Whitmire, C. Pannetier, P. Kourilsky, B. Evavold, A. Sette, R. Ahmed. 1998. Conserved T cell receptor repertoire in primary and memory CD8 T cell responses to an acute viral infection. J. Exp. Med. 188:71.[Abstract/Free Full Text]
28. Wilson, J. D. K., G. S. Ogg, R. L. Allen, P. J. R. Goulder, A. Kelleher, A. K. Sewell, C. A. O’Callaghan, S. L. Rowland-Jones, M. F. C. Callan, A. J. McMichael. 1998. Oligoclonal expansions of CD8⁺ T cells in chronic HIV infection are antigen specific. J. Exp. Med. 188:785.[Abstract/Free Full Text]
29. Busch, D. H., E. G. Pamer. 1999. T cell affinity maturation by selective expansion during infection. J. Exp. Med. 189:701.[Abstract/Free Full Text]
30. Savage, P. A., J. J. Boniface, M. M. Davis. 1999. A kinetic basis for T cell receptor repertoire selection during an immune response. Immunity 10:485.[Medline]
31. Bousso, P., A. Casrouge, J. D. Altman, M. Haury, J. Kanellopoulos, J.-P. Abastado, P. Kourilsky. 1998. Individual variations in the murine T cell response to a specific peptide reflect variability in naïve repertoires. Immunity 9:169.[Medline]
32. Harding, F., J. G. McArthur, J. A. Gross, D. H. Raulet, J. P. Allison. 1992. CD28 mediated signalling costimulates murine T cells and prevents the induction of anergy in T cell clones. Nature 356:607.[Medline]
33. Williams, I. R., E. R. Unanue. 1990. Costimulatory requirements of murine Th1 clones: the role of accessory cell-derived signals in responses to anti-CD3 antibody. J. Immunol. 145:85.[Abstract]
34. Malefyt, R. W., S. Verma, M.-T. Bejarano, M. Ranes-Goldberg, M. Hill, H. Spits. 1993. CD2/LFA-3 or LFA-1/ICAM-1 but not CD28/B7 interactions can augment cytotoxicity by virus-specific CD8⁺ cytotoxic T lymphocytes. Eur. J. Immunol. 23:418.[Medline]
35. Parra, E., A. G. Wingren, G. Hedlund, T. Kalland, M. Dohlsten. 1997. The role of B7-1 and LFA-3 in costimulation of CD8⁺ T cells. J. Immunol. 158:637.[Abstract]
36. Krummel, M. F., W. R. Heath, J. Allison. 1999. Differential coupling of second signals for cytotoxicity and proliferation in CD8⁺ T cell effectors: amplication of the lytic potential by B7. J. Immunol. 163:2999.[Abstract/Free Full Text]
37. Bachmann, M. F., E. Sebzda, T. M. Kündig, A. Shahinian, D. E. Speiser, T. W. Mak, P. S. Ohashi. 1996. T cell responses are governed by avidity and costimulatory thresholds. Eur. J. Immunol. 26:2017.[Medline]
38. Wang, B., R. Maile, R. Greenwood, E. J. Collins, J. A. Frelinger. 2000. Naïve CD8⁺ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells. J. Immunol. 164:1216.[Abstract/Free Full Text]
39. Guiner, S. L., E. L. Dréan, N. Labarriére, J.-F. Fonteneau, C. Viret, E. Diez, F. Jotereau. 1998. LFA-3 co-stimulates cytokine secretion by cytotoxic T lymphocytes by providing a TCR-independent activation signal. Eur. J. Immunol. 28:1322.[Medline]
40. Moingeon, P., H. C. Chang, P. H. Sayre, L. K. Clayton, A. Alcover, P. Gardner, E. L. Reinherz. 1989. The structural biology of CD2. Immunol. Rev. 111:111.[Medline]
41. Beyers, A. D., A. N. Barclay, D. A. Law, Q. He, A. F. Williams. 1989. Activation of T lymphocytes via monoclonal antibodies against rat cell surface antigens with particular reference to CD2 antigen. Immunol. Rev. 111:59.[Medline]
42. Bachmann, M. F., M. Barner, M. Kopf. 1999. CD2 sets quantitative thresholds in T cell activation. J. Exp. Med. 190:1383.[Abstract/Free Full Text]

This article has been cited by other articles:

				Y. Liu, J. McNevin, H. Zhao, D. M. Tebit, R. M. Troyer, M. McSweyn, A. K. Ghosh, D. Shriner, E. J. Arts, M. J. McElrath, et al. Evolution of Human Immunodeficiency Virus Type 1 Cytotoxic T-Lymphocyte Epitopes: Fitness-Balanced Escape J. Virol., November 15, 2007; 81(22): 12179 - 12188. [Abstract] [Full Text] [PDF]

				R. Fukumoto, M. Dundr, C. Nicot, A. Adams, V. W. Valeri, L. E. Samelson, and G. Franchini Inhibition of T-Cell Receptor Signal Transduction and Viral Expression by the Linker for Activation of T Cells-Interacting p12I Protein of Human T-Cell Leukemia/Lymphoma Virus Type 1 J. Virol., September 1, 2007; 81(17): 9088 - 9099. [Abstract] [Full Text] [PDF]

				K. A. Casey and M. F. Mescher IL-21 Promotes Differentiation of Naive CD8 T Cells to a Unique Effector Phenotype J. Immunol., June 15, 2007; 178(12): 7640 - 7648. [Abstract] [Full Text] [PDF]

