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The Journal of Immunology, 2004, 172: 4709-4716.
Copyright © 2004 by The American Association of Immunologists

B Cell-Dependent TCR Diversification1

Cristina João*, Brenda M. Ogle*,{dagger}, Carlota Gay-Rabinstein*, Jeffrey L. Platt*,{ddagger},§,¶ and Marilia Cascalho2,*,{ddagger},§,¶

* Transplantation Biology Program and Departments of {dagger} Physiology, {ddagger} Immunology, § Surgery, and Pediatrics, Mayo Clinic, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell diversity was once thought to depend on the interaction of T cell precursors with thymic epithelial cells. Recent evidence suggests, however, that diversity might arise through the interaction of developing T cells with other cells, the identity of which is not known. In this study we show that T cell diversity is driven by B cells and Ig. The TCR V{beta} diversity of thymocytes in mice that lack B cells and Ig is reduced to 6 x 102 from wild-type values of 1.1 x 108; in mice with oligoclonal B cells, the TCR V{beta} diversity of thymocytes is 0.01% that in wild-type mice. Adoptive transfer of diverse B cells or administration of polyclonal Ig increases thymocyte diversity in mice that lack B cells 8- and 7-fold, respectively, whereas adoptive transfer of monoclonal B cells or monoclonal Ig does not. These findings reveal a heretofore unrecognized and vital function of B cells and Ig for generation of T cell diversity and suggest a potential approach to immune reconstitution.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The generation of TCR diversity is initiated by recombination of the V, D, and J gene segments and originates the variable region of the TCR genes in T cell precursors in the thymus (1). Although V(D)J recombination generates billions of different TCRs, only a small fraction of these (5%) is expressed by the mature thymocytes (2). Thymocytes that fail to produce TCR or that produce TCR that fails to recognize MHC bearing self peptide die by neglect. Thymocytes bearing self-reactive TCR are eliminated (negative selection), leaving a small fraction of thymocytes surviving (positive selection) (2). Thus, positive and negative selection give rise to a primary T cell repertoire that recognizes self-MHC (restriction) with moderate avidity that is not self-reactive and, in turn, establishes the diversity of naive T cells. Therefore, assuming an equal contribution of V(D)J recombination, the diversity of newly made thymocytes reflects the efficiency of selection. T cell diversity has been estimated to be ~108 different T cells in humans and 106 in mice (3, 4).

Positive selection and T cell restriction have been thought to result from the interaction of developing T cells with thymic epithelial cells (5). This conclusion was deduced from experiments in which lethally irradiated recipient mice of different MHC haplotypes were reconstituted with bone marrow cells obtained from H-2b TCR transgenic mice. In these chimeras, T cells were positively selected only when the thymic MHC was of the H-2b haplotype, indicating that H-2b expression by bone marrow-derived cells alone was not sufficient to promote positive selection (6). The conclusion that thymic epithelial cells mediate positive selection was also indicated by the work of Benoist and Mathis (7), who showed that the expression of MHC class II on cortical thymic epithelium was sufficient to achieve positive selection of thymocytes, whereas the expression of the same MHC Ags on hemopoietic-derived cells was not.

Although thymic epithelium might be sufficient to mediate positive selection, some wondered whether cells other than thymic epithelial cells could also participate in positive selection. Pawlowski et al. (8) and Hugo et al. (9) showed in separate experiments that MHC class I- or MHC class II-bearing fibroblasts injected into the thymus of MHC class I-deficient or MHC class II-deficient mice were able to mediate positive selection. Bix and Raulet (10) showed that bone marrow-derived cells in MHC class I-deficient bone marrow chimeras promoted positive selection of CD8+ thymocytes. In contrast, MHC class II-positive bone marrow-derived cells did not rescue CD4+ T cells in MHC class II-deficient mice (11). If some experiments establish that positive selection can, in some cases, be mediated by nonthymic epithelial cells, the question of how physiologic these interactions may be is not resolved. Zinkernagel and collaborators (12) generated tetraparental aggregation chimeras in which thymic epithelial cells expressed one MHC, and hemopoietic-derived cells expressed another. In these chimeras, T cells were restricted to the MHC expressed on the thymic epithelial cells as expected, but also to the MHC expressed by the hemopoietic cells. These results indicated that nonthymic epithelial cells as well as thymic epithelial cells promote positive selection of thymocytes (12). Which hemopoietic-derived cells were responsible for the positive selection of thymocytes was not determined.

Besides the question of which cells mediate positive selection of thymocytes is the question of which cells provide the source of peptides. One speculation has been that the peptides originate in the thymic epithelium (13); however, the repertoire of peptides available from this source may not suffice. Selection of a diverse T cell repertoire requires diverse peptides presented in the context of self-MHC. Thus, mice that express MHC associated with a single peptide have a markedly constrained T cell repertoire (14, 15, 16).

