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Cutting Edge: TCR Revision Occurs in Germinal Centers¹

Department of Immunology, University of Washington, Seattle, WA 98195

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mouse CD4⁺V5⁺ T cells recognize a peripherally expressed superantigen encoded by an endogenous retrovirus. Ag encounter tolerizes the mature CD4 T cell compartment, either by deletion of autoreactive cells or by TCR revision. This latter process is driven by TCR rearrangement through RAG activity and results in the rescue of cells expressing novel TCRs that no longer recognize the tolerogen. Consistent with the notion that revising T cells represent a distinct peripheral T cell population, we now show that these lymphocyte blasts express a hybrid effector/memory phenotype and are not undergoing cell division. A population of revising T cells is CD40⁺, expresses the germinal center (GC) marker CXCR5, and is V5^lowThy-1^low. Histology reveals that, consistent with their surface Ag phenotype, T cells undergoing TCR revision are enriched in splenic GCs. These data demonstrate that TCR revision is a multistep tolerance pathway supported by the unique microenvironment provided by GCs.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Tolerance induction among mature T cells to self-Ags expressed in the lymphoid periphery occurs by mechanisms ranging from anergy and deletion to nutrient deprivation and active suppression (reviewed in Refs. 1 and 2). The risks inherent in one recently described mechanism, TCR revision, appear particularly large because it requires extrathymic Ag receptor gene rearrangement. Despite this implied risk, evidence from both mice and humans indicates that rearrangement of TCR genes by peripheral T cells can occur (3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Although TCR revision can tolerize T cells to the initiating Ag (3, 4, 7, 8), the danger of generating autoreactive cells through this mechanism is especially acute in autoimmune-prone mice (10, 13). It is therefore important that TCR revision be coupled with mechanisms that select against those T cells expressing newly generated autoreactive TCRs.

TCR revision represents one of two alternate tolerance pathways traveled by V5⁺CD4⁺ T cells from both V5 TCR transgenic (Tg)³ and non-Tg C57BL/6 (B6) mice upon recognition of a self-Ag-encoded by endogenous mouse mammary tumor virus (Mtv) 8 (14). After encounter with Mtv-8, most of these cells are rendered anergic and deleted, while a minority initiates TCR revision (15, 16). Approximately 3% of V5⁺CD4⁺ T cells undergo TCR revision every 4–5 days in mice carrying Mtv-8 (11). One hypothesis to explain how Mtv-8-experienced T cells enter one pathway vs the other suggests that when V5⁺CD4⁺ T cells encounter Mtv-8, some receive a weak or partial signal that induces the TCR revision pathway and some perceive a stronger Mtv-8 signal and are thereby deleted (16). In line with this hypothesis, TCR revision requires B cells and CD28 and ICOS molecules and is enhanced in the absence of functional Fas molecules, while the deletional pathway is B cell, CD28, Fas, and ICOS independent (3, 4, 17). Although these data hint that Mtv-8 expression by distinct cell types may deliver signals that instruct the partner T cell to die or to revise, little is known either about what triggers TCR revision or its mechanistic details.

Despite the paucity of information, we know that only CD4⁺ T cells are allowed to undergo TCR revision in Mtv-8⁺ V5 Tg mice, and that the end product of this pathway is a population of previously activated, self-tolerant, and functional T cells expressing a diverse TCR repertoire (4, 16). Furthermore, TCR revision can occur in V5⁺CD4⁺ T cells from mice thymectomized up to 2 mo previously (11). These results indicate that TCR revision does not target recent thymic emigrants with an immature transitional phenotype, cells known to disappear within 2 wk after removal of the thymus (18). It is clear that revising T cells are mature, peripheral T cells triggered to express RAG1, RAG2, and TdT, that together catalyze the formation of signal end intermediates of V to DJ rearrangement and the expression of diverse TCR chains with unusually short N regions (3, 4).

Using GFP as a readout of RAG2 promoter activity has allowed us to define the process of TCR revision and to locate revising cells within the periphery. We now show that revising CD4⁺ T cells express markers of germinal center (GC) T cells, are enriched within splenic GCs, and are large, nondividing lymphocytes with an unusual hybrid memory/effector cell phenotype. These data suggest that cells undergoing TCR revision are incompletely activated by tolerogen encounter and indicate that TCR revision is confined to a tightly regulated microenvironment known to support Ag receptor recombination in B cells. The GC also provides a microenvironment that selects the appropriate B cell products of receptor editing and may similarly select self-tolerant T cell products of TCR revision.

	Materials and Methods

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Mice

RAG2p-GFP Tg mice obtained from Dr. M. Nussenzweig (NG-BAC mice as used in Ref. 19) were bred to V5 TCR Tg B6 mice (11) and are Mtv-8⁺ and carry a GFP transgene driven by the RAG2 promoter. Five-week-old mice were anesthetized with tribromoethanol (Sigma-Aldrich, St. Louis, MO) and thymectomized as described previously (20). Thymectomy was verified by the absence of CD4⁺CD8⁺ lymphocytes in remaining tissue in the thymic region. All mice were maintained and procedures were performed according to the guidelines of the University of Washington Institutional Animal Care and Use Committee.

Cell surface staining and flow cytometry

Water-lysed PBL, splenocytes, lymph node (LN) cells, and bone marrow cells were stained as described previously (11). In experiments analyzing surface phenotype, splenocytes were depleted of B220⁺ cells using B220 microbeads and an AutoMACS column according to the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA). In the indicated experiments, isolated splenic B220⁺ B cells, bone marrow cells, unseparated splenocytes, or splenocytes activated for 24 h with 5 µg/ml Con A were used for comparison. BD Pharmingen (San Diego, CA) supplied streptavidin-allophycocyanin and Abs specific for V5 (MR9-4), CD4 (RM4-5 or RM4-4), CD16/32 (2.4G2), CD19 (1D3), CD25 (PC61), CD40 (3/23), CD44 (IM7), CD45RB (16A), CD62 ligand (CD62L; MEL-14), CD71 (transferrin receptor, C2), CXCR5 (2G8), Thy-1.2 (53-2.1), and Ter119/Ly76. Surface staining was limited to PE, PerCP, and allophycocyanin fluorochromes detected in the FL-2, FL-3, and FL-4 channels, respectively; FL-1 was used for GFP detection. Flow cytometric analysis of T cells was performed using CellQuest software on a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). At least 5000 CD4⁺GFP⁺ or GFP^– gated events were analyzed in each experiment.

DNA content was determined by pooling spleen and LN cells from three thymectomized V5 Tg RAG2p-GFP Tg mice and enriching for CD4⁺ T cells by negative selection using the CD4⁺ T cell isolation kit and an AutoMACS column (Miltenyi Biotec). Enriched CD4⁺ T cells were sorted into CD4⁺GFP⁺ and CD4⁺GFP^– populations with a FACSAria (BD Biosciences). Cells were fixed in ice-cold 70% ethanol and stained for >30 min with 50 µg/ml propidium iodide in PBS containing 100 U/ml RNase A and 0.1% glucose. DNA was visualized using a FACScan flow cytometer (BD Biosciences).

Immunohistochemistry and fluorescence microscopy

Spleens from V5 Tg RAG2p-GFP Tg mice thymectomized 5–6 wk previously were fixed in a fresh PBS solution containing 0.5% sucrose and 4% paraformaldehyde (Fisher Scientific, Pittsburgh, PA) for 2 h at 4°C (21). Fixed spleens were placed in a 10% sucrose solution until they sank, and the process was repeated with 20% and 30% sucrose solutions. Frozen tissue was cut into 8-µm sections, allowed to dry for 2 h, and fixed onto the slide with 4% paraformaldehyde/PBS for 30 min at room temperature. Background staining was blocked by incubating sections for 1 h in PBS containing 2.5% BSA, 1.25% normal rat serum, and 1.25% normal goat serum. GCs were localized by sequential staining of marginal zones with anti-mouse MOMA-1 (Cedarlane Laboratories, Hornby, Ontario, Canada) and biotinylated peanut agglutinin (PNA; Sigma-Aldrich), both diluted in 5% BSA/PBS. Sections were stained for 1 h at room temperature, washed three times with PBS, and incubated for 45 min with AlexaFluor 633-conjugated donkey anti-goat IgG (Molecular Probes, Eugene, OR) and CyChrome 5-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA). To visualize CD4⁺ cells, sections were counterstained with AlexaFluor 568-conjugated anti-CD4 (RM4-5; BD Pharmingen). Coverslips were mounted using Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). At the same time, spleens from V5 Tg B6 mice were processed and analyzed to assess the level of green fluorescence. Autofluorescence was excluded from further analysis on the basis of its punctate pattern and lack of association with CD4⁺ cells. Single-color staining was used to exclude bleed-through from other channels. Photographs were taken using a Nikon Microphot-SA microscope with an attached camera and Spot version 3.5 software (Diagnostic Instruments, Sterling Heights, MI). Images were processed using Photoshop software (Adobe, San Jose, CA). The total imaged areas were as follows: inside GC, 7257 µm²; close to GC, 2867 µm²; and outside GC, 6451 µm².

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
CD4⁺GFP⁺ cells from thymectomized V5 Tg RAG2p-GFP Tg mice are large and metabolically active but not dividing

In V5 Tg RAG2p-GFP Tg mice that have been thymectomized to eliminate GFP⁺ recent thymic emigrants (18), GFP⁺ T cells carry RAG1- and RAG2-specific RNA and V to DJ TCR recombination intermediates (11). A comparison between GFP⁺ and GFP^– CD4⁺ cells from thymectomized V5 Tg RAG2p-GFP Tg mice reveals several consistent distinctions. CD4⁺GFP^–cells are typical resting T cells, being small, quiescent lymphocytes that express low levels of transferrin receptor. In contrast, CD4⁺GFP⁺ cells are large lymphocytes that express high levels of transferrin receptor, indicating their high metabolic activity (Fig. 1). We assayed sorted populations for total DNA content to determine whether or not CD4⁺GFP⁺ T cells are dividing. Despite their size and metabolic activity, fewer CD4⁺GFP⁺ than CD4⁺GFP^– T cells are in the G₂-M phase of the cell cycle (0.3% compared with 1.5%, Fig. 1). Cells in S phase are not detectable in the CD4⁺GFP⁺ population. These data are consistent with the in vitro induction of RAG expression in CD4⁺V5⁺ T cells cultured in the presence of the cell cycle inhibitor mimosine (11) and indicate that CD4⁺GFP⁺ T cell blasts are not simultaneously rearranging and synthesizing DNA.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. RAG+ T cells undergoing TCR revision are large and metabolically active, but are not dividing. A, By analysis of unseparated T cell populations from V5 Tg RAG2p-GFP Tg mice thymectomized 3–6 wk previously, forward scatter was used to compare the size of splenic CD4+GFP– or CD4+GFP+ T cells (filled histograms) to that of GFP– or GFP+CD19+ B cells, respectively (open histograms). Data are representative of 10 mice. B, Transferrin receptor expression is shown for CD4+GFP– and CD4+GFP+ B cell-depleted splenocytes (filled histograms). The dashed line represents positive control staining of Ter119+ erythroid lineage bone marrow cells and the dark line represents unstained CD4+GFP– and CD4+GFP+ T cells. Results are representative of three mice. C, Splenocytes and LN cells were pooled from three mice, enriched for CD4+ T cells by negative selection, and sorted to >95% purity on the basis of CD4 and GFP. Total DNA content was visualized in fixed cells stained with propidium iodide. Markers denote cells in the G0-G1 (left) and G2-M (right) phases of the cell cycle. Cells falling between the two markers are in S phase, and are undetectable in the CD4+GFP+ population. Numbers denote the percentage of cells within the indicated markers.

Consistent with our previous findings, cells from V5 Tg mice that are undergoing TCR revision down-regulate surface levels of V5 (Fig. 2A) and eventually express a new, nonautoreactive TCR encoded by rearranged endogenous TCR genes (3, 4). It is important to note that both CD4⁺GFP^– and GFP⁺ cells continue to express T lineage markers (data not shown) and remain negative for B lineage markers, including CD19 (Fig. 2A). The age-dependent accumulation of activated V5^– T cells that express novel endogenous TCR chains (V^endo+) is just beginning in the young adult animals analyzed in Fig. 2, in which 78% of V5^highCD4⁺GFP^– cells express low levels of CD44, while 91% of the CD4⁺GFP⁺ cells are CD44^high (Fig. 2B). Although most of the V5^high CD4⁺GFP⁺ cells from these animals are CD62L^high, 90% of the V5^low cells are CD62L^low (Fig. 2B). Dual staining of CD4⁺GFP⁺ gated cells for CD44 and CD62L suggests that CD44 expression is up-regulated on CD62L^high cells, followed by down-regulation of CD62L (data not shown). The population of V5^–(V^endo+)CD62L^lowCD4⁺ GFP^– cells has likely completed TCR revision.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. Revising CD4+ T cells have a unique hybrid effector/memory phenotype. Spleen cells from V5 Tg RAG2p-GFP Tg mice thymectomized 3–6 wk previously were stained as indicated. Data are representative of at least six mice. The filled histograms (A and C) and dot plots (B) represent B cell-depleted splenocytes gated as CD4+GFP– or CD4+GFP+. The percentages of cells falling within each gate are indicated. A, Open histograms represent B220 positively selected cells stained for CD19 and gated as GFP– or GFP+. C, Open histogram represents anti-CD25 staining of splenocytes stimulated for 24 h with Con A.

Consistent with recent findings correlating CD40 expression on peripheral CD4⁺ T cells with RAG expression and TCR revision (10), 25% of CD4⁺GFP⁺ T cells in V5 Tg RAG2p-GFP Tg mice also express CD40 (Fig. 3A). It is interesting that the CD40⁺CD4⁺GFP⁺ population expresses predominantly high and intermediate levels of V5 (Fig. 3B), suggesting the involvement of V5 and CD40 coengagement in the initiation of TCR revision.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. A subset of revising T cells is Thy-1.2low, CXCR5+, and CD40+. Spleen cells from V5 Tg RAG2p-GFP Tg mice thymectomized 3–6 wk previously were surface stained for Thy-1.2, CXCR5, and CD40. Data are representative of at least five mice. A, The filled histograms are B cell-depleted cells gated as CD4+GFP– or CD4+GFP+ and the open histograms are the same populations stained with conjugated streptavidin alone. The dashed lines represent positive control staining of B220 positively selected cells gated as CD19+GFP– or CD19+GFP+. B, CD4+GFP+ cells are further gated as noted. The percentages of cells falling within each marker are indicated.

GCs are known to support somatic hypermutation of Ig genes and B cell receptor editing (Refs. 19 and 23 and reviewed in Ref. 24). Because GCs potentiate the selection of B cells on the basis of their expressed Ag receptors, these microenvironments could also provide niches for imposing self-tolerance on T cell populations expressing newly generated TCRs. In support of this notion, TCR revision in our system (4, 17, 25) is restricted to CD4⁺ T cells (as is entry into GCs) and is dependent on B cells and CD28 expression (as is GC formation). We now show (Fig. 3A) that a subset of CD4⁺GFP⁺ cells is Thy-1^low and CXCR5⁺, a phenotype associated with GC T cells (26, 27, 28). The cells that are Thy-1^low are also V5^low/–(Fig. 3B). Furthermore, those cells that are CXCR5⁺ and CD40⁺ may be in the process of down-regulating surface expression of V5 and Thy-1, being enriched for cells expressing intermediate levels of both markers (Fig. 3B and data not shown).

Although the CXCR5⁺Thy-1^low phenotype of revising T cells is suggestive, we directly examined whether CD4⁺GFP⁺ cells are enriched within and around splenic GCs. GFP⁺ T cells both in and close to GCs were counted in these tissue sections because although initial expression of GFP is a faithful reporter for RAG promoter activity, cells remain GFP⁺ after RAG expression is extinguished (18). Although most splenic T cells are located outside GCs, 83% of CD4⁺GFP⁺ cells are within or close to GCs (Fig. 4C). Taken together, these data indicate that TCR revision occurs preferentially in GCs.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. CD4+GFP+ cells are enriched in and around splenic GCs. A, GCs in splenic sections from thymectomized V5 Tg RAG2p-GFP Tg mice were identified by PNA staining (shown here in red) within a MOMA-1+ region (shown here in white). For actual experiments, PNA and MOMA-1 both fluoresce in the same channel (AlexaFluor 633 and CyChrome 5). B, CD4+GFP+ cells (yellow) were identified by overlaying images of CD4+ (red) and GFP+ (green) cells. C, From a total of four V5 Tg RAG2p-GFP Tg mice thymectomized 3–6 wk previously, CD4+GFP+ cells were counted within, immediately surrounding (<48 µm), or far away (>84 µm) from the GC. The indicated p value was determined by the two-tailed Student’s t test. A total of 224 GFP+CD4+ cells (50–60 per mouse) were counted.

Using GFP as a reporter of RAG expression has allowed us to visualize mature peripheral CD4⁺ T cells in the act of TCR revision and to define the phenotype of revising cells. A model of TCR revision that is compatible with our data is illustrated in Fig. 5. The process begins with naive CD4⁺ T cells that are small, RAG^–, and V5^highThy-1^highCD44^lowCD62L^highCD45RB^highCD69^– CD25^–. These cells encounter Mtv-8-expressing cells, presumably in the spleen or LNs, and enter the pathway at stage 1, eventually assuming the CD44^highCD62L^lowCD45RB^low phenotype of memory T cells, the phenotype of V5⁺CD4⁺ T cells remaining in aging V5 Tg mice (3, 15). A fraction of these cells expresses CXCR5 and CD62L and is drawn into the GC. Perhaps through coengagement of the TCR and CD40, these cells are triggered to express RAG genes and initiate TCR revision (stage 2), becoming large and CD45RB^high (Fig. 5). These cells that fall outside the accepted categories of naive, effector, and memory CD4⁺ T cells (22) begin to down-regulate surface expression of V5 and Thy-1. Further along the revision pathway, the RAG⁺ T cells enter stage 3 and become V5^lowThy-1^lowCD62L^low. We hypothesize that within the GC environment, revising T cells that successfully rearrange and express an alternate TCR chain gene are purged of cells expressing autoreactive TCRs. Eventually, the surviving cells extinguish RAG and CXCR5 expression and exit both the GC and the TCR revision pathway, having rearranged an endogenous TCR chain gene that encodes a functional, self-tolerant Ag receptor. These cellular products of TCR revision are small lymphocytes that display the V5^–V^endo+Thy-1^highCD44^highCD45RB^lowCD62L^high/low phenotype of typical memory T cells. Thus, by the tightly controlled and sequential expression of activation markers, chemokine receptors, costimulatory molecules, and recombinase machinery, the potentially risky process of TCR revision is confined to the selective environment of the GC and regulated to effect the rescue and rehabilitation of autoreactive T cells.

View larger version (36K): [in this window] [in a new window]   FIGURE 5. TCR revision is a multistep pathway. The partial activation of V5+CD4+ T cells by Mtv-8 (stage 1) triggers a series of processes that include blasting (indicated by an increase in cell size) without DNA synthesis, the induction of the lymphocyte-specific recombination machinery (indicated by the shading), T cell movement into the GC (stage 2), loss of V5 surface expression, and generation of a new TCR chain by recombination of the endogenous locus (stage 3). Presumably after elimination of cells that express newly generated autoreactive TCRs, surviving T cells are allowed to exit the GC and join the pool of immunocompetent recirculating T cells.

	Acknowledgments

 
We thank Fred Lewis for assistance with cell sorting and Drs. T. Boursalian for help with thymectomy, M. Nussenzweig for the RAG2p-GFP Tg mice, and M. K. Kaja for use of his microtome.

	Footnotes

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

¹ This work was supported by National Institutes of Health Grants AG 13078 (to P.J.F.), AI 24137 (to A.G.F.), and T32 AI 07411 (to C.J.C.) and the Juvenile Diabetes Research Foundation Chet Edmonson Postdoctoral Fellowship (to C.J.C.).

² Address correspondence and reprint requests to Dr. Pamela Fink, Department of Immunology, University of Washington, Campus Box 357650, Seattle, WA 98195. E-mail address: pfink{at}u.washington.edu

³ Abbreviations used in this paper: Tg, transgenic; Mtv, mammary tumor virus; GC, germinal center; LN, lymph node; PNA, peanut agglutinin.

Received for publication August 4, 2004. Accepted for publication September 29, 2004.

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