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Only a Subset of Phosphoantigen-Responsive ₉₂ T Cells Mediate Protective Tuberculosis Immunity¹

Charles T. Spencer^*,, Getahun Abate^*, Azra Blazevic^* and Daniel F. Hoft^2,*,

^* Department of Internal Medicine and Department of Molecular Microbiology and Immunology, Saint Louis University, St. Louis, MO 63104

	Abstract

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Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG) induce potent expansions of human memory V₉⁺V₂⁺ T cells capable of IFN- production, cytolytic activity, and mycobacterial growth inhibition. Certain phosphoantigens expressed by mycobacteria can stimulate ₉₂ T cell expansions, suggesting that purified or synthetic forms of these phosphoantigens may be useful alone or as components of new vaccines or immunotherapeutics. However, we show that while mycobacteria-activated ₉₂ T cells potently inhibit intracellular mycobacterial growth, phosphoantigen-activated ₉₂ T cells fail to inhibit mycobacteria, although both develop similar effector cytokine and cytolytic functional capacities. ₉₂ T cells receiving TLR-mediated costimulation during phosphoantigen activation also failed to inhibit mycobacterial growth. We hypothesized that mycobacteria express Ags, other than the previously identified phosphoantigens, that induce protective subsets of ₉₂ T cells. Testing this hypothesis, we compared the TCR sequence diversity of ₉₂ T cells expanded with BCG-infected vs phosphoantigen-treated dendritic cells. BCG-stimulated ₉₂ T cells displayed a more restricted TCR diversity than phosphoantigen-activated ₉₂ T cells. In addition, only a subset of phosphoantigen-activated ₉₂ T cells functionally responded to mycobacteria-infected dendritic cells. Furthermore, differential inhibitory functions of BCG- and phosphoantigen-activated ₉₂ T cells were confirmed at the clonal level and were not due to differences in TCR avidity. Our results demonstrate that BCG infection can activate and expand protective subsets of phosphoantigen-responsive ₉₂ T cells, and provide the first indication that ₉₂ T cells can develop pathogen specificity similar to β T cells. Specific targeting of protective ₉₂ T cell subsets will be important for future tuberculosis vaccines.

	Introduction

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Previously T cells were considered innate immune cells, responding to various generalized inflammatory markers. However, newer evidence suggests that the ₉₂ T cell subset may exhibit pathogen specificity (1) as well as immune memory (2, 3), two characteristics of adaptive immunity. We have demonstrated that ₉₂ T cells from mycobacteria-immune individuals expand after in vitro Mycobacterium bovis bacillus Calmette-Guérin (BCG)³ stimulation with a memory-like recall response (2). Consistent with our observations, enhanced secondary ₉₂ T cell responses have been observed in nonhuman primates infected with virulent Mycobacterium tuberculosis (3). In addition to activation by M. tuberculosis (4), ₉₂ T cells proliferate and develop relevant effector functions during infections with virulent M. bovis (5), Listeria monocytogenes (6), Plasmodia (7), and herpesviruses (8). Furthermore, depletion of circulating ₉₂ T cells has been associated with the exacerbation of susceptibility to infectious diseases (9, 10, 11).

Human ₉₂ T cells can be activated by nonpeptidic, phosphorylated biometabolites (phosphoantigens) and alkylamines. Natural phosphoantigens, including biosynthetic precursors of lipid and steroid synthesis (e.g., prenyl pyrophosphates) as well as phosphorylated nucleotides, are secreted as byproducts from infecting pathogens and are released from host cells damaged by infection (12). Although it is clear that optimal ₉₂ T cell activation requires the presence of APC (13), the specific Ags expressed or induced by human pathogens that stimulate protective ₉₂ T cell responses and the molecular details of how these Ags are presented to ₉₂ TCR are unknown.

Because optimal doses of two natural phosphoantigens, isopentenyl pyrophosphate (IPP) (14) and (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMB-PP) (15), and a synthetic phosphoantigen, bromohydrin pyrophosphate (BrHPP) (16), can induce the expansion of ₉₂ T cells in vitro at levels comparable to those induced by live BCG stimulation, it has been proposed that these phosphoantigens may be valuable components of next generation tuberculosis (TB) vaccines or immunotherapeutics (17, 18, 19). However, the possibility that vaccination with purified phosphoantigens can induce protective TB immunity has not been reported. Furthermore, we have shown in functional in vitro assays that IPP-stimulated PBMC from purified protein derivative-positive (PPD⁺) donors do not inhibit intracellular mycobacterial growth, whereas purified ₉₂ T cells from these same donors expanded by stimulation with BCG-infected APC can potently inhibit the growth of intracellular mycobacteria (20). In this study we expand these findings to show that BrHPP and HMB-PP also fail to stimulate ₉₂ T cells capable of inhibiting intracellular mycobacterial growth. In addition, costimulation through the TLR during phosphoantigen stimulation of ₉₂ T cells failed to induce mycobacterial inhibitory activity.

We hypothesized that live mycobacteria express Ags, other than the previously identified phosphoantigens, that are required to induce a subset of protective ₉₂ T cells. To analyze this protective subset, we have investigated differences in the diversity of the ₉₂ T cell immunorepertoire by TCR spectratypic and sequence analyses of the expressed CDR3. We found that live BCG induced a more restricted CDR3 spectratype and sequence diversity compared with IPP stimulation. These TCR studies, combined with further differential functional results, indicate that BCG-stimulated ₉₂ T cells comprise a specific subset of IPP-reactive ₉₂ T cells. These results have important implications for the development of prophylactic vaccines and immunotherapies designed to induce protective, pathogen-specific ₉₂ T cell responses.

	Materials and Methods

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Reagents

Whole cell lysates of M. tuberculosis (MtbWL) were generated as previously described (2). Ag concentrations for expansion were as follows: BCG multiplicity of infection (MOI) of 0.02–1 depending on the BCG strain used; 5 µg/ml M. tuberculosis Erdman lysate; 2–1000 nM BrHPP (provided by J.-J. Fournié, Institut National de la Santé et de la Recherche Médicale, Toulouse, France); 0.2 pM to 500 nM HMB-PP (HDMAPP; Echelon Bioscience); and 5–100 µM IPP (Echelon Bioscience) supplemented with 20 U/ml recombinant human IL-2 (rhIL-2; Hoffman-LaRoche). TLR1/2, TLR3, TLR4, and TLR9 agonists were tested, respectively, at the following ranges of concentrations: 1–100 µg/ml (S)-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys₄-OH, 3HCl (Pam₃Cys; Echleon Bioscience); 10–1000 µg/ml polyinosinic:polycytidylic acid (polyI:C; Sigma-Aldrich); 1–100 ng/ml LPS (Sigma-Aldrich); and 1–50 ng/ml CpG ODN (Coley Pharma).