As B cells are normal constituents of the thymus (17) and may present peptides derived from the Ig V regions (18) or peptides derived from Ags expressed endogenously (19), we hypothesized that B cells would potentially serve as a source of peptide diversity in the thymus. In fact, B cells are one of the major cell types expressing MHC class II and are capable of presenting Ags to T cells (20). We tested this hypothesis by comparing the diversity of the TCR repertoire in mice with varying B cell numbers and varying B cell diversity and by testing whether transfer of B cells or Ig could rescue TCR diversity. Our studies demonstrate that B cells and Ig generate a significant fraction of T cell diversity.


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

JH−/−, µMT, monoclonal B-T, and quasi-monoclonal (QM)3 mice were previously described (21, 22, 23, 24). The B cell-deficient strains consisted of JH−/− mice, obtained by gene-targeted deletion of the JH segments (21), and µMT mice, obtained by gene-targeted disruption of the µ Ig H chain membrane exons (22). C57BL/6 and µMT mice were purchased from The Jackson Laboratory (Bar Harbor, ME). JH−/−, monoclonal B-T, and QM mice were bred and all mice were housed in a specific pathogen-free facility at Mayo Clinic (Rochester, MN). All mice were between 6 and 16 wk of age and were age-matched. All animal experiments were conducted in accordance with protocols approved by Mayo Clinic Institutional Animal Care and Use Committee.

Adoptive transfer

Bone marrow cells were harvested from C57BL/6 mice or monoclonal B-T mice, and lymphocytes were isolated by Ficoll-Paque gradient (Amersham Pharmacia Biotech, Piscataway, NJ). Polyclonal B cells (1 x 107) purified with a Miltenyi Biotec isolation kit (Auburn, CA) and monoclonal B cells (6 x 106) purified with a high speed sorter FACSVantage SE (BD Biosciences, Mountain View, CA) were injected i.p. in newborn mice (20 µl). The polyclonal B cells contained, on the average, 1% CD3+CD4+ and 1.5% CD3+CD8+ cells, and the monoclonal B cells contained 0% CD3+CD4+ and 0.07% CD3+CD8+ cells.

Ig injections

JH−/− mice were injected i.p weekly with 250 µg of mouse polyclonal IgG (Serotec, Oxford, U.K.) or monoclonal anti-keyhole limpet hemocyanin IgG2b (C48-4; BD Biosciences) from birth. Serum levels of total Ig were tested 4 wk or more after the first injection.

FACS analysis

Thymocytes were obtained by mincing thymi through a 0.70-µm pore size mesh, followed by RBC hemolysis in a standard NH4Cl lysis buffer. Bone marrow cells were prepared by flushing femurs with cell suspension buffer, followed by RBC lysis, as previously described (24). Total thymocyte numbers were counted with a Coulter counter (Hialeah, FL). Cells were stained with one, two, or three of the following mAbs (all Abs were from BD PharMingen, San Diego, CA) as previously described (24): FITC-conjugated rat anti-mouse CD4 (GK 1.5), rat anti-mouse CD43 (Ly-48, leukosialin), and mouse anti-5-bromo-2'-deoxyuridine (anti-BrdU) Ab; PE-conjugated rat anti-mouse CD8{alpha} (53-6.7), rat anti-mouse CD19 (1D3), and rat anti-mouse IgMb (Igh-6b; AF6-78); and biotin-conjugated rat anti-mouse B220 (16A), rat anti-mouse CD62L (LECAM-1, Ly22), and rat anti-mouse CD3{epsilon} (145-2C11). Lymphocytes were gated on the light scatter plot by backgating onto CD4+CD3+ and CD8+CD3+ cells; the numbers of the thymocyte subpopulations were determined by multiplying the percentage, as defined by gating on the FACS plot, by their total number.

DNA analysis

Thymocytes (106/ml) were washed with ice-cold PBS and fixed in 70% ethanol at −20°C for at least 2 h. After fixation, cells were washed twice with PBS and incubated in 50 µl of DNA extraction buffer (0.2 M phosphate citrate buffer, pH 7.8) at 37°C for 30 min in a shaker. After DNA extraction, the cells were stained with propidium iodide in a solution containing 10 ml of 0.1% (v/v) Triton X-100 in PBS, 200 µl of 1 mg/ml propidium iodide (Molecular Probes, Eugene, OR), and 2 mg of DNase-free RNase A (Sigma-Aldrich, St. Louis, MO), for 30 min at room temperature. Detection of propidium iodide fluorescence was read at red wavelength in a FACScan flow cytometer (BD Biosciences) and was analyzed with ModFit LT software (Verity Software House, Topsham, ME).

