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The Journal of Immunology, 2008, 180, 7305 -7317
Copyright © 2008 by The American Association of Immunologists, Inc.
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Regulatory CD4+CD25+Foxp3+ T Cells Selectively Inhibit the Spontaneous Form of Lymphopenia-Induced Proliferation of Naive T Cells1

Colleen J. Winstead, Joanne M. Fraser and Alexander Khoruts2

Department of Medicine, University of Minnesota, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory CD4+CD25+Foxp3+ T cells play a critical role in controlling autoimmunity and T cell homeostasis. However, their role in regulation of lymphopenia-induced proliferation (LIP), a potential mechanism for generation of autoaggressive T cells, has been poorly defined. Currently, two forms of LIP are recognized: spontaneous and homeostatic. Spontaneous LIP is characterized by fast, burst-like cell-cycle activity, and may allow effector T cell differentiation. Homeostatic LIP is characterized by slow and steady cell cycle activity and is not associated with the acquisition of an effector phenotype. In this study, we demonstrate that CD4+CD25+Foxp3+ T cells suppress the spontaneous, but not homeostatic, LIP of naive CD8 and CD4 T cells. However, selective inhibition of spontaneous LIP does not fully explain the tolerogenic role of Tregs in lymphopenia-associated autoimmunity. We show here that suppression of LIP in the lymphoid tissues is independent of Treg-derived IL-10. However, IL-10-deficient Tregs are partially defective in their ability to prevent colitis caused by adoptive transfer of CD4 T cells into RAG–/– mice. We propose that Tregs may inhibit emergence of effector T cells during the inductive phase of the immune response in the secondary lymphoid tissues by IL-10-independent mechanisms. In contrast, Treg-mediated inhibition of established effector T cells does require IL-10. Both Treg functions appear to be important in control of lymphopenia-associated autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peripheral regulation of T cell homeostasis is a critical feature of the adaptive immune system, which ensures adequate T cell population size, TCR diversity, responsiveness to foreign Ags, and self-tolerance. Insults to the immune system that result in transient lymphopenia are common events in the lives of vertebrate organisms, and are typically followed by uneventful immune reconstitution. However, in some clinical settings the drive to re-establish the T cell population size may compromise the diversity of the TCR repertoire and allow emergence of autoreactive clones, which in turn may lead to immunodeficiency and autoimmunity (1, 2, 3, 4). Thus, understanding the mechanisms that regulate T cell homeostasis has important clinical applications.

Experimentally, lymphopenia-induced proliferation (LIP)3 of naive T cells is commonly studied using adoptive transfer systems with T cell-deficient recipients. Recently, two forms of LIP have been defined: homeostatic and spontaneous (5). Homeostatic LIP is slow and dependent on IL-7. In contrast, spontaneous LIP is rapid and independent of IL-7. Self-peptide/MHC ligand stimulation benefits both forms of LIP. However, the two forms of LIP may differ in the strength of TCR stimulation experience. In general, T cells with higher avidity for self-ligands undergo more vigorous LIP (6, 7, 8). Thus, it is possible that the experience of T cells undergoing spontaneous LIP may be comparable to full cognate antigenic stimulation, which benefits from CD28 costimulation (9, 10, 11, 12, 13). In fact, the highly divided T cells emerging from LIP preferentially acquire the ability to produce effector cytokines and migrate into peripheral tissues (5, 9, 10, 11, 12, 13). This phenotype, combined with the likely higher degree of self-reactivity among the high dividers, suggests that the majority of autoreactive pathogenic T cell clones are generated by spontaneous proliferation during LIP.

Although competition for positive resources such as cytokines and self-ligands in T cell homeostasis has been studied in some detail, much less is known about potential inhibitory factors. Regulatory CD4+CD25+Foxp3+ T cells (Tregs) have long been suspected to play a role in T cell homeostasis, and have been demonstrated to be suppressive in multiple animal models of autoimmunity that involve lymphopenic hosts. However, their role in controlling LIP has been unclear and controversial (14). Initial studies failed to demonstrate an effect of cotransfer of Tregs on the LIP of CFSE-labeled CD4+CD25– T cells as measured by dilution of the dye (15). Other studies noted that cotransfer of Tregs markedly decreased the homeostatic plateau population size of CD4+CD25– T cells, but allowed at least some LIP (16, 17). Finally, recent studies demonstrated that Tregs can inhibit proliferation, survival, and differentiation of CD4+CD25– responder T cells following adoptive transfer into lymphopenic hosts (9, 18).

Failure to distinguish between homeostatic and spontaneous forms of LIP is one potential reason for conflicting reports on the suppressive potential of Tregs. In this study, we report that Tregs can selectively inhibit the spontaneous form of LIP, but have little effect on the homeostatic form. This suppressive ability cannot be attributed to mere competition for TCR signals, because CD4 Tregs inhibit spontaneous LIP of CD8 T cells, which receive TCR signals from MHC class II and I molecules, respectively. Previously, IL-10-deficient Tregs have been reported to be unable to inhibit expansion of CD4 T cells in lymphocyte-deficient RAG–/– hosts (16). Therefore, we tested whether Treg-derived IL-10 could mediate inhibition of LIP. We found that IL-10-deficient and wild-type Tregs are indistinguishable in their ability to suppress LIP within secondary lymphoid tissues. Interestingly, IL-10-deficient Tregs were only partially effective in preventing colitis caused by CD4 T cell responders. Incomplete suppressive ability of IL-10-deficient Tregs is consistent with conflicting earlier reports on the requirements for Treg-derived IL-10 for the control of colitis (17, 19). Our results suggest a two-step model for the role of Tregs in lymphopenia-associated autoimmunity. First, by selectively inhibiting spontaneous LIP, Tregs limit the emergence of pathogenic T cells. Second, Tregs act within sites of inflammation to suppress activity of pathogenic T cells that do escape into the periphery. The two suppressive functions appear to be mediated by different mechanisms, and both may be required for optimal control of autoimmunity associated with lymphopenia.


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

C57BL6 (B6) and CD45.1 congenic mice were purchased from the National Cancer Institute (Frederick, MD). Recombinase-deficient (RAG-1–/–; hereafter referred to as RAG–/–), Thy1.1 congenic, and IL-10–/– mice on the B6 background were obtained from The Jackson Laboratory. RAG–/– OT-1 TCR-transgenic mice (Tg) (20) were bred onto CD45.1 and Thy1.1 congenic backgrounds. All mice used were generally 6–12 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. All experiments were approved by the University of Minnesota Institutional Animal Care and Use Committee.