Intracellular mycobacteria growth inhibition assay

We obtained PBMC from healthy volunteers with evidence of mycobacteria-specific, delayed-type hypersensitivity (10 mm of induration 48–72 h after intradermal placement of 5 IU of tuberculin) by density gradient purification from whole blood samples or leukapheresis products following Institutional Review Board-approved protocols. Frozen samples were thawed in the presence of 20% normal human serum (Sigma-Aldrich). For most experiments, target and effector cell populations were prepared as previously described (20, 21). Briefly, PBMC were plated to obtain macrophage targets that were infected with BCG (MOI = 3) the day before the addition of the T cells. Total PBMC and freshly purified T cells were added to infected macrophages at an E:T ratio of 10:1. Long-term T cell lines and T cell clones were added at an E:T ratio of 1:1. To test the direct inhibitory effects of T cell clones on intracellular mycobacterial growth without other T cells being present, target cells were prepared by plating 10-fold higher concentrations of PBMC and waiting until day 3 to wash away nonadherent cells. This latter method of preparing target macrophages was found to prevent the BCG growth-enhancing effects of the addition of 7-day-rested PBMC described earlier (20, 21), simplifying the interpretation of the results. Percentage of inhibition was calculated as 100 – (100 x (experimental cultures/control cultures)). Fold expansion of T cells was determined by flow cytometric staining 7 days after stimulation. Absolute numbers (AN) of T cells present were computed by multiplying the flow cytometric percentages times the numbers of viable cells present after expansion, and fold expansions were calculated as (AN of T cells after Ag expansion)/(AN of T cells after medium rest).

Generation of long-term Ag-specific T cell lines

PBMC were thawed and expanded with BCG or IPP as described above. After 7 days of stimulation, the cells were transferred to 24-well plates and expanded in fresh medium containing not more than 50 U/ml rhIL-2 replaced every 3–4 days as needed. After expansion with IL-2, the cells were cocultured with BCG-infected or IPP-pulsed autologous dendritic cells (DC) as APC. DC were generated by the incubation of adherent monocytes with GM-CSF (Immunex) and IL-4 (R&D Systems) and induced to mature with IL-6 (BD PharMingen), TNF- (Roche), IL-1β (BD PharMingen), and PGE₂ (Sigma-Aldrich) as previously described (22). DC were infected overnight with BCG at an MOI of 5 or pulsed with 10 µM IPP, purified by centrifugation over Ficoll-Paque PLUS (Amersham-Pharmacia) to remove extracellular BCG, and irradiated with 3000 rad before culture at a 1:5 APC:T cell ratio. Ten µM IPP was added to the culture medium of IPP-stimulated cultures to induce the optimal proliferation of IPP-reactive T cells. T cells and APC were cocultured for 14 days, replacing medium supplemented with rhIL-2 every 3–4 days. After a second Ag/DC expansion cycle, the resulting T cell populations were purified by immunomagnetic bead selection (Miltenyi Biotech), routinely resulting in >95% purity. ₉₂ T cells were purified as needed to maintain the purity of the populations as determined by flow cytometry. The Ag/IL-2 stimulation/expansion process was repeated every 2 wk. At least four cycles were completed before the cells were used in TCR spectratyping experiments. Some cells were frozen to preserve the populations for later use.

Limiting dilution cloning of Ag-specific T cells

T cell clones were generated from BCG and IPP primarily expanded T cells. Ag-expanded T cells were purified on day 7 as above with immunomagnetic beads and plated at 0.3, 3, 30, and 300 cells/well in 96-well round-bottom tissue culture plates. Irradiated autologous feeder cells (PBMC or nonadherent cells) were added at 1 x 10⁵ cells/well. The T cells were stimulated with 2 µg/ml PHA-L (leukoagglutinin; Sigma-Aldrich) for 24 h and then expanded by replenishing the medium with 5 U/ml rhIL-2 every 3–4 days as needed beginning on day 1. Potential clones were identified visually by increase in the size of the cell pellet. As predicted by the Poisson distribution, cell growth was assumed to be due to clonal expansion if less than one-third of the wells plated for a given dilution showed cell growth. Positive clones were removed to a separate plate and restimulated with 2 µg/ml PHA every 2–3 wk. Clonal T cells (shown to be 100% positive for TCR by FACS) were tested for the ability to inhibit intracellular mycobacterial growth by coculture with BCG-infected macrophages as described above. Clones able to inhibit mycobacterial growth were defined as those clones suppressing intracellular mycobacterial growth to levels less than the mean minus the SE of growth detected in control wells without T cells added.

Cytotoxicity assay

The cytolytic activities of BCG- and IPP-expanded T cells were assessed by a standard 4-h chromium release assay. Daudi cells (1 x 10⁶) (American Type Culture Collection) were labeled overnight with 100 µCi of Cr⁵¹ (GE Healthcare) in 750 µl of RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 10% FBS (Sigma-Aldrich). The following day, these Daudi cells were washed and incubated for 15 min in medium to reduce spontaneous release. Purified T cells and 5,000 Daudi cell targets were cultured at varying E:T ratios for 4 h at 37°C with 5% CO₂. Spontaneous release was measured by incubation in medium alone. Maximum lysis was measured by lysis of the Daudi cells with 2% Triton X-100. Supernatants were harvested and the amount of Cr⁵¹ released was quantified in a Micro-β scintillation counter (PerkinElmer). Percentage of lysis was calculated as follows: (experimental cpm – spontaneous cpm)/(maximal cpm – spontaneous cpm) x 100.

Flow cytometric analysis

The following Abs from BD PharMingen were used for flow cytometric analyses: anti- TCR PE (clone 11F2); anti-β TCR FITC (clone B3); anti-CD3 PerCP (clone SK7); anti- TCR FITC (clone WT31); anti-CD4 PE (clone SK3); anti-CD8 FITC (clone SK1); anti-2 TCR PE (clone B6); anti-9 TCR FITC (clone B1); anti-IFN- allophycocyanin or PE (clone B27); anti-perforin PE (clone G9); anti-granzyme A FITC (clone CB9); anti-CD134 PE (clone 1D11); anti-NKp46 PE (clone 9E2); anti-CD161 FITC (clone DX12); anti-KIR-NKAT2 PE (clone DX27); anti-CD94 FITC (clone HP-3D9); anti-NKB1 FITC (clone DX9); and anti-NKp44 PE (clone p44-8.1). The anti-granulysin PE (clone DH2) was purchased from eBioscience. Cells were stained for 20 min in 100 µl of PBS supplemented with 4% FCS (Sigma-Aldrich) and 1% NaN₃ at 4°C and washed with 1 ml of staining buffer. Cells were fixed in 1% methanol-free paraformaldehyde (Polyscience) in PBS and analyzed on a BD Immunocytometry Systems FACSCalibur at the Flow Cytometry Core Facility at Saint Louis University (St. Louis, MO). For intracellular cytokine studies, cells were stained as above for surface molecules and then fixed and permeabilized for 15 min using BD Cytofix/Cytoperm (BD Bioscience) using the manufacturer’s recommended dosage. Staining for intracellular molecules was performed for 20 min in 100 µl of PermWash (BD Bioscience) at 4°C and washed and analyzed on a BD Immunocytometry Systems FACSAria or LSR II at the Core Flow Facility at Saint Louis University. Flow cytometric analysis of clonal T cell responses to Ag titrations was preformed on Guava EasyCyte (Guava Technologies).

Spectratypic analyses

Total RNA was harvested from T cell populations following the Qiagen RNeasy protocol, including the optional DNase treatment. V₉- and V₂-specific CDR3 sequences were amplified by RT-PCR (Sigma JumpStart AccuTaq). First strand synthesis was primed by anchored oligo(dT) primers to generate a cDNA library representative of the entire cellular mRNA pool. Primer sequences complementary to upstream V regions and downstream C regions were used to amplify CDR3 regions (5'-CCAGTACTAAAACGCTGTC-3' and 5'-GTCGTTAGTCTTCATGGTGTTCCC-3' (23) for TCR₉ chains; 5'-GCACCATCAGAGAGAGATGAAGGG-3' and 5'-AAACGGATGGTTTGTATGAGGC-3' (24) for TCR₂ chains). The C region primers were 5'-end-labeled with the D2 dye from Beckman Coulter by Proligo. Using the Beckman Coulter CEQ8000 genetic analysis system, the labeled fragments were separated by capillary gel electrophoresis and compared against size standards to estimate the nucleotide length for each fragment. The resulting fragment peaks were analyzed using the fragment analysis software package included with the CEQ8000 system, using the default settings to define peak threshold.

