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The Journal of Immunology, 2004, 172: 248-259.
Copyright © 2004 by The American Association of Immunologists

T Cell Clonal Conditioning: A Phase Occurring Early after Antigen Presentation but before Clonal Expansion Is Impacted by Toll-Like Receptor Stimulation 1

Joseph R. Maxwell, Robert J. Rossi, Stephen J. McSorley and Anthony T. Vella2

Division of Immunology, University of Connecticut Health Center, Farmington, CT 06032


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
After in vivo immunization, Ag-specific T cells disappear from circulation and become sequestered in lymphoid tissue where they encounter Ag presented by dendritic cells. In the same site and just after Ag presentation, they "disappear" a second time and we investigated this process. Using a mouse model of T cell deletion (without Toll-like receptor (TLR) stimulation) vs survival (with TLR stimulation), Ag-specific T cells indeed became undetectable by flow cytometry, however were readily detected by immunohistochemistry. Thus, whether or not the activated T cells were destined to delete or survive, they were difficult to extract from lymphoid tissue and did not disappear but in fact were abundantly present. Nevertheless, profound differences were observed during this time period when tolerizing conditions were compared with immunizing conditions. TLR stimulation induced an increase in CD25 expression, acquisition of surface MHC class II, and abnormally high increases in forward and side scatter of the peptide-specific T cells. Using a modified adoptive transfer approach, we demonstrated by flow cytometry that in the presence of TLR stimulation the Ag-specific T cells were tightly coupled to dendritic cells, explaining the unusual increases in size and granularity. Ultimately, these events induced the specific T cells to differentiate into memory cells. We postulate that this is a stage where T cells are either conditioned to survive or to delete depending upon the activation status of the innate immune system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interaction between a TCR and its cognate Ag presented by MHC molecules is a crucial step in T cell activation. This exquisitely specific interaction can generate millions of identical Ag-specific T cells from a rare starting population. It is during this early phase of T cell activation that many of the signals determining T cell fate are provided. The dynamics of this initial phase are quite complex and despite intense research remain unclear.

Historical studies by Sprent and Miller (1, 2) documented that bulk lymphocytes taken from thoracic duct lymph, spleen, lymph nodes (LNs), 3 or Peyer’s patch of mice immunized 1 day previously do not adoptively transfer Ag reactivity. These unexpected and paradoxical results suggest that T cells are not functional before clonal expansion. In the last decade, a related early event has been documented in superantigen (SAg) models, which show that after migration to and trapping in LNs, the specific T cells "disappear" before clonal expansion (3, 4, 5). A number of studies have explained this phenomenon with TCR down-regulation (6, 7, 8, 9) and early deletion or death (4, 5, 10, 11) being the most common. Nevertheless, the reason this phase occurs in apparently every model of in vivo T cell activation, including memory recall responses (12), is unclear.

We investigated this process after in vivo peptide stimulation and the data indicate that although TCR down-regulation occurs on Ag-specific T cells, they are nevertheless difficult to extract from lymphoid tissue even though they are readily detectable by immunohistochemistry. Eventually, the hidden T cells emerge, clonally expand, and finally delete to near undetectable levels.

We cogitated whether this process was of any biological consequence for long-term T cell immunity and therefore tested the influence of Toll-like receptor 4 (TLR4) stimulation mediated by bacterial LPS, which is known to break T cell tolerance (13, 14). The data show that in the presence of TLR4 stimulation peptide-stimulated T cells are even more difficult to extract and undergo important physiological changes. The specific T cells up-regulate CD25, possess surface MHC class II, and massively increase forward and side scatter. We found that the peptide-reactive T cells were tightly coupled to innate APCs like dendritic cells (DCs), which was not observed in the absence of TLR4 stimulation and is the likely explanation for the increases in forward and side scatter. Therefore, after Ag presentation, the specific T cells appear hidden because they are difficult to extract from lymphoid tissue; however, only after TLR4 activation did the specific T cells undergo important physiological changes resulting in very tight coupling to innate APCs.

Thus, we have termed this phase T cell clonal conditioning because it occurs after Ag presentation in lymphoid tissue with or without TLR stimulation but precedes clonal expansion. There are at least two conditioning pathways: deletion programming, which occurs under tolerizing conditions, and a second that induces survival, which is dependent on appropriate stimulation of the innate immune system. Therefore, even though specific T cells are initially hidden after peptide-alone stimulation, they do not receive survival conditioning mandated by the innate immune system. We propose that T cell clonal conditioning is an intersection of information exchange between the innate and adaptive immune systems, rendering it an important therapeutic target for vaccine development.


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

B10.A and C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD) and The Jackson Laboratory (Bar Harbor, ME). The green fluorescent protein (GFP)-transgenic mice (C57BL/6-TgN(ACTbEGFP)1Osb) with enhanced GFP expression under the control of a chicken {beta}-actin promoter have been described previously (15) and were also purchased from The Jackson Laboratory. The SM1 TCR-transgenic recombination-activating gene (RAG) 2-deficient mice have been described previously (16) and were bred by our laboratory. All mice were maintained in the animal facility at the University of Connecticut (UCONN) Health Center under specific pathogen-free conditions.

Injection schedules

For the staphylococcal enterotoxin A (SEA) studies, B10.A mice were injected with 0.30 µg of SEA (Sigma-Aldrich, St. Louis, MO) at time 0. Two days before this, 0.1 mg of anti-CD40 mAb or control rat IgG (Sigma-Aldrich) was injected. The anti-CD40-producing hybridoma FGK45.5 (17) was a kind gift from Dr. A. Rolink (University of Basel, Basel, Switzerland). The mAb was purified from hybridoma supernatants over a protein G column (Invitrogen, Carlsbad, CA). Salmonella typhimurium LPS (Sigma-Aldrich) was injected 24 h after SEA at a dose of 10 µg. All reagents were administered to mice as i.p. injections in a total volume of 200 µl of either balanced salt solution (BSS) or PBS.

