The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Drake, D. R.
Right arrow Articles by Braciale, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Drake, D. R., III
Right arrow Articles by Braciale, T. J.
Right arrowPubmed/NCBI databases
*Gene
The Journal of Immunology, 2005, 175: 1507-1515.
Copyright © 2005 by The American Association of Immunologists

Transient Loss of MHC Class I Tetramer Binding after CD8+ T Cell Activation Reflects Altered T Cell Effector Function1

Donald R. Drake, III*, Rebecca M. Ream*,{dagger}, Christopher W. Lawrence*,{dagger} and Thomas J. Braciale2,*,{dagger},{ddagger}

* Beirne B. Carter Center for Immunology Research, {dagger} Department of Microbiology, and {ddagger} Department of Pathology, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Engagement of the Ag receptor on naive CD8+ T cells by specific peptide-MHC complex triggers their activation/expansion/differentiation into effector CTL. The frequency of Ag-specific CD8+ T cells can normally be determined by the binding of specific peptide-MHC tetramer complexes to TCR. In this study we demonstrate that, shortly after Ag activation, CD8+ T cells transiently lose the capacity to efficiently bind peptide-MHC tetramer complexes. This transient loss of tetramer binding, which occurs in response to naturally processed viral peptide during infection in vitro and in vivo, is associated with reduced signaling through the TCR and altered/diminished effector activity. This change in tetramer binding/effector response is likewise associated with a change in cell surface TCR organization. These and related results suggest that early during CD8+ T cell activation, there is a temporary alteration in both cell surface Ag receptor display and functional activity that is associated with a transient loss of cognate tetramer binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Induction of protective immunity against most intracellular pathogens requires the selective expansion of a small number of naive CD8+ T cell precursors into effector CTL that can clear the infection. Recent evidence suggests that between 2 and 24 h of antigenic stimulation under optimal conditions may be sufficient to trigger naive CD8+ T cells to undergo a step-wise and highly regulated differentiation program into competent effectors (1, 2, 3). Early changes following TCR ligation include the reorganization and modulation in expression of various membrane molecules that are associated with T cell function and signal transduction. Once activated, CD8+ T cells can proliferate rapidly and extensively, with one precursor generating up to several thousand daughter cells in a 2–3 day period (4). The induction (and expression) of distinct effector activities by naive CD8+ T cells after Ag encounter appears to require different activation thresholds, with the ability to produce cytokines often preceding the capacity to induce cytolysis, although both functions can be detected in recently activated CD8+ T cells (5). The outcome of CD8+ T cell expansion and differentiation is the generation of a large pool of Ag-reactive, mature CTL that can respond to TCR ligation rapidly and with increased sensitivity, such that only a few MHC-peptide complexes are necessary to trigger effector activities (6).

The development of novel flow cytometric-based techniques to quantify Ag-specific CD8+ T cell responses during the last decade has rapidly advanced our understanding of the breadth and tempo of CD8+ T cell responses against infection (7, 8). For instance, binding of tetrameric complexes of specific peptide and MHC class I molecules by TCR allows for the direct analysis of T cell specificity at the level of the Ag receptor (9). An interesting and unexpected finding in several such studies is that the extent of tetramer binding to CD8+ T cells can vary depending on the activation state of the lymphocytes. For instance, Fahmy et al. (10) found that MHC class I dimer binding increased 20- to 50-fold on effector CD8+ T cells and appears to be dependent on membrane TCR reorganization. In contrast, we previously described an activated subdominant influenza virus-specific CD8+ T cell population from immune mice that was unable to bind specific tetramer until stimulated repeatedly with cognate Ag in vitro (11). Subsequent reports documented similar tetramer-negative CD8+ T cell populations during chronic infection in humans (12) and tumor progression in mice (13). Although the significance of the tetramer-negative phenotype remains unclear, in all three cases it was associated with a lack of one or more functional activities that was also reversed after further Ag encounter in two of the studies (11, 13).

In this report, we examined the CD8+ T cell response to influenza virus in a TCR transgenic (Tg)3 model. Our results demonstrate that naive virus-specific CD8+ T cells undergo a rapid and transient loss of tetramer reactivity following Ag encounter. Our results and related observations4 suggest that this response is a normal feature of CD8+ T cell activation and maturation, which in our report occurs during natural influenza virus infection in vivo. This transient loss of tetramer binding was linked to the redistribution of cell surface TCR, which in turn was associated with altered TCR signaling for cytokine production and cytolytic activity. The implication of these findings to the generation of CD8+ T cell immunity will be discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/cAnNTac (H-2d) mice (10- to 12-wk-old) were obtained from Taconic Farms. The clone (CL)-4 TCR Tg mouse strain (14) was provided by R. W. Dutton (Trudeau Institute, Saranac Lake, NY). Thy-1.1+/+ BALB/c mice were a gift from R. I. Enelow (Yale University, New Haven, CT). The congenic mice were used at 8–12 wk of age.

Virus and virus inoculation

The preparation of influenza A/PR/8/34 (PR/8) virus (H1N1) and mouse inoculation protocol have been described (15).

CL-4 T cell stimulation

Naive CL-4 CD8+ T cells were purified to ~90% (data not shown) using CD8-conjugated magnetic beads and positive-selection columns from Miltenyi Biotec. Purified cells were labeled with 5 µM CFDA-SE (CFSE; Molecular Probes) and then used for in vitro or in vivo assays.

Adoptive transfer and preparation of tissue lymphocytes was performed as described (15). Cells were labeled with Ab to specific surface Ags or used in functional assays, as described below.

For in vitro cultures, purified, CFSE-labeled CD8+ T cells were stimulated with 20 Gy-irradiated, syngeneic spleen cells that had been pulsed with the indicated doses of hemagglutinin (HA)533–541 peptide (synthesized by University of Virginia Biomolecular Research Facility) or infected with PR/8 virus at a multiplicity of infection of 6.3. The cells were washed extensively and then cultured with CD8-purified CL-4 T cells at a stimulator to responder ratio of 10:1. Culture media consisted of IMEM (Invitrogen Life Technologies) supplemented with 10% FBS, 10 U/ml penicillin G, 10 µg/ml streptomycin sulfate, 2 mM L-glutamine, and 0.05% 2-ME. A total of 40 U/ml rhIL-2 was added to peptide cultures. At the indicated times, cells were analyzed by flow cytometry for surface marker expression and/or functional activity (see below).

Flow cytometric analyses

Tetramers were prepared as described (9) and used at a final concentration of 28 nM. The H-2Kd construct was a generous gift of J. D. Altman (Emory University, Atlanta, GA). PE-, PerCP-Cy5.5-, allophycocyanin-, or FITC-conjugated Abs specific for murine CD8{alpha} (53-6.7), CD62L (MEL-14), CD69 (H1.2F3), CD3{epsilon} (145-2C11), CD8{beta} (Ly-3.2), V{beta}8.1 (MR5-2), TCR{beta} (H57-597), Thy1.2 (53-2.1), CD16/32 (2.4G2; to block nonspecific FcR interactions), and CD25 (PC61) were purchased from BD Pharmingen and diluted as suggested by the manufacturer. A total of 5 x 105 mononuclear cells were labeled with Ab for 45 min. at 4°C in PBS containing 2% serum and 0.05% sodium azide, washed extensively, and fixed in 2% paraformaldehyde.