PCR cloning and sequencing

RNA harvested for spectratyping was reverse transcribed as above. PCR was performed with the same primer sequences as above using Taq polymerase (Sigma-Aldrich) to incorporate an adenosine at the 3'-end of the amplified DNA. The PCR fragments were ligated into the pCR2.1 TA cloning vector (Invitrogen) and the resulting plasmids were transfected by heat shock into TOP10 competent Escherichia coli (Invitrogen) for propagation. Glycerol stocks were frozen to maintain the clones. Colonies were picked and grown overnight in 1–2 ml of Luria-Bertani broth containing carbenicillin (50 mg/ml). Plasmids were purified using the DNeasy Miniprep kit (Qiagen). The plasmid DNA was sequenced using the Dye Terminator Cycle Sequencing Quick Start kit (Beckman-Coulter) and the M13 reverse primer (5'-CAGGAAACAGCTATGAC-3'). Sequences were determined using the Beckman-Coulter CEQ8000 genetic analysis system. CDR3 lengths were calculated as four less than the number of amino acids between the last conserved Cys residue in the V region to the conserved GXG or AXG motif in the joining segment as previously described (25).

Heterologous stimulation of long-term ₉₂ T cell lines and clones

A modification of a previously published method (26) to measure Ag-specific intracellular cytokine production was used to quantitate the degree of specificity of our long-term ₉₂ T cell lines and clones. DC were infected with a titration of the Danish strain of BCG (MOI = 1–100) or pulsed with 100 µM IPP or a titration of HMB-PP (10–1000 pM) for 2 h. Purified ₉₂ T cells were cultured with BCG-infected or IPP-pulsed DC at an APC:T cell ratio of 1:1.2 in the presence of anti-CD28 and anti-CD49d (both at 1 µg/ml; BD PharMingen). For the phosphoantigen stimulation, phosphoantigen was added to the culture medium to stimulate responsive T cells. After 3 h of stimulation at 37°C, 0.7 µl/ml GolgiStop (BD PharMingen) was added and the cultures were incubated for five more hours at 37°C. Cells were surface-stained with anti-β TCR, anti- TCR, anti-V9 TCR, or anti-CD3. Then cells were fixed and permeabilized with Cytofix/Cytoperm (BD PharMingen) and stained for intracellular IFN- before four-color analysis on a FACSCalibur flow cytometer or three-color analysis on a Guava EasyCyte. A minimum of 10,000 events were acquired and analyzed using CellQuest or Guava EasyCyte software. For analyses of long-term T cell lines, the percentages of T cells staining positively for IFN- expression after homologous stimulation (i.e., BCG stimulation of BCG-expanded T cells and IPP stimulation of IPP-expanded T cells) were defined as 100% responses. Percentages of heterologous responses were calculated as the percentages of IFN--positive T cells after heterologous stimulation (i.e., IPP stimulation of BCG-expanded T cells and BCG stimulation of IPP-expanded T cells) divided by the percentages of IFN--positive T cells after homologous stimulations, multiplied times 100. For analyses of T cell clones, the percentages of T cells staining positively for IFN- expression are reported.

Graphics, statistics, and correlations

Graphics and statistical results were generated using Microsoft Excel, Statistica, or SPSS. Percentage of inhibition is displayed as mean ± SE. Mann-Whitney U tests were used to compare peak number distributions from TCR spectratyping. Wilcoxon matched pairs tests were used to compare intracellular BCG growth inhibition for the following: 1) matched primary cultures of PBMC stimulated with phosphoantigens and live BCG; 2) phosphoantigen- and BCG-expanded long-term T cell lines generated from the same individuals; and 3) stimulations of ₉₂ T cell lines with homologous and heterologous Ags (Ags used to expand long-term lines or not, respectively). The percentages of BCG- and IPP-expanded ₉₂ T cell clones able to inhibit intracellular mycobacterial growth were compared using ² tests. To control for differential cloning efficiency, Spearman rank correlations were used to compare spectratyping and sequencing data. Both spectratyping and sequencing data were normalized by multiplying each nucleotide fragment length detected times the observed frequency to generate continuous variables for comparison by the Spearman rank test.

	Results

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BCG-expanded ₉₂ T cells inhibit intracellular mycobacterial growth, whereas IPP-expanded ₉₂ T cells do not

Despite the ability of mycobacteria and phosphoantigens to expand ₉₂ T cells (Fig. 1A), we have found that BCG- and IPP-expanded PBMC have differential effects on intracellular mycobacterial growth. Results for the four volunteers studied in Fig. 1A demonstrate that IPP was at least as potent as BCG in stimulating the expansion of ₉₂ T cells. However, PBMC expanded with BCG were capable of inhibiting the growth of intracellular mycobacteria by 65% compared with cultures containing only medium-rested PBMC (Fig. 1B; _*, p < 0.005; n = 12). In contrast, PBMC from the same volunteers expanded with IPP failed to inhibit intracellular mycobacterial growth (p = 0.81; n = 12).

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Differential effector functions of BCG- and IPP-expanded 92 T cells. A, Both BCG and IPP expand V9+V2+ T cells. After 1 wk of rest in medium (MR) alone or expansion with either BCG or IPP, T cells present were identified by staining with anti-CD3 and either a pan- TCR-specific mAb or V9- and V2-specific mAb. Virtually all T cells expanded with either BCG or IPP expressed both V9 and V2. None of the expanded T cells expressed detectable V1 or V1 chains (data not shown). Vol, Volunteer. B, BCG- but not IPP-expanded PBMC inhibit intracellular mycobacteria. PBMC were cultured in vitro with BCG, IPP plus IL-2, or in medium alone for 7 days and then cocultured with autologous BCG-infected macrophages. BCG viability was determined 3 days later by [3H]uridine incorporation. PBMC expanded with BCG could inhibit intracellular mycobacterial growth (n = 12/group; *, p < 0.005). PBMC expanded with IPP did not inhibit intracellular BCG (not significant (n.s.), p = 0.81 by Wilcoxon matched-pairs tests). C, Purified BCG-expanded 92 T cells inhibit intracellular mycobacteria. BCG expanded and purified T cells (purity >98% -TCR+CD3+ T cells) or total BCG-expanded PBMC, were cocultured with infected macrophages at an E:T ratio of 10:1. Both total BCG-expanded PBMC and purified BCG-expanded T cells significantly inhibited intracellular mycobacterial growth compared with cultures containing medium-rested cells alone (n = 9/group; *, p < 0.01 by Wilcoxon matched pairs test), and the inhibitory effects of purified T cells were significantly greater than the effects of total BCG-expanded PBMC (n = 9/group; **, p = 0.011). D, Both BCG- and IPP-expanded T cells express cytolytic effector functions. T cells were purified from PBMC after 7 days of expansion with BCG or IPP plus IL-2. Cr51-labeled Daudi cells and purified T cells were cultured at various E:T ratios for 4 h at 37°C. Supernatants were harvested and the percentage of lysis was calculated as described. E, Mycobacteria and phosphoantigen stimulation induce similar levels of key effector molecules. PBMC from two PPD+ volunteers (Vol #1 and Vol #2) were rested in medium (open histogram with thin line), stimulated with 5 µg/ml mycobacterial lysate (open histogram with thick line), or stimulated with 250 pM HMB-PP (filled histogram) for 7 days. The levels of intracellular perforin, granzyme A, granulysin, and IFN- were assessed by intracellular cytokine staining. Results shown are gated on CD3+V2+ T cells.