Adoptive transfer and cell preparations

Unless otherwise specified, donor T cells were collected from secondary lymphoid tissues (axillary, brachial, cervical, mesenteric, and inguinal lymph nodes, and spleen). CD44low CD8 and control CD44low CD4 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, and B220 (all Abs labeled with FITC) using anti-FITC BioMag particles (Polysciences), and in some cases also anti-CD25-, glucocorticoid-induced TNFR (GITR), and CD103-FITC, followed by depletion of CD44high cells using magnetic microbeads (Miltenyi Biotec), as previously described (21). Briefly, the purified CD8 or CD4 T cells were suspended in labeling buffer (2% FCS in PBS), incubated with 0.004 µg of anti-CD44-FITC (eBioscience) per 106 cells for 15 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). RAG–/– OT-1 lymph nodes and spleens or lymph nodes alone were harvested, and purified CD8 T cells were labeled with CFSE and i.v. injected into host mice. Labeling with CFSE was done using a technique described previously (22).

FACS analysis

Mice were sacrificed and perfused with PBS before removal of lymph nodes, spleen, liver, lungs, and colons. All secondary lymphoid tissues were disrupted by mashing with a syringe. Livers and lungs were digested in collagenase D (Roche Applied Science), 400 U/ml–1 for 30 min at 37°C. In some cases, the collagenase medium was supplemented with 10 µg/ml–1 brefeldin A (Sigma-Aldrich) to prevent lymphokine secretion. Liver cells were separated using a 40/100 Percoll gradient, and the hematopoietic cells were collected from the interface. Colons were washed with PBS, cut into small segments, and digested before staining for FACS. Intracellular staining for cytokines was done following stimulation with PMA/ionomycin and fixation. Specific T cell subsets were identified using fluorochrome-labeled Abs against CD4, CD8, and congenic markers such as anti-Thy1.1/Thy1.2 and anti-CD45.1/CD45.2. All anti-cytokine Abs (IL-2, IL-17A, IFN-{gamma}, and isotype controls) were purchased from eBioscience. Granzyme B was stained intracellularly using anti-human granzyme B Abs (Caltag Laboratories). Staining for Foxp3 was done using an eBioscience kit and instructions provided by the manufacturer. Absolute numbers of T cells within various tissues were calculated using PKH reference beads (Sigma-Aldrich). Relative numbers of CD4 T cells of specific subtype were calculated in comparison to the number of total CD4 T cells in recipient groups that did not receive Tregs.

Induction and assessment of colitis

Colitis was induced by i.v. adoptive transfer of 1 x 105 CD4+CD25–CD103–CD44lowGITRlow cells purified using magnetic microbeads from wild-type donor mice, as described above, into RAG–/– animals. For some of the recipient mice, 0.5–2 x 106 CD4+CD25+ Tregs were transferred 1 wk before the transfer of naive CD4 T cells, unless indicated otherwise. The animals were monitored clinically by weights and signs of colitis and systemic toxicity. After sacrifice, a 1-cm segment of the distalmost part of the colon was removed and fixed in 10% buffered formalin. Paraffin-embedded sections were cut and stained with H&E. Severity of colitis was scored in a blinded fashion according to a scale similar to that previously described (19): grade 0, normal histologic appearance; grade 1, minimal scattered inflammatory cell infiltrates within the lamina propria, with or without minimal epithelial hyperplasia; grade 2, mild to moderate, scattered inflammatory infiltrates, sometimes extending into the submucosa, with mild to moderate epithelial hyperplasia and mild to moderate depletion of mucin from goblet cells; grade 3, moderate inflammation in the lamina propria, sometimes transmural, occasional crypt abscesses, moderate to severe epithelial hyperplasia and mucin depletion; grade 4, severe inflammatory infiltration of lamina propria, transmural inflammation, abundant crypt abscesses, occasional ulceration, marked epithelial hyperplasia, and mucin depletion; grade 5, severe inflammation with diffuse loss of epithelium.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Tregs inhibit spontaneous LIP of naive CD8 T cells

Adoptive transfer of naive CD4 or CD8 T cells into lymphocyte-deficient recipient animals initiates their proliferation. The use of CFSE-labeled T cells in adoptive transfer protocols allows their proliferation to be easily measured by dilution of the CFSE dye (22). Two forms of LIP are currently recognized and are typically observed in animals where lymphopenia is caused by genetic disruption of T cell development: "homeostatic" and "spontaneous" (5). The rapid, burst-like proliferation characteristic of spontaneous LIP leads to loss of detectable CFSE in responder T cells (CFSE–). In contrast, T cells undergoing homeostatic LIP, which is slow and steady, have detectable CFSE (CFSE+). To demonstrate these two forms of LIP, we measured CFSE content subsequent to adoptive transfer of CFSE-labeled naive CD8 T cells into lymphocyte-deficient RAG–/– mice. We used two different responder CD8 T cell populations: naive polyclonal T cells and monoclonal OT-1 TCR Tg RAG–/– T cells (OT-1 T cells). The two distinct forms of LIP were exhibited by both responder populations. Ten days after transfer, two-thirds of polyclonal CD8 responder T cells had lost detectable CFSE signal, indicating that they were undergoing spontaneous LIP (Fig. 1A, left panel). A significant fraction of OT-1 T cells had also lost detectable CFSE signal 8 days following adoptive transfer into RAG–/– mice, although the majority of these responders retained measurable CFSE content characteristic of homeostatic LIP (Fig. 1A, right panel).


Figure 1
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  		FIGURE 1. Naive polyclonal and monoclonal transgenic OT-1 CD8 T cells undergo homeostatic and spontaneous forms of LIP. OT-1 RAG–/– or CD44lowCD8 T cells were CFSE-labeled and injected in varying numbers into RAG–/– recipients on day 0. Lymph nodes were harvested and analyzed by FACS on day 8 (OT-1 cells) or 10 (polyclonal cells). CD8 responders were gated using CD45.1 or Thy1.1 congenic markers and CD8. A, Representative CFSE profiles of CD8 responder T cells in the lymph nodes from individual animals following adoptive transfer of 1 x 106 cells. The arrows within histograms indicate the control location of the signal generated by control, undivided T cells 1 day after transfer. B, Data shown are the mean percentage of responder T cells that have lost detectable CFSE signal ± variance of two animals per group. Two separate experiments are shown for both CD8 responder populations.