For the SM1 studies, 100–200 µg of flagellin peptide 427–441 (18) was injected i.p. on day 0 into C57BL/6 or GFP-transgenic mice that had been adoptively transferred the previous day with between 0.5 and 7 x 106 SM1-transgenic T cells. LPS was injected 18 h after the peptide injection at a dose of 10–250 µg.

Cell processing and collagenase treatment

In our traditional cell isolation procedure, spleens and LNs (inguinal, axillary, brachial, and mesenteric) were crushed through nylon mesh cell strainers (Falcon; BD Biosciences, San Diego, CA) and RBC were lysed with ammonium chloride. After washing with BSS, cells were counted using a Z1 particle counter (Beckman Coulter, Miami, FL). In the SEA experiment in Fig. 1, splenic T cells were further purified over nylon wool columns as described previously (19). Liver and lung lymphocytes were isolated as described previously (20). Briefly, after perfusion, lung tissue was digested by separate incubations in the presence of EDTA and collagenase (Invitrogen) and then crushed through a cell strainer. Recovered cells were resuspended in 44% Percoll (Amersham Biosciences, Piscataway, NJ), layered on 67% Percoll, and isolated from the interface after centrifugation. Cell counts were obtained before staining. Liver tissue was crushed through a cell strainer and resuspended in 35% Percoll (Sigma-Aldrich). Pelleted cells were treated with ammonium chloride, washed, and counted before staining.



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  		FIGURE 1. LPS injection prolongs the early disappearance of SEA-specific T cells. B10.A mice were injected with SEA and control rat IgG ({circ}); SEA and anti-CD40 mAb ({square}); SEA, rat IgG, and LPS (•); or SEA, anti-CD40 mAb, and LPS ({blacksquare}) as described in Materials and Methods. At time points before (4 and 18 h), during (24 h), and after (30 and 36 h) LPS injection, LNs and spleens were removed from three mice per group and T cells were isolated, counted, and analyzed by flow cytometry. Results show the percentages and absolute numbers ± SEM of splenic CD4 (a, b, e, and f) and CD8 (c, d, g, and h) T cells expressing the SEA-specific TCR V{beta}3 and are from one representative experiment of six performed. Numbers were obtained by multiplying the percentage of CD4+V{beta}3+ or CD8+V{beta}3+ cells by the total cell number in each tissue. Similar data was obtained from LNs.

 
For the collagenase digestion procedure, pooled LNs or spleens were homogenized in 1 ml of a collagenase D (Roche, Indianapolis, IN) solution (3.3 mg/ml collagenase D, 10 mM HEPES, and 2% FBS in MEM). After a 30-min culture at 37°C and 5% CO2, 100 µl of 0.1 M EDTA in PBS was added to the well. The cell suspension was passed through a cell strainer and washed with calcium/magnesium-free BSS containing 5 mM EDTA and 2% FBS. After this, the RBC were lysed and the cells were washed, counted, and stained as described above. In experiments where collagenase-treated tissues were compared with untreated tissues, one-half of a pair of LNs and one-half of the spleen were prepared using our traditional procedure, while the other half was digested with collagenase.

Cell staining and flow cytometry

Anti-TCR V{beta}3, KJ25-607.7 (21), and anti-CD44,I42/5 (22) Abs were purified from hybridoma supernatants over a protein G column and conjugated to FITC (23, 24). FITC-conjugated anti-TCR V{beta}2, PerCP-conjugated anti-CD4, PE-conjugated anti-CD11c, anti-TNF, rat IgG1, hamster IgG1, and rat IgG2b and biotinylated anti-B220, anti-CD11c, anti-Thy1.1, and the control mouse IgG1 were purchased from BD Biosciences. Allophycocyanin-conjugated anti-Thy1.1 and streptavidin and PE-conjugated anti-CD11b and anti-IFN-{gamma} were purchased from eBioscience (San Diego, CA). PE-conjugated anti-CD25 and allophycocyanin-conjugated anti-CD4 and anti-CD8 were purchased from both BD Biosciences and eBioscience.

For four-color staining, cells were incubated for 30 min on ice with the primary Abs in staining buffer (BSS, 3% FBS, and 0.1% sodium azide) in the presence of Fc block. Fc block consists of 5% normal mouse serum (Sigma-Aldrich), culture supernatant from hybridoma cells producing an anti-mouse FcR mAb, 2.4.G2 (25), and 10 µg/ml human {gamma}-globulin (Sigma-Aldrich). After incubation, the cells were washed twice and analyzed by flow cytometry, or if a secondary reagent was necessary, the incubation and wash procedures were repeated.

For intracellular staining, 1 x 106 splenocytes were cultured with 1 µg of brefeldin A with or without 1 µg of flagellin peptide 427–441 in 200 µl of complete tumor medium (CTM) for 5 h. CTM consists of MEM with FBS, amino acids, salts, and antibiotics. After 5 h, the cells were washed and stained as described above with allophyocyanin-conjugated anti-Thy1.1 for 30 min. After washing in cold BSS, cells were fixed for 5 min at 37°C in BSS/2% formaldehyde. Cells were then washed in permeabilization buffer (staining buffer from above containing 0.25% saponin) and then stained with the appropriate anti-cytokine Ab for 10 min at room temperature. After washing with permeabilization buffer, the cells were analyzed by flow cytometry. For each stain, the cultures were done in triplicate. All flow cytometry was conducted on a BD FACSCalibur flow cytometer and the data were analyzed using CellQuest software (BD Biosciences) or FlowJo software (Tree Star, San Carlos, CA).