Another possible explanation for the failure of IPP to induce protective ₉₂ T cells could be that this purified phosphoantigen did not fully induce activated effector functions. Effector ₉₂ T cells have been shown previously to be capable of cytolytic activity directed against tumor cells such as the EBV-transformed Daudi cell line (27, 28). Although IPP-expanded ₉₂ T cells were unable to inhibit intracellular mycobacterial growth, they demonstrated higher levels of lytic activity directed against the Daudi cell line compared with BCG-expanded ₉₂ T cells (Fig. 1D). In addition, ₉₂ T cells stimulated with phosphoantigen had similar levels of intracellular staining for the effector molecules perforin, granzyme, granulysin, and IFN- (Fig. 1E), effectively ruling out the possibility that purified phosphoantigen stimulation alone failed to induce ₉₂ T cell effector functions. These results demonstrate that while 10 µM IPP does stimulate cytolytic activity and other effector molecules in ₉₂ T cells, these ₉₂ T cells fail to inhibit intracellular mycobacterial growth. In contrast, live BCG- and MtbWL-stimulated ₉₂ T cells expressing the same effector molecules and functions potently inhibit intracellular mycobacterial growth.

Failure of IPP-expanded ₉₂ T cells to inhibit mycobacterial growth is not related to the dose or phosphoantigen potency

Although the 10 µM IPP concentration used in Fig. 1 induced greater expansions of ₉₂ T cells than BCG stimulation in three of four volunteer samples (Fig. 1A), we considered the possibility that optimal IPP concentrations for the induction of ₉₂ T cell expansion and inhibitory function may be different, such that the concentrations used may have been optimal for expansion but not for induction of inhibitory functions in ₉₂ T cells. To address this possibility, we analyzed the inhibitory properties of PBMC expanded with a dose titration of IPP. As shown in Fig. 2B, T cell expansion (open bars) was maximal at an IPP concentration of 25 µM and then declined. Although BCG-expanded PBMC inhibited intracellular mycobacterial growth by >50%, none of the IPP doses, even at concentrations much higher than required for optimal T cell expansion, resulted in the generation of ₉₂ T cells able to inhibit intracellular mycobacterial growth (filled bars).

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Failure of IPP-expanded 92 T cells to inhibit mycobacterial growth is not related to IPP dose or phosphoantigen potency. A, Structure and synthesis of the three phosphoantigens used in these investigations. The mevalonate pathway of IPP biosynthesis (left side) is common to most organisms and converts acetyl CoA to IPP through the intermediate mevalonate. In contrast, only bacteria and lower eukaryotes (right side) can convert gylceraldehyde-3-phosphate and pyruvate to IPP through the intermediates MEP, DOXP, and HMB-PP. A synthetic phosphoantigen has been developed for clinical applications by converting IPP into BrHPP. These phosphoantigens all stimulate the expansion of 92 T cells with the indicated potencies (EC50). B–D, The lack of inhibitory effects by phosphoantigen-expanded PBMC is independent of phosphoantigen concentration. PBMC were expanded with various concentrations of IPP (5–75 µM) (B), BrHPP (2–1000 nM) (C), or HMB-PP (1 pM to 500 nM) (D) and tested for their ability to inhibit mycobacterial growth (filled bars; left axis). The fold changes in expansion of T cells detected by flow cytometry are depicted by open bars (right axis). Fold expansion was calculated as (percentage T cells in stimulated cultures)/(percentage T cells in medium rested cultures). Data shown are means ± SE (n = 5 volunteers). BCG, BCG-expanded PBMC; MR, medium-rested PBMC.

Although IPP is known to be produced in mycobacteria-infected cells, concentrations of IPP needed to stimulate ₉₂ T cell expansions are unlikely to be achieved in mycobacteria-infected cells. Furthermore, IPP is produced as a critical intermediate in the synthesis of isoprenoids in almost all living organisms via the mevalonate biosynthetic pathway (12). The ubiquitous nature of IPP and the suboptimal IPP concentrations present in mammalian cells strongly suggest that IPP is not the natural ligand responsible for inducing TB-protective ₉₂ T cells. The discovery of the 2-C-methyl-D-erythritol 4-phosphate (MEP)/1-deoxy-D-xylulose 5-phosphate (DOXP) pathway for IPP biosynthesis (Fig. 2A) present in bacteria (including mycobacteria) and lower eukaryotes (29), but not in mammalian cells, provided a potential alternative mechanism to explain mycobacteria-specific induction of TB-protective ₉₂ T cells. The intermediates produced in the MEP/DOXP pathway could be seen as "foreign" by the mammalian host. Furthermore, the penultimate metabolite in this pathway, HMB-PP, was shown to be at least 10,000-fold more potent for the induction of ₉₂ T cell expansion than IPP (15), such that physiologic concentrations of HMB-PP expressed in mycobacteria-infected cells could reasonably be expected to induce ₉₂ T cells in vivo. After finding that IPP and BrHPP failed to induce ₉₂ T cells inhibitory for intracellular mycobacteria, it was critically important to test whether HMB-PP could stimulate ₉₂ T cells with mycobacterial inhibitory effects (Fig. 2D). T cells expanded (Fig. 2D, open bars) in a HMB-PP concentration-dependent manner with maximal proliferation seen at 500 pM. However, intracellular mycobacterial growth was unaffected by T cells expanded with all doses of HMB-PP (filled bars). The failure of these previously identified phosphoantigens to induce inhibitory ₉₂ T cells was therefore not related to the doses used or the relative phosphoantigen potency.

Absence of secondary "danger signals" provided by viable mycobacteria or TLR signaling does not explain the failure of phosphoantigen-expanded ₉₂ T cells to inhibit mycobacterial growth

We also considered the possibility that the induction of inhibitory ₉₂ T cells requires additional signals provided by the metabolism of live BCG, which would not be stimulated by purified phosphoantigens. We first tested whether viable mycobacteria were necessary for the induction of inhibitory T cells. PBMC were stimulated for 7 days with live BCG or a whole killed lysate prepared from the M. tuberculosis Erdman strain, and then the expanded T cells were purified and assayed for their ability to inhibit intracellular mycobacterial growth. T cells stimulated with mycobacterial lysate and live BCG mediated similar inhibition of intracellular mycobacterial growth (Fig. 3A).

View larger version (17K): [in this window] [in a new window]   FIGURE 3. The induction of inhibitory 92 T cells does not require viable mycobacteria or involve known TLR ligands expressed by mycobacteria. A, PBMC were either rested in medium or stimulated with live BCG or soluble M. tuberculosis (Mtb) lysates for 7 days. The expanded T cells were immunomagnetically purified and then cocultured at a 10:1 E:T ratio with BCG-infected macrophages to determine inhibitory effects against intracellular mycobacteria. *, p < 0.05 comparing the effects of BCG- or MtbWL-expanded T cells with control cultures by Wilcoxon matched pairs tests (n = 5/group). B, TLR costimulation during IPP expansion does not generate inhibitory 92 T cells. PBMC were expanded with the indicated concentrations of TLR agonists in the presence or absence of 10 µM IPP for 7 days. Shown are fold expansions of 92 T cells (open bars; right axis) and percentage inhibition of intracellular mycobacteria (filled bars; left axis). ODN2336, CpG containing oligodeoxynucleotide 2336.