To detect intracellular cytokines, cultured CL-4 T cells were stimulated for 6 h with H-2Kd-expressing P815 mastocytoma targets in a 1:1 ratio in the absence or presence of the indicated doses of HA533–541 peptide and brefeldin A (1 µg/ml). The cells were surface labeled with Ab, as described, and then intracellularly labeled with Ab specific for murine IFN-{gamma} (XMG1.2; BD Pharmingen) using BD Pharmingen Cytofix/Cytoperm and perm/wash reagents. A similar procedure was used to detect intracellular granzyme B (GB7; Caltag Laboratories), although cycloheximide (10 µg/ml) was included in the assay media to inhibit new protein synthesis.

Granule exocytosis (surface CD107a mobilization) was examined using a modification of the protocol described by Betts et al. (16). Briefly, activated CL-4 T cells were cultured with P815 targets, specific peptide, and monensin (1 µg/ml). PE-conjugated anti-CD107a (1D4B; Santa Cruz Biotechnology) was included at a final concentration of 5 µg/ml during the culture period. Following T cell stimulation, the cells were labeled with Ab specific for surface molecules and fixed in 2% paraformaldehyde. To measure total CD107a expression, the nonstimulated cells were fixed, permeabilized, and labeled with anti-CD107a Ab, as described for intracellular IFN-{gamma} analysis.

All samples were acquired on a FACSCalibur flow cytometer using CellQuest software (both by BD Pharmingen). Data analysis was performed with FlowJo software (TreeStar).

Confocal analysis

CFSE-labeled cells were cultured in vitro for 2 days with different doses of specific peptide and then the proliferating (CFSE dilute) cells were isolated on a FACSVantage cell sorter (BD Pharmingen). The purified cells were surface-labeled with anti-TCR{beta} Ab, fixed in 2% paraformaldehyde, and cytospun onto glass slides. The cells were analyzed on a Zeiss LSM 510 confocal microscope (Zeiss).

Calcium flux

CD8-purified CL-4 T cells were cultured for 2 days with peptide-pulsed stimulators. The cells were then washed extensively in column wash buffer (phenol red-free HBSS, 2% FBS, 1.3 mM CaCl2, and 20 mM HEPES). The cells were labeled with column wash buffer containing 5 µM fluo-4, 5 µM fura-red, and 0.06% pluronic acid (reagents from Molecular Probes). The cells were washed extensively and then labeled with anti-CD8{alpha} Ab. Approximately 2 min before flow cytometric analysis, the cells were warmed to 37°C. After 45 s of event collection (to establish a baseline signal), biotin-conjugated anti-CD3{epsilon} or anti-V{beta}8 Ab was added at 20 µg/ml, and then streptavidin (100 µg/ml) was added 1 min later. Alternatively, ionomycin was added at 10 µg/ml.

51Cr release assay

CTL assays were performed and values for the percentage of specific release was calculated as described (11). Data were acquired on an ICN Isomedic 4/600 HE gamma counter. Spontaneous release was never greater than 10% of the total release.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Diminished peptide/MHC class I tetramer binding following T cell activation

In an effort to characterize early events in the response of naive CD8+ T cells to influenza virus, we analyzed the response of CD8+ T cells derived from Tg CL-4 mice in vitro and in vivo. As previously reported (14), these T cells express a TCR using the V{beta}8/V{alpha}10 variable region gene segments that recognizes an epitope (residues 533–541) within the transmembrane domain of the PR/8 virus HA protein in association with H-2Kd. Splenic CD8+ T cell from CL-4 mice express uniformly high levels of TCR {beta}-chain and ~85–95% of these CD8+ T cells bind the cognate HA533–541 tetramer but not another H-2Kd tetramer containing an irrelevant (HA204–212) peptide (Fig. 1). As expected, these freshly isolated CD8+ T cells predominantly express the CD62LhighCD44low phenotype characteristic of naive CD8+ T cells (Fig. 1).



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 1. Phenotypic analysis of naive CD8+ CL-4 T cells. Freshly isolated CD8-purified, naive CL-4 T cells were directly labeled ex vivo with control (HA204–212) or cognate (HA533–541) tetramers or Abs specific for markers of T cell activation. Data shown are representative of over five experiments.

 
To evaluate the tempo of activation and proliferation of CL-4 T cells following Ag encounter, purified splenic CD8+ T cells were labeled with CFSE and cultured with syngenic, irradiated splenocyte APC pulsed with 10–6 M synthetic HA533–541 peptide. In parallel (and in all in vitro experiments), dye-labeled purified CL-4 T cells were cultured with untreated splenocytes for the indicated periods to determine baseline levels of expression of tetramer and cell surface markers (Fig. 2A). Proliferation (incremental loss of CFSE dye intensity) was first detected at day 2 and continued through days 4–6 of in vitro stimulation (Fig. 2B and data not shown). The onset of T cell proliferation (days 2–3) was preceded by the up-regulation of CD25 and the loss of CD62L on a significant fraction of the T cells during the first 24 h of in vitro stimulation. The percentage of cells displaying elevated or diminished levels of marker expression (or tetramer staining) was based on quadrants established using staining of cultured unstimulated CL-4 T cells on the indicated day in culture (Fig. 2A).



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 2. Naive CD8+ T cells have a diminished capacity to bind cognate tetramer following antigenic stimulation. CD8-purified CL-4 T cells were labeled with CFSE and cultured with untreated syngeneic splenocytes (A) or cultured with syngeneic splenocytes (B) that had been pulsed with 10–6 M HA533–541 peptide. Daily cultured cells were analyzed for the expression of CD25, CD62L, tetramer, TCR{beta}, CD8{alpha}, and CD8{beta}. CD8+CFSE+ cells are represented. Quadrant markers (solid horizontal and vertical line) are established based on the intensity of staining of cultured unstimulated CL-4 T cells for the indicated marker on the indicated day. A, Baseline HA533 tetramer staining by cultured unstimulated CL-4 T cells over time is displayed. B, Quadrant demarcations (based on staining intensity of cultured unstimulated CL-4 T cells) are defined so that the majority of unstimulated CL-4 T cells are located in the upper right quadrant for tetramer, CD62L, TCR{beta}, CD8{alpha}, and CD8{beta} staining or lower right quadrant for CD25 staining. Background control tetramer or isotype Ab labeling intensity (dotted line) are shown. Numbers indicate the percentage of cells in each quadrant. Data shown are representative of over five experiments. In these replicate experiments, the percentage of cells displaying decreased or absent tetramer staining at the nadir of tetramer staining (day 2) ranged from 55 to 68%.