Immunohistological analysis

Thymi removed from 6- to 8-wk-old mice were oriented, covered with OCT (Sakura, Torrance, CA), snap-frozen by precooled isopentane, and stored at −85°C. Four-micron-thick frozen sections were mounted on positively charged microscope slides (SuperFrost Plus; Fisher Scientific, Pittsburgh, PA) and stored at −85°C. Before processing, sections were air-dried at room temperature, fixed 10 min in 4°C acetone, air-dried for an additional 10 min, then postfixed for 2 min in 100 mM Tris-buffered 1% paraformaldehyde containing 1 mM EDTA, pH 7.2, and rinsed with PBS (pH 7.2). Before staining, the specimens were incubated in 0.3% hydrogen peroxide in 0.1% sodium azide (aq) solution to quench the presence of endogenous peroxidase. Specimens were incubated with rat mAbs to murine CD19 (clone 1D3; BD PharMingen) and to CD45R/B220 (clone RA3-6B2; BD PharMingen), rinsed with PBS, then detected by mouse serum preabsorbed, affinity-purified, biotinylated goat IgG anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), followed by PBS rinses and the tertiary application of horseradish-conjugated streptavidin (DAKO, Carpinteria, CA). Slides were developed by incubation with a peroxidase substrate NovaRED (Vector Laboratories, Burlingame, CA), which resulted in an insoluble reddish-brown precipitate, followed by counterstaining with a progressive alum-hematoxylin, dehydrated in graded ethanols, cleared in xylene changes, and coverslipped with Cytoseal-Xyl (Stephens Scientific, Kalamazoo, MI). Digital images were obtained using a brightfield microscope equipped with a CCD digital camera (SPOT II; Diagnostic Instruments, Sterling Heights, MI).

TUNEL

Apoptotic cells were detected in cryostat sections of thymi by in situ TUNEL, performed according to the manufacturer’s instructions (ApopTag Plus Peroxidase Kit; Serologicals, Norcross, GA).

Determination of TCRV{beta} diversity

Isolation of RNA. Thymi harvested from mice were placed in RPMI 1640 and pushed through a 70-µm pore size cell strainer. Lymphocytes were isolated by Ficoll-Paque (Amersham Pharmacia Biotech) gradient. Total RNA was obtained with RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.

Generation of diversity standards. Diversity standards were prepared by creating oligonucleotide mixtures of known diversity. For example, to generate a standard diversity of 106, 18-mer oligonucleotides were synthesized with 10 sites of random assignment, generating 410 = 1,040,526 different oligomeres. Similarly, we created oligomer mixtures with 1, 103, and 109 variants. Oligonucleotides were biotin-labeled and hybridized to the gene chips as explained below.

Generation of lymphocyte receptor-specific cRNA. First-strand cDNA was obtained by RT with a mouse TCR C{beta} reverse primer, T7+C{beta} (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGCTTGGGTGGAGTCACATTTCTC-3'). Second-strand synthesis and preparation of biotin-labeled cRNA were conducted according to standard protocols (Affymetrix, Santa Clara, CA).

Application of cRNA to the gene chip. Equal amounts of cRNA from different samples and diversity standards were hybridized to U95B gene chips (Affymetrix). Gene chips were processed at the Microarray Core Facility (Mayo Clinic).

Data analysis

For each gene chip experiment, we obtained raw data corresponding to oligo location and hybridization intensity. Data were arranged in order of ascending hybridization intensity. The number of oligo locations with intensity above background (i.e., number of hits) was summed. The standard curve was generated from hybridization of samples with known numbers of different oligomeres. The standard oligonucleotide mixtures were 18-mer oligomeres synthesized to obtain mixtures containing 1, 103, 106, and 109 different oligonucleotides. The diversity of cRNA obtained from monoclonal T cells was used to establish the background, and the diversity of the test samples was extrapolated directly from the standard curve. We controlled for the TCR specificity of the C{beta} reverse primer by determining the diversity of cRNA obtained from purified polyclonal B cells with the C{beta} reverse primer, which was found to be three per 10 µg of RNA and indistinguishable from background.