 
T cells undergoing spontaneous LIP have been shown to promptly acquire properties of memory T cells as defined by surface markers, cytokine production potential, and enhanced ability to traffic into peripheral tissues (9, 11, 12, 23, 24). It is likely that this fraction of T cells experiences activation similar to that of cognate Ag stimulation, and is enriched for autoreactive specificities. Therefore, we wished to test whether Tregs have different effects on the two different forms of LIP. We focused the initial studies on CD8 responder T cells to avoid effects of Tregs that could be attributed to competition for self-ligand stimulation.

To measure LIP, we transferred varying numbers of CFSE-labeled polyclonal naive CD8 T cells or monoclonal OT-1 T cells into RAG–/– recipients. Eight or 10 days later, we measured the CFSE dye content of adoptively transferred T cells in the secondary lymphoid tissues. Interestingly, regardless of the number of transferred polyclonal CD8 T cells, we always observed a similar percentage of CFSE– T cell responders. (Fig. 1B, left panel). However, in the case of OT-1 T cells, the emergence of the CFSE– fraction was more sensitive to the input number, and it could not be detected if we transferred >5 x 106 T cells per recipient (Fig. 1B, right panel). The high input of OT-1 T cells only allowed homeostatic LIP to take place. This observation is consistent with the idea that spontaneous LIP requires strong TCR stimulation, which can become limiting for large numbers of monoclonal T cells competing for the same peptide/MHC signals.

Next, we tested the effects of Treg cells on LIP by first transferring Treg cells into RAG–/– recipients, then 1 wk later transferring CFSE-labeled naive CD8 T cell responders into the same recipients, RAG–/– mice that had not received a pretransfer of Treg cells, or wild-type mice. Presence of Tregs greatly decreased the proportion of CFSE– T cells arising from the polyclonal input CD8 T cell population or OT-1 cells transferred in relatively low numbers (Fig. 2, A, left and middle panels, and B, top left and middle panels). In contrast, Tregs had little effect on the CFSE content of T cells undergoing the slower homeostatic form of LIP (Fig. 2B, lower left and middle panels). Furthermore, Tregs did not inhibit LIP of OT-1 T cells transferred at high input number (Fig. 2, A, right panel, and B, bottom right panel), a condition that only allows the homeostatic form of LIP. These results indicated that Tregs selectively inhibited emergence of the CFSE– fraction, but had no effect on the profile of the CFSE+ responders.


Figure 2
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  		FIGURE 2. Tregs selectively suppress the spontaneous form of LIP experienced by naive CD8 T cells. CD25+ cells were injected via tail vein into RAG–/– recipients on day –7 (2 x 106 cells/animal). Approximately 1 x 107 OT-1 RAG–/– (high transfer) or 1 x 106 (low transfer) each of OT-1 or naive polyclonal CD8 T cells were CFSE-labeled and injected on day 0 into the same recipients, RAG–/– mice that had not received CD25+ cells on day –7, or wild-type mice. Lymph nodes were harvested for FACS analysis on day 10. Responder T cells were gated using staining for CD45.1 or Thy1.1 congenic markers and CD8. A, Representative CFSE profiles of responding CD8 T cells in the lymph nodes from individual animals. Solid line histograms show CFSE content of CD8 T cells in RAG–/– animals in absence of Tregs. Stippled line histograms show CFSE content of CD8 T cells in RAG–/– animals in the presence of Tregs. Shaded histograms show CFSE content of CD8 T cells transferred into wild-type recipients. Similar results were obtained from more than two independent experiments (two or more individual mice per experiment). B, The data for the RAG–/– recipient mice were analyzed for percentage of CFSE– fractions in different responder CD8 T cell populations, as well as the CFSE content in the CFSE+ CD8 T cells, a measure of overall cell cycle activity in this subpopulation of responders, represented by the mean fluorescence intensity (MFI). Each bar represents the mean ± SD of four animals. Statistical significance was determined using the two-tailed Student’s t test, comparing the group with pretransfer of Tregs with the control group without pretransfer of Tregs. Stars represent a statistical significance with p < 0.05.

 
In addition, we tested the effects of Tregs on some functional characteristics of responder CD8 T cells undergoing LIP. This included expression of granzyme B and homing into peripheral tissues. We first transferred Treg cells into RAG–/– recipients, then one week later transferred CFSE-labeled naive polyclonal CD8 T cell responders into the same recipients or into RAG–/– mice that had not received a pretransfer of Treg cells. In the absence of Tregs, the CFSE– fraction formed the majority in the secondary lymphoid tissues and was overwhelmingly dominant in nonlymphoid tissues such as liver and lung (Fig. 3A, top panels). In the presence of Tregs the fraction of CFSE– T cells significantly diminished (Fig. 3, A, bottom panels, and B, top panels), and the total numbers of CFSE– T cells decreased more than an order of magnitude in both lymphoid and nonlymphoid tissues (Fig. 3C, left panel). In contrast, the numbers of CFSE+ T cells were not altered by the presence of Tregs (Fig. 3C, right panel). Furthermore, the presence of Tregs did not alter the CFSE content of CFSE+ T cells in the lymphoid tissues, although it did seem to increase the CFSE content of CFSE+ T cells slightly in the nonlymphoid tissues (Fig. 3B, bottom panel). Notably, only the CFSE– fraction of polyclonal CD8 T cells expressed significant levels of granzyme B (Fig. 3A), suggestive of their more differentiated, effector phenotype (25). Interestingly, both low and high dividers found in the liver in the presence of Tregs expressed granzyme B, which was not the case for other peripheral tissue sites, such as lungs (Fig. 3A, rightmost lower panel). This may relate to the observation of liver being a site of accumulation for effector T cells destined to undergo apoptosis (26).


Figure 3
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  		FIGURE 3. Tregs suppress expression of effector characteristics by CD8 T cells undergoing spontaneous LIP. Approximately 2 x 106 CD25+ cells were transferred into RAG–/– recipients on day –7. Approximately 3 x 106 naive polyclonal CD8 T cells were CFSE-labeled and injected on day 0 into the same recipients (or into RAG–/– mice that had not received CD25+ cells on day –7). Tissues were harvested on day 10, stained for granzyme B, and analyzed by FACS. A, Each plot is gated on the responder CD8 T cells and represents data from a single animal. The vertical line in each plot represents separation between CFSE– and CFSE+ responder T cells. Numbers in plots represent percentages of CFSE– cells in the sample. B, Data from two independent experiments were pooled and analyzed for percentage of CFSE– cells in different responder CD8 T cell populations. CFSE content in the CFSE+ fraction is represented by the MFI. Each bar represents the mean of four animals ± SD. Statistical significance was determined using the two-tailed Student’s t test. Stars represent a statistical significance with p < 0.05. C, Absolute number of CFSE– and CFSE+ responder CD8 T cells in the indicated tissues. Green bars represent the number of responder cells in absence of Tregs, while red bars represent the number of responder cells in the presence of Tregs.