 
At day 1 of in vitro stimulation, there was a decrease/loss of surface TCR expression on ~50% of the T cells (Fig. 2B), which is consistent with the phenomenon of Ag-dependent TCR down-regulation (17). However, by day 2 of culture, surface TCR{beta} expression had returned to levels comparable or slightly higher than observed for unstimulated CL-4 T cells. CD3{epsilon} levels were comparable between stimulated and naive CL-4 T cells at this time (data not shown). Cognate HA533–541 tetramer binding was likewise reduced at 24 h poststimulation, but remained at low levels (300% decrease in mean fluorescence intensity, MFI) for 2–3 days despite the high cell surface expression of TCR{beta} during this period (Fig. 2B). Tetramer binding by the activated CD8+ T cells was nearly completely restored by day 4 of in vitro culture. The level of CD8{alpha}{beta} heterodimer expression is thought to affect the efficiency of the interaction of TCR with tetramer in certain circumstances (18, 19). However, we observed that the surface expression of both CD8{alpha} and CD8{beta} was unchanged or even slightly elevated on CL-4 T cells undergoing antigenic stimulation in vitro (Fig. 2B).

We considered the possibility that the loss of tetramer binding might represent an artifact of the nonphysiologic stimulation of CL-4 T cells with a relatively high concentration of synthetic peptide in vitro. To rule out this option, we next characterized the in vitro response of purified CL-4 CD8+ T cells to PR/8 virus-infected splenocyte stimulators. As Fig. 3 demonstrates, the onset of proliferation and differential expression of activation markers on the CD8+ T cells in response to virally infected APC was slightly delayed when compared with CL-4 T cells stimulated with high-dose peptide (Fig. 2B). As observed with peptide stimulation, there was a transient down-regulation of surface TCR on a fraction (64%) of the T cells at day 1 poststimulation that returned to normal by the second day of culture. Again, however, tetramer binding by the T cells was significantly diminished (370% reduction in MFI) at day 3 and was only partially restored by day 4 of culture on these activated (CD69highCD62lowCD25high) T cells (Fig. 3). This finding suggests that the loss of tetramer reactivity to activated CD8+ T cells in the face of high levels of surface TCR expression, at least in vitro, occurs in response to naturally processed viral peptide.



View larger version (65K):
[in this window]
[in a new window]
  		FIGURE 3. Naive CD8+ T cells lose the ability to bind HA533–541 tetramer following stimulation with virus-infected stimulators. Purified, CFSE-labeled CL-4 T cells were cultured with syngeneic splenocytes that had been infected with PR/8 virus. At the indicated days, cultured cells were harvested and analyzed for surface marker expression and their capacity to bind specific tetramer by flow cytometry. Quadrant demarcations (with staining of cultured unstimulated CL-4 T cells; data not shown) are defined so that the majority of unstimulated CL-4 T cells are located in the upper right quadrant for tetramer, CD62L, TCR{beta}, CD8{alpha}, and CD8{beta} staining or lower right quadrant for CD25 staining. Plots represent CD8+CFSE+ cells. Data shown are representative of three independent experiments. In these replicate experiments, the percentage of cells displaying decreased or absent tetramer staining at the nadir of tetramer staining (day 3) ranged from 64 to 73%.

 
Transient loss of peptide-MHC class I tetramer binding during viral infection in vivo

In view of these findings, it was important to establish whether this transient loss of tetramer binding was a feature of the response of CD8+ T cells to natural infection in vivo. To this end, we adoptively transferred CD8-purified, CFSE-labeled CL-4 T cells into naive recipient mice that were infected intranasally with a sublethal dose of PR/8 virus 24 h later. The fate of the transferred dye-labeled Tg CD8+ T cells in the draining mediastinal lymph nodes (MLN) and lungs was evaluated on successive days after infection. Although donor T cells present in these sites could be readily distinguished by dye intensity early after infection (days 1–3), Thy1.2+ (CD90.2) T cells were transferred into recipient Thy1.1+ congenic mice for analyses at later times (days 4, 5, and 8) postinfection (p.i.) to distinguish donor and recipient T cells based on CD90 expression.

An analysis of the kinetics of the response of CL-4 T cells to intranasal influenza infection has been reported (15). For the present experiments, we restricted the analysis to the kinetics and magnitude of TCR expression and HA533–541 tetramer binding. In the draining lymph node (Fig. 4B), the onset of donor T cell proliferation was delayed 24–36 h when compared with the stimulation of these cells in vitro, which presumably reflects the need for virus replication in the lungs and migration of Ag bearing APC from the lungs to the draining lymph node following infection (20). As reported (15), T cell proliferation in the MLN proceeded through days 4–5, with the majority of the Thy1.2+ donor CD8+ T cells undergoing at least six divisions. Throughout the course of infection, surface TCR expression on undivided and dividing cells remained high and comparable to that of MLN resident donor T cells from uninfected mice (Fig. 4A) and at day 1 of infection (Fig. 4B). At the initiation of T cell activation in the MLN (days 1–2 p.i.) there was minimal loss of tetramer staining or TCR down-regulation. With the onset of cell division in the nodes (days 3–4 p.i.), there was a marked reduction (450% decrease in MFI) in staining with the cognate tetramer on a majority of both the undivided T cells (80%) and dividing cells (89%), with no change in the TCR level. Tetramer binding was partially restored on the CL-4 T cells in the draining nodes by day 5 p.i. and had returned to normal on most of the lymph node-resident Thy1.2+ donor CD8+ T cells by day 8 following infection (Fig. 4B).



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 4. Ag encounter during natural virus infection triggers decreased tetramer binding to adoptively transferred CD8+ T cells. Purified splenic CL-4 T cells were labeled with CFSE and injected i.v. into recipient mice that were left uninfected or infected intranasally with PR/8 virus 1 day later. A, At 5 days posttransfer, MLN or pooled popliteal, inguinal, and cervical lymph nodes (NDLN) were harvested from uninfected mice and labeled with specific tetramer. Alternatively, at the indicated times p.i., MLN (B) and lungs (C) were harvested and analyzed for tetramer binding and TCR{beta} expression by flow cytometry. Plots show CD8+CFSE+ events for days 1 and 3, and CD8+Thy1.2+ cells for days 4, 5, and 8. Quadrant demarcations are defined so that the majority of unstimulated CL-4 T cells are located in the upper right quadrant for tetramer, CD62L, TCR{beta}, CD8{alpha}, and CD8{beta} staining or lower right quadrant for CD25 staining, except that staining intensity of CL-4 T cells in MLN/NDLN of uninfected mice that had received transferred T cells (data not shown), were used to set quadrants. Data shown are representative of three independent experiments. In these replicate experiments, the percentage of cells displaying decreased or absent tetramer staining at the nadir of tetramer staining (day 4) ranged from 78 to 83%.