Statistical analysis

Statistical analysis for group comparison of means of TCR V{beta} diversity of thymocytes was performed using log transformation of the data, followed by one-way ANOVA. Groups of two comparisons were performed by unpaired, two-sided Student’s t test. Comparisons of thymocyte numbers were performed using the Kruskal-Wallis test for global differences, followed by the Wilcoxon rank-sum test. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Thymocyte development is perturbed in mice that lack B cells and Ig

To test whether B cells and/or Ig might contribute to thymic selection, we compared the numbers of thymocytes and thymocyte subpopulations in mice that lack B cells and Ig (JH−/−) with the numbers in wild-type mice (C57BL/6). Because the thymus atrophies with age, the mice in each group were age-matched. Our results show that JH−/− mice had significantly fewer total thymocytes (6.5-fold) than C57BL/6 mice (Fig. 1). The smaller number of thymocytes in JH−/− mice was mainly due to a 3.9-fold decrease in the number of CD4+CD8+ thymocytes (Fig. 1), but also reflected a significant decrease in the numbers of CD4− CD8−, CD4+ CD8−, and CD4− CD8+ populations compared with wild type. These results suggest that B cells and/or a B cell product such as Ig might influence thymocyte development by various direct or indirect means.



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  		FIGURE 1. Thymocyte numbers in C57BL/6, µMT, and JH−/− mice. The number of thymocytes corresponding to each subpopulation was calculated by multiplying the respective percentage of the total events as defined in the flow cytometric dot plot analysis by the total number of WBC obtained by counting on a Coulter counter. The number of thymocytes (average ± SD) was 1.3 x 108 ± 5.1 x 107 in C57BL/6, 8.9 x 107 ± 6.4 x 107 in µMT, and 3.1 x 107 ± 1.7 x 107 in JH−/− mice. The average number of CD4−CD8− thymocytes was 5.5 x 106 ± 2.1 x 106 in C57BL/6, 3 x 106 ± 2 x 106 in µMT, and 2.2 x 106 ± 2.7 x 106 in JH−/− mice; the average number of CD4+CD8+ thymocytes was 9 x 107 ± 4.4 x 107 in C57BL/6, 7.3 x 107 ± 4.7 x 107 in µMT, and 2.3 x 107 ± 2.7 x 107 in JH−/− mice; the average number of CD4−CD8+ thymocytes was 1.2 x 107 ± 1.2 x 107 in C57BL/6, 7.7 x 106 ± 1.3 x 107 in µMT, and 1.5 x 106 ± 1.4 x 106 in JH−/−mice; and the average number of CD4+CD8− thymocytes was 1.9 x 107 ± 2.8 x 107 in C57BL/6, 4.9 x 106 ± 2.8 x 106 in µMT, and 3.5 x 106 ± 3 x 106 in JH−/− mice. Error bars represent the SD. Data were obtained from 10 C57BL/6, 8 µMT, and 10 JH−/− mice for the total number of thymocytes and from 8 C57BL/6, 8 µMT, and 7 JH−/− mice for thymocyte subpopulations. Mice were between 6 and 16 wk of age. Comparisons of the number of cells in the three strains of mice (indicated by global p) were performed by the Kruskal-Wallis test, and comparisons between two groups of mice (p values indicated below the diagrams) were made using the Wilcoxon rank-sum test.

 
To determine whether Ig might on its own contribute to T cell development, we examined thymocytes in the µMT mouse, which has a serum Ig concentration 4.5% that of the wild-type mouse, but very few B cells (25, 26) (Table I). We found that the number of thymocytes in µMT mice was increased significantly by 3.2-fold compared with that in JH−/− mice (Fig. 1). These results are consistent with a role for Ig in the development of T cells.


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  		Table I. Mean concentration of serum Ig ± SD in C57BL/6, QM, µMT, JH−/− mice and in JH−/− mice reconstituted with B cells or after administration of IgG

 
Increased apoptosis in the thymic cortex of mice that lack B cells and Ig

Next we asked whether the fewer thymocytes in JH−/− mice were the result of higher levels of cell death. Consistent with that concept, TUNEL, which detects DNA strand breaks in cells undergoing apoptosis, revealed increased apoptosis (at least 2.5-fold) in the thymic cortex of JH−/− mice compared with µMT or to C57BL/6 mice (Fig. 2). Increased cell death could be the consequence of decreased positive selection and/or increased negative selection. Because defective positive selection is accompanied by cortical thymocyte apoptosis (27), our results are compatible with B cells and/or Ig promoting thymic positive selection. As apoptosis in the thymic cortex of µMT mice is comparable to apoptosis detected in C57BL/6 mice, and as µMT mice produce serum Ig, but few B cells (25, 26), our results suggest that serum Ig promotes thymocyte survival.



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  		FIGURE 2. Apoptosis in the thymus. Apoptotic cells are stained brown and were detected in cryostat sections of thymi by in situ TUNEL. Photographs are representative of four mice per genotype analyzed. The number of apoptotic spots in JH−/− sections was at least 2.5-fold greater than the number of spots counted in equivalent areas of C57BL/6 or µMT sections. C, Cortex; M, medulla.