IPP and live BCG induce ₉₂ T cells with different TCR spectratypes

Because we were unable to generate inhibitory ₉₂ T cells by stimulation with purified phosphoantigens in the presence or absence of TLR ligands, we hypothesized that BCG infection may in fact be stimulating a different subset of ₉₂ T cells. To study this possibility, we generated long-term BCG- and IPP-expanded ₉₂ T cell lines as described in Materials and Methods. By serial stimulation and expansion using DC infected with live BCG or cultured with IPP, we were successful in generating stable BCG- and IPP-stimulated ₉₂ T cell lines, respectively. These BCG- and IPP-stimulated ₉₂ T cell lines retained the differential inhibitory properties of the corresponding 7-day expansion cultures (Fig. 4). Therefore, we reasoned that we could use these paired ₉₂ T cell lines to examine whether the TCR sequences expressed by inhibitory BCG-stimulated and noninhibitory IPP-stimulated ₉₂ T cells were different. Ag specificity is largely determined by the sequences encoded by the hypervariable CDR3 regions of the TCR. To study differences in the specificities of the ₉₂ T cell lines, we analyzed the diversity of the V₉ and V₂ TCR CDR3 loops expressed by these populations of T cells. TCR spectratyping generates a spectrum of PCR fragments of different lengths representative of the ₉₂ TCR CDR3 length diversity in the cellular population.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. Long-term BCG- and IPP-stimulated 92 T cell lines also display differential inhibitory effector functions. Long-term 92 T cell lines were generated by serial stimulation with IPP-pulsed or BCG-infected autologous DC every 2 wk adding fresh IL-2 every 2–3 days. Because DC cannot retain IPP following washing, 10 µM IPP was added to the culture medium during stimulation of IPP-expanded lines. 92 T cell lines at the end of a stimulation cycle were cocultured with BCG-infected macrophages. *, p < 0.05 comparing BCG-stimulated and IPP-stimulated inhibitory responses by Wilcoxon matched pairs test (n = 5). Comparisons of IPP-stimulated inhibitory responses and medium-rested control cultures were not significantly different (p = 0.89; n = 5).

View larger version (42K): [in this window] [in a new window]   FIGURE 5. TCR Spectratyping profile of BCG- and IPP-stimulated long-term 92 T cell lines. A, Total RNA was harvested from T cell lines after a minimum of four stimulations with BCG-infected or IPP-pulsed DC. RT-PCR was used to specifically amplify the V9 and V2 chain CDR3 regions of the T cell receptors. The sizes and frequencies of different PCR fragments were detected with the CEQ8000 genetic analysis system. The number of peaks detected by the CEQ8000 software is displayed in the upper right corner of each graph. B, A comparison of the number of CDR3 fragment peaks obtained with BCG- and IPP-stimulated V9 and V2 TCR spectratypic profiles from all four volunteers. BCG-stimulated V9 and V2 TCR spectratypes contained significantly fewer peaks compared with IPP-stimulated V9 and V2 TCR spectratypes (*, p < 0.04 by Mann-Whitney U tests), and the predominant peaks identified in BCG- and IPP-stimulated spectratypes were distinct.

Sequencing of the CDR3 regions confirm that BCG and IPP induce different populations of ₉₂ T cells

We have shown that BCG stimulation and IPP stimulation expand T cell populations that differ in their expressed CDR3 lengths. However, a single CDR3 length can encode multiple amino acid sequences and, thus, TCR spectratyping alone may not reveal the full levels of diversity present within T cell populations. To confirm that BCG stimulates a limited diversity of ₉₂ T cells compared with IPP, we cloned the ₂ (Table I) and ₉ (Table II) CDR3 PCR fragments from selected pairs of BCG- and IPP-stimulated long-term ₉₂ T cell lines generated from PPD⁺ volunteers and sequenced at least 10 randomly chosen clones for each to obtain a more complete understanding of the variations within the CDR3 regions of the expressed TCR.

View this table: [in this window] [in a new window]   Table I. Sequencing of the CDR3 regions of the TCR 2 chaina

View this table: [in this window] [in a new window]   Table II. Sequencing of the CDR3 regions of the TCR 9 chaina

Similarly, Table II demonstrates that BCG-expanded ₉₂ T cell lines expressed V₉ CDR3 regions with much less diversity than IPP-expanded ₉₂ T cell lines. In agreement with data previously published showing that the J1.2 segment was associated with phosphoantigen responsiveness (37), the majority of our expanded ₉₂ T cells expressed the J1.2 segment, whereas few ₉ TCR expressed the J1.3/2.3 segments. In addition, all of the sequenced J regions contained the conserved adjacent lysine motifs (in bold font) previously shown to be critical for phosphoantigen recognition (38). Although these residues may be critical for binding of the phosphoantigen to the TCR, they do not appear to correlate with mycobacterial inhibitory activity.

These data demonstrate that IPP can stimulate a larger subset of ₉₂ T cells inclusive of BCG-activated ₉₂ T cells, also consistent with IPP having a mitogen-like effect. These results further support our hypothesis that BCG-infected APC stimulate a subset of ₉₂ T cells, while IPP stimulates the nonspecific expansion of more polyclonal ₉₂ T cells. A high degree of correlation between the frequencies of the specific CDR3 fragment lengths detected was observed by comparing our TCR spectratyping and sequencing results, indicating that the clones sequenced were highly representative of the total heterogeneity present within the ₉₂ T cell populations and not skewed by biased cloning efficiency (Fig. 6).

View larger version (31K): [in this window] [in a new window]   FIGURE 6. High correlations between sequencing and spectratyping data rule out artifactual in vitro skewing during the cloning techniques used to obtain sequencing results. Continuous variables for comparative analysis were generated for both sequencing and spectratyping data by multiplying the nucleotide size of each fragment times its observed frequency within the population. Spearman-rank correlations were analyzed between these sequence and spectratype data for each individual’s paired data as described in Materials and Methods. R2 (R Sq) coefficients were 0.704, 0.983, and 0.951 for the three different volunteers (all p < 0.0001 by Spearman-rank tests).