 
As recently reported (15), at day 5 p.i., when the CL-4 T cells had undergone greater than six divisions in the MLN, there was a dramatic influx of Thy1.2+ donor T cells (which had likewise undergone six or more divisions) from the nodes into the infected lungs. These day 5 lung-infiltrating Thy1.2+ CD8+ T cells expressed high levels of TCR{beta} (Fig. 4C), comparable to the corresponding donor T cell population in the draining MLN (Fig. 4B). Importantly, as demonstrated for donor T cells in the MLN, a significant fraction (77%) of the lung-infiltrating donor T cells failed to bind the HA533–541 tetramer. Again, tetramer binding was restored on a majority of the lung-infiltrating donor CD8+ T cells by day 8 p.i. (Fig. 4C). Taken together, these results suggest that the transient loss of tetramer binding early after CD8+ T cell activation, which occurs in the face of high levels of surface TCR expression, represents a normal (physiologic) response of naive CD8+ T cells to Ag encounter in vivo.

Loss of tetramer staining is dependent on the strength of initial TCR signaling

T cell activation/differentiation is an incremental process intimately tied to the strength of TCR signaling (17, 21). Our findings to this point suggest that the transient loss of tetramer binding to the CL-4 T cells is a consequence of TCR engagement by Ag in vivo and in vitro and may represent a transition state of naive CD8+ T cells differentiating into fully activated effectors. It was therefore of interest to us to determine whether the transition of activated CD8+ T cells from a tethigh to tetlow phenotype was linked to the strength of TCR signaling. To address this issue, we examined the kinetics of the in vitro response of CFSE-labeled CL-4 T cells to splenocytes pulsed with varying concentrations (10–5–10–9 M) of the synthetic HA533–541 peptide (Fig. 5, A and B, and data not shown). T cell stimulation with the highest concentration of peptide (10–5 M) resulted in an acute down-regulation of TCR on 72% of the labeled, responding T cells at day 1 poststimulation that was associated with the nearly complete loss of tetramer binding (Fig. 5A) and up-regulation of CD25 expression (data not shown) on these cells. As observed (Figs. 2 and 3), TCR levels were rapidly restored by day 2 following stimulation and were sustained at high levels for the duration of T cell culture. By contrast, the MFI of cognate tetramer staining was reduced up to 700% through days 2–3 of culture and was partially restored at day 4 of in vitro stimulation. Furthermore, the tetlow phenotype of activated CL-4 T cells (those stimulated for 2 days with 10–5 M peptide) was unaffected by either the binding temperature or the concentration of tetramer used in this analysis (Fig. 5C).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 5. The extent of loss of tetramer binding to activated CD8+ T cells is dependent on the strength of TCR stimulation during initial Ag encounter. Purified, CFSE-labeled CL-4 T cells were cultured with syngeneic splenocytes that had been pulsed with 10–5 (A) or 10–8 M (B) HA533–541 peptide. At the indicated days, cultured cells were harvested and analyzed for surface marker expression and capacity to bind specific tetramer by flow cytometry. Plots showing CD8+CFSE+ cells are analyzed. Quadrant demarcations (with staining of cultured unstimulated CL-4 T cells; data not shown) are defined so that the majority of unstimulated CL-4 T cells are located in the upper right quadrant for tetramer, CD62L, TCR{beta}, CD8{alpha}, and CD8{beta} staining or lower right quadrant for CD25 staining. C, Purified, CFSE-labeled CL-4 CD8+ T cells were left unstimulated or cultured with 10–5 M cognate peptide for 2 days and then labeled with anti-CD8{alpha} Ab-specific tetramer at the indicated doses and temperatures for 45 min. The graph shows the MFI of tetramer binding to cultured CD8+CFSE+ T cells. Data shown are representative of over five experiments.

 
At progressively lower concentrations of stimulatory HA533–541 peptide (e.g., 10–8 M; Fig. 5B and data not shown), the fraction of T cells that responded progressively decreased and the onset of proliferation was slightly delayed. Importantly, although the kinetics of acute TCR down-regulation and recovery were independent of the peptide dose used for stimulation, a divergent pattern of tetramer responsiveness was observed as a function of peptide dose. At the highest peptide dose (i.e., 10–5 M), T cell activation was linked to a concomitant and transient loss of tetramer binding. By contrast, at the 10–8 M peptide dose, only a fraction (~10–15% of the CD8+ T cells) activated and proliferated (Fig. 5B). Importantly, the cells responding to low doses of peptide were able to undergo at least six divisions (and partially up-regulate CD25, data not shown), but exhibited minimal loss of tetramer binding over time in culture (Fig. 5B). Thus, under conditions of suboptimal stimulation (i.e., low peptide dose), the responding CD8+ T cells were stimulated to activate and divide, but were not able to undergo the transient loss of tetramer reactivity. Together, these results suggest that a transient loss of tetramer binding may be linked to optimal signaling through the TCR on naive CD8+ T cells.

Functional activity of tetlow T cells

We previously reported that memory CD8+ T cells directed to a subdominant epitope in the influenza A/JAPAN/57 HA (HA210–219) proliferated vigorously to specific Ag but exhibited a tetlow phenotype that was associated with a defect in the expression of CD8+ T cell effector activities (11). By contrast, memory effector CD8+ T cells generated in response to other dominant A/JAPAN/57 epitopes exhibited a conventional tethigh phenotype with normal expression of effector functions when sampled at days 5–6 after stimulation. It was of interest, therefore, to determine whether the loss of tetramer binding to activated CD8+ T cells specific for the dominant PR/8 HA epitope was associated with a change in the functional activity of these cells. As a convenient source of tetlow T cells for these analyses, we used CL-4 T cells stimulated with a high dose of peptide (10–5–10–6 M) in vitro. These T cells were harvested during active proliferation (i.e., days 2–3 of in vitro culture) when TCR expression on the cells was at normal levels, but specific tetramer staining was low (Fig. 5A and Fig. 2B). For comparison, we used CL-4 T cells stimulated in vitro with a low dose of agonist peptide (10–8 M) as a source of activated proliferating T cells, which retained the tethigh phenotype (Fig. 5B). In these experiments, the T cells were labeled with CFSE before in vitro culture/stimulation to distinguish the actively proliferating cells.

A hallmark of the early response of activated T cells to TCR engagement by Ag is the rapid and sustained mobilization of intracellular Ca2+ stores (22). Suboptimal Ca2+ flux has been reported for defective (anergic) T cell populations (reviewed in Ref. 23). As Fig. 6 demonstrates, when activated tetlow (10–5 M peptide-stimulated) and tethigh (10–8 M peptide-stimulated) CD8+ T cells were stimulated with the Ca2+ ionophore, ionomycin, both T cell populations rapidly mobilized Ca2+ stores to a similar degree (i.e., 4- to 5-fold above background), and the mobilization of Ca2+ was sustained for greater than 5 min. By contrast, when Ca2+ mobilization was triggered through the TCR (that is, by treatment with biotinylated anti-CD3{epsilon} with or without streptavidin cross-linking), a different pattern of Ca2+ mobilization emerged. The tethigh T cells again rapidly mobilized Ca2+ stores after anti-CD3{epsilon}/streptavidin cross-linking (Fig. 6). As has been observed for activated, fully differentiated effector CD8+ T cells (24), the tethigh T cell population could partially mobilize Ca2+ after treatment with anti-CD3{epsilon} Ab alone, without streptavidin cross-linking (Fig. 6). By contrast, the tetlow T cells exhibited only a modest mobilization of Ca2+ stores in response to anti-CD3{epsilon}/streptavidin cross-linking, and were unable to flux calcium in response to anti-CD3{epsilon} Ab alone (Fig. 6). Comparable results were observed when TCR engagement and cross-linking were conducted using biotinylated anti-V{beta}8 Ab (data not shown).