 
Numbers of recent thymic emigrants are maintained and thymocyte proliferation is increased in mice that lack B cells and Ig

We next wondered whether decreased numbers and increased apoptosis of thymocytes in JH−/− mice would change the numbers of recent thymic emigrants. Recent thymic emigrants were identified based on their tendency to take up relatively low levels of BrdU according to the method of Tough and Sprent (28, 29). We defined the recent thymic emigrants gate by comparing thymectomized and nonthymectomized mice treated with BrdU. The recent thymic emigrants gate includes the population of naive T cells lost by thymectomy (Fig. 3, B and D). Using these gates we found no differences in the proportions of recent thymic emigrants in CD4+ or CD8+ naive (CD62L-positive) T cells analyzed in JH−/−, µMT, and C57BL/6 mice, suggesting that thymic output is maintained despite decreased number of thymocytes (Fig. 3, A and C).



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  		FIGURE 3. Recent thymic emigrants. The plots represent BrdU incorporation by peripheral blood CD4+CD62L+ (naive) T cells (A and B) or CD8+CD62L+ T cells (C and D). The x-axis shows BrdU staining fluorescence intensity; the y-axis shows CD62L staining fluorescence intensity. The recent thymic emigrants are the naive CD4+ or CD8+ lymphocytes that incorporate low levels of BrdU (28 29 ). The rarity of CD4+CD62L+ and CD8+CD62L+ T cells incorporating low levels of BrdU in thymectomized mice defined the recent thymic emigrants gates (B and D). The figure shows that thymic output did not differ significantly in C57BL/6, µMT, and JH−/− mice. The dot plots shown are representative of three independent experiments with C57BL/6 and JH−/− mice and two experiments with µMT mice.

 
To reconcile the decreased number of thymocytes in JH−/−mice with the maintenance of thymic output, we hypothesized that these mice have increased proliferation of thymocytes. Increased thymocyte proliferation would allow JH−/− mice to maintain thymic output despite increased cell death. Cell cycle analysis of thymocytes of JH−/−, µMT, and C57BL/6 mice revealed a 1.5-fold increase in the number of cycling (S+G2/M) thymocytes in JH−/− compared with µMT and C57BL/6 mice (Fig. 4). Our results thus suggest that lack of B cells and Ig leads to increased cell death and to thymocyte proliferation, perhaps as a compensatory mechanism to maintain T cell production.



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  		FIGURE 4. DNA content of thymocytes from C57BL/6, µMT, and JH−/− mice. Histograms of one representative experiment of a total of four per genotype are shown. The x-axis shows the DNA content; the y-axis shows the number of cells. Diagrams depict the number of cells in the G1, S, and G2/M phases of the cell cycle. The fraction of cells in the S+G2/M phase of the cell cycle (±SD) were 23 ± 0.12% for C57BL/6, 19 ± 0.14% for µMT, and 35 ± 0.19% for JH−/− mice. Twenty thousand events were collected for each analysis.

 
Contraction of the TCR repertoire in mice that lack B cells and/or Ig

If mice that lack B cells and Ig have fewer thymocytes and normal T cell egress, one might predict that the TCR repertoire would be contracted due to clonal expansion of the fewer surviving thymocytes. As a direct test of this idea, we assayed TCR diversity in JH−/−, µMT, and C57BL/6 mice.

Until recently, quantification of TCR diversity has been accomplished by generalization from exemplary sequencing of CDR3 regions obtained from spectratyping analysis, a method that has been referred to as quantitative immunoscope analysis (30). Although this method yielded useful estimates of TCR diversity in human and mouse (3, 4), it is only accurate as long as the number of different sequences obtained for the exemplary V(D)J joints chosen is a good estimate of the number for any other V(D)J joint. The extraordinary effort needed to perform this method in multiple individuals or mice renders it impractical for quantification of TCR diversity. These problems prompted us to develop a novel method to directly measure TCR diversity. Our method allows direct quantification of TCR diversity and was recently published (31).