 
Naive CD4+CD25– T cells enhance spontaneous LIP of naive CD8 T cells

We considered that the observed inhibition of spontaneous LIP by Tregs may be due to consumption of some limiting resources, such as cytokines, that are common to all T cells. Thus, we next tested the effects of conventional CD4 T cells on LIP of naive CD8 T cells by first transferring naive CD4+CD25– T cells into RAG–/– recipients, then 1 wk later transferring CFSE-labeled naive polyclonal CD8 or OT-1 responders into the same recipients or into RAG–/– mice that had not received a pretransfer of CD4 cells. The conventional CD4 T cells rapidly expanded during the week before the transfer of CD8 T cells to match or exceed the numbers of cells we observed following Treg transfers (data not shown). Notably, the mice did not display any clinical signs of autoimmunity, such as colitis, that may be seen over a longer time course (27). In contrast to the effects we observed with Tregs, we found that the conventional CD4 T cells actually enhanced spontaneous proliferation of CD8 T cells. In fact, on day 10 the CFSE– fraction dwarfed the CFSE+ population in mice that received conventional CD4 T cells (Fig. 4A), and the total numbers of CD8 T cells were considerably greater in these animals compared with animals that did not receive a pretransfer of conventional CD4 T cells (Fig. 4B, center panel). The results were similar for polyclonal and OT-1 CD8 T cell responders. Furthermore, CD8 T cells that underwent spontaneous LIP in the presence of conventional CD4 T cells also benefited in terms of differentiation, as measured by greater expression of granzyme B (Fig. 4B, right panel). This differentiation was evident even for highly divided OT-1 cells, which express only minimal levels of granzyme B in absence of CD4 help (Fig. 4B, right panel). Interestingly, the CFSE content of the CFSE+ CD8 T cells was greater in the presence of conventional CD4 T cells (Fig. 4, A and B, left panel), suggesting that the two T cell populations do compete for common resources that are important for the homeostatic form of LIP.


Figure 4
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  		FIGURE 4. CD4+CD25– T cells enhance spontaneous LIP of CD8 T cells. Approximately 2.5 x 105 naive CD4+CD25– T cells were transferred into RAG–/– recipients on day –7. Approximately 1 x 105 OT-1 RAG–/– or naive polyclonal CD8 T cells were CFSE-labeled and injected into the same recipients on day 0 (or into RAG–/– mice that had not received CD4+CD25– cells on day –7). Lymph node cells were harvested on day 10 and stained for granzyme B, and analyzed by FACS. A, Representative CFSE profiles of responding CD8 T cells in the lymph nodes from individual animals. Histograms are shown for total (upper panels) and only the CFSE+ (lower panels) responder CD8 T cells. Solid line histograms show CFSE content of responders in RAG–/– animals in the absence of CD4 T cells, and stippled line histograms show CFSE content of responders in RAG–/– animals in the presence of CD4 T cells. B, Left panel, The CFSE content of CD8 T cells undergoing homeostatic proliferation (CFSE+ cells), represented by the MFI, in the presence or absence of CD4 T cells. Center panel, The absolute number of total CD8 T cells in the presence or absence of CD4 T cells. Right panel, The fraction of CD8 responders undergoing spontaneous proliferation (CFSE–) (Figure 4), and the subfraction of these that express granzyme B ({blacksquare}). Each bar represents the mean ± SD of three animals. Statistical significance was determined using the two-tailed Student t test comparing groups with pretransfer of CD4 T cells with the control groups without pretransfer of CD4 T cells. Stars represent a statistical significance with p < 0.05.

 
IL-10 production by Tregs is not required to inhibit LIP of CD8 T cells

Next, we wished to probe the mechanism of Treg-mediated inhibition of spontaneous LIP. Although multiple mechanisms for suppressive function of Tregs have been proposed to operate in different systems, these mechanisms remain very poorly understood. We decided that IL-10 production by Tregs is one important mechanism to explore, because it has been reported that IL-10-deficient Tregs cannot inhibit expansion of CD4 T cells following adoptive transfer into RAG–/– hosts (16). Thus, we tested whether IL-10-deficient Tregs could inhibit LIP of naive CD8 T cells. Because IL-10-deficient mice develop inflammatory bowel disease over time, we exclusively used relatively young mice at 6 wk of age or less as donors of Tregs. Analyses of Tregs from the lymph nodes and spleens of IL-10-deficient mice and wild-type mice indicated that the purity of Tregs isolated from both donors and the levels of Foxp3 expression in both types of mice were comparable (Fig. 5A). In addition, wild-type and IL-10-deficient Tregs expressed equivalent levels of CTLA-4, GITR, and CD69 (Fig. 5B), and had similar ability to undergo their own homeostatic proliferation as evidence by comparable CFSE profiles (Fig. 5C, left panel), and numbers of cells recovered following expansion (Fig. 5C, right panel).


Figure 5
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  		FIGURE 5. Tregs from young wild-type and IL-10–/– mice are phenotypically similar. A, FACS analysis of suspensions of pooled lymph nodes and spleens from four wild-type and four IL-10–/– mice before and after purification by positive selection with anti-CD25 mAb (PC61). Numbers in plots represent gated cells as a percent of total. B, Histograms show surface staining for CD69, GITR, and intracellular staining for CTLA-4 in wild-type (blue lines) and IL-10–/– (red lines) Tregs isolated as described in A, and Foxp3– CD4 T cells (shaded). C, Approximately 6 x 105 wild-type or IL-10–/– CD25+ cells were CFSE-labeled and injected into RAG–/– recipients on day 0. Lymph nodes were harvested on day 7 and analyzed by FACS. Histograms show representative CFSE profiles of wild-type (blue histograms) and IL-10–/– (red histograms) Tregs transferred into RAG–/– recipients. The bar graph represents absolute numbers of CD4+Foxp3+ cells in the lymph nodes (pooled axial, brachial, inguinal, and mesenteric) of RAG–/– animals that received either wild-type or IL-10–/– CD25+ cells.