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 6. Tetlow CD8+ T cells have reduced signaling through the TCR. Purified CL-4 T cells were cultured in the presence of 10–5 M or 10–8 M HA533–541 peptide for 2 days and then labeled with 5 µM fluo-4 and 5 µM fura-red. The cells were labeled with anti-CD8{alpha} Ab and then the proliferating (CFSElow) cells were analyzed for calcium flux following cross-linking with anti-CD3{epsilon} to mimic TCR engagement or treatment with the ionophore ionomycin. For ionomycin treatment, arrow indicates time of drug addition; for anti-CD3{epsilon} cross-linking, the first arrow indicates the time of biotin-conjugated anti-CD3{epsilon} Ab addition and the second arrow indicates the time of addition of streptavidin. Data are representative of three experiments.

 
These results suggest that early in the course of CD8+ T cell activation/differentiation, the T cells displaying a transient tetlow phenotype might not efficiently trigger through the TCR and therefore could exhibit a suboptimal/defective effector response to Ag. To further explore this possibility, we examined the capacity of activated tetlow and tethigh T cells to secrete IFN-{gamma} after acute peptide stimulation. As Fig. 7A demonstrates, both T cell populations could respond with IFN-{gamma} synthesis at peptide doses as low as 10–11 M. However, although the majority of tethigh T cells (>80%) responded with cytokine synthesis, less than one-half (<45%) of the tetlow T cells produced detectable levels of IFN-{gamma}. Furthermore, the average level of IFN-{gamma} production by the responding tetlow T cells (i.e., the MFI of anti-cytokine Ab staining) was likewise about one-half the intensity of the tethigh T cell population (Fig. 7B). Given the transient expression and relative instability of cytokine message (25), this result likely reflects a reduced capacity of these cells to induce the transcription of the IFN-{gamma} gene following TCR ligation. As expected, fully differentiated CL-4 CD8+ T cells derived from cultures stimulated with a high dose of synthetic peptide for 8 days (i.e., transitioned from the early tetlow to a tethigh phenotype) regained full IFN-{gamma} responsiveness (data not shown).



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 7. Tetlow CL-4 T cells do not efficiently synthesize IFN-{gamma} following acute TCR ligation. Purified and CFSE-labeled Tg T cells were cultured for 3 days with a high or low dose of peptide and then assayed for IFN-{gamma} production following 6 h stimulation with the indicated doses of synthetic peptide. A, FACS plots showing the IFN-{gamma} response in activated tetlow and tethigh cells (plots are gated on CD8+ T cells and the number indicates the percentage of the dividing cells that are IFN-{gamma}high). B, The MFI of the IFN-{gamma}high cells (the upper left quadrant of the FACS plots) are represented graphically. Data shown are representative of three experiments.

 
Cytotoxicity is a hallmark of mature effector CD8+ T cells, and the capacity for in vitro cytolytic activity is linked to the expression of lytic granule-associated molecules, such as perforin and certain granzymes (e.g., granzyme B). We recently reported that the expression of granzyme B is rapidly up-regulated in CL-4 TCR Tg CD8+ T cells in vivo during the initial activation of these lymphocytes in response to pulmonary influenza infection (15). To evaluate the lytic potential of the activated CL-4 T cells, we analyzed the granzyme B content (by intracellular staining) of both the tetlow and the tethigh T cells in conjunction with a standard in vitro cytotoxicity assay. The tetlow T cells generated after day 2–3 of in vitro stimulation with the optimal peptide-pulsed (10–5 M; Fig. 8A) or PR/8 virus-infected APC (data not shown), expressed high levels of intracellular granzyme B on a majority of the cells (>80% of cells with an MFI of 77). Unexpectedly, the tethigh T cells expressed substantially lower levels of granzyme B, with a MFI of 31 by the positive cells (Fig. 8A). However, when the two T cell populations were tested for in vitro cytotoxicity, the level of the lytic activity was substantially higher for the tetlow T cells over several E:T ratios using target cells pulsed with a range of peptide doses (Fig. 8B).



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 8. Tetlow CD8+ T cells do not efficiently degranulate following antigenic stimulation. Purified, CFSE-labeled CL-4 T cells were cultured with peptide-pulsed stimulators for 3 days and then assayed for intracellular granzyme B (A) in the absence (solid line) or presence (dotted line) of 1 µM specific peptide for 6 h or lytic activity (B) in a standard 51Cr release assay. The populations were also assessed for total (surface and intracellular) CD107a expression (C) (with no antigenic stimulation) or up-regulation of surface CD107a (D) at various times poststimulation with the indicated dose of synthetic peptide. The FACS histograms and the graph represent the dividing (CFSE-low) CD8+ T cells. Data shown are representative of three experiments.

 
The above cytotoxicity assay results could imply that the tetlow T cells were fully functional with respect to cytolytic potential. However, it is possible that the in vitro lytic activity exhibited by the tetlow T cells was conducted by only a fraction of these cells, that is, those with the capacity to undergo granule exocytosis (and granzyme release) after TCR engagement. By contrast, the lytic activity exhibited by the tethigh T cells reflected the activity of the majority of cells responding in the assay, albeit with a lower overall efficiency because of the decreased level of granzyme expression. In support of this possibility, we observed that the coculture of these T cells with peptide-pulsed target cells for 6 h, in the presence of cycloheximide (to inhibit new protein synthesis), resulted in a substantial loss of intracellular granzyme B stores from tethigh T cells, but almost no change in granzyme B levels from the tetlow T cells (Fig. 8A). After further progression from the tetlow to tethigh phenotype in vitro (i.e., days 7–8 in culture), the T cells from the 10–5 M culture also efficiently degranulate (data not shown). These results suggested that only the tethigh CD8+ T cells efficiently degranulated following TCR engagement.