Immunohistochemistry

Thin sections (6 µm) of frozen inguinal LNs from C57BL/6 mice injected with SM1 TCR-transgenic cells were cut using a cryostat and fixed onto slides for 5 min in acetone. After rehydration in PBS and further fixation in 1% formaldehyde for 15 min, endogenous peroxidase activity was quenched by incubating the tissues with 1% H2O2 in PBS/0.1% sodium azide for 30 min. Subsequent 20-min blocking steps were performed using an Fc block described above and an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA) before addition of biotinylated anti-Thy1.1 mAb for 30 min. To amplify the biotin signal, the tissues were incubated for 30 min using the Vectastain Elite ABC kit (Vector Laboratories) and deposited biotin visualized using the diaminobenzidine substrate kit (Vector Laboratories) to give a brown color. After washing with PBS, the avidin/biotin block was repeated and the second Ab, either biotinylated anti-B220 or anti-CD11c, was added to the tissues for 30 min. The Elite ABC kit was repeated and the second stain visualized with the Vector SG Substrate kit (Vector Laboratories) to give a blue color. After washing with water, the tissue was counterstained briefly in 5% methyl green (Sigma-Aldrich) in methanol and dehydrated by dipping in 70, 95, and 100% ethanol. Finally, the tissues were cleared using Citrisolv (Fisher, Pittsburgh, PA) and then mounted with DPX mountant for histology (Sigma-Aldrich). Tissues were photographed with a Spot RT Slider digital camera (Diagnostic Instruments, Sterling Heights, MI) interfaced with a Nikon E400 microscope (Nikon, Melville, NY) using a x20 or x40 objective.

In vivo T cell and APC coupling

To visualize T cell/APC coupling by flow cytometry, at least 2 x 106 cells from SM1 RAG-/- TCR-transgenic mice were injected i.v. into GFP-transgenic mice. One day later, 100 µg of flagellin peptide 427–441 was injected and, after 18 h, 10–150 µg of LPS was injected. At 37 h after Ag injection, LNs and spleens were removed and treated with collagenase D as described above. For the GFP coupling assay, cells were washed and stained with anti-CD4 and anti-Thy1.1 (mouse IgG1 as a control) Abs and either anti-CD11c or anti-CD11b (hamster IgG1 and rat IgG2b isotype controls, respectively) Abs. Cells were analyzed by gating on CD4+ Thy1.1+ double-positive cells, and from this population GFP+ Thy1.1+ cells were analyzed.

For cell sorting, cells recovered after collagenase digestion were washed and stained in BSS containing 3% FBS (no azide). After staining for Thy1.1, cells were sorted on a BD FACSVantage Se DIVA cell sorter using a 130-µm nozzle under digital conditions. GFP+Thy1.1+ (coupled cells) and GFP-Thy1.1+ (uncoupled Ag-specific T cells) populations were dual sorted and, when necessary, GFP+Thy1.1- (host cells) were separately obtained. For H&E images, cytospins were performed on half of the sorted cell populations, followed by a 1-min fixation in methanol and 1-min stains in H&E. Bright-field images of the resulting slides were obtained with a Nikon E400 microscope (Nikon) as described above using a x40 objective. For the GFP images, the second half of the sorted cell populations were concentrated by centrifuging and resuspending them in the residual buffer left over after decanting. Ten microliters of the cells was placed uncovered onto a microscope slide and the cells allowed to settle. Bright-field and GFP fluorescence images were photographed through a x20 objective using a Zeiss Axiocam HRC digital color camera (Zeiss, Thornwood, NY) linked to a Zeiss Axioplan 2 IE microscope.

5-bromo-2'-deoxyuridine (BrdU) staining

Mice were injected with SEA, anti-CD40 mAb, and LPS as described above. Additionally, the mice were injected with 1 mg of BrdU (Sigma-Aldrich) dissolved in PBS at 24 and 30 h after SEA and the cells were isolated at 36 h. T cells from peripheral LNs and spleens of treated mice were stained with biotinylated anti-TCR V{beta}3 mAb and then with PE-conjugated streptavidin. The cells were stained using a modified BrdU staining protocol (26). Briefly, the cells were dehydrated and fixed in ice-cold 95% ethanol, then fixed in BSS containing 1% paraformaldehyde and 0.01% Tween 20. Next, cellular DNA was lightly digested with 50 Kunitz units of DNase I (Sigma-Aldrich) and the cells were stained with anti-BrdU-FITC (BD Biosciences) before flow cytometric analysis.

In vitro IL-2 restimulation assay

Mice were injected with SEA, anti-CD40 mAb and LPS as described above. At 24, 30, or 36 h after SEA injection, LN and spleen cells were isolated for culture. Cells from each in vivo treatment group were plated at 250,000 cells/well in CTM. Recombinant murine IL-2 (Intergen, Purchase, NY) was added to the wells at a maximum concentration of 100 U/ml and successive 3-fold dilutions were made from this. The cultures were left for 72 h with 1 µCi of [3H]thymidine (ICN, Costa Mesa, CA) being added for the last 8 h. Incorporation of [3H]thymidine was measured on a 1450 Microbeta Trilux Scintillation Counter (Wallac, Turku, Finland). cpm from cultures with no IL-2 added were subtracted from the cpm determined for the treated cultures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Injection of SEA into mice causes clonal expansion of both CD4 and CD8 T cells expressing the V{beta}3 chain of the TCR (27, 28). Using this in vivo model system, we have previously shown that the TLR4 agonist LPS can promote long-term T cell survival (14) and that coadministration of agonistic mAbs to OX40, CD40, or 4-1BB can synergistically enhance survival (24, 29, 30). With this knowledge, we reasoned that early time points after immunization might provide clues as to how survival was being generated.

Injection of SEA and control rat IgG caused a decline in SEA-specific T cells for ~18 h, followed by their reappearance and clonal expansion to nearly three times the starting population by 36 h (Fig. 1, {circ}). A very similar trend was observed after SEA and anti-CD40 treatment (Fig. 1, {square}).

The most profound results were observed with LPS treatment. Injecting LPS 24 h after SEA/IgG injection slightly delayed the expansion of the specific V{beta}3 T cells at 30 h, but by 36 h expansion was similar to that of the control group (Fig. 1, •). Additionally, it should be noted that there is a greater delay in expansion if a larger dose of LPS is used (data not shown). Most interestingly, when LPS was given after CD40 stimulation and SEA, T cell accumulation was dramatically inhibited for at least 36 h (Fig. 1, {blacksquare}). Control V{beta}14 T cell populations, which do not respond to SEA, did not change significantly with any treatment (data not shown).