 
After confirming that Tregs from IL-10-deficient mice were phenotypically similar to those from wild-type mice, we examined the effects of IL-10–/– Tregs on LIP of naive polyclonal CD8 T cells in RAG–/– mice. We first transferred wild-type or IL-10–/– Treg cells into RAG–/– recipients, then 1 wk later transferred CFSE-labeled naive CD8 T cell responders into the same recipients or into RAG–/– mice that had not received a pretransfer of Treg cells. We found that wild-type and IL-10-deficient Tregs were comparable in their ability to suppress spontaneous LIP of CD8 T cells, as evidenced by similarly decreased fraction of CFSE– responders in presence of either type of Tregs (Fig. 6A). The suppression resulting in decreased total numbers of CD8 T cells in the presence of either wild-type or IL-10-deficient Tregs in the secondary lymphoid tissues was even more dramatic in the periphery, where in absence of Tregs the overwhelming majority of responders belonged to the CFSE– fraction (Fig. 6B). The experiments suggest that inhibition of spontaneous LIP by Tregs within the lymphoid tissues limits emergence of effector T cells capable of trafficking into the periphery.


Figure 6
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  		FIGURE 6. Suppression of CD8 T cell spontaneous LIP by Tregs is not IL-10 dependent. Approximately 2 x 106 wild-type or IL-10–/– CD25+ cells were transferred into RAG–/– recipients on day –7. Approximately 3 x 106 naive polyclonal CD8 T cells were CFSE-labeled and injected on day 0 into the same recipients (or into RAG–/– mice that had not received Treg cells on day –7). Tissues were harvested on day 10 and analyzed by FACS. A, Representative CFSE profiles of CD8 responder T cells in indicated groups of mice and tissues. The numbers are percentages of responder CD8 T cells undergoing spontaneous LIP (CFSE– cells). B, Total numbers of CD8 responders recovered from different tissues in indicated groups of mice. Each bar shows the average of four animals pooled from two separate experiments. Stars represent a statistical significance with p < 0.05 by Student’s two-tailed t test when comparing the number of cells isolated from tissues of animals receiving a pretransfer of Tregs with those from animals without pretransfer of Tregs.

 
IL-10 production by Tregs is not required to inhibit LIP of CD4 T cells

We considered that the lack of any defect in suppressive effects of IL-10-deficient Tregs on LIP of CD8 T cells may be due to a fundamental difference between CD8 and CD4 responders. This includes their relative pathogenicity. Adoptive transfer of naive polyclonal CD4 T cells can lead to fatal autoimmunity or immunopathology, which includes colitis and pneumonitis (27, 28). In contrast, we observed RAG–/– recipients of naive polyclonal CD8 T cells for 3 mo and did not note any disease (data not shown). Thus, we tested the ability of Tregs to inhibit LIP of polyclonal CD4 T cells using the identical experimental design we used for CD8 responders above. We first transferred wild-type or IL-10–/– Treg cells into RAG–/– recipients, then 1 wk later transferred CFSE-labeled naive CD4 T cell responders into the same recipients or into RAG–/– mice that had not received a pretransfer of Treg cells. CFSE content was measured 7 days after the adoptive transfer of CD4 responders (Fig. 7A). Presence of either IL-10-deficient or wild-type Tregs resulted in a decreased proportion of CFSE– CD8 T cell responders (Fig. 7B, upper panels). Interestingly, there was a trend toward greater CFSE content in the CFSE+ fraction in the presence of Tregs, at least in the mesenteric lymph nodes (Fig. 7B, lower right panel). This suggests that there may be some competition for common resources among Tregs and conventional CD4 T cells. However, the most marked effect of Tregs, either IL-10-deficient or wild type, was on the numbers of CFSE– responders, which were significantly decreased in the presence of both types of Tregs (Fig. 7C). These data indicated that Tregs selectively inhibited the spontaneous form of LIP of naive CD4 cells, and that wild-type and IL-10-deficient Tregs were no different in their suppressive potency.


Figure 7
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  		FIGURE 7. Suppression of CD4 T cell spontaneous LIP by Tregs is not IL-10 dependent. Approximately 5 x 105 wild-type or IL-10–/– CD25+ cells were transferred into RAG–/– recipients on day –7. Approximately 2.5 x 106 naive polyclonal CD4 T cells were CFSE-labeled and injected on day 0 into the same recipients (or into RAG–/– mice that had not received Treg cells on day –7). Lymph nodes were harvested on day 7 and analyzed by FACS. A, Representative CSFE profiles of responding CD4 T cells in the lymph nodes. Solid line histograms represent CFSE content of CD4 responders in the absence of Tregs. Stippled line histograms represent CFSE content of CD4 responders in the presence of Tregs. B, Percentages of CFSE– responder CD4 T cells in indicated groups of mice, as well as the CFSE content in the CFSE+ CD4 T cell responders represented by the MFI. C, Relative numbers of indicated CD4 T cells responders in different groups of mice. Each bar represents the mean ± SD of three animals. Statistical significance was determined using the two-tailed Student t test. One star represents a statistical significance with p < 0.05; Two stars represent a statistical significance with p < 0.01. Skin-draining lymph nodes (LN) include axial, brachial, cervical, and inguinal.

 
Treg-derived IL-10 plays an important role in control of colitis induced by CD4 T cells in RAG–/– mice

Although our data demonstrated that Treg-derived IL-10 is not involved in control of LIP within lymphoid tissues in the absence of disease, we questioned whether it may become important after onset of inflammatory disease, which itself may perpetuate expansion of CD4 T cells if left uncontrolled. To test this possibility, we first transferred wild-type or IL-10–/– Treg cells into RAG–/– recipients, then 1 wk later transferred naive CD4 T cell responders into the same recipients or into RAG–/– mice that had not received a pretransfer of Treg cells. We then assessed the severity of colitis following adoptive transfer. RAG–/– mice that received no Tregs developed severe colitis and had to be sacrificed at 6–9 wk after transfer of conventional CD4 T cells, according to preset criteria. RAG–/– mice that received IL-10-deficient Tregs before the transfer of CD4 T cells developed colitis of intermediate severity, while RAG–/– mice that received wild-type Tregs before the transfer of CD4 T cells developed no disease (Fig. 8). Significantly different weights were noted among the groups, expressed as percent of initial weight (Fig. 8A, left panel), and they correlated well with the colitis histology scores (Fig. 8, A, right panel, and B).