Heterologous stimulations of BCG- and IPP-expanded ₉₂ T cell lines confirm that BCG-activated inhibitory T cells are a subset of IPP-responsive ₉₂ T cells

Our results have led us to believe that BCG-infected DC can induce a pathogen-specific subset of IPP-responsive ₉₂ T cells, whereas IPP acts more like a polyclonal mitogenic stimulus, broadly stimulating most if not all peripherally circulating ₉₂ T cells. If this hypothesis is correct, BCG-expanded ₉₂ T cell lines should be fully responsive to IPP while only a fraction of the more heterogeneous population of IPP-expanded ₉₂ T cell lines would respond optimally to BCG, unless of course BCG-specific ₉₂ T cells have been expanded in vivo to represent the predominant ₉₂ T cells circulating at the time of our PBMC harvests. However, all volunteers studied here were healthy PPD⁺ individuals with remote histories of BCG vaccination and/or chronic latent TB infection. Therefore, none of these volunteers would be expected to have had ongoing high level mycobacterial replication at the time of their lymphocyte harvests, which might be expected to expand mycobacteria-specific ₉₂ T cells in vivo as the predominant activated/memory population among circulating ₉₂ T cells. Shown in Fig. 7 are comparisons of the intracellular IFN- response to homologous and heterologous stimulations of paired BCG- and IPP-expanded long-term ₉₂ T cell lines generated from five different PPD⁺ individuals. The response to the homologous stimulus was defined as the "100%" response for each T cell line. The percentage heterologous response was defined as the fraction of the response detected after stimulation with the heterologous Ag divided by the homologous response times 100. The mean heterologous response of BCG-expanded T cells to IPP stimulation was 90 ± 5% (not significantly different from the mean of homologous responses). In contrast, the mean heterologous response of IPP-expanded T cells to BCG stimulation was only 26 ± 7% of the homologous IPP responses (_*, p < 0.05 compared with homologous responses). These results indicate that virtually all ₉₂ T cells expanded with BCG can respond to IPP stimulation, but only a minor fraction of the ₉₂ T cells expanded with IPP can recognize BCG-infected DC.

View larger version (18K): [in this window] [in a new window]   FIGURE 7. Only a subset of IPP-responsive 92 T cells recognize BCG. A, BCG-stimulated long-term 92 T cell lines respond to both BCG and IPP, whereas IPP-stimulated long-term 92 T cell lines preferentially respond to IPP. These data are consistent with the hypothesis that BCG induces pathogen-specific 92 T cells, while IPP has a polyclonal, nonspecific mitogenic effect. Long-term 92 T cell lines were incubated with BCG-infected or IPP-pulsed DC for 16 h. IFN- production was detected by intracellular cytokine staining. The homologous response (the response to the same Ag used for generation of the lines) was defined as the "100%" response for each 92 T cell line. Responses in BCG-stimulated long-term 92 T cell lines stimulated with BCG and IPP were not significantly different by the Wilcoxon matched pairs test (n = 5). *, p < 0.05 comparing IPP-stimulated long-term 92 T cell lines stimulated with BCG and IPP by Wilcoxon matched pairs test (n = 5). B, Clonal analysis confirms differential functions in the majority of BCG- and IPP-expanded 92 T cells, providing further evidence that protective 92 T cells are a subset of IPP-responsive 92 T cells. 92 T cell clones were generated from matched BCG- and IPP-stimulated PBMC, and the inhibitory effects of these clones on intracellular mycobacterial growth were measured. Clones able to inhibit mycobacterial growth were defined as those clones resulting in mycobacterial growth less than the mean minus SE of growth detected in control wells without T cells added. *, p < 0.04 by 2 test (n = 11 and n = 21 for BCG- and IPP-stimulated 92 T cell clones, respectively). **, p < 0.004 by 2 test (n = 21 for both BCG- and IPP-stimulated 92 T cell clones).

Limiting dilution cloning analyses further confirm that ₉₂ T cells capable of inhibiting intracellular mycobacterial growth are only a subset of IPP-responsive ₉₂ T cells

Our results indicated that BCG-stimulated ₉₂ T cells represent a subset of IPP-responsive ₉₂ T cells that mediate inhibition of intracellular mycobacterial growth. These data imply that BCG-stimulated ₉₂ T cells able to inhibit intracellular mycobacterial growth should be present as a minor fraction of phosphoantigen-reactive ₉₂ T cells in vivo. To test this hypothesis, we generated ₉₂ T cell clones by limiting dilution from matched BCG- and IPP-stimulated PBMC from two PPD⁺ volunteers and tested the ability of individual clonal populations of ₉₂ T cells to inhibit intracellular mycobacterial growth (Fig. 7B). Clones able to inhibit mycobacterial growth were defined as those clones that suppressed mycobacterial growth to levels less than the mean minus SE of growth detected in control wells without T cell clones added. As predicted, only a minor fraction of the ₉₂ T cell clones generated following IPP-expansion (33 and 23% for volunteers 1 and 2, respectively) were able to inhibit intracellular mycobacterial growth. In contrast, significantly more of the ₉₂ T cell clones generated following BCG expansion of PBMC (72 and 76% for volunteers 1 and 2, respectively) were able to inhibit intracellular mycobacterial growth (_*, p <0.04 by ²; _**, p < 0.004 by ²). These data confirm at the clonal level that ₉₂ T cells capable of inhibiting intracellular mycobacterial growth are a minor fraction of IPP-responsive ₉₂ T cells present in circulating lymphocytes.

T cell clones capable of inhibiting intracellular mycobacterial growth do not have a higher TCR avidity for phosphoantigens than the noninhibitory T cell clones

Our results indicate that BCG-stimulated ₉₂ T cells represent a subset of the more broadly reactive phosphoantigen-responsive ₉₂ T cells. However, this apparent "specificity" for BCG could be explained by selection for ₉₂ TCR that have a higher affinity for the phosphoantigens produced in low concentrations by BCG-infected cells. To determine whether ₉₂ T cells capable of inhibiting intracellular mycobacterial growth respond to lower concentrations of phosphoantigen (i.e., have a higher avidity), we stimulated mycobacterial inhibitory and noninhibitory ₉₂ T cell clones with a dose titration of BCG and HMB-PP and compared the intracellular IFN- responses induced. Table III reports the percentages of ₉₂ T cells that produced IFN- after overnight stimulation with DC infected with a dose titration of BCG (MOI = 1–100) or incubated with a dose titration of HMB-PP (10–1000 pM). Similar to the results obtained with the long-term heterogeneous ₉₂ T cell lines (Fig. 7A), BCG-inhibitory ₉₂ T cell clones produced IFN- after stimulation with BCG-infected DC, while noninhibitory ₉₂ T cell clones did not produce IFN- responses detectable above background levels after stimulation with BCG-infected DC. Also similar to the results presented in Fig. 7A, both populations responded similarly to HMB-PP stimulation. Importantly, BCG-inhibitory ₉₂ T cell clones did not respond to lower concentrations of HMB-PP than did noninhibitory ₉₂ T cell clones. If anything, the noninhibitory ₉₂ T cell clones seemed to have a higher avidity for HMB-PP than the inhibitory ₉₂ T cell clones. These data confirm our previous results showing that BCG-stimulated ₉₂ T cells are a subset of phosphoantigen-reactive ₉₂ T cells and exclude the possibility that BCG-stimulated ₉₂ T cells are selectively expanded due to a higher TCR avidity for phosphoantigens.