Since LPS caused significant disappearance of the specific T cells, it was possible that LPS inhibited T cell proliferation. IL-2 responsiveness was not defective, since cells from SEA/anti-CD40/LPS-treated mice cultured with increasing concentrations of IL-2 proliferated in vitro as well as or better than cells taken from SEA/anti-CD40-treated mice (Fig. 2a). To examine this issue in vivo, mice were injected with BrdU 24 and 30 h after SEA (Fig. 2b). The percentage of V{beta}3 T cells possessing BrdU at 36 h after SEA was similar regardless of whether or not LPS was injected (Fig. 2b). These data suggest that upon LPS injection, recently stimulated specific T cells can initiate DNA replication yet do not accumulate.



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  		FIGURE 2. SEA-specific T cells stimulated with LPS have normal proliferatory capacity. a, LN and spleen cells from B10.A mice injected with nothing ({diamond}); SEA and anti-CD40 mAb ({square}); or SEA, anti-CD40 mAb, and LPS ({blacksquare}) were isolated at different times after SEA injection and cultured with decreasing doses of IL-2 as described in Materials and Methods. Results show mean cpm ± SEM of incorporated [3H]thymidine from triplicate cultures and are from one of three experiments performed. b, B10.A mice treated with nothing (), SEA and anti-CD40 mAb ({square}), or SEA, anti-CD40 mAb and LPS ({blacksquare}) were injected with BrdU as described in Materials and Methods. Shown is the percentage ± SEM of V{beta}3 T cells incorporating BrdU between 24 and 36 h after SEA (within 12 h after LPS). Results are one experiment of four performed.

 
Our hypothesis for the disappearance of the specific T cells after LPS injection was that they were being retained within secondary lymphoid tissues. To explore this idea, we used the SM1 TCR-transgenic adoptive transfer model (16). SM1 mice possess a transgenic TCR that recognizes a peptide from Salmonella flagellin presented by IAb. Injection of the transgenic T cells into a B6 host allows tracking of these cells in response to peptide injection not only by their TCR (V{beta}2), but, more importantly, by the Thy1.1 congenic marker. Thy1.1 is not internalized like the TCR (31), thus eliminating the complication of TCR down-regulation. We conducted a time course using this model and showed that after immunization, specific T cells declined in the LNs and spleens by 24 and 30 h to <0.3% and accumulated in large numbers by 42 h (Fig. 3a). Analysis of V{beta}2 TCR expression on the recovered CD4 Thy1.1 cells showed significant TCR down-regulation (data not shown), demonstrating the difficulty that techniques relying solely on anti-TCR mAbs would have in detecting specific T cells during this time.



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  		FIGURE 3. Responding T cells disappear from lymphoid tissues by flow cytometric analysis, yet are present histologically. C57BL/6 mice received 1.5–2 x 106 SM1 RAG-/--transgenic T cells, and 1 day later these mice were injected with flagellin peptide 427–441 alone ({square}) or with LPS ({blacksquare}) as described in Materials and Methods. a) At 24, 30, and 42 h postpeptide injection, T cells were isolated from the LNs and spleens from three mice per group. Shown are the percentages ± SEM of CD4 cells that express the congenic marker Thy1.1. The 24- and 42-h time points are from one experiment where mice received 200 µg of peptide and 100 µg of LPS, while the 30-h time point is from a separate experiment where mice received 150 µg of peptide and 200 µg of LPS. b–i, Inguinal LNs removed during the time course and stained for Thy1.1 (brown) and B220 (blue). Images were taken with a x20 objective and show peptide-only-treated mice at 24 (b), 30 (c), and 42 (d) h after Ag, and peptide/LPS-treated mice at 24 (f), 30 (g), and 42 h (h) after Ag. e, LN from a transfer only mouse. i, LN from a peptide/LPS mouse stained with Thy1.1 and B220 isotype controls. Results are from one experiment of two performed at each time point.

 
Since TCR internalization cannot account for the "disappearance," we used a direct in situ staining strategy to confirm our hypothesis that the specific T cells were actually present in the tissue. Thy1.1 staining of inguinal LN sections revealed that at 24 and 30 h, when few T cells were detected by flow cytometry (Fig. 3a), many Ag-specific T cells were present in the LNs from peptide-treated (Fig. 3, b and c) and peptide/LPS-treated (Fig. 3, f and g) mice. The significance of this finding is underscored by the fact that transfer-only mice had a larger percentage of Ag-specific T cells at 24 and 30 h (~0.5%), as measured by flow cytometry, than treated mice, but far fewer transfer-only T cells could be observed histologically at these same time points (Fig. 3e). By 42 h, the LNs from treated mice were filled with Thy1.1+ cells, showing the massive expansion that had occurred (Fig. 3, d and h). Taken together, these data demonstrate that T cells responding to Ag are indeed present within secondary lymphoid tissue even when they cannot be easily identified by flow cytometry.

To examine this issue in more detail, we improved tissue disruption of the LNs and spleens. Treatment with collagenase D for 30 min enhanced Ag-specific T cell recovery in LNs and spleens at all time points examined, whereas the absence of Ag did not enhance recovery (Fig. 4). The largest fold increase was observed at 24 h after Ag, but by 30 h smaller increases were observed. At 42 h, the increase was <2-fold, a somewhat misleading number considering that the percentage of CD4 cells expressing Thy1.1 often increased by >1% (3% CD4 Thy1.1+ before digestion to 4 or 5% afterward). Increased percentages of Thy1.1-bearing splenic CD4 cells were consistently observed, but the fold increase was generally lower than in LNs. Overall, what is striking about this data is the preferential recovery of Ag-specific CD4 T cells after collagenase treatment.



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  		FIGURE 4. Collagenase digestion preferentially improves Ag-specific T cell recovery from lymphoid tissues. Half of the LNs or half of a spleen was either crushed without collagenase digestion using our traditional techniques or digested for 30 min at 37°C with collagenase D as described in Materials and Methods. a, Ag-specific T cell recovery at 24, 30, 37, and 42 h after Ag from untreated or collagenase-treated peripheral LNs. Each line represents the percentage of CD4 cells expressing Thy1.1 before and after collagenase treatment for a single mouse. b, Mean fold increase ± SEM of LN CD4 cells expressing Thy1.1 in tissues treated with collagenase compared with untreated tissues. Results in a and b are the complete data from two or three separate experiments.