To further explore this latter possibility, we used a recently described flow cytometry-based assay that employs the mobilization of CD107a (LAMP-1a) from intracellular granule stores to the cell surface of CD8+ T cells in response to TCR engagement as a surrogate for granule exocytosis-dependent cytotoxicity (16). For this analysis, we first examined the total CD107a content (both in intracellular stores and at the cell surface) of the tethigh and tetlow T cell populations before the in vitro cocultivation with target cells. Intracellular CD107a levels were comparable for the two T cell populations (Fig. 8C), and neither T cell population expressed cell surface CD107a before antigenic stimulation (data not shown). We then monitored cell surface expression of CD107a after exposure to peptide-pulsed target cells by flow cytometry in a modified 6 h in vitro cytotoxicity assay (see Materials and Methods). As Fig. 8D demonstrates, the tethigh T cells up-regulated cell surface CD107a expression two to three times more efficiently than the tetlow T cells, suggesting that acute TCR ligation does not efficiently trigger granule exocytosis in the tetlow CL-4 T cells.

Efficiency of tetramer binding is linked to cell surface TCR distribution

We have previously demonstrated that the loss of tetramer binding by activated CD8+ T cells is association with an alteration in the organization of cell surface TCR from a clustered/aggregated arrangement to a diffuse cell surface TCR display (26). Therefore we examined the cell surface arrangement of TCR on proliferating tetlow and tethigh CL-4 T cells (labeled with CFSE before in vitro activation) by confocal microscopy. As Fig. 9 demonstrates, the majority of tethigh cells had a polarized, crescent-shaped TCR display with large regions of membrane containing little or no detectable Ag receptor. By contrast, TCR organization on the tetlow CL-4 cells was much more diffuse and symmetrical, with few regions of discrete receptor aggregation. These results are in line with our previous observations that clustered TCR appear to be most efficient at binding tetramer (26), and agree with the claim of Stefanová et al. (27) that clustered TCR induces a stronger action potential after ligand encounter than Ag receptors organized in a diffuse arrangement.



View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 9. The loss of tetramer reactivity to naive CD8+ T cells following Ag encounter is associated with the reorganization of surface TCR from a polarized to symmetrical display. CFSE+ CL-4 T cells were cultured with stimulators pulsed with 10–5 M or 10–8 M peptide. Two days poststimulation, FACS sorting was performed to isolate the dividing cells, which were then labeled with anti-TCR{beta} Ab and analyzed by confocal microscopy. Data shown are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we demonstrate, both in vivo and in vitro, that following initial TCR engagement by Ag, naive HA-specific TCR CD8+ T cells transiently enter an activation state where cognate peptide-MHC tetramer binding is markedly reduced, despite high level TCR expression. A consequence of this transition is altered TCR signaling (and diminished effector activity) and a transient redistribution of cell surface TCR display. Independently, researchers have described an identical loss of cognate peptide-MHC tetramer binding (and noncognate TCR-tetramer interactions) for two unrelated TCR Tg CD8+ T cell populations.4 These results as well as similar preliminary evidence obtained by us on the response of wild-type mice to influenza infection suggest that this transition to a tetlow state is a physiologic consequence of CD8+ T cell activation.

Both intrinsic TCR affinity and cell surface TCR expression levels affect the efficiency of cognate peptide/MHC binding to CD8+ T cells (28). We did observe the characteristic (17) rapid, transient TCR internalization early (within 24 h) after in vitro stimulation with virus (Fig. 3) or high/optimal (10–5 M, 10–6 M) peptide doses (Figs. 2B and 5A). However, surface TCR levels were restored by day 2 and remained high on days 2–4 and 5 (Figs. 2B, 3, and 5A), when the activated, proliferating T cells exhibited the tetlow phenotype. Similarly, there was a rapid and dramatic loss of peptide-MHC binding by specific CD8+ T cells in vivo in the draining nodes of virus-infected mice without any change in the high level of TCR expression (Fig. 4B). In this connection, we (11) and others (12, 13) previously characterized activated CD8+ T cell populations that express high levels of cell surface TCR, but fail to bind cognate peptide-MHC tetramer. Thus, we believe that the activation-induced loss of tetramer binding is not due to transient loss of cell surface TCR expression, but rather (as discussed below) reflects an activation state-dependent change in the display of cell surface TCR.

Daniels et al. (19) demonstrated that the efficiency of both cognate and noncognate interactions between cell surface TCR and tetrameric peptide-MHC ligand can be markedly influenced by the interaction of the MHC tetramer complex with the CD8 coreceptor. Likewise, Kambayashi et al. (29) showed that high dose IL-2 stimulation could trigger a loss of tetramer binding by CD8+ T cells that was attributed to a decrease in surface CD8{alpha} and CD8{beta} expression. In our analyses, we detected no significant activation-associated change in T cell surface expression of CD8{alpha} or CD8{beta} expression, whether in the absence or presence of IL-2, to account for the activation-dependent loss of cognate peptide-MHC tetramer binding (Fig. 2B and data not shown). Although we did not explore the impact of T cell activation on noncognate peptide interactions, researchers provide evidence that the efficiency of CD8 coreceptor interaction with peptide-MHC complexes may vary with T cell activation because they observed a dramatic loss of noncognate tetramer binding to two Tg T cell populations following Ag encounter.4 In this connection, we find that the binding of cognate peptide-MHC tetramer to both naive and mature, fully activated effector CL-4 T cells is inhibited by Ab directed to the CD8 {beta}-chain (data not shown). Thus, alteration in the display of TCR, as well as the efficiency of CD8 interaction (but not the cell surface expression of either molecule), could contribute to the activation-dependent, transient loss of T cell-MHC tetramer interaction reported in this study.

An intriguing finding in our analysis was the observation that the loss of peptide-MHC binding after T cell activation is associated with a diminished capacity of the activated CD8+ T cells to express effector activity during this early phase of differentiation. What might be the physiological significance of this diminished effector response? In considering this question, it is important to note that, under in vivo conditions of Ag exposure such as the pulmonary influenza infection examined in this study, Ag migrates (as virions or more likely as Ag-loaded, activated, mature respiratory dendritic cells) from the site of infection (e.g., the lungs) to the draining nodes (20). These Ag-bearing APC must be available to present Ag directly or indirectly (30, 31) to the recirculating pool of naive specific CD8+ T cells that traffic through the lymph node. According to our findings, these newly activated T cells then transition to a state of diminished responsiveness to antigenic stimulation that is associated with a loss of peptide-MHC tetramer binding. If, as several lines of evidence suggest (1, 2, 3), efficient CD8+ T cell activation leading to full differentiation requires limited stimulation (that is, contact between the naive T cells and Ag-bearing APC on the order of hours), then the transition of the activated T cells into this diminished responsiveness state would ensure that the activated T cells would not prematurely destroy the Ag-bearing APC before the subsequent recruitment and activation of additional specific naive T cells from the circulation to the lymph node.

The mechanism to account for this transient loss of tetramer binding is yet to be defined. We have previously reported (26) that the tetramer tetlow phenotype can be induced by the pharmacological disruption of membrane lipid rafts, which causes the redistribution of TCR into a diffuse cell surface array. Confocal data presented in this report (Fig. 9) support this view and raise the possibility that activation-associated alteration in TCR display on the cell surface, possibly through altered interactions with the underlying cytoskeleton (32, 33), could account for this transient loss of cognate peptide-MHC tetramer binding. In this connection, research provides evidence that activation-induced changes in cell surface glycosylation, possibly involving the CD8 coreceptor, may affect the efficiency of peptide-MHC tetramer staining.4 Whether these two alterations in the T cell surface are distinct or reflect different facets of the same underlying mechanism awaits experimental determination.