				H. S. Bashyam, S. Green, and A. L. Rothman Dengue Virus-Reactive CD8+ T Cells Display Quantitative and Qualitative Differences in Their Response to Variant Epitopes of Heterologous Viral Serotypes. J. Immunol., March 1, 2006; 176(5): 2817 - 2824. [Abstract] [Full Text] [PDF]

				V. Lennerz, M. Fatho, C. Gentilini, R. A. Frye, A. Lifke, D. Ferel, C. Wolfel, C. Huber, and T. Wolfel The response of autologous T cells to a human melanoma is dominated by mutated neoantigens PNAS, November 1, 2005; 102(44): 16013 - 16018. [Abstract] [Full Text] [PDF]

				E. Elkord, P. E. Williams, H. Kynaston, and A. W. Rowbottom Differential CTLs specific for prostate-specific antigen in healthy donors and patients with prostate cancer Int. Immunol., October 1, 2005; 17(10): 1315 - 1325. [Abstract] [Full Text] [PDF]

				Y. Zhao, Z. Zheng, P. F. Robbins, H. T. Khong, S. A. Rosenberg, and R. A. Morgan Primary Human Lymphocytes Transduced with NY-ESO-1 Antigen-Specific TCR Genes Recognize and Kill Diverse Human Tumor Cell Lines J. Immunol., April 1, 2005; 174(7): 4415 - 4423. [Abstract] [Full Text] [PDF]

				T. Ueno, H. Tomiyama, M. Fujiwara, S. Oka, and M. Takiguchi Functionally Impaired HIV-Specific CD8 T Cells Show High Affinity TCR-Ligand Interactions J. Immunol., November 1, 2004; 173(9): 5451 - 5457. [Abstract] [Full Text] [PDF]

				M. R. Betts, D. A. Price, J. M. Brenchley, K. Lore, F. J. Guenaga, A. Smed-Sorensen, D. R. Ambrozak, S. A. Migueles, M. Connors, M. Roederer, et al. The Functional Profile of Primary Human Antiviral CD8+ T Cell Effector Activity Is Dictated by Cognate Peptide Concentration J. Immunol., May 15, 2004; 172(10): 6407 - 6417. [Abstract] [Full Text] [PDF]

				J. M. Slavik, D.-G. Lim, S. J. Burakoff, and D. A. Hafler Rapamycin-resistant Proliferation of CD8+ T Cells Correlates with p27kip1 Down-regulation and bcl-xL Induction, and Is Prevented by an Inhibitor of Phosphoinositide 3-Kinase Activity J. Biol. Chem., January 9, 2004; 279(2): 910 - 919. [Abstract] [Full Text] [PDF]

				D.-G. Lim, J. M. Slavik, K. Bourcier, K. J. Smith, and D. A. Hafler Allelic Variation of MHC Structure Alters Peptide Ligands to Induce Atypical Partial Agonistic CD8+ T Cell Function J. Exp. Med., July 7, 2003; 198(1): 99 - 109. [Abstract] [Full Text] [PDF]

				B. Wang, A. Sharma, R. Maile, M. Saad, E. J. Collins, and J. A. Frelinger Peptidic Termini Play a Significant Role in TCR Recognition J. Immunol., September 15, 2002; 169(6): 3137 - 3145. [Abstract] [Full Text] [PDF]

				H. Pilch, H. Hohn, K. Freitag, C. Neukirch, A. Necker, P. Haddad, B. Tanner, P. G. Knapstein, and M. J. Maeurer Improved Assessment of T-Cell Receptor (TCR) VB Repertoire in Clinical Specimens: Combination of TCR-CDR3 Spectratyping with Flow Cytometry-Based TCR VB Frequency Analysis Clin. Vaccine Immunol., March 1, 2002; 9(2): 257 - 266. [Abstract] [Full Text] [PDF]

				K. D. Bourcier, D.-G. Lim, Y.-H. Ding, K. J. Smith, K. Wucherpfennig, and D. A. Hafler Conserved CDR3 Regions in T-Cell Receptor (TCR) CD8+ T Cells That Recognize the Tax11-19/HLA-A*0201 Complex in a Subject Infected with Human T-Cell Leukemia Virus Type 1: Relationship of T-Cell Fine Specificity and Major Histocompatibility Complex/Peptide/TCR Crystal Structure J. Virol., October 15, 2001; 75(20): 9836 - 9843. [Abstract] [Full Text] [PDF]

				M. J. Dobrzanski, J. B. Reome, and R. W. Dutton Role of Effector Cell-Derived IL-4, IL-5, and Perforin in Early and Late Stages of Type 2 CD8 Effector Cell-Mediated Tumor Rejection J. Immunol., July 1, 2001; 167(1): 424 - 434. [Abstract] [Full Text] [PDF]

				R. M. Welsh Assessing CD8 T Cell Number and Dysfunction in the Presence of Antigen J. Exp. Med., March 5, 2001; 193(5): F19 - F22. [Full Text] [PDF]

				J. M. Slavik, D.-G. Lim, S. J. Burakoff, and D. A. Hafler Uncoupling p70s6 Kinase Activation and Proliferation: Rapamycin-Resistant Proliferation of Human CD8+ T Lymphocytes J. Immunol., March 1, 2001; 166(5): 3201 - 3209. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lim, D.-G. Articles by Hafler, D. A. Search for Related Content PubMed PubMed Citation Articles by Lim, D.-G. Articles by Hafler, D. A.