 
Upon closer inspection of the specific T cells recovered from the LNs and spleens, we observed several physiological changes (Fig. 5). In the SAg model, CD25 was expressed on almost 100% of the V{beta}3 T cells 4 h after SEA/IgG and SEA/anti-CD40 stimulation (Fig. 5a, {circ} and {square}). Subsequently, the percentage of V{beta}3 T cells expressing CD25 declined to ~25% at 24 h. However, if LPS was injected at 24 h after SEA, the percentage of CD4 and CD8 V{beta}3 T cells expressing CD25 increased a second time to ~80% (Fig. 5a, • and {blacksquare}). Augmented increases of CD25 on CD4 Thy1.1+ T cells after LPS injection were also observed in the SM1 adoptive transfer model (Fig. 5b). CD4 Thy1.1+ T cells expressed MHC class II (Fig. 5c). Only 5–10% of resting and peptide-stimulated Thy1.1+ cells expressed IAb 37 h after peptide stimulation; however, nearly 80% of the Thy1.1+ cells in peptide/LPS-treated mice stained positive for MHC class II. Further analysis showed that CD40 expression was increased on Thy1.1+ cells to a greater degree after peptide/LPS treatment vs peptide alone (data not shown).



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  		FIGURE 5. LPS treatment induces changes in the responding T cell population. a, CD4 V{beta}3 T cells from the experiment in Fig. 1 were stained for CD25 expression. B10.A mice were injected with SEA and control rat IgG ({circ}); SEA and anti-CD40 mAb ({square}); SEA, rat IgG, and LPS (•); or SEA, anti-CD40 mAb, and LPS ({blacksquare}). Results show the percentage ± SEM of CD4 V{beta}3 T cells that expressed CD25. b, CD4 Thy1.1 T cells from the time course described in Fig. 3a were stained for CD25. Mice were treated with peptide ({square}) or peptide and LPS ({blacksquare}). Results are the average percentage ± SEM of CD4 Thy1.1+ T cells expressing CD25. c, MHC class II expression on CD4 Thy1.1 cells 37 h after peptide treatment. Results show IAb expression on Thy1.1 cells after gating on CD4+Thy1.1+ T cells. The number in the upper right is the percentage of Thy1.1 cells expressing IAb. d, Forward scatter and side scatter profiles of LN and splenic Thy1.1 gated cells from transfer-only, peptide-treated, or peptide/LPS-treated mice. T cells were analyzed 37 h after peptide injection. Arrows depict regions of increased forward and side scatter where primarily peptide/LPS-treated cells accumulated. Results are from one representative experiment of three performed.

 
In addition to the above surface receptor changes, the CD4 Thy1.1 T cells also developed unexpected shifts in their size and granularity (Fig. 5d). At 37 h after Ag, Thy1.1+ cells from transfer-only mice had forward scatter and side scatter profiles consistent with a resting phenotype. Upon injection of peptide, the T cells underwent blastogenesis; however, upon coadministration of LPS, Thy1.1+ cells became even larger and more granular than blasting cells, with the largest effect observed in the spleen (Fig. 5d, see arrows). The median fluorescence intensity (MFI) of side scatter was 805 for splenic T cells in the peptide/LPS-treated mouse and 495 in the peptide-treated mouse compared with 386 in the transfer-only mouse. Forward scatter MFI increased from 441 in the transfer-only mouse to 732 in the peptide-treated mouse and further to 932 in the peptide/LPS-treated mouse. Comparable shifts in forward and side scatter were observed in the LNs. Overall, from these plots, we reasoned that in the presence of adjuvant, the responding T cells were being isolated from the tissues bound up either in multicellular aggregates or to large and granular cells such as innate APCs.

To test this hypothesis, we developed an in vivo assay in which SM1 TCR-transgenic cells were adoptively transferred into GFP-transgenic mice (Fig. 6). One possibility is that the Thy1.1+ T cells would also be "GFP+," suggesting that these T cells were bound to another cell population from the host. In a naive mouse, we detected a low percentage of Thy1.1+ GFP+ cells within the host spleen (3.3%). Treatment with peptide increased the percentage slightly to ~4.5%, but LPS with peptide induced 25% Thy1.1+ GFP+ cells. Some of this increase was due to LPS, since injection of LPS alone induced 9% Thy1.1+ GFP+ cells. Still, it remained to be determined whether these "double-positive" populations were indeed conjugates of T cells and APCs.



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  		FIGURE 6. LPS prolongs and enhances T cell coupling to innate APCs. a, Diagram of the procedure used in the in vivo GFP coupling assay. One day after adoptive transfer of SM1 RAG-/--transgenic T cells, peptide and LPS injections were given. Peptide injections were given at hour 0 and LPS injections were given at hour 18. Data were obtained at 37 h after peptide (19 h after LPS). b, GFP-transgenic mice received 3 x 106 SM1 RAG-/--transgenic T cells, and the next day mice were treated as follows: with nothing (Transfer only), vehicle at 0 h and 150 µg of LPS at 18 h (LPS), 100 µg of peptide at 0 h and vehicle at 18 h (Peptide), or peptide and LPS (Peptide/LPS). After 37 h, spleens and LNs were treated with collagenase D. Initially, CD4+Thy1.1+ cells were gated, and the percentage of Thy1.1+ cells that were GFP+ from this gate was determined. b, Density plots showing GFP fluorescence on splenic CD4+Thy1.1+ gated cells. The number in the upper right quadrant represents the percentage of Thy1.1+ cells that are positive for GFP. Similar data were obtained for LNs. Results are from one representative experiment of six performed.

 
The GFP+Thy1.1+ cells from the peptide/LPS-treated mice were sorted by flow cytometry and examination by light microscopy revealed frequent clusters of cells, many of which, when imaged for GFP fluorescence, consisted of mixtures of GFP+ cells (host cells) and GFP- cells (Thy1.1 donor T cells, Fig. 7a). Sorted GFP-Thy1.1+ T cells showed few clusters of cells and little to no GFP fluorescence (Fig. 7b). Separate sorting of GFP+Thy1.1- populations were predominantly GFP+ and rarely clustered (Fig. 7c). Fixed H&E-stained cells from the GFP+Thy1.1+ sorted population again showed frequent clusters of cells, many of which were T cells, yet which also included large DCs (Fig. 7d). Cells resembling myeloid DCs were frequently observed along with some resembling other innate APCs. Conversely, GFP-Thy1.1+ sorted populations consisted almost exclusively of T cells (data not shown). Thus, using this in vivo GFP coupling assay, T cells responding to peptide/LPS could be isolated and directly visualized in clusters with DCs.



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  		FIGURE 7. Specific T cell survival conditioning mediated by the innate immune system occurs after TLR4 stimulation. GFP+Thy1.1+ (a), GFP-Thy1.1+ (b), and GFP+Thy1.1- (c) cells were sorted as described in Materials and Methods. a–c compare bright-field images (top) to GFP fluorescence (bottom) for each cell population using a x20 objective. The images in a and c were cropped to enlarge regions of interest. Image b was not cropped to show the lack of cell clustering. d, Representative H&E-stained cells from the GFP+Thy1.1+ sorted population taken with a x40 objective. Each image not necessarily to scale since some have been enlarged to emphasize morphological features. Results in a–d were selected from data from four sorts. e and f, Histograms showing the percentage of CD4 Thy1.1 cells coexpressing CD11b or CD11c (gray histograms). The black histograms represent isotype control staining. Positive staining percentages were determined by setting the isotype control at around 1%. g, Inguinal LN section stained for Thy1.1 (brown) and CD11c (blue). No counterstain was used. Original magnification, x40.

 
To further confirm which cells the T cells were binding to, CD4+Thy1.1+ cells were costained for CD11c and CD11b (Fig. 7, e and f). Little staining was observed in transfer-only or peptide-treated mice, yet LPS treatment alone caused an increase in CD11b staining and a moderate increase in CD11c staining. This effect was dramatically pronounced in peptide/LPS-treated mice where nearly 50% of the Ag-specific T cells were associated with CD11b+ and CD11c+ cells. In situ staining for Thy1.1 and CD11c further confirmed that the T cells were clustering around DCs within the LNs (Fig. 7g).

Finally, to determine whether the above data had any relevance for T cell survival and memory development, SM1 T cells were isolated from adoptively transferred C57BL/6 mice 20 days after immunization (Fig. 8 and Table I). A small percentage of CD4+Thy1.1+ T cells from transfer-only mice could be detected in the spleen, liver, and lung (Fig. 8a). T cells from peptide alone mice were rarely detected 20 days after immunization. No treatments matched the level of survival observed in peptide/LPS-treated mice, which had 30- to 50-fold increases in the CD4+Thy1.1+ population compared with normal or peptide-treated mice. Similar trends were observed when absolute numbers were calculated (Fig. 8b), and the tabulated numbers from six experiments are given in Table I. In the blood, 20-day-old surviving cells had elevated CD44 expression (MFI 18) compared with CD4+Thy1.1- cells (MFI 7). Additionally, the peptide/LPS-treated cells underwent a blasting phase shown on day 4, but reverted to a size equivalent to resting cells by day 20 (forward scatter MFI of 338 vs 321 for Thy1.1+ and Thy1.1- cells, respectively), consistent with a memory T cell phenotype (Fig. 8c). Furthermore, peptide/LPS-treated T cells produced copious amounts of IFN-{gamma} and TNF upon restimulation, confirming that they possessed potent recall potential (Fig. 8d). This is in contrast to cells taken from transfer-only or LPS-alone mice, which produced very little IFN-{gamma} upon stimulation (Table II).



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  		FIGURE 8. LPS injection promotes long-term survival and memory development of specific CD4 T cells. C57BL/6 mice received 2 x 106 SM1 RAG-/--transgenic cells, and the next day these mice were injected with nothing, 50 µg of LPS, 100 µg of peptide, or peptide/LPS. Blood, LNs (inguinal, axillary, brachial, and mesenteric), spleen, liver, and lung were isolated from each mouse on day 20 and resident T cells analyzed. a, Dot plots depicting the percentage of CD4+Thy1.1+ T cells in the spleen, liver, and lung. b, Graphs of the absolute numbers of CD4+Thy1.1+ T cells in the spleen and liver. Numbers were obtained by multiplying the percentage of CD4+Thy1.1+ cells by the total cell number in the tissues. c, CD44 and forward scatter plots comparing CD4 Thy1.1+ (gray histogram) and CD4 Thy1.1- (black histogram) cells from peptide/LPS-treated mice on day 20 or comparing day 4 post-Ag injection of peptide/LPS-treated cells (dotted line) to naive transfer-only cells (solid line). All graphs are data taken from experiments using PBLs. d, Intracellular cytokine staining of day 20 T cells that were restimulated for 5 h as described in Materials and Methods. Shown are representative percentages of Thy1.1+ cells from a peptide/LPS-treated mouse staining positive for IFN-{gamma}, TNF, or an isotype control. Each long-term experiment performed had one mouse per group and was repeated six times (see Tables I and II).

 

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  		Table I. Combined absolute number data from all day 20 experiments performeda

 

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  		Table II. Combined intracellular cytokine data from all day 20 experiments performeda

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Specific T cells sequester into lymphoid tissue and after Ag presentation disappear regardless whether or not an adjuvant is used. In this study, we present new evidence that stimulated T cells do not disappear but actually are difficult to extract even though they are readily detected by immunohistochemistry. We have termed this phase T cell clonal conditioning and propose that T cells can be conditioned to delete or survive based on the activation state of the innate immune system. Under survival conditioning, adjuvants such as the TLR4 agonist LPS induce specific T cells to communicate differently with DCs compared with tolerizing conditions (Fig. 9).