Figure 8
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  		FIGURE 8. IL-10-deficient Tregs cannot completely prevent colitis caused by adoptive transfer of CD4 T cells into RAG–/– mice. Approximately 5 x 105 wild-type or IL-10–/– CD25+ cells were transferred into RAG–/– recipients on day –7. Approximately 1 x 105 naive polyclonal CD4 T cells were injected on day 0 into the same recipients (or into RAG–/– mice that had not received Treg cells on day –7). Mice were sacrificed when the group of RAG–/– mice without pretransfer of Tregs was showing obvious clinical signs of disease, including diarrhea and hunched posture. Three separate experiments were harvested 6, 8, or 9 wk after transfer and the results were pooled here. Histologic scores were determined on H&E sections of distal colons blinded to the identity of the experimental groups. A, Mean changes ± SD in weight of the animals and histologic scores ± SD in indicated groups of mice. The pooled experiments together totaled 13 mice per group. Statistical significance was determined using the two-tailed Student t test. B, Representative H&E sections from each of the experimental groups.

 
In addition to measuring the effects of wild-type and IL-10-deficient Tregs on clinical colitis caused by CD4 T cells in RAG–/– mice, we analyzed the effector cytokine profiles generated by CD4 T cells in the same animals. T cells taken from mesenteric lymph nodes or colons were stimulated with PMA/ionomycin in vitro, and cytokine production was measured by intracellular staining. Most cells produced either IFN-{gamma} or IL-17, although some cells produced both cytokines simultaneously (Fig. 9, A and black bars in B). Tregs, regardless of their ability to produce IL-10, markedly inhibited expansion of IFN-{gamma}-producing CD4 T cells. This was evidenced by smaller fractions and total numbers of IFN-{gamma}-producing CD4 T cells in the mesenteric lymph nodes (Fig. 9B, top left panels). However, IL-10-deficient Tregs were less effective than wild-type Tregs in suppressing infiltration of colons by IFN-{gamma}-producing CD4 T cells (Fig. 9B, bottom left columns). Interestingly, both types of Tregs were only modestly effective in reducing the numbers of Th17 cells compared with Th1 cells. In fact, the presence of wild-type or IL-10-deficient Tregs inverted the IFN-{gamma}-producer/IL-17-producer ratio in favor of Th17 T cells. Nevertheless, similarly to the effect seen on IFN-{gamma}-producing CD4 T cells, wild-type Tregs effectively prevented infiltration of colons by IL-17-producing CD4 T cells, while IL-10-deficient Tregs failed to do so. These findings, combined with no demonstrable difference in the ability of wild-type and IL-10-deficient Tregs to control LIP within lymphoid tissues, suggest that Treg-derived IL-10 is important primarily in control of established pathogenic effectors in the peripheral tissues.


Figure 9
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  		FIGURE 9. IL-10-deficient Tregs are impaired in their ability to suppress effector CD4 T cells in the colon. Approximately 5 x 105 wild-type or IL-10–/– CD4+CD25+ T cells were transferred into RAG–/– recipients on day –7. Approximately 1 x 105 naive polyclonal CD4 T cells were injected on day 0 into the same recipients (or into RAG–/– mice that had not received Treg cells on day –7). Mice were sacrificed at 9 wk, and cells isolated from the lymph nodes and colons were stimulated with PMA/ionomycin in vitro, stained for cytokine expression, and subjected to FACS analysis. A, Representative FACS plots of gated CD4 T cells in the mesenteric lymph nodes of individual animals. Percent numbers show fractions of total CD4 T cells expressing indicated cytokines. B, Number of effector cytokine-producing CD4 T cells in mesenteric lymph nodes and colons. Relative CD4 T cell numbers were calculated in comparison to the group that did not receive a pretransfer of Tregs. Each bar represents the mean ± SD of at least six animals. Statistical significance was determined using the two-tailed Student t test. The black and red stars indicate statistical significance with p < 0.05 and p < 0.01, respectively. WT, Wild type.

 
It is important to note that suppressive function of Tregs in experimental colitis experiments is typically measured following a cotransfer with ~105 conventional CD4 T cells in a 1:1 or even 1:10 Treg-responder ratio. This contrasts with our staggered protocol using more Treg cells. However, we had determined early in our work that cotransfer of Tregs in relatively small numbers does not inhibit LIP (data not shown). We did the above colitis experiments specifically trying to tease out the potential contribution of Treg-mediated inhibition of spontaneous LIP in pathogenesis of experimental disease. However, we did attempt to compare our results reported above (Fig. 8) with those that would be obtained using a cotransfer protocol. We ran pretransfer and cotransfer Treg/responder T cell adoptive transfer protocols in parallel. In one set of mice, we first transferred 1 x 106 wild-type or IL-10–/– Treg cells into RAG–/– recipients (on day –7), then 1 wk later transferred naive CD4 T cell responders into the same recipients (on day 0). A second set of RAG–/– mice that had not received a pretransfer of Treg cells received a cotransfer of 5 x 105 or 1 x 105 wild-type or IL-10–/– Treg cells and 1 x 105 naive CD4 T cell responders on day 0. A control set of RAG–/– mice received only naive CD4 T cell responders on day 0. When assayed simply by weight, the mice benefited from both wild-type and IL-10-deficient Tregs received through both the pretransfer or the cotransfer protocols (Fig. 10A, left panel). However, in both adoptive transfer protocols, IL-10-deficient Tregs failed to prevent significant histological colitis activity, while wild-type Tregs completely abolished histologic colitis (Fig. 10A, right panel). As found above, these data indicate that IL-10-deficient Tregs at all doses tested were less successful in prevention of disease than wild-type Tregs. Higher histology scores correlated with higher total numbers of responder T cells isolated from the colon (Fig. 10B, left panel), as well as with higher numbers of T cells capable of producing IFN-{gamma} and/or IL-17 (data not shown).


Figure 10
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  		FIGURE 10. When cotransferred, large numbers of Tregs are required to suppress infiltration of effector CD4 T cells in the colon. In one set of mice, indicated numbers of wild-type or IL-10–/– CD4+CD25+ T cells were transferred into RAG–/– recipients on day –7, then ~1 x 105 naive polyclonal CD4 T cells were injected into the same recipients on day 0. In a second set of mice, indicated numbers of wild-type or IL-10–/– CD4+CD25+ T cells were cotransferred with ~1 x 105 naive polyclonal CD4 T cells on day 0. Control RAG–/– mice received only 1 x 105 naive CD4 T cell responders on day 0. Mice were sacrificed at 12 wk following adoptive transfer of CD4 T cell responders. A, Mean changes in weight of the animals and histologic scores for colon sections for the indicated groups of mice. Histologic scores were determined on H&E sections of distal colons blinded to the identity of the experimental groups. Each bar represents the mean ± SEM of four animals. B, Relative CD4 responder T cell and Treg numbers were calculated based on total lymphocyte numbers. WT, Wild type; MLN, mesenteric lymph nodes.