View this table: [in this window] [in a new window]   Table III. Responses of BCG-inhibitory and noninhibitory 92 T cell clones to dose titrations of BCG and phosphoantigena

	Discussion

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IPP, one of the first phosphoantigens shown to induce ₉₂ T cell expansion, is known to be produced in mycobacteria-infected cells and was the only purified ₉₂ T cell stimulus readily available when we began the investigations reported in this article. However, concentrations of IPP needed to stimulate ₉₂ T cell expansions are unlikely to be achieved in mycobacteria-infected cells. Furthermore, IPP is already produced as a critical intermediate in the synthesis of isoprenoids in almost all living organisms via the mevalonate biosynthetic pathway (12). The ubiquitous nature of IPP and suboptimal IPP concentrations present in mammalian cells strongly suggested that IPP alone is not the ligand responsible for inducing TB-protective ₉₂ T cells. The discovery of a second pathway for IPP biosynthesis (MEP/DOXP pathway) present in bacteria (including mycobacteria) and lower eukaryotes (29), but not in mammalian cells, provided a potential alternative mechanism to explain mycobacteria-specific induction of TB-protective ₉₂ T cells. The intermediates produced in the MEP/DOXP pathway could be seen as "foreign" by the mammalian host. Furthermore, the penultimate metabolite in this pathway, HMB-PP, was shown to be at least 10,000-fold more potent for the induction of ₉₂ T cell expansion than IPP (15), such that physiologic concentrations of HMB-PP expressed in mycobacteria-infected cells could reasonably be expected to induce ₉₂ T cells in vivo. After finding that IPP failed to induce ₉₂ T cells inhibitory for intracellular mycobacteria, it was critically important to test whether ₉₂ T cells stimulated with HMB-PP or another highly potent synthetic ₉₂ T cell agonist currently in clinical development, BrHPP (16), could generate ₉₂ T cells with mycobacterial inhibitory effects. Similar to our results with IPP stimulation, neither HMB-PP nor BrHPP were able to stimulate the development of ₉₂ T cells capable of inhibiting intracellular mycobacterial growth despite being much more potent in inducing expansion of ₉₂ T cells even when compared with BCG stimulation. These combined results indicated that none of the previously identified phosphoantigens by themselves could induce ₉₂ T cells relevant for protective mycobacterial immunity.

A possible explanation for the failure of IPP, BrHPP, and HMB-PP to induce protective ₉₂ T cells could be that these purified phosphoantigens were not fully inducing activated effector functions. Concentrations of IPP, BrHPP, or HMB-PP optimal for T cell expansion were unable to induce ₉₂ T cells capable of inhibiting intracellular mycobacterial growth. However, ₉₂ T cells stimulated with IPP expressed similar levels of the effector molecules perforin, granzyme A, granulysin, and IFN- and displayed more efficient cytolytic activity for Daudi target cells compared with mycobacteria-stimulated ₉₂ T cells, effectively ruling out the possibility that purified phosphoantigen stimulation alone failed to induce ₉₂ T cell effector functions. Recently, ₉₂ T cells capable of lysing BCG-infected macrophages were identified by cell surface staining to belong to a subset of T cells identified as CD45RA^– and CD27⁺ (39). If this population of T cells was uniquely responsible for mycobacterial inhibitory effects, it could be hypothesized that BCG induced the differentiation of ₉₂ T cells into this subset while IPP failed to do so. Therefore, we analyzed the frequency of CD45RA^–CD27⁺ ₉₂ T cells induced by stimulation with mycobacteria compared with phosphoantigen and observed that both stimuli induced similar frequencies of this ₉₂ T cell subset (data not shown). Several reports have implicated receptors typically displayed on NK cells in the modulation of ₉₂ T cells (40, 41, 42, 43). We considered that these receptors may act as costimulatory molecules recognizing BCG-infected macrophages (especially NKp44, which binds mycobacterium directly; Ref. 43). Flow cytometric staining revealed no differences in the levels of NKp44, NKp46, CD94/NKG2D, KIR-NKAT2, NKB1, or CD161 expressed on BCG-expanded ₉₂ T cell clones compared with IPP-expanded ₉₂ T cell clones (data not shown). In addition, costimulation through TLR 1/2, TLR3, TLR4, and TLR9 during IPP expansion also failed to induce inhibitory ₉₂ T cells. These combined results indicate that the failure of purified phosphoantigens to induce TB-protective ₉₂ T cells could not be explained by inadequate phosphoantigen dose or potency, failure to induce effector functions or specific effector subsets, or lack of TLR-mediated costimulation.

Another possible mechanism for the differential ability of mycobacteria- and phosphoantigen-stimulated ₉₂ T cells to inhibit intracellular mycobacterial growth could be related to the recently published Ag-presenting functions mediated by activated ₉₂ T cells (44). Phosphoantigen-stimulated ₉₂ T cells could become potent APC capable of activating protective effector functions in mycobacterial peptide-specific memory β T cells. Because the mycobacterial peptides are absent from purified phosphoantigen-stimulated cultures, protective memory β T cell responses would not be induced. Indeed, we have shown that ₉₂ T cells can enhance the effector functions provided by mycobacteria-specific β T cells (C. T. Spencer, G. Abate, S. M. Truscott, A. Blazevic, and D. F. Hoft, submitted for publication). However, we have also demonstrated that ₉₂ T cells purified to >98% after 7-day expansions with live mycobacteria and BCG-stimulated long-term ₉₂ T cell lines and clones not contaminated with β T cells can directly inhibit intracellular mycobacteria. Therefore, APC and/or helper functions cannot explain the differential effector functions provided by highly purified ₉₂ T cells stimulated with live BCG and purified phosphoantigens.

Selective expansion of a subset of ₉₂ T cells expressing high affinity TCR also could explain the inhibitory effects mediated by BCG-stimulated, but not purified phosphoantigen-stimulated, ₉₂ T cells. Lower levels of phosphoantigens expressed during mycobacterial infection might be capable of stimulating only a subset of ₉₂ T cells with the highest TCR affinity, and the possibility exists that only ₉₂ T cells expressing TCR with the highest affinity can mediate protective functions against intracellular mycobacteria. In contrast, exogenous phosphoantigen added at optimal concentrations might stimulate all ₉₂ T cells regardless of TCR affinity. If it was true that only the ₉₂ T cells with the highest affinity TCR were capable of inhibiting intracellular mycobacteria, we would predict that limiting doses of exogenously added purified phosphoantigen should preferentially stimulate high affinity ₉₂ T cell subsets capable of mycobacteria-protective effects. However, in our dose titration experiments with the purified phosphoantigens IPP, BrHPP, and HMB-PP we did not observe inhibitory effects mediated by ₉₂ T cells stimulated with suboptimal concentrations that still led to ₉₂ T cell expansion. More directly, BCG-inhibitory ₉₂ T cell clones stimulated with titrations of HMB-PP did not produce IFN- at lower HMB-PP concentrations compared with noninhibitory ₉₂ T cell clones. In fact, a greater percentage of BCG noninhibitory ₉₂ T cell clones produced IFN- at lower HMB-PP concentrations than did BCG-inhibitory ₉₂ T cell clones. It also is conceivable that purified phosphoantigens could induce a selective depletion of inhibitory ₉₂ T cells expressing high affinity TCR similar to the recently reported effects of repeated BrHPP injection in monkeys (18). However, we observed that a fraction of ₉₂ T cell clones from IPP-expanded PBMC were capable of inhibiting intracellular mycobacterial growth, demonstrating that inhibitory ₉₂ T cells can be expanded with phosphoantigen. This suggests that phosphoantigen stimulation of ₉₂ T cells does not result in the selective depletion of inhibitory ₉₂ T cells. Therefore, while differences in affinity of ₉₂ TCR for relevant mycobacterial Ags likely exist, selective expansion of ₉₂ T cells based on TCR affinity does not appear to explain the differential inhibitory effects of mycobacteria-stimulated and purified phosphoantigen-stimulated ₉₂ T cells.