This activation-induced loss of peptide-MHC tetramer binding would imply that, in this activation state, T cells are unable to efficiently interact with processed Ag displayed by APC. We (11) and others (12, 13) have previously demonstrated that activated, tetramer-negative T cell populations, although lacking the capacity to express one or more effector activities in response to Ag-pulsed APC, are fully capable of proliferating and further differentiating into tetramer-binding cells with full effector activity following in vitro antigenic stimulation. Our observation in this report of suboptimal Ca2+ mobilization after TCR cross-linking (Fig. 6) suggests that, even after full TCR engagement (i.e., by cross-linking Ab), the signaling through this receptor may be suboptimal. Thus, loss of peptide-MHC tetramer binding may not represent the inability of T cells to engage APC, but may reflect an altered activation state associated with suboptimal signaling. This level of signaling through the TCR may be sufficient to drive cell proliferation, but not expression of effector activity (11).

In our initial report on the lack of cognate peptide-MHC tetramer staining by activated CD8+ T cells directed to a subdominant viral epitope, we speculated that a transient loss of tetramer binding may be part of the normal activation/differentiation program for CD8+ T cells following Ag encounter (11). The findings reported in our study further support this view. In the case of the several reports that identify tetramer negative CD8+ T cells directed to tumors or in chronic viral infections (12, 13), Ag persistence resulting in chronic antigenic stimulation has been suggested to account for this abnormal T cell phenotype. An alternative explanation consistent with the findings reported is that immune dysregulation (e.g., induced by infection with immunosuppressive agents like hepatitis B virus) results in the generation of activated CD8+ T cells that are unable to fully differentiate, that is, they are "frozen" in this tetramer-negative effector-deficient, early activation state.

The development of tetramer technology (9) has enormously advanced our ability to identify specific T cells and to quantitate T cell responses. Our findings suggest that, in applying this technology, the activation state of the responding CD8+ T cells should be taken into consideration. For example, the in vivo analysis of CD8+ T cell activation in response to virus infection in the TCR Tg adoptive transfer model (Fig. 4) implies that, in the response to pulmonary virus infection, the ability to identify and quantitate responding T cells in the draining nodes early after infection by cognate peptide-MHC tetramer staining may be limited. Using a combined set of activation/differentiation markers for Ag-driven CD8+ T cell stimulation, we have observed a higher frequency of responding T cells in the draining nodes early (days 2–4) after pulmonary influenza infection of wild-type mice than was detected by conventional tetramer staining (C. W. Lawrence and T. J. Braciale, unpublished observations). An appreciation of both the utility and limitations of powerful technologies like tetramer staining should help us to obtain new insights into the process of T cell activation and the control of T cell responses to infections, tumors, and autoaggressive responses.


   Acknowledgments
 
We thank Charlly Kao and Dr. Stephen Jameson for sharing unpublished observations and helpful discussions, and Drs. Richard Dutton and Linda Sherman for providing us the clone-4 transgenic mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work is supported by U.S. Public Health Service Grants AI-15608 and HL-33391 (to T.J.B.) and D.R.D. is the recipient of a Cancer Research Institute Postdoctoral Fellowship award. Back

2 Address correspondence and reprint requests to Dr. Thomas J. Braciale, Beirne B. Carter Center for Immunology Research, Medical Research Building 4, University of Virginia Health System, Box 801386, Charlottesville, VA 22908. E-mail address: tjb2r{at}virginia.edu Back

3 Abbreviations used in this paper: Tg, transgenic; MLN, mediastinal lymph node; p.i., postinfection; HA, hemagglutinin; MFI, mean fluorescence intensity. Back

4 C. Kao, M. A. Daniels, and and S. C. Jameson. Loss of CD8 and TCR binding to class I MHC ligands following T cell activation. Submitted for publication. Back