 
An interesting caveat of these titration colitis experiments, which were performed in the later phase of this project, was that a longer period was required for colitis induction than in earlier studies. In our early experiments shown in previous figures, diarrhea was evident by 3–4 wk after transfer of responder T cells. Interestingly, we saw that the ability of Tregs to prevent colitis in early experiments was relatively inefficient, when simultaneously cotransferred with responders in 1:1 ratio (data not shown). However, in these later experiments (Fig. 10), the colitis induction period was 9–10 wk. This longer induction period was consistently observed by several individual workers in the laboratory in various individual experiments, and may reflect a change in enteric flora in our animal facility. During this long induction period, Tregs likely have sufficient time to expand even from low input numbers to occupy their homeostatic niche, which is reflected by similar numbers of Tregs found in the lymphoid tissues at the end of the experiment (Fig. 10B, right panel).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although regulatory CD4+CD25+ T cells were originally discovered in the context of autoimmunity associated with lymphopenia (29, 30, 31), surprisingly they were also initially reported to have no inhibitory effects on T cell LIP (14, 15). At least two distinct forms of LIP have been recognized since: spontaneous and homeostatic (5). These are readily distinguished in short-term experiments by the content of CFSE dye detected subsequent to the adoptive transfer of CFSE-labeled T cells. The spontaneous form is characterized by rapid, burst-like T cell proliferation that results in complete loss of CFSE. It is also associated with acquisition of potentially pathogenic effector phenotypes. In contrast, the homeostatic form is characterized by a slow and steady rate of cell division and does not usually lead to acquisition of effector functions. We show here that Tregs selectively inhibit the spontaneous form of LIP, but spare the homeostatic form of LIP. This finding does resolve some of the conflict in the literature on the potential role of Tregs in controlling LIP. First, the spontaneous form of LIP can be easily missed if CFSE is used as the sole marker of adoptively transferred cells. Second, not all models of lymphopenia support spontaneous LIP. Although spontaneous LIP is readily seen following adoptive transfer of T cells into animals with a complete congenital deficiency of T cells, it is largely absent in irradiation-induced lymphopenia (5, 10, 32). In fact, our results provide one explanation for the latter finding: residual endogenous Tregs may be sufficient to block spontaneous LIP following irradiation.

After our original observation that Tregs can inhibit LIP of CD4 T cells (9), we considered that Tregs may simply compete for TCR signals with the CD4 responders. Indeed, Treg TCRs are generally thought to have higher affinities for self-peptide/MHC complexes compared with TCRs of conventional CD4 T cells (33). To test the hypothesis of competition for peptide/MHC signals, we focused our initial studies here on CD8 T cells as responders, because it is unlikely that there is substantial overlap between peptide/MHC signals seen by CD8 T cells and CD4 Tregs. Indeed, a different mechanism is likely responsible for the ability of Tregs to inhibit the spontaneous form of LIP experienced by CD8 T cells. However, competition for peptide/MHC signals may be one reason for the very modest inhibition of homeostatic LIP experienced by naive CD4 T cells (Fig. 7B). It might also explain complete Treg-mediated inhibition of LIP by several monoclonal CD4 T cell populations reported previously (9, 18). However, our results here show that Tregs inhibit the spontaneous form of LIP experienced by polyclonal CD4 T cells more than the homeostatic form, and it is reasonable to speculate that this inhibition is mediated by a mechanism common for both CD4 and CD8 responders.

Molecular mechanisms of Treg-mediated suppression are still poorly understood. It is likely that multiple mechanisms operate simultaneously, and those that are critical in vitro may play only secondary roles in vivo. For example, Tregs were described to suppress IL-2 production and expression of CD25 by responder T cells during in vitro culture by a mechanism that required direct cell-cell contact (34). However, direct in vivo imaging studies failed to detect physical interactions between Tregs and responder T cells. Instead, Tregs were seen to limit stable conjugation between responder T cells and dendritic cells (DCs) (35, 36). Interestingly, these effects do not interfere with early activation events in the responder T cells, including production of IL-2 (37, 38). Some of the inhibitory mechanisms mediated by Tregs that have been shown to play important roles in vivo include production of immunosuppressive cytokines, such as IL-10 (39, 40), and TGF-β (41, 42), induction of IDO via CTLA-4:B7 engagement in certain DC subsets (43, 44), and generation of antiproliferative molecules such as adenosine (45, 46, 47).

We decided to focus our initial exploration of Treg-mediated suppression of LIP on IL-10 because it has been reported previously that IL-10-deficient Tregs fail to inhibit expansion of CD4 T cells in RAG–/– hosts (16). However, we found no defect in the ability of IL-10-deficient Tregs to control LIP of CD8 or CD4 T cells measured within the secondary lymphoid tissues. Interestingly, IL-10-deficient Tregs were significantly impaired in their ability to control colitis. This result can be understood if we recognize that Tregs function differently in different anatomic locations. We propose the following model (Fig. 11). During the inductive phase of the immune response, which takes place within the secondary lymphoid tissues, Tregs limit oligoclonal expansion of T cells experiencing cognate Ag stimulation. This function is IL-10 independent; in fact, Tregs localized within secondary lymphoid tissues produce little IL-10 (37, 48). However, the suppressive effects of Tregs in the lymphoid tissues are incomplete, and some effector T cells are generated even in their presence. These effectors leave the lymphoid tissues and migrate into the periphery. Once there, established effectors can become activated again and trigger inflammation. At this phase of the immune response, Tregs suppress the activity of established pathogenic effectors by mechanisms that at least partially depend on their ability to produce IL-10. This model fits with the observation that Tregs produce IL-10 within inflamed tissues, but not within secondary lymphoid tissues (37, 48). This model predicts that wild-type Tregs limit emergence of pathogenic T cells from the lymphoid tissues and inhibit their activity in the periphery. Therefore, no colitis is observed in their presence. However, IL-10-deficient Tregs are only partially effective. Although they limit emergence of pathogenic T cells in the lymphoid tissues, they fail to suppress pathogenic activity of effector T cells in the periphery. The model explains the intermediate severity of colitis observed in our experiments with IL-10-deficient Tregs (Figs. 8–10). The model is also consistent with failure to rescue scurfy mice (Foxp3sf) from fatal autoimmunity by adoptive transfer of Tregs after the immediate neonatal period. Adoptive transfer of wild-type Tregs into neonatal Foxp3sf mice fully protects them from disease (49, 50, 51). However, adoptive transfer of Tregs into Foxp3sf mice on day 3 of life or later merely delays their eventual death from autoimmune disease (52). We suspect that in the first case, Tregs limit emergence of pathogenic T cells and further suppress activity of the few that do escape into the peripheral tissues. However, in the second case, the large number of differentiated pathogenic effectors cannot be completely controlled in the periphery.