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  		FIGURE 9. T cell clonal conditioning precedes clonal expansion and either development into memory or deletion. The time period after Ag presentation but before clonal expansion has largely been understudied due to the disappearance of activated T cells. Our data show that activated T cells are actually difficult to extract before clonal expansion. We have termed this period T cell clonal conditioning because it is the stage where T cells exchange information or communicate with cells of the innate immune system. TLR4 stimulation, for example, as a result of the release of a pathogen-associated molecular pattern (PAMP), alters clonal conditioning by enhancing T cell interactions with innate APCs and this is postulated to determine a T cell’s fate to forego deletion and develop into memory. Line thickness refers to the relative intensity of the process.

 
Upon injection of SEA, specific T cells became rapidly activated, and despite an apparently normal capacity to proliferate (Fig. 2) still seemed to disappear or undergo early deletion (Fig. 1). We attempted to determine whether the early "deletion" was a cell death effect and our results were equivocal (data not shown), although in the past we detected TUNEL-positive cells in situ just after T cell clonal expansion (32). Thus, before expansion there are fewer specific T cells and these may be engulfed rapidly after the initiation of apoptosis, thereby precluding our ability to detect them. Nevertheless, it is possible that some of the disappearance is due to cell death or potentially due to migration to other sites in the body.

We conclude based on the observations obtained with the SM1 model that TCR internalization occurs (data not shown), but is not the explanation for this early phenomenon. Just as collagenase treatment preferentially enhanced SM1 T cell recovery (Fig. 4), we also found that collagenase enhanced SAg-specific T cell recovery at early time points (data not shown). These data suggested that after the specific T cells are stimulated in LNs they become difficult to detect by flow cytometry. Consistent with this idea are classic data from Sprent and Miller (2) suggesting that T cells lose Ag reactivity possibly because of a loss of receptor. These early perceptions were indeed correct but the loss of reactivity may have more to do with inefficient extraction of T cells from lymphoid tissue, which many current studies are likely to suffer from as well.

To better understand this process, we developed a useful in vivo assay to quantitate and analyze T cell/APC clustering and coupling (Figs. 6 and 7). Traditional coupling experiments have primarily been conducted in vitro by examining increases in forward/side scatter or by labeling two populations of cells with different dyes or Abs and looking for double-positive populations by flow cytometry (33, 34). The GFP coupling assay described here requires no cell labeling and can be done in vivo by direct surface staining of at least one of the target populations. Using this assay, we uncovered the striking observation that LPS induces a niche where specific T cells couple to DCs, which is not apparent in the absence of LPS (Figs. 6 and 7). Under tolerizing conditions, peptide-specific T cells initially became undetectable (in the conditioning phase), but this was not sufficient to confer long-term specific T cell survival (compare Figs. 3 and 8). Accordingly, peptide and anti-CD40 stimulation, which does not confer survival (Table I), induced 8% of the Thy1.1 cells to become conjugated in the GFP coupling assay (data not shown), which was 3.2-fold less than LPS (Fig. 6b). Also, a low dose of LPS (10 µg) delivered after peptide injection results in low amounts of coupling (data not shown) and memory (Table I) (24); however, increasing the dose of LPS not only produces enhanced T cell coupling, but greatly augmented T cell memory as well (Fig. 6b and Table I). Additionally, it should be noted that some of the clustering may not be completely due to Ag-induced interactions alone since LPS only induced an increase of GFP Thy1.1 double-positive cells over transfer only (Fig. 6b). Thus, T cell clonal survival conditioning depends on a threshold of TLR stimulation or appropriate activation of the innate immune system.

It is important to consider that the GFP coupling assay probably underestimates the frequency of cell conjugates induced by LPS treatment. The fact that we retain cell coupling after multiple rounds of centrifugation, washing, and cell sorting is a testament to how strong these peptide/adjuvant-induced T and DC interactions are. A second finding from the GFP coupling assay is that with peptide/LPS stimulation, the GFP fluorescence on uncoupled Thy1.1 cells also increases (Fig. 6b, lower right quadrant). This could simply be due to an increase in autofluorescence or, alternatively, support the idea that responding T cells acquire and internalize surface molecules such as MHC from an APC (35), and, in this case, GFP as well. This would explain why we observed up to 80% of the Thy1.1 cells from peptide/LPS-treated mice "expressing" MHC class II during this conditioning phase (Fig. 5c). Mechanistically, LPS treatment likely enhances T cell scanning of APC surfaces, allowing more T cells to interact with and acquire pieces of the APC.

Since the responding Thy1.1+ cells possessed traditional APC surface molecules, it was important to determine what type of APCs were interacting with the specific T cells. Sorting of the GFP+Thy1.1+ conjugates from peptide/LPS-treated mice yielded a mixed population of DCs and T cells (Fig. 7). We observed what appeared to be myeloid DCs, but did find other innate cells as well. Myeloid DCs have been shown to be excellent initiators of T cell responses (36, 37, 38, 39). Recently, CD11b+ DCs have been observed exclusively interacting with OVA-specific T cells in situ after s.c. injection of soluble Ag (40). Our work (Fig. 7g) and that of other laboratories observed Ag-specific T cells interacting with CD11c+ DCs in situ following Ag injection (41, 42, 43, 44, 45). Thus, DCs continue to be the significant APC for initiating T cell responses, a point that is further underscored by the recent study documenting the inability of CD8 T cell responses to be mounted in vivo in the absence of CD11c+ DCs (46).

