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The Journal of Immunology, 1999, 163: 5978-5988.
Copyright © 1999 by The American Association of Immunologists

Antigen-Driven Selection of TCR In Vivo: Related TCR {alpha}-Chains Pair with Diverse TCR ß-Chains1

John A. Mikszta, Louise J. McHeyzer-Williams and Michael G. McHeyzer-Williams2

Department of Immunology, Duke University Medical Center, Durham, NC 27710


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ag-driven selection mediates effective T cell help and the development of Th cell memory in vivo. To analyze the dynamics of interclonal competition during the selection process in vivo, we use the I-Ek-restricted murine response to pigeon cytochrome c (PCC). The dominant PCC-specific clonotype expresses V{alpha}11Vß3 V regions with preferred sequence features in the third hypervariable regions (CDR3). In the current study we define and quantitatively monitor four subdominant PCC-specific clonotypes that express V{alpha}11 paired with non-Vß3 TCR ß-chains (Vß6, Vß8.1/8.2, Vß8.3, and Vß14). The subdominant clonotypes emerge with similar dynamics to the dominant clonotype and together amount to similar numbers as the dominant clonotype in vivo. These subdominant clonotypes do not efficiently enter germinal centers, although they enter the memory compartment and rapidly re-emerge upon secondary challenge. Analysis of CDR3 diversity in the TCR {alpha}-chains identifies many preferred sequence features expressed by the dominant clonotype. These studies quantitatively demonstrate selection for diverse Th cells in vivo and highlight TCR {alpha}-chain dominance in Ag-driven selection for best fit.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The adaptive immune response depends on the recognition of foreign antigenic peptides presented in the context of self-encoded MHC molecules by specific T lymphocytes. These specific T cells express a heterodimeric TCR complex of {alpha}- and ß-chains assembled by the random rearrangement of variable (V), diversity (D; in ß-chain), and joining (J) gene segments (1). A broad preimmune repertoire of TCR is established through the processes of positive and negative selection of developing T cells in the thymus (2, 3, 4). In most Ag systems studied, the repertoire of T cells that respond to a given Ag is quite diverse in terms of TCR ß-chain V region usage and fine epitope specificity (5, 6, 7, 8, 9, 10, 11). In some cases clonal dominance prevails, and T cells with preferred TCR motifs are selectively expanded for primary responses and appear selectively preserved for memory responses (12, 13, 14).

The I-Ek-restricted murine response to pigeon cytochrome c (PCC)3 remains the best-characterized example of clonal dominance in the Th compartment (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). The majority of the Th response to PCC is directed against a single immunodominant epitope encompassing aa residues 88–104 (15, 16). The Th cells responding to this epitope predominantly express V{alpha}11 and Vß3 TCR variable regions with restricted CDR3 features (12, 13, 14, 17, 18, 19, 20). Early studies using Th clones and hybridomas demonstrate the predominance of a glutamic acid residue at position {alpha}93 and serine or the conservative threonine at {alpha}95 (17, 18, 22). In the ß-chain, asparagine is predominant at position ß100 in conjunction with alanine or glycine at ß102 (17, 19). Studies by Jorgensen et al. suggest that the glutamic acid at position {alpha}93 and the asparagine at ß100 are involved in critical interactions with specific residues of the antigenic peptide (21). Our recent studies directly ex vivo demonstrate a rapid and progressive selection for the dominant clonotype driven by Ag after priming (12, 13, 14). There is a narrowing of CDR3 diversity in PCC-specific V{alpha}11Vß3-expressing Th cells between primary and memory responses. This narrowing of diversity is rapid and largely complete during the first week after initial exposure to Ag (14). Savage et al. (23) provide evidence for an increase in average affinity of TCR for peptide/MHC that drives the selection for the preferred TCR in vivo.

Even within this highly restricted Th response, there is evidence for additional PCC-specific clonotypes. A stronger bias for V{alpha}11 over Vß3 can be found in panels of I-Ek-restricted PCC-specific Th clones and hybridomas (17, 18, 19, 22). While 90–100% of these cells have been reported to express V{alpha}11, only 50–60% express Vß3. When Vß3 is artificially ablated in vivo, non-Vß3-expressing clonotypes arise in response to PCC immunization. Liang et al. show that B10.BR mice injected from birth with Staphylococcus aureus enterotoxin (to delete Vß3-expressing cells) still generate a T cell proliferative response to the dominant peptide epitope; the great majority of PCC-specific T cell hybridomas derived from these mice express Vß8 (24). Likewise, PCC-specific T cell lines and hybridomas derived from mouse strains expressing Mls-2a also use alternate Vß-chains, including Vß8 and Vß1 (24, 25, 26). The expression of wild-type or substituted analogue peptides of PCC in the thymus also affects the preimmune Vß repertoire and reveals PCC-responsive Th cells expressing V{alpha}11 paired with alternate Vß-chains, including Vß8, Vß14, and Vß16 (27, 28). Our recent studies implicate a preimmune bias in B10.BR mice for TCR {alpha}-chains that express CDR3 features associated with PCC specificity (14). Thus, a cohort of subdominant V{alpha}11+ non-Vß3 clonotypes can emerge in response to PCC immunization.

The presence of subdominant PCC-specific clonotypes provides an opportunity to study the dynamics and outcomes of interclonal competition in the Th response. Using high resolution flow cytometry, we identify four V{alpha}11+ non-Vß3 clonotypes that emerge specifically in response to PCC. All PCC-specific responders expand and decline with similar kinetics, and the four subdominant clonotypes together contribute close to half the total PCC-specific cellular response in the draining lymph nodes (LN). Although V{alpha}11Vß3-expressing clonotypes predominantly enter LN germinal centers (GC) by day 9 after initial challenge (14), cells expressing the subdominant TCR rarely migrate to this specialized microenvironment. The ratio of dominant to subdominant clonotypes at the peak of the memory response is similar to that found after initial priming and suggests that no inherent growth advantage is conferred through expression of the dominant TCR. To more closely assess repertoire differences in the subdominant clonotypes, we amplify and sequence TCR {alpha}-chains from many single cells after primary and secondary challenge. Despite the obvious diversity in TCRß V region usage, the TCR {alpha}-chain CDR3 are similar in many ways to the dominant clonotype; most express glutamic acid at position {alpha}93 and serine or threonine at position {alpha}95 with a CDR3 length of 8 or 9 aa. These results underscore a dominant role for V{alpha}11 and its particular CDR3 in PCC-specific recognition and provide a more comprehensive analysis of Ag-specific TCR diversity in vivo.