Figure 11
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  		FIGURE 11. Model of Treg-mediated control of responder T cells in lymphopenia. The top half of the figure illustrates control of LIP by Tregs within the secondary lymphoid tissues. Responder T cells and Tregs are shown to exist in different compartments, although it is possible that Tregs (shaded circles) and responder T cells (black circles) undergoing spontaneous LIP compete for some common resources. The presence of Tregs limits spontaneous LIP, which in turn limits the number of potentially autoreactive T cells capable of causing autoimmunity. This function of Tregs is independent of their ability to produce IL-10. The bottom half of the figure illustrates the second phase of Treg-mediated suppression, which happens in the periphery. Some effector T cells generated in the course of spontaneous LIP leave the lymphoid tissues. However, their local activity and ability to trigger inflammation in the periphery is inhibited by Tregs in an IL-10-dependent manner. Finally, we speculate at this time that T cells undergoing spontaneous proliferation compete for resources with T cells undergoing homeostatic proliferation (white circles). Therefore, uncontrolled spontaneous LIP may limit homeostatic proliferation and potentially impair overall TCR diversity during immune reconstitution.

 
It is important to note that our experimental system differs somewhat from the usual protocols for adoptive transfer with Tregs for disease suppression. Typically, Tregs are cotransferred simultaneously along with the responder T cells into the RAG–/– recipients. In contrast, in most experiments here we transferred 0.5–2 x 106 Tregs 1 wk before the responder T cells were transferred. Tregs occupy a homeostatic compartment separate from other T cells (17, 53, 54, 55) and during this week they expand to reach near plateau numbers (data not shown). Our staggered adoptive transfer protocol is similar to one used by Shen et al. (18) who were also able to demonstrate control of LIP by Tregs. The relatively large number of polyclonal Tregs is important for revealing their ability to suppress LIP. In contrast, coinjection of a relatively small number of Tregs fails to control the initial proliferation and expansion of CD4 responders (56). Therefore, it is likely that in cotransfer experiments, Tregs exert most of their suppressive effects directly in the tissues. This may explain the ability of Tregs to cure established CD4 T cell-induced colitis in RAG–/– mice, and the failure of IL-10-deficient Tregs to treat this disease (48).

It is reasonable to ask whether inhibition of LIP by Tregs plays a significant role in inhibition of experimental colitis and other types of autoimmunity. We did see a trend toward less efficient disease suppression when the lowest numbers of Tregs were used, as evidenced by greater numbers of responder T cells in the colon (Fig. 10B) and greater numbers of polarized Th1 and Th17 effectors (data not shown). Nevertheless, in their totality our results can also be interpreted to suggest that inhibition of LIP within lymphoid tissues is not an essential mechanism of disease suppression. Instead, suppression of established effector T cells may be sufficient. However, this would contradict recent demonstration of poor suppression of colitis by CCR7-deficient Tregs, which fail to migrate into lymph nodes and fail to inhibit generation and expansion of colitogenic T cell clones (57). These data do support our interpretation: Tregs do play an important role during the inductive phase of the immune response within the lymphoid tissues, and they have to be present in large numbers to be effective during this stage. This condition was likely unmet in simultaneous cotransfer experiments where there was insufficient time for Treg expansion before colitis induction.

It should be noted that clinically important lymphopenic states with relative sparing of the Treg compartment are common. We have already mentioned that irradiation-induced lymphopenia, common in cancer clinics, is rarely associated with autoimmunity. Similarly, Tregs may be spared in lymphopenia caused by viral infections, which results in impaired viral clearing (58). Increased numbers of Tregs have been observed in patients with HIV infection (59, 60). In this case, we speculate that residual Tregs may protect patients being treated by highly active antiretroviral therapy against the immune reconstitution syndrome.

Finally, it is interesting to consider that selective inhibition of spontaneous LIP may also benefit the overall TCR diversity achievable during recovery from a lymphopenic state by LIP. Currently, it is not known whether responders undergoing spontaneous and homeostatic forms of LIP compete for any overlapping resources. If they do, then it is possible for a relatively small number of T cells prone to spontaneous LIP to dominate the T cell repertoire following recovery from a lymphopenic state. For example, although T cells undergoing spontaneous LIP do not require IL-7, they still express similar levels of the IL-7R{alpha} as T cells undergoing homeostatic LIP, and theoretically might steal IL-7 away from them (5). It is also possible that T cells undergoing spontaneous LIP may compete for TCR signals. In fact, it has been shown that at least some TCR Tg T cells with relatively high avidity for self-ligands can inhibit homeostatic proliferation of other TCR Tg T cells with lower avidity for self-ligands following cotransfer into lymphopenic hosts (7). Furthermore, memory T cells generated by immunization with Ag or LIP in RAG–/– recipients can inhibit LIP of naive responders if the two populations have an overlap in TCR specificity (61, 62). Therefore, it is possible that the expansion of T cells by spontaneous proliferation can occur at the expense of homeostatic proliferation. In this case, the spontaneous form of LIP may be detrimental not only because it is more likely to give rise to pathogenic T cells, but also because it may also limit immune fitness by constraining the homeostatic form of LIP. Future studies are needed to test this hypothesis.


   Acknowledgments
 
We are grateful to Drs. Erik Peterson, Daniel Mueller, Stephen Jameson, and Marc Jenkins for a critical review of the manuscript. We are also thankful for the editorial assistance provided by Linda Raab.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 R01 DK061961 and DK061961-03S1. Back

2 Address correspondence and reprint requests to Dr. Alexander Khoruts, Department of Medicine, University of Minnesota, Mayo Mail Code 334, Nils Hasselmo Hall Building, 312 Church St. SE, Minneapolis, MN 55455. E-mail address: khoru001{at}umn.edu Back

3 Abbreviations used in this paper: LIP, lymphopenia-induced proliferation; Treg, regulatory CD4+CD25+Foxp3+ T cell; Tg, transgenic; GITR, glucocorticoid-induced TNFR; DC, dendritic cell. Back

Received for publication November 29, 2006. Accepted for publication March 19, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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