Because none of the potential mechanisms discussed above can explain the differential protective effects of mycobacteria-stimulated and purified phosphoantigen-stimulated ₉₂ T cells, we examined the antigenic specificity of BCG-reactive and IPP-reactive T cells. Crystallographic studies of the ₉₂ TCR suggest that the same hypervariable regions critical for β TCR stimulation by Ag/MHC are important for ₉₂ TCR stimulation (45). In addition, phosphoantigen reactivity has been shown to be dependent upon the sequence of the rearranged V9/J1.2 CDR3 gene segment (37) and coexpression of the V9 and V2 TCR chains (46). Therefore, we examined the Ag specificity of BCG- and IPP-stimulated ₉₂ T cells by comparing the diversity of the V9 and V2 CDR3 regions of the TCR expressed by differentially expanded ₉₂ T cell long-term lines. Our results demonstrate that BCG-expanded ₉₂ T cells undergo a phenomenon consistent with "antigenic focusing" similar to β T cells, which in some cases resulted in the appearance of a single peak detected by TCR spectratyping. This apparent antigenic focusing indicates that only certain subsets of ₉₂ T cells present in persons with mycobacterial immunity are relevant for protection. Consistent with this conclusion, we demonstrated that only a fraction of IPP-reactive ₉₂ T cells were able to respond to BCG-infected DC with the production of intracellular IFN-. In addition, only a minority of ₉₂ T cell clones expanded with IPP were found to inhibit intracellular mycobacterial growth, whereas most BCG-expanded ₉₂ T cell clones could inhibit intracellular mycobacterial growth.

Previous sequencing of multiple human phosphoantigen-responsive ₉₂ T cell clones, in combination with mutagenesis and transfection studies focused on a single phosphoantigen-responsive TCR, collectively demonstrated that two adjacent and highly conserved lysine residues within the J1.2 regions of the V9 CDR3 regions were critical for functional recognition of phosphoantigens (36, 38). Molecular modeling studies suggested that these conserved lysines might form a positively charged pocket in which the negatively charged pyrophosphate moieties present on ₉₂ T cell stimulatory phosphoantigens could dock (36). Although a high frequency of our expanded ₉₂ T cells expressed J1.2, we also observed that both BCG- and IPP-expanded ₉₂ T cell lines included clones expressing J1.3. These J1.3 expressing V9 CDR3 regions also encode a pair of adjacent lysine residues that may provide for the docking of pyrophosphate in the ₉₂ TCR. The previous structural studies also suggested the importance of a conserved hydrophobic or neutral residue within the 5'-portion of the V2 CDR3 region expressed by phosphoantigen-responsive ₉₂ T cells (36). Consistent with this previous finding, almost all of the V2 CDR3 clones we identified in BCG- and IPP-expanded ₉₂ T cells expressed a 5' hydrophobic or neutral amino acid residue.

The previous ₉₂ TCR sequencing studies suggested that besides the conserved lysines in the V9 CDR3 region and the conserved hydrophobic/neutral residue within the V2 CDR3, phosphoantigen-responsive ₉₂ TCR are highly diverse. This is also reflected in our findings with IPP-expanded ₉₂ T cell lines. Thus, purified phosphoantigens may interact with only a very small region of the ₉₂ TCR. Our findings of the more highly restricted diversity expressed by BCG-expanded ₉₂ T cell lines suggest that mycobacterial Ags other than the previously identified phosphoantigens may be expressed that induce inhibitory ₉₂ T cells. Alternatively, more complex antigenic structures containing phosphoantigen cores or intermolecular combinations of phosphoantigens with unknown presenting elements could alter the antigenic face of the presented phosphoantigens and induce the CDR3 focusing observed in inhibitory ₉₂ T cell subsets. However, at this point we cannot rule out the possibility that phosphoantigen alone may contact the ₉₂ TCR while an additional activating receptor present on only a subset of ₉₂ T cells must be concurrently engaged to induce the more restricted populations with inhibitory activity for intracellular mycobacteria.

Our results also are reminiscent of the demonstration that two types of β T cells exist that appear to respond to different conformations of peptide epitopes generated by the following: 1) epitopes processed intracellularly and loaded onto class II MHC molecules with the aid of HLA-DM molecules in MIIC compartments; and 2) synthetic peptide epitopes loaded onto empty MHC molecules at the cell surface or in endosomes without the need for intracellular processing (47). So-called type A T cells respond both to epitopes processed intracellularly from native Ag as well as to purified peptide epitopes pulsed onto extracellular MHC. In contrast, type B T cells only respond to peptide-pulsed APC and not native Ags requiring intracellular processing. IPP-expanded ₉₂ T cells may be similar to type B β T cells, responding to purified phosphoantigens supplied exogenously and directly loaded onto unknown APC surface elements required for presentation, bypassing the normal processing of ₉₂ T cell-stimulatory Ags that are presented during the natural infection of APC by mycobacteria. ₉₂ T cells expanded with BCG-infected DC may be analogous to type A β T cells that are capable of responding to naturally processed Ags expressed in mycobacteria-infected cells. The natural processing pathways and restriction elements required for presenting mycobacterial Ags to ₉₂ T cells in infected cells are unknown but likely are distinct from the lysosomal processing and MHC presentation involved in the stimulation of β T cells. Future research will be required to further explore the possibility that BCG- and IPP-expanded ₉₂ T cells are analogous to type A and type B β T cells, respectively.

Our previous and current data suggest that ₉₂ T cells can develop the characteristics of adaptive memory immunity (2) and now Ag specificity similar to that of β T cells. Although phosphoantigens have been used to study ₉₂ T cell responses in nonhuman primates (18, 19, 48, 49), their ability to protect against mycobacterial infection has not been reported. Using an in vitro growth inhibition assay, we have demonstrated that although stimulation with the previously purified phosphoantigens alone can induce the expansion of ₉₂ T cells, these cells are unable to protect against intracellular mycobacterial replication. Our data indicate that mycobacterial protective immunity can be mediated by ₉₂ T cells, but only by an Ag-specific subset of ₉₂ T cells. These data also suggest that additional antigenic components, presenting elements, and/or activating receptors specifically involved in the induction of memory immune ₉₂ T cells must be identified to harness the full potential of ₉₂ T cells for pathogen-specific immunity.

	Acknowledgments

 
We thank Dr. Dana Oliver for assistance with spectratyping and sequencing correlations, Dr. John Tavis and Jeff Simmons for assistance in acquiring and analyzing spectratyping data, and the gift of BrHPP from Dr. Jean-Jacques Fournié.

	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 investigation was supported by National Institutes of Health Vaccine Treatment and Evaluation Unit Contract NO1-AI-25464 (to D.F.H., Co-investigator) and National Institutes of Health R01-AI-48391 (to D.F.H., Principal Investigator).

² Address correspondence and reprint requests to Dr. Daniel F. Hoft, Division of Immunobiology, Departments of Internal Medicine and Molecular Microbiology, Saint Louis University Health Sciences Center, 1100 South Grand Boulevard. DRC-807, St. Louis, MO 63104. E-mail address: hoftdf{at}slu.edu

³ Abbreviations used in this paper: BCG, M. bovis bacillus Calmette Guérin; AN, absolute number; BrHPP, bromohydrin pyrophosphate; DC, dendritic cell; DOXP, 1-deoxy-D-xylulose 5-phosphate, HMB-PP, (E)-4-hydroxy-3-methyl-but-enyl-pyrophosphate; IPP, isopentenyl pyrophosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; MtbWL, M. tuberculosis whole lysate; MOI, multiplicity of infection; Pam₃Cys, (S)-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys₄-OH, 3HCl; polyI:C, polyinosinic:polycytidylic acid; PPD, purified protein derivative; rhIL-2, recombinant human IL-2; TB, tuberculosis.

Received for publication September 4, 2007. Accepted for publication July 31, 2008.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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