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

Specific pathogen-free B10.BR mice (The Jackson Laboratory, Bar Harbor, ME), 6–10 wk of age, were housed under reverse barrier conditions at the Duke University vivarium. Whole PCC (Sigma, St. Louis, MO) was dissolved in PBS and emulsified in RAS adjuvant (RIBI Immunochem Research, Hamilton, MT). As previously described (12, 14) mice were injected at the base of tail with 400 µg of PCC in RAS for initial priming, rested for at least 8 wk, then reinjected with the same dose of Ag in adjuvant at the base of tail for the memory response. An equivalent volume of PBS emulsified in RAS was used for the adjuvant only controls.

Flow cytometry

Draining inguinal and periaortic LN were harvested at varying times, and single-cell suspensions were prepared as previously described (12, 14). Cells were stained for flow cytometry at 2.0 x 108/ml on ice for 45 min with predetermined optimal concentrations of FITC-anti-V{alpha}11 (RR8-1; PharMingen, San Diego, CA), allophycocyanin-anti-Vß3 (KJ25), PE-anti-CD62L (Mel14) (PharMingen), Cy5PE-anti-CD8 (53-6.7), Cy5PE-anti-CD11b (M1/70.15; Caltag, Burlingame, CA), Cy5PE-anti-B220 (6B2; PharMingen), and Texas Red (TR)-anti-CD44 (IM7). For detection of the non-Vß3-expressing clonotypes, cells were stained with Cy5PE-labeled Abs as described above plus allophycocyanin-anti-CD44 (IM7), PE-anti-V{alpha}11 (RR8-1; PharMingen), TR-anti-CD62L (Mel14), and each of the 14 FITC-labeled Abs to the different Vß (PharMingen) displayed in Fig. 2GoA. After staining, cells were washed twice in PBS plus 5% FCS, then resuspended for analysis in the same buffer containing propidium iodide (2 µg/ml).



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  		FIGURE 2. Determination of TCR Vß expression by the subdominant PCC-specific Th clonotypes. A, Seven days after immunization, draining LN cells were stained for seven-parameter flow cytometric detection of the subdominant clonotypes as described in Materials and Methods. Cells were gated as intermediate for forward and side scatter; negative for PI, CD8, B220 and CD11b; V{alpha}11+VßX+; and CD44high,CD62Llow, as depicted in Fig. 1Go. The frequencies of V{alpha}11VßX-expressing Th cells that are CD44high and CD62Llow and total cell numbers in the draining LNs from each animal at sacrifice were used to calculate the displayed numbers. Data represent the mean ± SEM for at least three individual mice per clonotype. A very low frequency (<0.02%) of V{alpha}11+ T cells in B10.BR mice coexpresses Vß5, -7, -9, -11, or -17; total cell numbers were <500 in all cases. B, Activated cell numbers for the dominant V{alpha}11+Vß3+ are compared with the combined cell numbers for the V{alpha}11+ PCC-specific subdominant clonotypes (Vß6, Vß8.1/8.2, Vß8.3, and Vß14 combined).

 
Samples were analyzed using a FACStarPlus flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) modified with dual lasers (a primary argon laser and a secondary tunable dye laser) and capable of simultaneous detection of seven parameters. PI is excluded before file acquisition in the Cy5PE detection channel. For all analyses, we collect two files for each sample; the first file contains 100,000 PI-negative events and is used to calculate the frequency of Cy5PE-negative cells coexpressing the TCR of interest in the total LN population. The second file contains between 250 and 1000 events gated as PI-Cy5PE- and positive for the TCR of interest to evaluate the fraction of cells that have up-regulated CD44 and down-regulated CD62L. Files were acquired using CellQuest software (Becton Dickinson) and were analyzed using FlowJo software (Tree Star, San Carlos, CA). All profiles are presented as 5% probability contours with outliers.

Laser scanning confocal microscopy (LSCM)

Draining LN were quick frozen in OCT embedding compound (Miles, Elkhart, IN). Cryostat microtome (Leica, Heidelberg, Germany)-cut sections (6 µm thick) were mounted onto gelatin-coated slides, air-dried, and fixed in acetone for 10 min at 4°C, then stored at -80°C. For staining, sections were rehydrated with PBS, then blocked with PBS containing 10% FCS, 10% skim milk (w/v) powder, and 2.4G2 (anti-FcR) culture supernatant (50%, v/v) for 30 min at room temperature. For visualization of non-Vß3-expressing clonotypes, sections were stained with FITC-conjugated Abs (PharMingen) to Vß8.3 (1B3.3), Vß14 (14-2) or Vß6 (RR4-7), allophycocyanin-anti-IgD (11.26), and avidin-rhodamine red (Molecular Probes, Eugene, OR)/biotin-anti-V{alpha}11 (RR8-1). The Vß8.1/8.2-expressing clonotype was visualized by staining with FITC-anti-V{alpha}11 (RR8-1), allophycocyanin-anti-IgD (11.26), and rhodamine red-avidin/biotin-anti-Vß8.1/8.2 (MR5-2). The dominant clonotype was revealed by staining with FITC-anti-V{alpha}11 (RR8-1), allophycocyanin-anti-Vß3 (KJ25), and TR-anti-IgD (11.26) as previously described (14). All stains were performed at room temperature for 1 h. Stained sections were washed in PBS, then mounted in VectorShield (Vector Laboratories, Burlingame, CA). The excitation wavelengths and collection filters used for FITC, allophycocyanin, and TR have been previously described (14). Rhodamine red was excited at 568 nm and collected using a 580- to 630-nm band-pass filter. Data were acquired using a Zeiss Axiovert LSM 410 confocal microscope (Zeiss, Thornwood, NY). Each color was collected individually and serially in the first detector using LSM 3.95 software (Zeiss), then reassembled using Adobe Photoshop (Adobe Systems, San Jose, CA) for quantitation and localization as previously described (14).

Single-cell repertoire studies

Complementary DNA synthesis. Cells were sorted using the automatic cell dispensing unit of the FACStarPlus (Becton Dickinson) and CloneCyt software (Becton Dickinson) as previously described (14). Briefly, single cells were sorted into individual wells of low profile 72-well microtiter trays (Robbins Scientific, Sunnyvale, CA), with each well containing 5 µl of oligo(dT)-primed cDNA reaction mix: 4 U/ml murine leukemia virus-reverse trancriptase in recommended reaction buffer (Life Technologies, Gaithersburg, MD), 0.5 nM spermidine (Sigma), 100 µg/ml BSA (Boehringer Mannheim, Indianapolis, IN), 10 ng/ml oligo(dT) (Becton Dickinson), 200 µM of each dNTP (Boehringer Mannheim), 1 mM DTT (Promega, Madison, WI), 220 U/ml RNAsin (Promega), 100 µg/ml Escherichia coli transfer RNA (Boehringer Mannheim), and 1% Triton X-100 (Sigma). After sorting, trays were incubated at 37°C for 90 min, then stored at -80°C until PCR. Cells were only sorted into the center 60 wells of each tray; the first and last wells of each row served as negative controls and were processed together with positive samples throughout the entire experimental procedure. Controls for the sorting include nested PCR for actin mRNA (70–100% of the wells are positive) and the direct visualization of single sorted hybridoma cells by light microscopy (60–80% of the wells contain a visible single cell, doublets were never observed).

Nested PCR. For the first rounds of PCR, 2 µl of cDNA from each single cell was used in a 25-µl reaction to amplify the TCR V{alpha}11 using primers specific for the variable and constant regions as previously described (14). The first-round PCR mix consisted of 2 U/ml Taq polymerase in the recommended reaction buffer (Promega), 0.1 mM of each dNTP (Boehringer Mannheim), 2 mM MgCl2, 1.2 µM primer V{alpha}11.L1 (5'-ATGCAGAGGAACCTGGGAGC-3'), and 1.2 µM primer C{alpha}.2 (5'-AATCTGCAGCGGCACATTGATTTGGGA-3'). Reactions begin with 5 min at 95°C, then 40 cycles of 95°C for 15 s, 50°C for 45 s, and 72°C for 90 s and ending with 72°C for 5 min on a 9600 GeneAmp PCR system (Perkin-Elmer, Foster City, CA). For the second rounds of PCR, 1 µl of first-round PCR product was used for an additional 25-µl amplification reaction, using primers nested medially to those used in the first round. The second-round PCR mix consisted of 2 U/ml Taq polymerase in the recommended reaction buffer (Promega), 0.1 mM of each dNTP (Boehringer Mannheim), 2 mM MgCl2, 0.8 µM primer V{alpha}11.L2 (5'-AATCTGCAGTGGGTGCAGATTTGCTGG-3'), and 0.8 µM primer C{alpha}.ext (5'-GAGTCAAAGTCGGTGAACAGG-3'). Reactions begin with 5 min at 95°C, then 35 cycles of 95°C for 15 s, 55°C for 45 s, and 72°C for 90 s, and ending with 72°C for 5 min. At least two negative cDNA samples were processed per 10 single-cell samples. Negatives were interspersed with positives to control for contamination during sample preparation. The frequency of obtaining a V{alpha}11 PCR product from single cells was 38 ± 4%.

DNA sequencing. Five microliters of second-round PCR product was run on a 1.5% agarose gel; positive bands were visualized under UV using ethidium bromide. PCR product was separated from primers using CL-6B Sepharose (Pharmacia, Piscataway, NJ) columns and then directly sequenced using 3 µl of product, 4 µl of Dye Terminator Ready Reaction Mix (Perkin-Elmer, Palo Alto, CA), and 1.5 pmol primer V{alpha}11.seq (5'-CAGGAACAAAGGAGAATGGGAG-3') on a linear amplification protocol of 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min as previously described (14). Samples were separated on a 6.5% acrylamide gel after ethanol precipitation of sequencing reaction products, run on an ABI 373 sequencing system, and processed using ABI Prism sequence 2.1.2 (Perkin-Elmer) for collection and analysis. The nomenclature for single cells used in the figures reflects primary (P), memory (M), or RIBI only control response (R) followed by the day of the response (day 0, 7, or 9 of the primary; day 7 RIBI adjuvant only; or day 3 memory), followed in parentheses by the mouse number (1 through 3) and Vß expression (A = Vß14, B = Vß8.1/8.2, C = Vß6, D = Vß8.3), then the clone number. For example, P7(1A).1 indicates a single cell isolated from day 7 of the primary response (P7) from mouse number 1 (1A); the clone expresses Vß14 (1A) and is the first clone listed from this mouse (.1).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Subdominant clonotypes in the Ag-specific Th response to PCC

Using a five-color flow cytometric strategy we had developed to monitor the dominant clonotype (14), we could estimate the size of the total V{alpha}11+ non-Vß3-expressing PCC-responsive Th cell compartment in normal B10.BR mice (Fig. 1Go). We focus analysis on (CD8, B220, CD11b)- V{alpha}11+Vß3- cells that increase CD44 and decrease CD62L expression in response to PCC. The frequency of CD44highCD62Llow V{alpha}11+Vß3- Th cells 7 days after immunization with PCC is significantly greater than that in mice injected with adjuvant only (13.7 ± 2.6% (n = 7) vs 3.7 ± 0.6% (n = 4), respectively; p = 0.005; Fig. 1Go). Mice injected with an unrelated protein Ag (hen egg lysozyme) in adjuvant or unimmunized controls displayed equally low background levels of activated V{alpha}11+Vß3- cells (4.4 ± 1.0% (n = 4) and 3.9 ± 0.9% (n = 4), respectively). The lack of response to hen egg lysozyme is another critical control that argues against any significant contribution of a bystander effect to the CD44highCD62Llow V{alpha}11+Vß3- Th cells that arise in response to PCC immunization. Thus, all the relevant in vivo specificity controls indicate a sizable compartment of V{alpha}11+ non-Vß3-expressing Th cells that emerge specifically in response to PCC, with a minimal bystander contribution to the flow cytometric analysis.



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  		FIGURE 1. Subdominant clonotypes in the Ag-specific Th response to PCC. Draining LN cells were stained for flow cytometry as described in Materials and Methods. Propidium iodide-negative cells are initially gated for light scatter properties and exclusion criteria (CD8, B220, and CD11b negative). Light scatter gates are set to exclude most macrophages and neutrophils, but to include resting and blasting lymphocytes. TCR expression is assessed by V{alpha}11 and Vß3 expression. The inset boxes display gating used to isolate the dominant (V{alpha}11+Vß3+) and subdominant (V{alpha}11+Vß3-) Th clonotypes. Panels at the right display CD44 and CD62L staining on the dominant and subdominant clonotypes on day 7 of the primary response to PCC or adjuvant only.

 
To directly examine Vß usage by the V{alpha}11+Vß3- PCC-specific Th cells, we substituted anti-Vß3 Abs in our general schema with a panel of the 14 additional Vß-specific Abs. Fig. 2GoA presents total cell numbers within the draining LN of activated (CD44highCD62Llow), V{alpha}11+ Th cells that coexpress the indicated Vß-chains. We identify four V{alpha}11+ Th clonotypes expressing alternate Vß-chains (Vß6, Vß14, Vß8.1/8.2, and Vß8.3) that consistently expand to levels significantly above day 0 control values (p = 0.002–0.01). The frequencies of these four clonotypes are also significantly above adjuvant only controls (p = 0.0003–0.01). Individually, each of the non-Vß3-expressing clonotypes remains subdominant to the V{alpha}11Vß3-expressing Th cells, with total activated cell numbers 3- to 9-fold lower than those of the dominant clonotype. However, when these four subdominant clonotypes are considered together, they expand to similar cell numbers as the dominant clonotype and thus make up a sizable proportion of the PCC-specific Th repertoire (Fig. 2GoB). Similar analyses on days 5 and 9 revealed the same four non-Vß3 clonotypes emerging above the other Vßs (data not shown). Many of these ß-chains have been reported in V{alpha}11-expressing PCC/moth cytochrome c-responsive T cell clones and hybridomas (18, 24, 27, 28), although the frequency or number in normal mice in vivo has not previously been examined.

Other V{alpha}11+ ß-chain-expressing clonotypes (Vß4, Vß2, Vß13, and Vß10) occasionally expand in some mice (Fig. 2GoA); however, mean cell numbers were not significantly above baseline levels in unimmunized controls (p > 0.05). The remaining ß-chains examined (Vß5, Vß7, Vß9, Vß11, Vß12, and Vß17) were expressed by a very low frequency (0.02%) of V{alpha}11+ Th cells in B10.BR mice and did not expand in response to PCC (Fig. 2GoA). Thus, we will focus on four subdominant V{alpha}11+ PCC-specific Th clonotypes (Vß6, Vß14, Vß8.1/8.2, and Vß8.3) that can be quantitatively monitored directly ex vivo by flow cytometry.

Cellular dynamics of the primary immune response

Although total numbers of the subdominant clonotypes on day 7 are similar to those of the dominant clonotype, it is possible that the expression of different TCR may affect the dynamics of cellular expansion and decline in vivo. Representative probability contours for two of the subdominant clonotypes are displayed in Fig. 3GoA, and the change in total cell numbers over time for all four clonotypes together are shown in Fig. 3GoB or separately in Fig. 3GoC. When considered together, the subdominant PCC-specific Th clonotypes expand and contract to a similar extent and with similar kinetics as the dominant clonotype over the course of the primary immune response (Fig. 3GoB). Each V{alpha}11+ PCC-specific Th clonotype reaches peak cell numbers by day 7 with a gradual decline that is clearly evident by day 13 (Fig. 3GoC). Peak cell numbers on day 7 represent a 31-fold increase over those on day 0 for Vß8.3, 18-fold for Vß8.1/8.2, 17-fold for Vß14, and 9-fold for Vß6. Therefore, it appears that expression of subdominant TCR affects the maximum number of cells formed but not the rate of their expansion or decline in vivo.



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  		FIGURE 3. Similar primary response kinetics of the dominant and subdominant PCC-specific Th clonotypes. A, Probability contour plots display percentages (±SEM) of representative subdominant clonotypes (V{alpha}11+Vß14+, V{alpha}11+Vß8.1/8.2+) that are CD44high and CD62Llow before immunization (day 0) and 7 days after injection with adjuvant only (Day 7-Adj.) or PCC in adjuvant (Day 7-PCC). B, Activated cell numbers for all four V{alpha}11+ subdominant Th clonotypes combined in comparison to the dominant V{alpha}11Vß3-expressing clonotype. Frequencies of V{alpha}11+VßX+, CD44high, CD62Llow Th cells and total cell numbers in the draining LNs from each animal were used to calculate the changes in total PCC-responsive cell numbers over the primary response time course. C, Activated cell numbers for each of the subdominant Th clonotypes, displayed individually. Cell numbers 7 days after injection of adjuvant only are shown in the boxed insets. Between three and six mice were analyzed for each clonotype at all time points except days 7 and 9 of the PCC-specific response, when 9–17 individual mice were examined. Data represent the mean ± SEM.

 
Subdominant clonotypes remain predominantly outside germinal centers

One clear fate of PCC-specific Th cells expressing the dominant clonotype is migration into the follicles and GCs (14, 29, 30, 31, 32). By day 9 of the primary LN response, nearly 75% of the PCC-responsive (CD44highCD62Llow) V{alpha}11+Vß3+ cells are found within GC (14). Here, we demonstrate that very few of the four subdominant clonotypes localize to the GC (Fig. 4Go). First, we evaluated the efficiency of the confocal quantitation in comparison with flow cytometric estimations of V{alpha}11+VßX+ cells. Although each clonotype was slightly underestimated by confocal microscopy compared with flow cytometry (Fig. 4GoA, right), we were still able to detect >80% of all the different clonotypes on tissue sections. Using this approach, we counted and localized ~100–200 single cells for each clonotype (Fig. 4GoB). Very few of the subdominant clonotypes localize to the GC microenvironment compared with non-GC regions of the follicles and T cell zones (representative staining for one of the subdominant clonotypes is shown in Fig. 4GoA, while the entire dataset for all of the clonotypes is displayed in Fig. 4GoB).



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  		FIGURE 4. The subdominant PCC-specific Th clonotypes remain predominantly non-GC. Cryosections were prepared from draining LNs of B10.BR mice 9 days after primary immunization and were stained for LSCM as described in Materials and Methods. A, Representative LSCM staining (x40 objective) for the subdominant clonotypes (V{alpha}11Vß8.3 staining is shown) in the B zone/GC and T zone; IgD staining is displayed in cyan, V{alpha}11 in green, and Vß8.3 in red. GC are identified as IgD- regions within the IgD bright B zone. Yellow arrows indicate V{alpha}11+Vß8.3+ cells, which are frequent in the T zone but rare in the GC. The table at the right displays quantitation of TCR staining on day 9 of the primary response for the dominant clonotype and each subdominant clonotype, comparing percentages determined by flow cytometry (100,000 event files; mean ± SEM from three or four separate experiments) and LSCM (mean ± SEM for entire LN cross-sectional area of four to seven sections derived from three or four individual mice per clonotype). Total numbers of V{alpha}11- and/or VßX-staining cells are considered 100%; percentages shown represent the frequency of cells positive for both V{alpha}11 and the indicated Vß. B, The table at the left displays the total number of cells counted for each clonotype and the distribution of V{alpha}11VßX-expressing cells in the T zone, B zone, and GC. Data for V{alpha}11Vß3 distribution has been reported previously (14 ). The bar graph at the right displays the percentage of each V{alpha}11VßX-expressing clonotype that localizes to the GC. C, Vß expression by V{alpha}11+ GC T cells. Sections derived from three or four different mice were analyzed for each clonotype. Indicated are the total number of V{alpha}11+ Th cells identified and the percentage of these that costain with the indicated Vß. n indicates the total number of GC analyzed for each clonotype. The image at the right (x40 objective) displays IgD (cyan), V{alpha}11 (green), and Vß3 (red) staining, with V{alpha}11+Vß3+ T cells in yellow. Green arrows indicate V{alpha}11+Vß3- T cells in the GC.

 
Although many non-GC Th cells with the defined TCR V regions may not be responding to the particular Ag, GC are dynamic microenvironments dependent on specific Ag and thereby are highly enriched for Ag-specific Th cells (14, 29, 30, 31, 32). In 12–29 different GC examined for each clonotype, ~65% of all V{alpha}11+ Th cells coexpress the dominant Vß3, while Vß8.1/8.2, Vß8.3, and Vß14 are each expressed by <2% of the V{alpha}11+ cells, and Vß6 is expressed by only 5% (Fig. 4GoC). Thus, even though the four subdominant Th clonotypes accumulate to similar cell numbers as the dominant clonotype in the draining LN (Fig. 3Go), expression of different TCRß V region genes largely precludes them from GC entry. These data suggest that these subdominant TCR may deliver qualitatively different signals in vivo.

Subdominant clonotypes enter the memory compartment in vivo

Although expansion after initial priming indicates a significant role for the subdominant clonotypes in the primary effector response, it was not clear whether these cells could still enter the memory compartment. An accelerated cellular response to secondary challenge is characteristic of the LN memory response to PCC (12, 13, 14). This is also true for the subdominant clonotypes (Fig. 5Go). On day 3 after secondary challenge, PCC-specific cell numbers for each of the subdominant clonotypes are significantly greater than those on day 0 (at least 8 wk postprimary; p = 0.04–0.002) and in adjuvant-only controls (day 3 secondary; p = 0.04–0.002; Fig. 5Go, A and B). In all cases, the day 3 memory response cell numbers for the additional clonotypes are significantly greater than those on day 3 of the primary response (p = 0.04–0.001) and are also similar to those on day 7 of the primary response (compare to Fig. 3Go). These patterns are consistent with a bonafide memory response to PCC and not simply a second primary response. Further, PCC-specific cells expressing these different TCR are able to compete effectively with the dominant clonotype in this memory response.



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  		FIGURE 5. The subdominant clonotypes enter the Th memory compartment. B10.BR mice were immunized with PCC as described in Materials and Methods, rested for 8 wk, then challenged with the same dose of Ag in adjuvant. A, Indicated on the probability contour plots are percentages (±SEM) of V{alpha}11Vß8.1/8.2- expressing Th cells that are CD44high and CD62Llow before challenge (day 0) or 3 days after challenge with adjuvant only (Day 3-Adj.) or PCC in adjuvant (Day 3-PCC). B, Frequencies of V{alpha}11+VßX+, CD44high,CD62Llow Th cells and total cell numbers in the draining LNs from each animal were used to calculate total PCC-responsive cell numbers for each of the subdominant clonotypes. C, Activated cell numbers for the dominant clonotype compared with all four of the V{alpha}11+ subdominant clonotypes combined. Data represent mean cell numbers ± SEM (n = 3 individual mice for day 3 adjuvant only, n = 2 for day 0) Between five and nine individual mice were analyzed for each clonotype on day 3 after challenge with PCC.

 
The V{alpha}11+Vß8.1/8.2+ and V{alpha}11+Vß14+ clonotypes consistently expand more vigorously after secondary challenge (mean cell numbers 15- and 11-fold greater than prechallenge, respectively) than the V{alpha}11+Vß8.3+ and V{alpha}11+Vß6+ clonotypes (9- and 3-fold greater than prechallenge, respectively; Fig. 5GoB). Other V{alpha}11+Vß-expressing clonotypes that do not expand in the primary response (e.g., V{alpha}11Vß2) also fail to expand in the memory response (data not shown). As in the primary response, when considered together, the subdominant clonotypes contribute almost identical numbers of memory responders to the recall response as the dominant clonotype (Fig. 5GoC).

Clonal restriction in the TCR {alpha}-chain CDR3

To more closely examine the extent of TCR diversity in the repertoire of the subdominant clonotypes, we next isolated, amplified, and sequenced TCR {alpha}-chains from single cells expressing V{alpha}11 paired with the different Vß-chains. We sorted single PCC-specific Th cells from the primary (days 7 and 9) and memory (day 3) responses to determine whether there were any discernible patterns of restriction in the TCR{alpha} CDR3 regions (Figs. 6Go and 7). Preferred CDR3 features are defined as sequence features present in the majority of cells from individual animals and across different animals (three different mice analyzed per group).



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  		FIGURE 6. TCR {alpha}-chain CDR3 sequences from the V{alpha}11+ Th clonotypes expressing Vß14 and Vß8.1/8.2. Single cells were sorted as (FSC, SSC)int,(PI; CD8; B220; CD11b)neg,V{alpha}11+,VßX+,CD44high,CD62Llow directly into cDNA reaction mix, subjected to two rounds of PCR to amplify TCR V{alpha}11, then directly sequenced. Sequence analyses were performed on days 7 and 9 of the primary response and day 3 of the memory response. Controls represent {alpha}-chain sequences of resting cells (CD44low,CD62Lhigh) from unimmunized mice and activated (CD44high,CD62Llow) cells from mice injected 7 days previously with adjuvant only (no significant differences between the two control groups). A, A representative set of nucleotide and predicted aa sequences from 10 single cells obtained from PCC-specific V{alpha}11+Vß14+ cells. Positions {alpha}93 and {alpha}95 are highlighted in each sequence. The TCRJ region is displayed with the CDR3 loop (defined as 2 aa downstream of the conserved C and 2 aa upstream of the conserved GXG motif) presented between the V and J elements. N sequence insertions are underlined. Total n = 29 single cells from the primary response, n = 15 from the memory response, n = 21 control cells. B, Summary of all the sequence data for the V{alpha}11+Vß14+ clonotype. Displayed are the percentage of single cells expressing each of the four preferred CDR3 features and the restriction index (percentage of single cells expressing three or more features). C, Representative sequences for the V{alpha}11+Vß8.1/8.2+ Th clonotype. Total n = 14 single cells from the primary response, n = 24 from the memory response, n = 17 controls. D, Summary of all the sequence data for the V{alpha}11+Vß8.1/8.2+ clonotype as described above.

 
Despite the expression of diverse TCRß V regions, the TCR{alpha} CDR3 regions for the subdominant clonotypes are very similar to those found in the dominant clonotype (12, 13, 14). The V{alpha}11+Vß14+ and V{alpha}11+Vß8.1/8.2+ clonotypes are grouped together as the most numerous of the memory responders (Fig. 6Go). The predominance of glutamic acid at {alpha}93 and serine/threonine at {alpha}95 is clearly evident in both clonotypes from the primary or memory response to PCC (representative sequences in Fig. 6Go, A and C). Preferred J{alpha} usage can also be defined for V{alpha}11+Vß14+ (J{alpha}16, -21, -22, -43, and -44) and V{alpha}11+Vß8.1/8.2+ Th (J{alpha}22, -40, and -44), some of which are expressed by the dominant clonotype (J{alpha}16, -17, -22, and -34) (12, 13, 14). CDR3 length of both 8 and 9 aa appears tolerated by the subdominant clonotypes (Fig. 6Go) in contrast to the highly restricted 8-aa length preferred by the dominant clonotype (12, 13, 14). The change in penetrance for each of these features between the primary and memory responses for all TCR sequenced is summarized in Fig. 6Go, B and D. Control sequences in these figures comprise T cells expressing both TCR V regions from naive animals and some TCR from infrequent cells that emerge after adjuvant only priming. Although individual CDR3 features are useful to consider when defining preferred TCR, the penetrance of cells expressing at least three of the preferred CDR3 features provides a more accurate estimate of the extent of Ag-driven selection in vivo (presented as the restriction index in Fig. 6Go, B and D).

The CDR3 sequence analyses for V{alpha}11+Vß6+ and V{alpha}11+Vß8.3+ clonotypes are presented together in Fig. 7Go. In the primary response, the preferred CDR3 for the V{alpha}11+Vß6+ clonotype is similar to the first two subdominant clonotypes, with a glutamic acid at {alpha}93; serine/threonine at {alpha}95; J{alpha}16, -17, -32, -44, and -50 usage; and CDR3 length of 8 or 9 aa (Fig. 7GoA). The penetrance of clones with three or more preferred CDR3 features is >70% at this stage of the response (Fig. 7GoB). In contrast, the penetrance of this preferred CDR3 in the V{alpha}11+Vß6+ memory response drops to 37% (Fig. 7GoB). It is important to note the efficacy of the in vivo specificity controls to reemphasize that the TCR sequenced are from T cells that not only express the V regions described, but also emerge specifically in response to PCC and not adjuvant only (Figs. 3Go and 5Go). The preferred CDR3 features for the primary response V{alpha}11+Vß8.3+ clonotype contain asparagine or glycine instead of serine/threonine at {alpha}95. The glutamic acid at {alpha}93, a CDR3 length of 8 or 9 aa, and J{alpha} usage (J{alpha}13, -16, -21, -37, and -43) are similar to the other clonotypes. Once again, the percentage of clones with this preferred CDR3 (with the asparagine/glycine at {alpha}95) decreases during the memory response, although more V{alpha}11+Vß8.3+clones expressing a serine/threonine at {alpha}95 do emerge after recall than in the primary response. Taken together, these data indicate that the primary response repertoire does not necessarily represent the memory response repertoire.



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  		FIGURE 7. TCR {alpha}-chain CDR3 sequences from the V{alpha}11+ clonotypes expressing Vß6 and Vß8.3. TCR {alpha}-chain CDR3 sequences were obtained from single cells and are displayed as in Fig. 6Go. A and B, For the V{alpha}11Vß6-expressing clonotype: total n = 21 single cells from the primary response, n = 19 from the memory response, n = 16 control cells. C and D, For the V{alpha}11Vß8.3-expressing clonotype: n = 17 single cells from the primary response, n = 20 memory, n = 18 control cells.

 
To summarize the penetrance of the common V{alpha}11-associated CDR3, we define the preferred features for PCC specificity encompassing all five clonotypes (glutamic acid at {alpha}93, serine/threonine at {alpha}95, CDR3 length 8/9, and broad array of 9/50 different J{alpha}; Table IGo). The increase in the percentage of cells with all four features is clearly evident between primary and memory responses for the dominant clonotype as well as those expressing Vß14 and Vß8.1/8.2. This common TCR {alpha}-chain is also present among primary responders expressing Vß6 and memory responders expressing Vß8.3. These data underscore the importance of the TCR {alpha}-chain CDR3 in recognition of PCC in vivo.


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  		Table I. Penetrance of common TCR {alpha}-chain CDR31

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent direct ex vivo studies of CD8+ CTL responses to infectious agents suggest diversity in the total available Ag-specific T cell repertoire (9, 10, 11). The present study establishes the PCC system as an appropriate model to study complex T cell responses in the CD4+ helper cell compartment. Although TCR diversity in the PCC-specific response has previously been suggested through analyses of hybridomas and T cell lines, this is the first quantitative analysis of subdominant PCC-specific Th clonotypes directly ex vivo. Our results demonstrate that the Th response to PCC consists of multiple clonotypes expressing different TCR. We extend a five-color flow cytometric strategy to isolate V{alpha}11+ non-Vß3-expressing Th cells specific for PCC. This direct approach allows us to assay some functional consequences associated with expression of diverse TCR specific for the same Ag. We monitor the cellular dynamics of the primary response, entry into the GC microenvironment during the primary response, and the kinetics and extent of the recall response in vivo. Overall, it appears that the dominant clonotype does not have a growth advantage over the subdominant clonotypes (especially evident in the memory response) but may express distinct functions (e.g., GC localization). Single-cell repertoire studies reveal a great deal of similarity at the level of TCR {alpha}-chain CDR3 sequence across the different PCC-specific clonotypes. These molecular analyses highlight the importance of the TCR {alpha}-chain in PCC recognition and the Ag-driven selection of best fit in vivo.

Diverse TCR usage in vivo

The total available peripheral T cell repertoire specific for a given Ag is, in many cases, quite diverse in TCR Vß region usage and fine epitope specificity (5, 6, 7, 8, 9, 10, 11). In the PCC system, although V{alpha}11 expression is clearly dominant, non-Vß3-expressing Th clonotypes often appear in panels of V{alpha}11+ T cell clones and hybridomas (17, 18, 19, 22, 24, 27, 28). Until now, it has been unclear whether such clonotypes make up a significant proportion of the normal PCC-specific Th repertoire in vivo or are the result of biases imposed during the selection and cloning of T cells in vitro (33, 34). We find that PCC-specific Th clonotypes expressing alternate Vß-chains (Vß6, Vß8.1/8.2, Vß8.3, and Vß14) make up a very significant proportion of the PCC-specific peripheral repertoire (~50%). Our recent results implicate thymic selection in shaping the TCR {alpha}-chain bias in the preimmune repertoire of normal mice (14); the current study lends further weight to the importance of the TCR {alpha}-chain CDR3 in PCC fine specificity. Our results clearly demonstrate that subdominant clonotypes are a significant part of the normal peripheral repertoire and compete effectively for Ag during primary and memory responses in vivo.

Fine specificity of subdominant Th clonotypes

Previous studies have shown that the vast majority of the T cell proliferative response to PCC is directed against the C-terminal dominant peptide epitope (PCC88–104), although a weak response against a polypeptide encompassing aa residues 1–65 has also been noted (15). The preferred {alpha}-chain CDR3 of the dominant V{alpha}11Vß3-expressing Th clonotype contains glutamic acid at position {alpha}93, serine at {alpha}95, a CDR3 loop length of 8 aa, and J{alpha}16, -17, -22, or -34 (12, 14, 17, 18). The TCR {alpha}-chain CDR3 for many of the subdominant clonotypes express all four of these preferred features (Figs. 6Go and 7Go and Table IGo); in fact, several single cells express identical (at the aa level) {alpha}-chain CDR3 as those expressed by the dominant clonotype. Jorgensen et al. demonstrated a critical role for the glutamic acid at TCR{alpha}93 in a charge-charge interaction with the lysine residue at position 99 of the immunodominant peptide (21). In the recent study by Nakano et al., the {alpha}-chain CDR3 sequence of a V{alpha}11Vß14-expressing T cell hybridoma specific for the dominant peptide epitope also contains all four of the preferred features defined for the V{alpha}11+Vß14+ clonotype in the current study (EASGSWQL; hybridoma 29-3 in Ref. 28). These data argue that many subdominant TCR are also selected on the same dominant peptide epitope of PCC. Like the preferred CDR3 of the dominant clonotype, we also argue that the preferred CDR3 features of the subdominant clonotypes emerge through Ag-driven selection.

Certain {alpha}-chain CDR3 expressed by the subdominant clonotypes display differences in preferred features compared with the dominant clonotype. Although the {alpha}-chain CDR3 of all the subdominant clonotypes retain expression of glutamic acid at {alpha}93, several single cells express a CDR3 loop of 9 aa in length, with threonine rather than serine preferred at position {alpha}95 (Figs. 6Go and 7Go and Table IGo). These features have also been identified previously as dominant in I-Ek-restricted T cell clones and hybridomas specific for PCC88–104 that express V{alpha}11 paired with non-Vß3; specifically, a number of V{alpha}11Vß16-expressing T cell hybridomas also express an {alpha}-chain CDR3 of 9 aa in length with threonine at position {alpha}95 (17, 18, 22). Differences in {alpha}-chain CDR3 loop length and J{alpha} usage probably reflect changes among the clonotypes to accommodate the different TCR ß-chains. Overall, the results further testify to the specificity of the subdominant clonotypes for the dominant peptide epitope and to the accuracy of the frequency estimates generated in this study.

Ag-driven selection of the memory Th repertoire

Several studies indicate that MHC class-I restricted CD8+ T cell responses to viral infection are of much greater magnitude than previously anticipated, with very little contribution of bystander activation (35, 36, 37, 38). Using tetrameric complexes of antigenic peptide/MHC class I, a number of recent studies have addressed the issue of Vß repertoire selection in CD8+ T cells (39). Pamer and colleagues demonstrate that T cells specific for both dominant and subdominant Listeria epitopes follow similar kinetics of expansion and decline in vivo (10) and suggest a narrowing of the Vß repertoire between the primary and memory responses (9). Such repertoire narrowing appears to occur through the selective expansion of clonotypes with increasing affinity for Ag (40). In other class I-restricted responses, however, the Vß repertoire of the primary responders is conserved in the memory response (11). Although such extensive Vß repertoire studies have been conducted in the CD8+ CTL compartment, until now very little information has been available for the corresponding CD4+ Th compartment. The extent of diversity in TCR-Vß usage we observe in the PCC-specific Th response is similar to the recent observations of repertoire in CTL responses. At the level of V region usage, our results generally favor a conserved repertoire between the primary and memory Th responses. The overriding TCR {alpha}-chain CDR3 across different PCC-specific clonotypes provides some level of consistency to the TCR selected for the memory compartment.

Using tetramer binding and an assay devised to estimate TCR-peptide/MHC dissociation rates, Savage et al. demonstrate the loss of PCC-specific Th cells with faster dissociation rates between the primary and memory responses (23). These studies also indicate a higher average affinity of memory response TCR for peptide/MHC. To date, there has not been a direct analysis of the range of CDR3 structures that can be detected using the moth cytochrome c/I-Ek tetramers in the response to PCC. It is not yet clear whether all PCC-specific TCR will bind the class II tetramers. Further, the much lower frequencies of Ag-specific class II-restricted Th cells (compared with the class I-restricted models studied) creates a significant technical hurdle to both direct quantitative estimates of responder cells and single-cell repertoire studies. Nevertheless, the cellular dynamics of the subdominant memory clonotypes suggest that expression of the dominant TCR does not confer an obvious growth advantage. Therefore, the subdominant clonotypes described in this study must also have reasonably high affinity to effectively compete for Ag in the recall response. Although some of the {alpha}-chain CDR3 preferred in the primary response did not re-emerge in the memory response, this may have been due to a lower average affinity for peptide/MHC. Thus, it is most likely that the extent of subdominance seen in the PCC-specific response reflects the frequency of specific precursors in the preimmune repertoire.

Functional consequences of TCR diversity

We have argued that there are no clear differences in TCR affinity between dominant and subdominant clonotypes; however, there appear to be at least some functional consequences that correlate with the expression of subdominant clonotypes. The one difference between the dominant and subdominant clonotypes revealed in our analysis is the ability to migrate to the GC microenvironment. Several investigators in a number of different systems have shown that Ag-specific Th cells accumulate in B cell-rich follicles and GC (14, 29, 30, 31, 32, 41, 42); however, the function of the GC Th cell remains unclear. Kelsoe and colleagues demonstrate that V{alpha}11Vß3-expressing cells in the GC show an increased sensitivity to TCR- and steroid-induced apoptosis (31), and express reduced levels of the Thy-1 molecule (30). Several studies suggest that T-B cell interactions in the GC are involved in supporting the development of B cell memory (43, 44, 45, 46). Whatever the precise differences in function, it appears that PCC-specific cells that express the subdominant TCR have a greatly reduced ability to enter GC (Fig. 4Go). As previously demonstrated for the dominant clonotype, Ag-driven selection is largely complete before formation of GC in the LN (14). This may also be true for the subdominant clonotypes, but has not been examined directly. Nevertheless, the capacity of the subdominant clonotypes to enter the memory compartment suggests that the GC microenvironment does not contribute in a major way to this critical Th function.

Implications

The immune response to infectious agents or complex protein Ags often consists of a diverse repertoire of potentially responsive T cells. The present study establishes the Th response to PCC as a relevant model system to investigate complex T cell responses in vivo. Using this model system, we quantitatively monitor interclonal competition and can define directly the penetrance of dominant and subdominant primary and memory responders in vivo. These studies provide an ideal model to directly examine the relationship between TCR usage and Th cell function in vivo. Quantitative analyses of specificity, repertoire, and function directly ex vivo are of critical importance to the development of more effective therapeutic interventions in settings of infectious diseases and autoimmunity.


   Acknowledgments
 
We thank Gabriel Bikah, Rebecca Caley, David Driver, Joanne Fanelli-Panus, and Carolyn Doyle for useful suggestions and critical review of the manuscript. We also thank J. Michael Cook and the Duke University Comprehensive Cancer Center Flow Cytometry Facility.


   Footnotes
 
1 J.A.M. is recipient of a National Research Service Award from the National Institutes of Health. This work is also supported by an Arthritis Foundation Biomedical Sciences Grant and National Institutes of Health Grant AI40215. Back

2 Address correspondence and reprint requests to Dr. Michael G. McHeyzer-Williams, Department of Immunology, Duke University Medical Center, Durham, NC 27710. E-mail address: Back

3 Abbreviations used in this paper: PCC, pigeon cytochrome c; GC, germinal center; LN, lymph node; LSCM, laser scanning confocal microscopy; PI, propidium iodide; TR, Texas Red. Back

Received for publication July 16, 1999. Accepted for publication September 9, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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