The mechanism of how adjuvants induce T cell clonal conditioning for survival is unknown. It may provide a sustained signaling process similar to that suggested by Lanzavecchia and Sallusto (47) for T cell activation. Although T cell NF-{kappa}B activation is not necessary for long-term specific T cell survival in response to LPS injection (24), such sustained signaling may optimally induce survival molecules such as Bcl-3 (48). Additionally, the tight interactions may allow DCs to better focus a collection of cytokines directly onto the specific T cell. Inflammatory cytokines are important by-products of adjuvant administration and have been shown to assist in T cell activation and memory development (14, 49, 50, 51). Alternatively, later acting costimulatory signals such as OX40 may be central to this process. One recent article suggested that OX40 signaling can contribute to circumvention of tolerance by acting later in the T cell activation process (52). We have previously shown that LPS can prolong OX40 expression on activated T cells (29), and this may be a possible mechanism of survival induction. Or, LPS may induce a specific type of DC that provides survival signals. Recent studies have found that T cells themselves play important roles in the maturation and survival of DCs (53, 54). Thus, in the presence of adjuvant-induced responses, T cells and DCs may be continually exchanging packets of information which enhance DC function and promote optimal T cell expansion and survival.

Taken together, after Ag-specific T cells are initially stimulated within lymphoid tissues but before clonal expansion, there is a phase we have termed T cell clonal conditioning where the specific T cells become very difficult to extract from these tissues. In the absence of appropriate activation of the innate immune system, the T cells will release, clonally expand, and delete (Fig. 9). In the presence of LPS, the specific T cells become conditioned to survive by activated innate APCs resulting in clonal expansion and differentiation into memory T cells (Fig. 9). Therefore, we postulate that T cell clonal conditioning is an important target for vaccine development as well as therapeutic intervention for the treatment of autoimmunity.


   Acknowledgments
 
We thank the Center for Histomorphometry and Histology and the Center for Immunotherapy of Cancer and Infectious Diseases for the use of their microscopes (UCONN Health Center), Dr. T. V. Rajan (UCONN Health Center) for assistance with histological analysis, E. Pizzo and D. Gran for help with cell sorting, and Drs. L. Lefrancois and A. Adler (UCONN Health Center) for helpful discussions.


   Footnotes
 
1 This work was funded by National Institutes of Health Grants AI142858 and AI52108. Back

2 Address correspondence and reprint requests to Dr. Anthony T. Vella, Division of Immunology, MC1319, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032. E-mail address: vella{at}uchc.edu Back

3 Abbreviations used in this paper: LN, lymph node; SAg, superantigen; DC, dendritic cell; TLR4, Toll-like receptor 4; GFP, green fluorescent protein; RAG, recombination-activating gene; SEA, staphylococcal enterotoxin A; BSS, balanced salt solution; CTM, complete tumor medium; BrdU, 5-bromo-2'-deoxyuridine; MFI, mean fluorescence intensity. Back

Received for publication July 24, 2003. Accepted for publication October 20, 2003.


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 Results
 Discussion
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Kupffer cell heterogeneity: functional properties of bone marrow derived and sessile hepatic macrophages
Blood, December 1, 2007; 110(12): 4077 - 4085.
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J. P. McAleer, D. J. Zammit, L. Lefrancois, R. J. Rossi, and A. T. Vella
The Lipopolysaccharide Adjuvant Effect on T Cells Relies on Nonoverlapping Contributions from the MyD88 Pathway and CD11c+ Cells
J. Immunol., November 15, 2007; 179(10): 6524 - 6535.
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R. Yadav, D. J. Zammit, L. Lefrancois, and A. T. Vella
Effects of LPS-mediated bystander activation in the innate immune system
J. Leukoc. Biol., December 1, 2006; 80(6): 1251 - 1261.
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J. S. Levine, R. Subang, S. H. Nasr, S. Fournier, G. Lajoie, J. Wither, and J. Rauch
Immunization with an Apoptotic Cell-Binding Protein Recapitulates the Nephritis and Sequential Autoantibody Emergence of Systemic Lupus Erythematosus
J. Immunol., November 1, 2006; 177(9): 6504 - 6516.
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M. Long and A. J. Adler
Cutting Edge: Paracrine, but Not Autocrine, IL-2 Signaling Is Sustained during Early Antiviral CD4 T Cell Response
J. Immunol., October 1, 2006; 177(7): 4257 - 4261.
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A. Jabbari, K. L. Legge, and J. T. Harty
T cell conditioning explains early disappearance of the memory CD8 T cell response to infection.
J. Immunol., September 1, 2006; 177(5): 3012 - 3018.
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J. R. Maxwell, R. Yadav, R. J. Rossi, C. E. Ruby, A. D. Weinberg, H. L. Aguila, and A. T. Vella
IL-18 Bridges Innate and Adaptive Immunity through IFN-{gamma} and the CD134 Pathway
J. Immunol., July 1, 2006; 177(1): 234 - 245.
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B. Koehn, S. Gangappa, J. D. Miller, R. Ahmed, and C. P. Larsen
Patients, pathogens, and protective immunity: the relevance of virus-induced alloreactivity in transplantation.
J. Immunol., March 1, 2006; 176(5): 2691 - 2696.
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J. K. Whitmire, N. Benning, and J. L. Whitton
Precursor Frequency, Nonlinear Proliferation, and Functional Maturation of Virus-Specific CD4+ T Cells.
J. Immunol., March 1, 2006; 176(5): 3028 - 3036.
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C. M. Bosio, A. W. Goodyear, and S. W. Dow
Early Interaction of Yersinia pestis with APCs in the Lung
J. Immunol., November 15, 2005; 175(10): 6750 - 6756.
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C. M. Bosio and S. W. Dow
Francisella tularensis Induces Aberrant Activation of Pulmonary Dendritic Cells
J. Immunol., November 15, 2005; 175(10): 6792 - 6801.
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 JEMHome page
B. H. Zinselmeyer, J. Dempster, A. M. Gurney, D. Wokosin, M. Miller, H. Ho, O. R. Millington, K. M. Smith, C. M. Rush, I. Parker, et al.
In situ characterization of CD4+ T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance
J. Exp. Med., June 6, 2005; 201(11): 1815 - 1823.
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 Int ImmunolHome page
R. J. Rossi, G. Muralimohan, J. R. Maxwell, and A. T. Vella
Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation
Int. Immunol., December 1, 2004; 16(12): 1751 - 1760.
[Abstract] [Full Text] [PDF]

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