To measure TCR diversity directly, we determined the number of hybridization spots (hits) of TCR V{beta}-chain RNA on a gene chip (U95B; Affymetrix, Santa Clara, CA). In brief, TCR V{beta}-chain cDNA is produced by RT from total RNA with a C{beta}-specific primer. After second-strand synthesis, biotinylated cRNA is produced by in vitro transcription according to Affymetrix protocols and hybridized to the gene chip (U95B; Affymetrix). The diversity of the TCR V{beta}-chain in a population is proportional to the number of hits above background (defined by the number of hits corresponding to hybridization of monoclonal TCR V{beta}-chain RNA) of TCR V{beta}-chain-specific RNAs on the gene chip (31). Diversity is calculated by plotting the number of hits onto a standard curve obtained for each experiment by hybridizing oligonucleotide mixtures of known diversity to individual gene chips. The number obtained varies proportionally to the actual TCR V{beta} diversity, even though it does not represent the number of different TCR V{beta}-chains, as each TCR V{beta}-chain may generate more than one hit (31). To determine whether the TCR C{beta}-specific primer cross-hybridized with B cell RNA, we determined the diversity of cRNA obtained from purified B cells with the C{beta} reverse primer. We found that the number of hits obtained from B cell cRNA prepared with the TCR C{beta}-specific primer was three per 10 µg of RNA and was indistinguishable from background, indicating that the TCR C{beta}-specific primer does not cross-hybridize with B cell RNA. In addition, the T cell diversity of C57BL/6 splenocytes, which include mostly B cells, was 1000-fold lower than the T cell diversity of C57BL/6 thymocytes (results not shown). Thus, the diversity of TCR C{beta}-specific cRNA does not reflect contamination with B cell receptor messages.

Fig. 5 shows that the TCR V{beta} diversity of JH−/− thymocytes was 6.0 x 102/10 µg of RNA compared with that of wild type, which was 1.1 x 108/10 µg of RNA (p = 0.0002). By showing profoundly decreased thymocyte diversity in mice that lack B cells and Ig (JH−/−), our results indicate that B cells and/or Ig promote thymocyte diversity. In this experiment we compared age-matched JH−/− and C57BL/6 mice that were between 5 and 12 wk old. The decreased thymocyte diversity in JH−/− mice was thus not due to age-dependent atrophy of the thymus.



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  		FIGURE 5. TCR V{beta} diversity. A, TCR V{beta} diversity of thymocytes obtained from C57BL/6, µMT, and JH−/− mice. The x-axis shows mouse strains; the y-axis shows TCR V{beta} diversity. Thymocytes were obtained from 5- to 12-wk-old mice as previously described (24 ), and mononuclear cells were isolated on Ficoll-Paque gradient (Sigma-Aldrich). Total RNA was obtained with the RNeasy kit (Qiagen). First-strand cDNA was synthesized with a reverse primer containing a T7 polymerase promoter 3' overhang that annealed to the TCR {beta} constant region, with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). The second strand was generated by nick translation. The double-strand cDNA was processed for gene chip hybridization according to Affymetrix protocols. Hybridization hits were summed, and the total number was used to calculate TCR V{beta} diversity by comparison with known diversity oligomer mixtures (standards) run at the same time. Measurement of TCR V{beta} diversity was specific, because when it was applied to purified diverse B cells (with <0.9% of T cells), it yielded background values (three per 10 µg of RNA). Each open circle represents one experiment, and the values indicate the mean of TCR V{beta} diversity for each strain of mice. Statistical analysis was performed on each of the log-transformed numeric values by unpaired two-sided t test.

 
Next, we asked whether Ig could promote T cell diversity under the condition of B cell deficiency. To this end we analyzed the TCR V{beta} diversity in thymocytes of µMT mice that have serum Ig, but very few B cells. We found that TCR V{beta} diversity in thymocytes of µMT mice was 4.2 x 104/10 µg of RNA and 70-fold greater than TCR V{beta} diversity in thymocytes of JH−/− age-matched mice (6.0 x 102/10 µg of RNA; p = 0.0004). Our results indicate that Ig and/or the few B cells in µMT mice contribute to T cell diversity. The reduced TCR diversity in B cell-deficient mice was maintained in the periphery as JH−/−, and µMT mice splenocytes had 1000- and 10-fold reduced TCR V{beta} diversity, respectively, compared with wild-type mice (results not shown).

Selection of a diverse T cell repertoire requires TCR recognition of diverse self-peptides in the context of self-MHC (2). Because the V regions of H and L chains of Abs are a potential source of diverse self-peptides, we wondered whether T cell diversity depended on the diversity of the B cells. To address this question, we determined TCR V{beta}-chain diversity of thymocytes obtained from QM mice that have 80% of B cells from a single clone and polyclonal serum Ig (24, 32). In QM mice, the diversity of the JH4-containing H chains is only 0.01% that in wild-type (31). Fig. 5 shows that the TCR V{beta} diversity of QM thymocytes was, on the average, 1.2 x 104/10 µg of RNA, not significantly different from that of µMT and 0.01% that of WT thymocyte diversity (p = 0.006). Our findings of comparable TCR diversity in QM and µMT age-matched mice indicate that oligoclonal B cells do not promote diversification of T cells.

B cell precursors in C57BL/6, QM, JH−/− and µMT thymi

Our results imply that B cells and/or Ig promote the selection of a diverse T cell repertoire, presumably in the thymus. As both mature B cells and B cell precursors are found in the thymus (33, 34), we wondered which B cell populations were present in the thymus of C57BL/6, QM, JH−/−, and µMT mice. Fig. 6A shows that JH−/− and µMT thymi had fewer CD19+ cells in the thymic cortex and fewer CD19+ and B220+ cells in the medulla compared with C57BL/6 mice. In contrast, the numbers of B220+ cells were comparable in the thymic cortexes of JH−/−, µMT, and C57BL/6 mice (Fig. 6A). To discriminate B cell precursors from mature B cells, we analyzed thymocytes of JH−/−, µMT, QM, and C57BL/6 mice by flow cytometry. Fig. 6B shows that mature B cells (IgM+ and B220+) are missing from JH−/− and µMT thymi and are reduced by half in QM thymi compared with those in C57BL/6 thymi. There were very few pre-B cells (IgM−CD43−B220+) (35) in the thymi of mice of all genotypes, whereas they were present in the bone marrow (Fig. 6C). Pro-B cells (IgM−CD43+B220+) (35) did not differ significantly in JH−/−, µMT, QM, or C57BL/6 thymi (Fig. 6C). Our findings thus suggest that mature B cells, rather than B cell precursors, promote thymocyte selection and the generation of T cell diversity.



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  		FIGURE 6. Immunohistochemical and flow cytometric analyses of thymic B cells and B cell precursors. A, Immunohistochemical staining of thymic sections of C57BL/6, µMT, and JH−/− mice. Positive cells are stained brown. JH−/− and µMT thymi had fewer CD19+ cells in the thymic cortex and fewer CD19+ and B220+ cells in the medulla compared with C57BL/6 mice. The numbers of B220+ cells were comparable in the thymic cortexes of JH−/−, µMT, and C57BL/6 mice. B, Flow cytometric analysis of mature B cells in the thymi of C57BL/6, QM, µMT, and JH−/− mice. The x-axis shows IgM staining fluorescence intensity; the y-axis shows B220 staining fluorescence intensity. The dot-plot diagrams identify mature thymic B cells that are B220+ and IgM+. Mature B cells are missing from the thymi of µMT and JH−/−, but are present in the thymi of C57BL/6 and QM mice. Percentages represent the proportion of thymocytes that are mature B cells. The results shown are representative of three mice per genotype that were between 6 and 10 wk of age. C, Flow cytometric analysis of pro- and pre-B cells in the thymi or bone marrow of C57BL/6, QM, µMT, and JH−/− mice. The x-axis shows the CD43 staining fluorescence intensity; the y-axis shows the B220 staining fluorescence intensity. The plots represent IgM− cells. Upper diagrams represent thymocytes; lower diagrams show bone marrow cells. There were no significant differences in the proportions of pre-B cells (IgM−CD43−B220+) and pro-B cells (IgM−CD43+B220+) in JH−/−, µMT, QM, or C57BL/6 thymi. The percentages refer to the fraction of IgM− cells that corresponds to the indicated phenotype. Pre-B cells were nearly absent in the thymi of mice of all genotypes, whereas they were present in the bone marrow.

 
T cell diversity in JH−/− mice reconstituted by adoptive transfer of B cells or administration of Ig

Next we tested whether providing B cells and/or Ig could increase TCR diversity in JH−/− mice lacking both B cells and Ig. To this end, we injected newborn JH−/− mice with bone marrow-derived wild-type or monoclonal B cells or with polyclonal or monoclonal IgG and measured TCR diversity after 4 wk. The presence of adoptively transferred B cells was verified by flow cytometric analysis; recipient mice had between 10 and 20% of B cells in PBL at the time of sacrifice (our unpublished observations). Mice injected with Ig had, on the average, serum concentrations >4.7-fold those in wild-type mice at the time of sacrifice (Table I and our unpublished observations).

Transfer of wild-type B cells in JH−/− mice increased thymocyte TCR V{beta} diversity by 8-fold; however, adoptive transfer of monoclonal B cells did not, indicating that B cell diversity is required for the generation of thymocyte diversity. Similarly, injection of polyclonal IgG in JH−/− mice increased thymocyte TCR V{beta} diversity by 7-fold, whereas injection of monoclonal IgG or OVA did not (Table II). These results show that diverse Ig promotes TCR V{beta} diversity. In agreement with this function for Ig in promoting T cell diversity, µMT mice with serum Ig 4.8% that in wild-type mice had 70-fold greater T cell diversity than JH−/− mice that had no serum Ig (Tables I and II).


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  		Table II. Reconstitution of TCR diversity in JH−/− mice after adoptive transfer of bone marrow B cells or administration of IgGa

 
As injection of polyclonal IgG alone increases TCR diversity, we wondered whether production of Ig was the mechanism by which adoptively transferred B cells promoted T cell diversity. To find out, we quantified serum Ig in mice recipients of wild-type B cells 4 wk or later post-transfer. Table I shows that JH−/− mice with adoptively transferred B cells had no measurable serum Ig, whereas JH−/− mice injected with polyclonal IgG had levels of Ig in the serum 4.8-fold greater than those in wild-type mice. One possible conclusion is that polyclonal B cells promote TCR diversification in the absence of serum Ig, but we cannot exclude the possibility that Abs transiently present in the serum promoted TCR diversification.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we report that diverse B cells and polyclonal Ig drive the selection of a diverse repertoire of T cells. This finding questions the long-standing idea that T and B cells develop independently (36). Generation of a diverse TCR repertoire relies on the quasi-random recombination of the TCR V region gene segments and on positive and negative selection (2). Positive selection and MHC restriction are generally thought to depend on interaction of T cell precursors with thymic epithelial cells (37); however, this concept is at odds with experimental evidence indicating that mice bearing T cells and thymic epithelial cells that are haplo-incompatible are also restricted to the MHC of nonthymic epithelial cells (12, 38, 39). These observations could suggest that B cells colonizing the thymus promote thymocyte selection and MHC restriction.

Our results showing reduced TCR diversity in mice that lack B cells (JH−/−) or that have very few B cells (µMT) could be explained if B cells promoted positive selection of thymocytes by decreasing death by neglect. In agreement with the concept that B cells promote positive selection is the finding of a decreased number of thymocytes and increased cell death in the thymic cortex of mice that lack B cells. We cannot, however, rule out the possibility that B cells decrease negative selection of thymocytes. Against this possibility are reports that thymic B cells promote, rather than decrease, negative selection of T cells to murine mammary tumor virus, minor lymphocyte-stimulating Ags (40), and I-E expressed only on B cells (41). Another possibility is that B cells may promote colonization of the thymus by thymic precursors or early thymocyte development before the expression of TCR and thus before selection.

As selection of a diverse T cell repertoire in the thymus is thought to depend at least in part on the diversity of peptides presented by self-MHC (2), we hypothesized that B cells and Ig may provide an alternative source of diverse peptides derived from the V region of Ig or from captured peripheral Ags. We tested this hypothesis in mice with QM B cells that are 1000-fold less diverse than wild-type B cells (31) and have normal numbers of T cells. Our finding of severely reduced T cell diversity in QM mice indicates that diversity of B cells may be more important than the number of B cells in the generation of a diverse T cell repertoire.

Our results showing increased diversity of T cells after injection of polyclonal IgG indicate that some of the effects of B cells in the selection of T cells can be mediated by secreted Ig. However, adoptive transfer of polyclonal B cells also increased TCR diversity in JH−/− mice in the absence of detectable serum Ig (Tables II and I). One possible interpretation is that polyclonal B cells promote TCR diversification in the absence of serum Ig, but we cannot exclude that Abs present transiently in the serum may have contributed to the increased T cell diversity following adoptive transfer of B cells. Thus, whether B cells may contribute to a mature T cell repertoire by means other than by producing Ig is not known.

Thymic epithelial cells may produce some of the peptides required for positive selection, but these may not be diverse enough to assure the survival of thymocytes representing wild-type TCR diversity. Our findings reveal a heretofore unrecognized and vital function of B cells and Ig in promoting TCR diversification, perhaps by contributing to the diversity of the peptides that developing T cells encounter. This function of B cells may help to explain the phenotype of B cell-deficient human subjects and suggests a potential approach to immune reconstitution.


   Acknowledgments
 
We thank Michelle Rebrovich, Peter Ouillette, and Tim Plummer for their excellent technical support. We also thank Dr. Walter K. Kremers (Mayo Clinic) for help with statistical analysis.


   Footnotes
 
1 This work was supported by the grants from the National Institutes of Health (AI48602, AI41570, and HL46810) and a grant (SFRH/BD/6797/2001) from the Fundação para a Ciência e Tecnologia, Portugal. The authors declare that they have no competing financial interests. Back

2 Address correspondence and reprint requests to Dr. Marilia Cascalho, Transplantation Biology Program, Mayo Clinic, 200 First Street SW, Medical Sciences Building 2-63, Rochester, MN 55905. E-mail address: cascalho.marilia{at}mayo.edu Back

3 Abbreviations used in this paper: QM, quasi-monoclonal; BrdU, 5-bromo-2'-deoxyuridine. Back

Received for publication November 12, 2003. Accepted for publication February 2, 2004.


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 Discussion
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