Received for publication December 21, 2004. Accepted for publication May 13, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Wong, P., E. G. Pamer. 2001. Cutting edge: antigen-independent CD8 T cell proliferation. J. Immunol. 166: 5864-5868.[Abstract/Free Full Text]
  2. van Stipdonk, M. J., E. E. Lemmens, S. P. Schoenberger. 2001. Naive CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation. Nat. Immunol. 2: 423-429.[Medline]
  3. Kaech, S. M., R. Ahmed. 2001. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat. Immunol. 2: 415-422.[Medline]
  4. Blattman, J. N., R. Antia, D. J. Sourdive, X. Wang, S. M. Kaech, K. Murali-Krishna, J. D. Altman, R. Ahmed. 2002. Estimating the precursor frequency of naive antigen-specific CD8 T cells. J. Exp. Med. 195: 657-664.[Abstract/Free Full Text]
  5. Opferman, J. T., B. T. Ober, P. G. Ashton-Rickardt. 1999. Linear differentiation of cytotoxic effectors into memory T lymphocytes. Science 283: 1745-1748.[Abstract/Free Full Text]
  6. Purbhoo, M. A., D. J. Irvine, J. B. Huppa, M. M. Davis. 2004. T cell killing does not require the formation of a stable mature immunological synapse. Nat. Immunol. 5: 524-530.[Medline]
  7. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. 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: 177-187.[Medline]
  8. Lukacher, A. E., J. M. Moser, A. Hadley, J. D. Altman. 1999. Visualization of polyoma virus-specific CD8+ T cells in vivo during infection and tumor rejection. J. Immunol. 163: 3369-3378.[Abstract/Free Full Text]
  9. Altman, J. D., P. A. H. Moss, P. J. R. Goulder, D. H. Barouch, M. G. McHeyzer-Williams, J. I. Bell, A. J. McMichael, M. M. Davis. 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274: 94-96.[Abstract/Free Full Text]
  10. Fahmy, T. M., J. G. Bieler, M. Edidin, J. P. Schneck. 2001. Increased TCR avidity after T cell activation: a mechanism for sensing low-density antigen. Immunity 14: 135-143.[Medline]
  11. Spencer, J. V., T. J. Braciale. 2000. Incomplete CD8+ T lymphocyte differentiation as a mechanism for subdominant cytotoxic T lymphocyte responses to a viral antigen. J. Exp. Med. 191: 1687-1698.[Abstract/Free Full Text]
  12. Reignat, S., G. J. Webster, D. Brown, G. S. Ogg, A. King, S. L. Seneviratne, G. Dusheiko, R. Williams, M. K. Maini, A. Bertoletti. 2002. Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B virus infection. J. Exp. Med. 195: 1089-1101.[Abstract/Free Full Text]
  13. Blohm, U., E. Roth, K. Brommer, T. Dumrese, F. M. Rosenthal, H. Pircher. 2002. Lack of effector cell function and altered tetramer binding of tumor-infiltrating lymphocytes. J. Immunol. 169: 5522-5530.[Abstract/Free Full Text]
  14. Lo, D., J. Freedman, S. Hesse, R. D. Palmiter, R. L. Brinster, L. A. Sherman. 1992. Peripheral tolerance to an islet cell-specific hemagglutinin transgene affects both CD4+ and CD8+ T cells. Eur. J. Immunol. 22: 1013-1022.[Medline]
  15. Lawrence, C. W., T. J. Braciale. 2004. Activation, differentiation, and migration of naive virus-specific CD8+ T cells during pulmonary influenza virus infection. J. Immunol. 173: 1209-1218.[Abstract/Free Full Text]
  16. Betts, M. R., J. M. Brenchley, D. A. Price, S. C. De Rosa, D. C. Douek, M. Roederer, R. A. Koup. 2003. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J. Immunol. Methods. 281: 65-78.[Medline]
  17. Valitutti, S., S. Muller, M. Cella, E. Padovan, A. Lanzavecchia. 1995. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature 375: 148-151.[Medline]
  18. Daniels, M. A., S. C. Jameson. 2000. Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers. J. Exp. Med. 191: 335-346.[Abstract/Free Full Text]
  19. Daniels, M. A., L. Devine, J. D. Miller, J. M. Moser, A. E. Lukacher, J. D. Altman, P. Kavathas, K. A. Hogquist, S. C. Jameson. 2001. CD8 binding to MHC class I molecules is influenced by T cell maturation and glycosylation. Immunity 15: 1051-1061.[Medline]
  20. Legge, K. L., T. J. Braciale. 2003. Accelerated migration of respiratory dendritic cells to the regional lymph nodes is limited to the early phase of pulmonary infection. Immunity 18: 265-277.[Medline]
  21. Gett, A. V., F. Sallusto, A. Lanzavecchia, J. Geginat. 2003. T cell fitness determined by signal strength. Nat. Immunol. 4: 355-360.[Medline]
  22. Zweifach, A.. 2000. Target-cell contact activates a highly selective capacitative calcium entry pathway in cytotoxic T lymphocytes. J. Cell Biol. 148: 603-614.[Abstract/Free Full Text]
  23. Schwartz, R. H.. 2003. T cell anergy. Annu. Rev. Immunol. 21: 305-334.[Medline]
  24. Esser, M. T., D. M. Haverstick, C. L. Fuller, C. A. Gullo, V. L. Braciale. 1998. Ca2+ signaling modulates cytolytic T lymphocyte effector functions. J. Exp. Med. 187: 1057-1067.[Abstract/Free Full Text]
  25. Gillis, P., J. S. Malter. 1991. The adenosine-uridine binding factor recognizes the AU-rich elements of cytokine, lymphokine, and oncogene mRNAs. J. Biol. Chem. 266: 3172-3177.[Abstract/Free Full Text]
  26. Drake, D. R., III, T. J. Braciale. 2001. Cutting edge: lipid raft integrity affects the efficiency of MHC class I tetramer binding and cell surface TCR arrangement on CD8+ T cells. J. Immunol. 166: 7009-7013.[Abstract/Free Full Text]
  27. Stefanová, I., J. R. Dorfman, R. N. Germain. 2002. Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes. Nature 420: 429-434.[Medline]
  28. Busch, D. H., E. G. Pamer. 1999. T cell affinity maturation by selective expansion during infection. J. Exp. Med. 189: 701-710.[Abstract/Free Full Text]
  29. Kambayashi, T., E. Assarsson, B. J. Chambers, H. G. Ljunggren. 2001. IL-2 down-regulates the expression of TCR and TCR-associated surface molecules on CD8+ T cells. Eur. J. Immunol. 31: 3248-3254.[Medline]
  30. Norbury, C. C., D. Malide, J. S. Gibbs, J. R. Bennink, J. W. Yewdell. 2002. Visualizing priming of virus-specific CD8+ T cells by infected dendritic cells in vivo. Nat. Immunol. 3: 265-271.[Medline]
  31. Bhardwaj, N., A. Bender, N. Gonzalez, L. K. Bui, M. C. Garrett, R. M. Steinman. 1994. Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8+ T cells. J. Clin. Invest. 94: 797-807.
  32. Wülfing, C., M. M. Davis. 1998. A receptor/cytoskeletal movement triggered by costimulation during T cell activation. Science 282: 2266-2269.[Abstract/Free Full Text]
  33. Dustin, M. L., J. A. Cooper. 2000. The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signaling. Nat. Immunol. 1: 23-29.[Medline]



This article has been cited by other articles:


Home page
 BloodHome page
J. R. Almeida, D. Sauce, D. A. Price, L. Papagno, S. Y. Shin, A. Moris, M. Larsen, G. Pancino, D. C. Douek, B. Autran, et al.
Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV-suppressive activity
Blood, June 18, 2009; 113(25): 6351 - 6360.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
R. B. Fulton, M. R. Olson, and S. M. Varga
Regulation of Cytokine Production by Virus-Specific CD8 T Cells in the Lungs
J. Virol., August 15, 2008; 82(16): 7799 - 7811.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
Z. Xiao, M. F. Mescher, and S. C. Jameson
Detuning CD8 T cells: down-regulation of CD8 expression, tetramer binding, and response during CTL activation
J. Exp. Med., October 29, 2007; 204(11): 2667 - 2677.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. Laugel, H. A. van den Berg, E. Gostick, D. K. Cole, L. Wooldridge, J. Boulter, A. Milicic, D. A. Price, and A. K. Sewell
Different T Cell Receptor Affinity Thresholds and CD8 Coreceptor Dependence Govern Cytotoxic T Lymphocyte Activation and Tetramer Binding Properties
J. Biol. Chem., August 17, 2007; 282(33): 23799 - 23810.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. S. Cush, K. M. Anderson, D. H. Ravneberg, J. L. Weslow-Schmidt, and E. Flano
Memory Generation and Maintenance of CD8+ T Cell Function during Viral Persistence
J. Immunol., July 1, 2007; 179(1): 141 - 153.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Yoon, K. L. Legge, S.-s. J. Sung, and T. J. Braciale
Sequential Activation of CD8+ T Cells in the Draining Lymph Nodes in Response to Pulmonary Virus Infection
J. Immunol., July 1, 2007; 179(1): 391 - 399.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. A. de Witte, M. Toebes, J.-Y. Song, M. C. Wolkers, and T. N. M. Schumacher
Effective graft depletion of MiHAg T-cell specificities and consequences for graft-versus-host disease
Blood, May 1, 2007; 109(9): 3830 - 3838.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
C. Kao, M. A. Daniels, and S. C. Jameson
Loss of CD8 and TCR binding to Class I MHC ligands following T cell activation
Int. Immunol., December 1, 2005; 17(12): 1607 - 1617.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Drake, D. R.
Right arrow Articles by Braciale, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Drake, D. R., III
Right arrow Articles by Braciale, T. J.
Right arrowPubmed/NCBI databases
*Gene

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS