The functional capacity of the adaptive immune system is dependent on the size and the diversity of the T cell population. In states of lymphopenia, T cells are driven to proliferate to restore the T cell population size. However, different T cell clones proliferate at different rates, and some T cells experience burst-like expansion called spontaneous lymphopenia-induced proliferation (LIP). These T cells are likely receiving stimulation from cognate Ags and are most responsible for inflammatory pathology that can emerge in lymphopenic states. Foxp3+ regulatory T cells (Tregs) selectively inhibit spontaneous LIP, which may contribute to their ability to prevent lymphopenia-associated autoimmunity. We hypothesized that another potential negative consequence of unrestrained spontaneous LIP is constriction of the total T cell repertoire. We demonstrate that the absence of Foxp3+ Tregs during the period of immune reconstitution results in the development of TCR repertoire “holes” and the loss of Ag-specific responsiveness to infectious microorganisms. In contrast, the presence of Tregs during the period of immune reconstitution preserves optimal TCR diversity and foreign Ag responsiveness. This finding contrasts with the generally accepted immunosuppressive role of Tregs and provides another example of Treg activity that actually enhances immune function.

Maintenance of peripheral T cell homeostasis is a critical feature of the adaptive immune system (1, 2). Strict control of T cell homeostasis ensures adequate size of the T cell population, TCR diversity, responsiveness to foreign Ags, and self-tolerance. If a state of peripheral T cell deficiency is created, the residual T cells are driven to undergo proliferation by a process termed lymphopenia-induced proliferation (LIP) to reconstitute the optimal T cell numbers (3). LIP can be observed in a number of physiological and pathological situations (4). Lymphopenia is physiologic during the prenatal and neonatal periods, and LIP may contribute to the generation of sufficient numbers of T cells with a memory phenotype (3). In addition, because the thymus involutes with increasing age, LIP assumes greater importance in maintaining T cell population size (5). Transient lymphopenia can accompany virtually any viral infection, and some viral infections (e.g., HIV) lead to chronic and progressive lymphopenia. In addition, there are multiple iatrogenic causes of lymphopenia, including cytodepletion by radiation, chemotherapy, and depleting Abs. Notably, lymphopenia can be a trigger to autoimmunity, which is explained, at least in part, by T cell resistance to tolerance induction during LIP (6, 7). Furthermore, because individual T cell clones expand at different rates during LIP, the emerging T cell population risks further loss of TCR diversity and diminished immune fitness. However, although lymphopenic states occur multiple times during the lifetimes of all individuals, autoimmunity and immune deficiency do not manifest in most people. Therefore, there must be mechanisms that help to maintain optimal immunologic tolerance and immune fitness during immune reconstitution.

Regulatory CD4+CD25+Foxp3+ T cells (Tregs) constitute 5–15% of peripheral CD4 T cells in healthy adult mice and humans (8) and are critical in the maintenance of immunologic tolerance and peripheral T cell homeostasis. These cells are able to suppress a range of immunologic responses in vitro and in vivo. Their discovery was aided by a number of animal models of autoimmunity associated with lymphopenia [e.g., neonatal thymectomy (9), colitis induced by adoptive transfer into SCID mice (10), and thyroiditis in the rat (11)]. However, early studies failed to demonstrate their ability to suppress LIP (12). Since then, at least two major forms of LIP were recognized: homeostatic and spontaneous (13). Homeostatic LIP is driven primarily by cytokines, such as IL-7 and -15, and is generally slow and steady. Spontaneous LIP is burst-like, likely driven by full agonist Ag stimulation, and it is the most likely source of pathogenic T cells specific for self- and commensal flora Ags. Importantly, the ultimate size of the T cell population is independent of the size of the input T cell population and relative proportion of homeostatic and spontaneous forms of LIP (14). We showed previously that although Tregs do not suppress homeostatic LIP, they do suppress the spontaneous form of LIP (15, 16). This likely contributes to their protective function against the development of autoimmunity during LIP. Because the expansion of T cells by spontaneous LIP is dependent on higher-affinity TCR stimulation, this T cell population is likely oligoclonal. However, it is possible that T cells undergoing either form of LIP, spontaneous and homeostatic, also compete for some of the same common resources (e.g., cytokines). Therefore, excessive spontaneous LIP may adversely affect the remainder of the T cell population, resulting in constriction of the TCR repertoire during immune reconstitution. This reasoning led us to speculate that selective suppression of spontaneous LIP by Tregs could benefit the remainder of the T cell population by allowing them greater access to those cytokine resources important for their survival and homeostatic LIP. Furthermore, we hypothesized that the presence of Tregs during immune reconstitution by LIP should lead to preservation of greater diversity of TCR repertoire and, consequently, better immune fitness. This is an important issue with significant clinical implications in various situations that involve T cell depletion (e.g., AIDS or intentional T cell depletion in the treatment of organ rejection, autoimmunity, or cancer).

In this study, we characterized the TCR repertoire in T cell populations expanded by LIP in the presence or absence of Tregs following adoptive transfer into RAG−/− or TCRα−/− recipients. Using TCR Vβ CDR3 spectratyping, we demonstrated significantly enhanced preservation of structural TCR diversity in the presence of Tregs during LIP. The reduction of TCR diversity in the absence of Tregs resulted in the development of functional “holes” in the repertoire that were revealed by enumerating Ag-specific T cells following infection with Listeria monocytogenes. Thus, markedly greater numbers of Listeria-specific responding T cells were found if Tregs were present than if they were absent during LIP. These findings suggest that Tregs preserve TCR diversity and immune responsiveness during immune reconstitution from lymphopenia. This positive effect of Tregs on immunity contrasts with their generally accepted immunosuppressive function.

C57BL6 (B6) and CD45.1 congenic mice were purchased from the National Cancer Institute (Frederick, MD). Some B6 mice used were wild-type littermates to IL-10−/− mice, originally obtained from The Jackson Laboratory (Bar Harbor, ME), and CTLA-4+/− mice, which were a generous gift from Dr. J. Allison (Memorial Sloan-Kettering Cancer Center, New York, NY), respectively. RAG-1−/−, TCRα−/−, and Thy1.1 congenic mice on the B6 background were obtained from The Jackson Laboratory. B6 RAG-1−/− mice were bred onto the CD45.1 congenic background in our facility. Thy1.1 BALB/c mice were originally provided by Dr. L. Turka (University of Pennsylvania, Philadelphia, PA) and were crossed with BALB/c RAG-2−/− mice in our facility. All mice used were generally 4–20 wk of age. All animals were maintained in a specific pathogen-free facility in microisolator cages with filtered air, according to the National Institutes of Health guidelines.

Donor T cells were collected from secondary lymphoid tissues (axillary, brachial, cervical, mesenteric, and inguinal lymph nodes and spleen). CD44lowCD4, CD8, and bulk (CD4 and CD8) T cells were purified in two stages. First, CD8 or CD4 T cells were prepared by negative selection against CD8 or CD4, MHC class II, CD11b, B220, CD25, and, in some cases, CD103 (all Abs labeled with FITC) using anti-FITC BioMag particles (Polysciences, Warrington, PA). Second, CD44high cells were depleted (B6 only) using magnetic microbeads (Miltenyi Biotec, Auburn, CA), as previously described (17). Briefly, the purified T cells were suspended in labeling buffer (2% FBS in PBS), incubated with 0.004 μg anti–CD44-FITC (eBioscience, San Diego, CA) per 106 cells for 20 min, washed, and labeled with anti-FITC magnetic microbeads. The negative fraction was collected following Miltenyi Biotec magnetic column separation. CD25+ T cells were prepared using positive selection with anti-CD25 biotinylated mAb, PC61, and streptavidin-labeled magnetic microbeads (Miltenyi Biotec). An example FACS profile showing the purity and Foxp3 expression of cells in this preparation was published previously (16). Purified cells were transferred into RAG−/− or TCRα−/− hosts via i.v. tail injection. Cells were labeled with 7 μM CFSE or SNARF-1 prior to transfer. Tregs were depleted in vivo using a single i.p. injection of 400–450 μg anti-Thy1.1 mAb (clone 1A14) (18).

Regulatory and conventional T cells collected from secondary lymphoid tissues were MACS sorted by positive selection for Thy1.1, as described above. Total RNA was extracted from positive (Thy1.1+/Treg) and negative (Thy1.1/conventional T cell) fractions with an RNeasy Mini Kit (Qiagen, Valencia, CA), and first-strand cDNA was generated from total RNA with Oligo(dT)20 and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). Each cDNA sample was checked for integrity and for detectable TCR Cβ-chain using β actin (5′-GTG GGC CGC TCT AGG CAC CAA; 3′-CTC TTT GAT GTC ACG CAC GAT TTC) and Cβ-specific primers (5′-Cβ1A; 3′-Cβ3C), under PCR-amplification conditions described below (19). The CDR3 size-distribution PCR assay was performed in duplicate for each individual sample, as described (20, 21). Briefly, each TCR Vβ was amplified with Vβ8.1-, Vβ8.3-, and/or Vβ10-specific sense primers and a Cβ antisense primer (22). For the generation of Vβ spectratypes, the Cβ antisense primer was labeled with 6-fluorescein phosphoramidite on the 5′ end. One microliter of the previous synthesis, corresponding to the reverse transcription of 0.05–1 μg total RNA, was used in the amplification. cDNA was added to tubes already containing a mixture of Taq Master Mix (Qiagen), DNAse/RNAse-free water, and Vβ- and Cβ-specific primers (10–20 μM), and amplification was performed as follows: 30 cycles of 95°C for 1 min/52°C for 1 min/72°C for 1 min, followed by 5 min at 72°C. A second, nested amplification for 12 specific Jβ sequences (Jβ1.1, Jβ1.2, Jβ1.3, Jβ1.4, Jβ1.5, Jβ1.6, Jβ2.1, Jβ2.2, Jβ2.3, Jβ2.4, Jβ2.5, and Jβ2.7) was performed using 1 μl Vβ-specific product (22). Cycling conditions remained the same, with the exceptions of Master Mix containing 5′ 6-fluorescein phosphoramidite–labeled Jβ-specific sense primers and 20 cycles of amplification. The size distribution of each fluorescent PCR product was determined by electrophoresis on an Applied Biosystems 3130xl Genetic Analyzer (Foster City, CA), and data were analyzed using Genoprofiler 2.1 (23, 24).

To assess each sample diversity profile as normal or limited, we used a variation of a previously reported complexity scoring system (25) [i.e., complexity score = (sum of all peak areas/sum of the major peak areas) × (number of major peaks)]. Major peaks were defined as those on the spectratype histogram with an area ≥10% of the sum of all peak areas.

Wild-type B6 mice were immunized i.v. with ∼1 × 107 CFU of a ∆ActA strain of L. monocytogenes transformed by a plasmid containing OVA and an I-Ab–specific mutant epitope of I-Eα (“2W1S”) (26). Bacteria were grown in Luria-Bertani media with 24 μg/ml chloramphenicol to an absorbance ∼0.1 at 600 nm. The actual number of bacteria injected was confirmed by dilution and growth on Luria-Bertani agar plates containing chloramphenicol. Kb-OVA and I-Ab–2W1S tetramers were produced in the laboratories of Drs. Stephen Jameson and Marc Jenkins, respectively, as previously described (27, 28). Lymph nodes and spleens were harvested together and processed for Kb-OVA and I-Ab–2W1S double-tetramer enrichment, according to the protocol established by Moon et al. (28, 29).

Mice were sacrificed by CO2 asphyxiation before spleen and lymph nodes were removed. All secondary lymphoid tissues were disrupted by mashing with a syringe. In some cases, complete media (10% FBS) was supplemented with 10 μg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO) to prevent lymphokine secretion. Fixation and intracellular staining for cytokines was done following 2–3 h in vivo challenge with ∼5 × 107 CFU LM-2W1S–OVA. All Abs used for cell surface staining, negative selection, and tetramer enrichment were purchased from eBioscience, with the exceptions of anti-B220 and anti-CD11c (Biolegend, San Diego, CA), anti-CD8α (Invitrogen), and anti-CD4 single-stain controls (BD Biosciences, San Jose, CA). Specific T cell subsets were identified using fluorochrome-labeled Abs against a panel of TCR Vβ receptors (15 in total), CD3ε, CD4, CD8, CD44, and congenic markers, such as anti-Thy1.1/Thy1.2 and anti-CD45.1/CD45.2. Anti–IFN-γ Abs were purchased from eBioscience. Staining for Foxp3 was done using an eBioscience kit and instructions provided by the manufacturer. Absolute numbers of T cells were calculated using phycoerythrin-compatible fluorescent linker dye reference beads (Sigma-Aldrich). All flow cytometry data were acquired on a FACSCalibur, an LSR, or an LSRII (BD Immunocytometry Systems, San Jose, CA) and analyzed with FlowJo software (Tree Star, Eugene, OR).

All error bars represent the SEM, unless noted otherwise. For data in Figs. 2, 3, and 5, a two-tailed, unpaired Student t test was used for assessment of the differences between groups. Prism (GraphPad, San Diego, CA) was used for graphs and statistical analysis. Differences were considered significant at a p value < 0.05. Twelve primers were used in the two-factor ANOVA, and Bonferroni correction was applied for multiple hypothesis testing; the cutoff for statistical significance in that analysis was 0.05/12 = 0.004.

FIGURE 2.

Assessing T cell diversity at the level of TCR Vβ-chain usage after completion of LIP. A, The experiment protocol for assessment of structural T cell diversity post-LIP in the presence or absence of Tregs involves two separate measurements from each sample: TCR Vβ surface staining for FACS and RNA isolation and spectratyping. B, RAG−/− T cell recipients were sacrificed, and lymphoid tissue samples were processed on day 30 after adoptive transfer. Half of each processed sample underwent cell surface staining with Abs specific to T cell markers (CD4, CD8, and Thy1.2) and FACS analysis. The panels demonstrate the percentages of CD4 T cells (top panel) and CD8 T cells (bottom panel) expressing individual Vβ-chains in the input T cell populations (white bars) and post-LIP T cell populations that emerged in the absence of Tregs (striped bars) or the presence of Tregs (black bars). C, The remaining half of each sample was processed for RNA preparation and TCR Vβ spectratyping. The same conditions (i.e., Tregs absent in the recipient mice [no Tr], Tregs present in the recipient mice [w/Tr], and the input T cell population [before transfer]) were tested. Each graphed data bar or point represents an average of three or four animals combined from two independent experiments. Complexity scores were calculated from spectratyping of Vβ 10 and 8.1 (experiment #1) and from Vβ 10 and 8.3 (experiment #2). The top panel shows the complexity scores of the CD4 T cell population; the middle panel shows the complexity scores of the CD8 T cell population, and the bottom panel shows the complexity scores of the total, unseparated T cell population (bulk Tc).

FIGURE 2.

Assessing T cell diversity at the level of TCR Vβ-chain usage after completion of LIP. A, The experiment protocol for assessment of structural T cell diversity post-LIP in the presence or absence of Tregs involves two separate measurements from each sample: TCR Vβ surface staining for FACS and RNA isolation and spectratyping. B, RAG−/− T cell recipients were sacrificed, and lymphoid tissue samples were processed on day 30 after adoptive transfer. Half of each processed sample underwent cell surface staining with Abs specific to T cell markers (CD4, CD8, and Thy1.2) and FACS analysis. The panels demonstrate the percentages of CD4 T cells (top panel) and CD8 T cells (bottom panel) expressing individual Vβ-chains in the input T cell populations (white bars) and post-LIP T cell populations that emerged in the absence of Tregs (striped bars) or the presence of Tregs (black bars). C, The remaining half of each sample was processed for RNA preparation and TCR Vβ spectratyping. The same conditions (i.e., Tregs absent in the recipient mice [no Tr], Tregs present in the recipient mice [w/Tr], and the input T cell population [before transfer]) were tested. Each graphed data bar or point represents an average of three or four animals combined from two independent experiments. Complexity scores were calculated from spectratyping of Vβ 10 and 8.1 (experiment #1) and from Vβ 10 and 8.3 (experiment #2). The top panel shows the complexity scores of the CD4 T cell population; the middle panel shows the complexity scores of the CD8 T cell population, and the bottom panel shows the complexity scores of the total, unseparated T cell population (bulk Tc).

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FIGURE 3.

Tregs prevent constriction of the T cell repertoire during LIP. RNA samples from CD4 T cells (left panel), CD8 T cells (middle panel), and unseparated T cells (bulk Tc; right panel) from the indicated experimental groups were PCR amplified for Vβ10 expression and spectratyped for CDR3 diversity by further PCR amplification for different Jβ segments. Each dot represents the complexity score for each individual Jβ-chain from two separate experiments. A mean value representing data from all 12 chains under each treatment condition (post-LIP in the absence [no Tr] or presence [w/Tr] of Tregs and input T cell populations labeled as “before transfer”) was used to determine statistical significance. Statistical significance (p < 0.05) between indicated groups is represented by the horizontal bars at the top of each panel.

FIGURE 3.

Tregs prevent constriction of the T cell repertoire during LIP. RNA samples from CD4 T cells (left panel), CD8 T cells (middle panel), and unseparated T cells (bulk Tc; right panel) from the indicated experimental groups were PCR amplified for Vβ10 expression and spectratyped for CDR3 diversity by further PCR amplification for different Jβ segments. Each dot represents the complexity score for each individual Jβ-chain from two separate experiments. A mean value representing data from all 12 chains under each treatment condition (post-LIP in the absence [no Tr] or presence [w/Tr] of Tregs and input T cell populations labeled as “before transfer”) was used to determine statistical significance. Statistical significance (p < 0.05) between indicated groups is represented by the horizontal bars at the top of each panel.

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FIGURE 5.

Ag-specific T cells were enumerated in the secondary lymphoid tissues following L. monocytogenes infection. The Ag-specific T cells were enriched using tetramer-bound magnetic beads. The gating strategy is shown. First, a tight lymphocyte gate is imposed that excludes doublet events (SSC-W). Next, non-T cells are excluded by using a “dump” channel. Finally, Ag-specific T cells are gated using fluorochrome-bound tetramers. As expected, Ag-specific T cells are rare before the infection and mostly CD44low. After the infection, their numbers increase dramatically, and they become CD44high.

FIGURE 5.

Ag-specific T cells were enumerated in the secondary lymphoid tissues following L. monocytogenes infection. The Ag-specific T cells were enriched using tetramer-bound magnetic beads. The gating strategy is shown. First, a tight lymphocyte gate is imposed that excludes doublet events (SSC-W). Next, non-T cells are excluded by using a “dump” channel. Finally, Ag-specific T cells are gated using fluorochrome-bound tetramers. As expected, Ag-specific T cells are rare before the infection and mostly CD44low. After the infection, their numbers increase dramatically, and they become CD44high.

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Tregs are well established in their ability to prevent and treat immunopathology arising in the course of immune reconstitution from lymphopenia by suppression of the emergence and reactivity of pathogenic T cells. In this study, we set out to test the idea that another function of Tregs during immune reconstitution is preservation of maximal immune fitness by maintaining optimal structural TCR diversity of the residual T cell population. To establish LIP, we used the RAG−/− adoptive-transfer model. First, we titrated numbers of bulk (CD4 and CD8), CD44loCD25 T cells transferred on day 0 and measured the number of T cells expressing each TCR Vβ-chain on days 7 and 30. Similarly to what was reported previously (14), we found that the ultimate number of T cells in the lymphoid periphery was independent of the initial input cell number (Fig. 1). Specifically, the T cell population size at 30 d was the same for inputs of 105–107 T cells, and it was still increasing following the adoptive transfer of 104 T cells. We decided to exclude 104–105 T cell transfers from future experiments because any potential TCR repertoire that could emerge from these would be, by definition, severely limited. Furthermore, adoptive transfers of very low numbers of T cells are associated with the development of more pronounced immunopathology (30, 31). The distribution of different Vβ usage by the emerging T cell populations seemed indistinguishable between 106 and 107 T cell transfers (data not shown). However, we excluded 107 T cell transfers because these filled the T cell niche within a mere 7 d, largely preserving the TCR complexity of the input T cell repertoire (14). We chose adoptive transfer of 106 T cells as optimal in allowing an experimental window into TCR repertoire changes that might emerge during LIP. Notably, this number of input cells is generally not optimal for the induction of immunopathology, such as colitis; fewer T cells are more efficient, presumably because of greater availability of resources for pathogenic T cell clones (31). We observed only the development of mild dermatitis in the course of these experiments and no clinical or histopathologic colitis.

FIGURE 1.

T cell reconstitution in RAG−/− mice following adoptive transfer. Varying numbers of naive (CD25CD44lo) T cells (104–107) taken from wild-type C57BL/6 mice were transferred into C57BL/6 RAG-1−/− recipients on day 0 (two recipients per condition). Major lymph nodes (cervical, brachial, axillary, inguinal, and mesenteric) and spleens were harvested on days 7 and 30, stained for T cell markers (CD4 and CD8) and a panel of Abs reactive to 14 mouse TCR Vβ-chains, and analyzed by FACS. The y-axes indicate the absolute numbers of recovered T cells expressing individual Vβ-chains ± variance.

FIGURE 1.

T cell reconstitution in RAG−/− mice following adoptive transfer. Varying numbers of naive (CD25CD44lo) T cells (104–107) taken from wild-type C57BL/6 mice were transferred into C57BL/6 RAG-1−/− recipients on day 0 (two recipients per condition). Major lymph nodes (cervical, brachial, axillary, inguinal, and mesenteric) and spleens were harvested on days 7 and 30, stained for T cell markers (CD4 and CD8) and a panel of Abs reactive to 14 mouse TCR Vβ-chains, and analyzed by FACS. The y-axes indicate the absolute numbers of recovered T cells expressing individual Vβ-chains ± variance.

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To test the effects of Tregs on the responder T cell population during LIP, we constructed the following experimental system (Fig. 2A). First, 106 CD25+ Tregs were purified from Thy1.1 BALB/c mice and transferred into RAG−/− recipients and allowed to expand for 7–10 d, after which 106 Thy1.2 CD44loCD25 responder T cells were adoptively transferred. The lymphoid tissues were harvested for T cell population analysis 30 d after the responder T cell transfer. We used this protocol previously to show that Tregs selectively suppress only the spontaneous form of LIP by the responder T cells (16). In agreement with previous reports (14), the population size of Foxp3 T cells within secondary lymphoid tissues at 30 d is the same, regardless of the presence or absence of the separate Treg adoptive transfer (data not shown).

As anticipated, the TCR Vβ usage of the reconstituted population was similar to the initial population, regardless of the presence or absence of Tregs in the host (Fig. 2B). Although simple and robust, flow cytometric measurements of TCR Vβ usage are a relatively insensitive measure of TCR diversity, in that they do not allow determination of clonal dominance or restriction within populations of cells expressing the same Vβ-chain. To probe the TCR repertoire further, we generated spectratypes at the level of a particular Vβ-chain (e.g., Vβ8.1, Vβ8.3, and Vβ10) (Fig. 2C). This assay suggested that CD4 T cells undergoing LIP in the absence of Tregs experience contraction in TCR diversity compared with that measured before LIP or after LIP in the presence of Tregs. However, we could not detect decreased TCR diversity when CD8 T cells or unfractionated naive (CD25CD44lo) responder T cells were allowed to undergo LIP under the same conditions.

Although Vβ spectratyping reflects some level of structural TCR diversity within a T cell population, it lacks sensitivity because it is not focused on the hypervariable regions of the TCR. In contrast, TCR Vβ-Jβ spectratyping is based on measurement of CDR3 size distribution within a population of T cells, and it provides a more accurate reflection of its clonality. We performed TCR Vβ10-Jβ spectratyping of naive responder T cells that had undergone LIP in the presence or absence of Tregs. We measured spectratypes of CD4, CD8, and unfractionated T cells (bulk) under these conditions. In all cases, the diversity of the TCR repertoire, at least as implied by CDR3 length size distribution, became severely constricted if Tregs were absent during LIP, as measured by the Vβ-Jβ complexity scores and tested with a two-sample t test (Fig. 3). The same result was seen upon additional analysis with a two-factor ANOVA applied to each primer and using the Bonferroni procedure to correct for multiple hypothesis testing. Visual inspection of spectratyping histograms from several TCR Vβ families also allowed us to see actual “holes” in the reconstituted repertoire, as defined by specific Jβ-chain representation below our spectratyping limit of detection (Fig. 4). These “holes” appeared only if Tregs were absent during LIP. In contrast, relative preservation of TCR complexity was noted if Tregs were present during LIP (Fig. 4).

FIGURE 4.

Repertoire “holes” develop during LIP in the absence of Tregs. Three T cell populations were adoptively transferred into individual RAG−/− mice: CD4 T cells (CD8CD44loCD25), CD8 T cells (CD4CD44loCD25), or bulk T cells (CD44loCD25). Representative CDR3 length histograms for individual Vβ10-Jβ TCR segments are shown. A and B represent different experiments. Histograms from the same individual animal are shown per experiment and the specific experimental condition.

FIGURE 4.

Repertoire “holes” develop during LIP in the absence of Tregs. Three T cell populations were adoptively transferred into individual RAG−/− mice: CD4 T cells (CD8CD44loCD25), CD8 T cells (CD4CD44loCD25), or bulk T cells (CD44loCD25). Representative CDR3 length histograms for individual Vβ10-Jβ TCR segments are shown. A and B represent different experiments. Histograms from the same individual animal are shown per experiment and the specific experimental condition.

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The value of a diverse, polyclonal T cell repertoire is best appreciated in the context of infection by a microbial pathogen in which pathogen-specific T cells are required for protection. Therefore, we wished to test the biological significance of LIP-associated TCR repertoire constriction in a model of infectious disease, such as infection with an intracellular pathogen L. monocytogenes. Specifically, we used the infection model with the attenuated L. monocytogenes engineered to express two nominal Ags: the 2W1S peptide along with a truncated portion of OVA containing the 257–264 epitope (ActA LM-2W1S-OVA) (26). The infection with this recombinant strain of L. monocytogenes allows tracking of Ag-specific CD4 and CD8 T cell responses using magnetic bead-based MHC tetramer enrichment and multiparameter flow cytometry within the endogenous T cell repertoire (26, 28, 29). Briefly, magnetic enrichment ensures that all of the epitope-specific T cells within the collected lymphoid tissues of the animal can be concentrated into a single sample and analyzed in its entirety on a flow cytometer. The technique is illustrated in Fig. 5; Ag-specific CD4 and CD8 are enumerated before and postinfection of wild-type C57BL/6 mice with ActA LM-2W1S-OVA.

Extensive TCR sequence analysis by Casrouge et al. (32) demonstrated that, in steady-state, a mouse spleen contains ∼2 × 106 distinct naive αβ TCR clones. Each T cell clone might be able to recognize more than one Ag, and it was estimated that lymphoid tissues of a mouse contain 50–500 distinct T cell clones in the naive T cell repertoire able to recognize a specific peptide/MHC epitope (33). Work published by Moon et al. (28), using tetramer-based enrichment of epitope-specific T cells, estimated the number of IAb-2W1S–specific CD4+ cells in a naive C57BL/6 mouse to be ≥200. Using the same basic methodology, the naive (precursor) frequency of Kb-OVA–specific CD8+ cells was found by Obar et al. (34) to be ∼100 cells per mouse. Therefore, one would expect that adoptive transfer of 1 × 106 naive donor T cells into RAG−/− or TCRα−/− recipients should introduce ∼2–4 T cells with specificity for Kb-OVA and IAb-2W1S, respectively. However, our hypothesis predicts that the fate of these naive precursor cells during LIP may be different, depending on the presence or absence of Tregs. Specifically, we thought that because these T cells have foreign Ag specificity, they would be less likely to undergo spontaneous LIP and would, in fact, benefit from suppression of spontaneous LIP by Tregs.

To test the hypothesis, we reconstituted RAG−/− or TCRα−/− mice by adoptive transfer of Thy1.2 CD25CD44low T cells. Some of the recipient animals were pretransferred with Thy1.1 CD25+ Tregs, and some were not. After 30 d of reconstitution, the mice were infected with ActA LM-2W1S-OVA. The effect of the variable that we wanted to test was the presence or absence of Tregs during LIP. Obviously, the presence or absence of Tregs during the infection could also alter the T cell response. Therefore, we added two additional experimental groups. Some of the mice that received Tregs before the adoptive transfer of naive responder T cells were treated with depleting anti-Thy1.1 Ab several days before the infection was introduced. In this group, transferred Tregs were present only during immune reconstitution but not during the infection. Another group of mice was adoptively transferred Tregs only after immune reconstitution was complete, so that Tregs would be absent during LIP but present during the infection (Fig. 6A).

FIGURE 6.

Presence of Tregs during LIP preserves the capacity of the T cell population to respond to foreign Ags. RAG−/− mice were adoptively transferred with naive Thy1.2 T cells on day 0 and infected with the attenuated L. monocytogenes bacteria expressing 2W1S and OVA peptides on day 30. The lymphoid tissues were harvested for analysis on day 40. The four experimental groups differed by the timing and presence of adoptively transferred Thy1.1 Tregs: no Tr (no Tregs were transferred); Tr late (Tregs transferred on day 28); Tr early + depletion (Tregs transferred on day −7 and depleted with anti-Thy1.1 mAb on day 28); and Tr early (Tregs transferred on day −7). A, Representative flow cytometry dot plots show the percentages of Ag-specific CD4 T cells (CD44highIAb2W1Stetramer+) and CD8 T cells (CD44highKbOVAtetramer+) in the left and middle columns, respectively. The right column represents Thy1.1 staining, which marks adoptively transferred Tregs. The dot plots show samples from one representative experiment in which two animals per group were used to enumerate the Ag-specific T cells by magnetic bead enrichment and FACS analysis. B, Enumeration of Ag-specific CD4 T cells (top panels) and CD8 T cells (bottom panels) from all individual experiments are shown. The left panels show the total numbers of non-Treg CD4 (top panel) and CD8 (bottom panel) T cells from the reconstituted RAG−/− animals and wild-type mice infected with the attenuated L. monocytogenes. The right panels show the numbers of Ag-specific CD4 (top panel) and CD8 (bottom panel) T cells. Statistical significance (p < 0.05) between indicated groups is represented by the horizontal bars at the top of each panel.

FIGURE 6.

Presence of Tregs during LIP preserves the capacity of the T cell population to respond to foreign Ags. RAG−/− mice were adoptively transferred with naive Thy1.2 T cells on day 0 and infected with the attenuated L. monocytogenes bacteria expressing 2W1S and OVA peptides on day 30. The lymphoid tissues were harvested for analysis on day 40. The four experimental groups differed by the timing and presence of adoptively transferred Thy1.1 Tregs: no Tr (no Tregs were transferred); Tr late (Tregs transferred on day 28); Tr early + depletion (Tregs transferred on day −7 and depleted with anti-Thy1.1 mAb on day 28); and Tr early (Tregs transferred on day −7). A, Representative flow cytometry dot plots show the percentages of Ag-specific CD4 T cells (CD44highIAb2W1Stetramer+) and CD8 T cells (CD44highKbOVAtetramer+) in the left and middle columns, respectively. The right column represents Thy1.1 staining, which marks adoptively transferred Tregs. The dot plots show samples from one representative experiment in which two animals per group were used to enumerate the Ag-specific T cells by magnetic bead enrichment and FACS analysis. B, Enumeration of Ag-specific CD4 T cells (top panels) and CD8 T cells (bottom panels) from all individual experiments are shown. The left panels show the total numbers of non-Treg CD4 (top panel) and CD8 (bottom panel) T cells from the reconstituted RAG−/− animals and wild-type mice infected with the attenuated L. monocytogenes. The right panels show the numbers of Ag-specific CD4 (top panel) and CD8 (bottom panel) T cells. Statistical significance (p < 0.05) between indicated groups is represented by the horizontal bars at the top of each panel.

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We found that the total numbers of conventional T cells in the lymphoid tissues after LIP and L. monocytogenes infection were identical (Fig. 6B, left panels). However, the percentages (Fig. 6A) and absolute numbers of IAb-2W1S–specific CD4 T cells and Kb-OVA–specific CD8 T cells (Fig. 6B, right panels) revealed that the presence of Tregs during LIP significantly enhanced the expansion of Ag-specific T cells. In fact, the percentage of Ag-specific T cells in reconstituted RAG−/− mice that had Tregs present during LIP was similar to that seen in wild-type animals, although the absolute numbers were somewhat lower, reflecting the fact that reconstituted RAG−/− animals lack the truly naive T cell compartment. Depletion of Tregs by anti-Thy1.1 mAb after completion of LIP did not impact the expansion of Listeria-specific responder T cells during the infection. In contrast, RAG−/− mice allowed to reconstitute in the absence of Tregs had very few Listeria-specific T cells, and there was no significant difference between animals that never received Tregs and those that received Tregs after LIP was completed. Virtually all measured pathogen-specific T cells derived from the naive responder T cell population and not the Thy1.1+ Tregs, as documented by tetramer staining (Fig. 7). This observation is consistent with the results reported previously, whereby infection by the same attenuated strain of Listeria used in our experiments did not cause expansion of Listeria-specific Foxp3+ T cells (26). Furthermore, the conversion rate of naive CD4 T cells into Foxp3+ T cells was minimal (Table I), and the Treg compartment was dominated by adoptively transferred Thy1.1 T cells.

FIGURE 7.

Few Tregs are specific for IAb-2W1S. Adoptively transferred Tregs were identified by congenic marker Thy1.1 in the indicated experimental groups of reconstituted RAG−/− mice infected with the attenuated L. monocytogenes (top panel). IAb-2W1S-specific T cells were identified using magnetic bead enrichment and tetramer staining for flow cytometry (bottom panel).

FIGURE 7.

Few Tregs are specific for IAb-2W1S. Adoptively transferred Tregs were identified by congenic marker Thy1.1 in the indicated experimental groups of reconstituted RAG−/− mice infected with the attenuated L. monocytogenes (top panel). IAb-2W1S-specific T cells were identified using magnetic bead enrichment and tetramer staining for flow cytometry (bottom panel).

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Table I.
LIP and subsequent bacterial infection do not induce significant peripheral conversion of conventional T cells to Foxp3+ cells
SamplesThy1.1+ That Are Foxp3+ (%)Thy1.1+Foxp3+ T Cells (n)Thy1.2+ That Are Foxp3+ (%)Thy1.2+Foxp3+ T Cells (n)Foxp3+ of Total CD4 T Cells (%)Foxp3+ (%)Foxp3+ T Cells (n)
LIP sample 
 Tr early 56.4 ± 6.0 247,526 ± 106,369 1.9 ± 0.1 19,666 ± 13,161 18.5 ± 3.0   
 Tr late 61.5 ± 9.6 93,409 ± 37,418 0.7 ± 0.4 12,463 ± 2,280 4.6 ± 0.4   
 No Tr NA NA 1.6 ± 0.1 50,595 ± 43,626 1.6 ± 0.1   
 Tr early + depletion 25.5 ± 4.4 358 ± 325 5.3 ± 2.8 247,743 ± 239,088 5.2 ± 2.8   
Wild-type sample 
 LM only 6.9 ± 2.5 637,252 ± 329,660 
 No Tx 10.0 ± 4.0 1,357,831 ± 939,317 
SamplesThy1.1+ That Are Foxp3+ (%)Thy1.1+Foxp3+ T Cells (n)Thy1.2+ That Are Foxp3+ (%)Thy1.2+Foxp3+ T Cells (n)Foxp3+ of Total CD4 T Cells (%)Foxp3+ (%)Foxp3+ T Cells (n)
LIP sample 
 Tr early 56.4 ± 6.0 247,526 ± 106,369 1.9 ± 0.1 19,666 ± 13,161 18.5 ± 3.0   
 Tr late 61.5 ± 9.6 93,409 ± 37,418 0.7 ± 0.4 12,463 ± 2,280 4.6 ± 0.4   
 No Tr NA NA 1.6 ± 0.1 50,595 ± 43,626 1.6 ± 0.1   
 Tr early + depletion 25.5 ± 4.4 358 ± 325 5.3 ± 2.8 247,743 ± 239,088 5.2 ± 2.8   
Wild-type sample 
 LM only 6.9 ± 2.5 637,252 ± 329,660 
 No Tx 10.0 ± 4.0 1,357,831 ± 939,317 

Data represent mean ± variance of two independent experiments with two animals per group, conducted as described in Fig. 6.

LM only, L. monocytogenes only; no Tr, no Tregs were transferred; no Tx, no treatment; Tr early, Tregs transferred on day −7; Tr early + depletion, Tregs transferred on day −7 and depleted with anti-Thy1.1 mAb on day 28; Tr late, Tregs transferred on day 28.

Ideally, we would have liked to correlate the adaptive immune response to Listeria with clinical outcomes, such as animal survival or bacterial counts. However, the adaptive immune response is dispensable for clearance of the primary infection by the attenuated organism (data not shown) (26). Nevertheless, production of IFN-γ by adaptive and innate immune systems contributes to clearance of intracellular pathogens, such as L. monocytogenes (3537). Therefore, it would be reasonable to presume that numbers of Listeria-specific CD4 T cells that can produce IFN-γ relate to the immune fitness of an individual animal challenged with such infection. To examine this question, we repeated some experiments described above using TCRα−/− recipients. Once again, animals were reconstituted with conventional naive T cells in the presence or absence of Tregs for 30 d and infected with 107 CFU of ActA LM-2W1S-OVA bacteria. IFN-γ production was stimulated in vivo on day 10 postinfection by i.v. injection of 5 × 107 CFU of live ActA LM-2W1S-OVA 2–3 h prior to harvest of the lymphoid tissues. We found that if Tregs were present during LIP, the reconstituted animals had more Ag-specific CD4 T cells overall, as well as more Ag-specific CD4 T cells capable of producing IFN-γ (Fig. 8).

FIGURE 8.

The presence of Tregs during LIP allows the enrichment of Ag-specific CD4 T cells capable of making IFN-γ following infection. Wild-type mice (right half of each panel) or 30-d reconstituted TCRα−/− mice (left half of each panel) were infected with the attenuated L. monocytogenes bacteria. IFN-γ production by IAb-2W1S–specific T cells was induced by injection of live L. monocytogenes bacteria 2–3 h prior to tissue harvest. The left panel shows the number of total CD4 T cells, the middle panel shows the number of IAb-2W1S–specific CD4 T cells, and the right panel shows the number of IAb-2W1S–specific CD4 T cells producing IFN-γ. Each dot represents lymph node and spleen cells from two animals processed together for each of the two independent experiments.

FIGURE 8.

The presence of Tregs during LIP allows the enrichment of Ag-specific CD4 T cells capable of making IFN-γ following infection. Wild-type mice (right half of each panel) or 30-d reconstituted TCRα−/− mice (left half of each panel) were infected with the attenuated L. monocytogenes bacteria. IFN-γ production by IAb-2W1S–specific T cells was induced by injection of live L. monocytogenes bacteria 2–3 h prior to tissue harvest. The left panel shows the number of total CD4 T cells, the middle panel shows the number of IAb-2W1S–specific CD4 T cells, and the right panel shows the number of IAb-2W1S–specific CD4 T cells producing IFN-γ. Each dot represents lymph node and spleen cells from two animals processed together for each of the two independent experiments.

Close modal

Finally, we tested whether Listeria-specific effector T cells generated within T cell populations that emerged in the course of immune reconstitution in the presence or absence of Tregs were capable of expansion when rechallenged with the pathogen (experiment is outlined in Fig. 9). RAG−/− mice were repopulated with naive Thy1.1 T cells in the presence or absence of Thy1.2 Tregs. After 30 d of reconstitution, all animals were infected with 107 ActA LM-2W1S-OVA bacteria. Ten days postinfection, lymphoid tissues of the animals were harvested, and T cells that derived from the conventional T cell inoculum (Thy1.2 T cells in this experiment) were purified by negative selection. New wild-type CD45.1 congenic recipients received 3 × 106 of these purified T cells (Thy1.1+CD45.2+), and half of the animals were challenged with 107 ActA LM-2W1S-OVA bacteria. Some wild-type recipients did not receive T cells from reconstituted RAG−/− animals and were used simply as wild-type controls. All mice were sacrificed on day 5 after the infection challenge, and lymphoid tissues were harvested for analysis. The results were in keeping with the previous results (Fig. 10). If Tregs were present during LIP, we could detect significant numbers of Thy1.1 CD4 T cells, most of which were specific for the 2W1S epitope. If Tregs were absent during LIP, the numbers of 2W1S-specific CD4 T cells were indistinguishable from the numbers in the preimmune repertoire.

FIGURE 9.

Experimental protocol to test Ag-specific memory T cell responses. In phase I of the experiment, two groups of RAG−/− mice were adoptively transferred with naive T cells: RAG−/− animals with or without pretransfer of Thy1.2 Tregs. After 30 d of reconstitution, these mice were infected with 107 attenuated L. monocytogenes bacteria. Ten days postinfection, the phase I animals were sacrificed, and their Thy1.1 T cells were retransferred into wild-type (Thy1.2/CD45.1 RAG+/+) mice (3 × 106 per recipient). The new recipients were infected or not with 107 attenuated L. monocytogenes bacteria. Magnetic bead enrichment and flow cytometric analysis were used to enumerate Thy1.1 (donor) and Thy1.2 (recipient) IAb-2W1S–specific T cells.

FIGURE 9.

Experimental protocol to test Ag-specific memory T cell responses. In phase I of the experiment, two groups of RAG−/− mice were adoptively transferred with naive T cells: RAG−/− animals with or without pretransfer of Thy1.2 Tregs. After 30 d of reconstitution, these mice were infected with 107 attenuated L. monocytogenes bacteria. Ten days postinfection, the phase I animals were sacrificed, and their Thy1.1 T cells were retransferred into wild-type (Thy1.2/CD45.1 RAG+/+) mice (3 × 106 per recipient). The new recipients were infected or not with 107 attenuated L. monocytogenes bacteria. Magnetic bead enrichment and flow cytometric analysis were used to enumerate Thy1.1 (donor) and Thy1.2 (recipient) IAb-2W1S–specific T cells.

Close modal
FIGURE 10.

Ag-specific memory T cells from reconstituted RAG−/− mice were detected only if Tregs were present during reconstitution. The figure shows the flow cytometric data from the experiment described in Fig. 9. Thy1.1 T cells originated from the reconstituted RAG−/− animals and were within the Thy1.2 wild-type secondary recipients. Ag-specific CD4 T cells are identified with the IAb-2W1S tetramers. Numbers in bold type represent the absolute numbers of indicated cells. The numbers in plain text in the lower right corners represent the percentages within the indicated gates.

FIGURE 10.

Ag-specific memory T cells from reconstituted RAG−/− mice were detected only if Tregs were present during reconstitution. The figure shows the flow cytometric data from the experiment described in Fig. 9. Thy1.1 T cells originated from the reconstituted RAG−/− animals and were within the Thy1.2 wild-type secondary recipients. Ag-specific CD4 T cells are identified with the IAb-2W1S tetramers. Numbers in bold type represent the absolute numbers of indicated cells. The numbers in plain text in the lower right corners represent the percentages within the indicated gates.

Close modal

Since their discovery and characterization, CD25+Foxp3+ Tregs have primarily been noted for their immunosuppressive functions and a critical role in the maintenance of immunologic tolerance (38, 39). However, in recent years, there has been increasing appreciation for more nuanced regulatory functions of these cells, which ultimately enhance the immune response. For example, Tregs can inhibit or delay pathogen clearance, which at first glance may seem detrimental to the host. However, in some models, such as leishmaniasis and schistosomiasis, Ag persistence ensured by Tregs contributes to the maintenance of immunity to reinfection (40, 41). In ocular HSV-1 infection, Tregs inhibited immunopathology caused by inflammatory responses that would otherwise severely disable the host (42). In vaginal HSV-2 infection, ablation of Tregs enhanced the entry and retention of effector immune cells in the draining lymph nodes and reduced their migration into the actual site of infection (vagina), which correlated with increased viral loads and fatality (43). In these examples of infections, Tregs may allow losing a battle with a pathogen, but at the same time they help to win the war. Of course, the paradigm of war does not apply well to most of the activity of the immune system. The majority of the immune effort is concentrated at the mucosal surfaces, where it helps to maintain homeostasis with the commensal microbial flora and aid the physical barrier to exclude pathogen entry. That is one of the main functions of the secreted IgA, for example, which requires TGF-β for class switching, one of the cytokines elaborated by Tregs (44).

At the outset of our experiments, we were intrigued by the differential ability of Tregs to inhibit spontaneous versus homeostatic forms of LIP (16). This finding suggested that Tregs could play another positive role in the function of the immune system. Specifically, we thought that they might optimize the immune fitness of the residual T cell population during its recovery from lymphopenia by shaping its TCR repertoire. Lymphopenia is a common occurrence during the lifetimes of individuals, and peripheral mechanisms of immune reconstitution assume increasing importance with age as the thymus involutes (45). Our experimental adoptive-transfer system models absence of the thymus and allows recovery of the T cell population exclusively by LIP.

We specifically wanted to start out with a minimally restricted potential TCR repertoire to mimic realistic clinical scenarios (e.g., recovery of the T cell population in AIDS patients treated with highly active antiretroviral therapy [HAART]) and to provide an experimental window to measure significant differences. A normal mouse contains ∼2 × 106 T cell clones within its naive TCR repertoire (32), and a starting population significantly below that would ensure severely restricted responsiveness to potential foreign Ags. In contrast, a starting population significant above that would limit LIP because there would be little lymphopenia. We used peptide-MHC tetramers to probe the responsiveness of T cell populations to two foreign peptides: the SIINFEKL peptide from OVA and 2W1S, expressed by an infectious pathogen, L. monocytogenes. Enumeration of Kb-OVA– and IAb-2W1S–specific T cells in the naive repertoire reported previously and reproduced in our own work suggested that adoptive transfer of 1 × 106 T cells would result in the transfer of ∼2–4 T cells per mouse specific for these Ags (28). In fact, it was shown that a single precursor T cell is capable of massive clonal expansion and functional subset diversification following immunization (46). Therefore, we expected to detect significant Ag-specific T cell expansion in all of our experimental conditions in the absence of significant distortion of the T cell repertoire during the period of immune reconstitution.

We found that the greatest numbers of responder foreign Ag-specific T cells recovered postinfection were seen only in mice that had Tregs during the period of LIP. The simplest explanation for this finding is that some Ag-specific precursors from the naive T cell inoculum did not survive the immune reconstitution period in the absence of Tregs. In fact, in some experiments when Tregs were absent during LIP we could not detect Ag-specific responder T cells at all following infection, whereas in others they were markedly decreased in numbers. This idea is also supported by results of characterization of the TCR repertoire obtained by TCR Vβ-Jβ spectratyping, which demonstrated that the absence of Tregs during immune reconstitution correlated with the development of a marked loss of structural TCR diversity. One would predict that the diversity of the few recovered Ag-specific responder T cells from populations established in the absence of Tregs would be extremely constricted. In fact, we performed several preliminary experiments using flow cytometric analysis of Vβ-chain usage by Ag-specific responders, and these were compatible with that prediction (data not shown). Specifically, the Vβ-chain usage of Ag-specific 2W1S-specific responders in mice reconstituted in the presence of Tregs was comparable to the repertoire of 2W1S-specific responders in wild-type mice, whereas, the Vβ-chain repertoire of 2W1S-specific responders in mice reconstituted in the absence of Tregs seemed distorted. However, because of the small numbers of Ag-specific T cells in these mice and their complete absence in some experiments, the total number of mice necessary to characterize the Vβ usage by these cells was prohibitive. It may be interesting to focus future studies on the characteristics of such residual tetramer-positive T cells that persist within the T cell population established by LIP in the absence of Tregs. We might anticipate, for example, that these cells have a greater affinity for self-Ags that provide them with more tonic survival signals. Such cells might have relatively low affinity for foreign Ags that the organism may encounter.

Our results indicate that the beneficial role of Tregs on the Ag-specific T cell response is exerted during the period of LIP. Tregs adoptively transferred after the completion of immune reconstitution had no beneficial effect. Furthermore, if Tregs were depleted following immune reconstitution, the beneficial effect persisted. It should be noted that Tregs were depleted using an Ab directed against a congenic marker, Thy1.1. Although that was very effective in depleting Thy1.1+ T cells, the early presence of Tregs also seemed to encourage some emergence of Foxp3+ T cells derived from the naive responder T cell population (Table I). This might represent an example of infectious tolerance and is consistent with the idea that factors produced by Tregs are somehow needed for de novo peripheral Treg induction (47). However, the effect was relatively small, and the numbers of induced Tregs in this group were considerably smaller than those produced by the late adoptive transfer of Tregs. In both cases, Tregs appearing after the completion of LIP did not enhance the diversity or immune fitness of the reconstituted responder T cell population.

The mechanisms by which Tregs may shape the TCR repertoire during LIP remain unclear. However, a reasonable speculative model can be constructed based on current knowledge of the specific signaling requirements of different T cell subpopulations (i.e., Tregs and T cells undergoing spontaneous versus homeostatic forms of LIP). Although these three T cell subpopulations require or benefit from TCR stimulation, inhibition of spontaneous LIP by Tregs cannot be explained by competition for TCR signaling (16). However, Tregs and T cells undergoing spontaneous LIP benefit from B7 costimulation (3, 15, 16, 48, 49). In contrast, B7 costimulatory signals are not needed for the homeostatic form of LIP, which, in turn, is driven mainly by mere tonic TCR signals and cytokines, such as IL-7 (50). Thus, it is possible that Tregs, which are known to express high levels of CTLA-4, interfere with B7 costimulation of the responder T cells by direct competition or negative signaling into the APCs, leading to B7 downregulation (51). In fact, Sojka et al. (52) recently reported that CTLA-4–deficient Tregs cannot inhibit the spontaneous form of LIP, whereas they remain highly suppressive in vitro. Similarly, we noted that residual spontaneous LIP experienced by CD28-deficient responder T cells cannot be suppressed in the absence of B7 expression on host APCs (data not shown). It is reasonable to speculate that T cells undergoing both forms of LIP use some common resources (e.g., IL-7) which may not be consumed significantly by Tregs (53). Therefore, suppression of the T cells undergoing spontaneous LIP would promote survival and homeostatic proliferation of all of the other T cells by allowing greater access to limiting amounts of IL-7. It is also possible that Tregs and at least some subsets of T cells undergoing spontaneous LIP may compete for additional common resources (e.g., IL-2 and -15) which are known to be important for Tregs and memory CD8 T cells (54, 55), and can drive spontaneous LIP of CD8 T cells (56). Of course, additional mechanisms are possible. It is conceivable that Tregs may selectively identify T cells undergoing spontaneous LIP and suppress them directly via cell-to-cell contact mechanisms, leading to arrest of their cell-cycle progression or the induction of apoptosis. In addition, we cannot exclude the less-likely possibility that Tregs may help to promote survival and homeostatic LIP of T cells that are not recruited into spontaneous LIP by facilitating the production of some positive factors by APCs and other immune cells. Further mechanistic dissection of Treg-mediated control of the T cell repertoire development during immune reconstitution is warranted.

We do not know whether Tregs are uniquely capable of preserving the functionality of the T cell population during immune reconstitution. We hypothesized that Tregs might have this ability because they can selectively inhibit spontaneous LIP, while sparing T cells undergoing homeostatic LIP. In a previous work we showed that bulk CD25 T cell competitors composed of memory and naive T cells could not inhibit spontaneous LIP efficiently (15) or even enhanced spontaneous LIP of CD8 T cell responders (16). However, we have not tested other more narrowly defined T cell subsets. It is now recognized that even Tregs are not uniform or stable in their phenotype (57, 58). For example, a small population of Foxp3+ T cells loses Foxp3 expression following adoptive transfer into lymphopenic hosts and expands (59). These ex-Foxp3+ T cells may come to represent ∼50% of the transferred population in the new hosts (59), which is consistent with our results (Table I), and may be pathogenic themselves (60). It may be interesting for mechanistic investigations to compare stable Foxp3+ T cells and ex-Foxp3+ T cells in their ability to suppress spontaneous LIP and regulate the diversity of the TCR repertoire.

The results presented have potential immediate clinical implications. Massive peripheral immune reconstitution occurs in a number of settings, including the treatment of AIDS with HAART and organ transplantation following cytoablative treatment. These interventions can be associated with potentially fatal inflammatory conditions, such as the immune reconstitution inflammatory syndrome and graft-versus-host disease, which are also associated with immunodeficiency caused by insufficient numbers of Ag-specific T cells and poor T cell function (61, 62). Interestingly, there is at least some evidence that CD4 Tregs are preferentially spared in HIV infection (63). This may be one of the reasons why the risk for immune reconstitution inflammatory syndrome is inversely related to pretreatment CD4 counts (64). In addition, delaying the initiation of HAART and lower pretreatment CD4 T cell counts result in poor functional immune restoration, despite normalization of CD4 T cell numbers (65). Our results suggest that the presence of sufficient numbers of Tregs during immune reconstitution should minimize the risk for triggering autoimmunity and immunopathology caused by opportunistic infections, as well as optimize the future functional capacity of the restored immune system in its responsiveness to infectious challenges. In fact, attempts are underway to introduce human Treg infusion as an adjunct therapy following bone marrow transplantation (66). Of course, multiple practical challenges need to be overcome before the acceptance of adoptive Treg transfers in the clinics. However, it is also possible that the administration of CTLA-4–Ig, which is already in clinical use, during the period of immune reconstitution may have similar beneficial effects on immune restoration.

We are grateful to Drs. Erik Peterson and Daniel Mueller for critical review of the manuscript.

Disclosures The authors have no financial conflicts of interest.

This work was supported in part by National Institutes of Health Grant RO1 DK061961 (to A.K.) and Institutional Immunology Training Grant 5T32AI007313-20 (to C.J.W.).

Abbreviations used in this paper:

HAART

highly active antiretroviral therapy

LIP

lymphopenia-induced proliferation

Treg

regulatory T cell.

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