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The Journal of Immunology, 2007, 179, 2143 -2152
Copyright © 2007 by The American Association of Immunologists, Inc.
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Chronic Immunodeficiency in Mice Lacking RasGRP1 Results in CD4 T Cell Immune Activation and Exhaustion1

John J. Priatel2,*, Xiaoxi Chen*, Lauren A. Zenewicz{dagger}, Hao Shen{dagger}, Kenneth W. Harder*, Marc S. Horwitz* and Hung-Sia Teh3,*

* Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada; and {dagger} Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Ras-guanyl nucleotide exchange factor RasGRP1 is an important link between TCR-mediated signaling and the activation of Ras and its downstream effectors. RasGRP1 is especially critical for the survival and differentiation of developing thymocytes whereas negative selection of thymocytes bearing an autoreactive TCR appears to be RasGRP1 independent. Despite apparently normal central tolerance, RasGRP1–/– mice spontaneously acquire an acutely activated and proliferating CD4 T cell population that exhibits characteristics of T cell exhaustion, including strong expression of programmed cell death-1. To elucidate the basis for RasGRP1–/– CD4 T cell immune activation, we initiated a series of adoptive transfer experiments. Remarkably, the copious amounts of cytokines and self-Ags present in hosts made lymphopenic through irradiation failed to induce the majority of RasGRP1–/– CD4 T cells to enter cell cycle. However, their infusion into either congenitally T cell- or T/B cell-deficient recipients resulted in robust proliferation and L-selectin down-regulation. These findings imply that the activation and proliferation of RasGRP1–/– CD4 T cells may be dependent on their residence in a chronically immunocompromised environment. Accordingly, bacterial and viral challenge experiments revealed that RasGRP1–/– mice possess a weakened immune system, exhibiting a T cell-autonomous defect in generating pathogen-specific T cells and delayed pathogen clearance. Collectively, our study suggests that chronic T cell immunodeficiency in RasGRP1–/– mice may be responsible for CD4 T cell activation, proliferation, and exhaustion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The two dominant forces maintaining homeostatic control on the size of naive T cell compartment are the availability of self-peptides/self-MHC ligands and the cytokine IL-7 (1, 2). Under normal T cell-sufficient conditions, low-avidity TCR interactions with self-peptides/self-MHC molecules along with IL-7R signaling promote T cell survival. During T cell lymphopenia, the increased abundance of these same elements is thought to induce slow T cell (homeostatic) proliferation in an attempt to increase peripheral T cell numbers. Therefore, homeostatic expansion is beneficial for preserving the size of the T cell population but it may pose risks because T cell proliferation is accompanied by acquisition of effector function, such as cytotoxicity and the capacity to rapidly secrete inflammatory cytokines (1, 2). Because this slow cellular division only applies to a subset of T cells, it is thought that the T cells recruited into cell cycle are the ones expressing TCRs with greater avidity for self-Ags (1, 2), Therefore, lymphopenia-induced T cell expansion could be dangerous as it may skew the TCR repertoire of the resident pool of T cells toward autoreactivity and, further, restrict its diversity.

The term "homeostatic proliferation" was originally coined to describe the slow T cell expansion observed when naive TCR-transgenic T cells were adoptively transferred into lymphopenic recipients, such as congenitally T cell-deficient RAG–/– mice or normal mice rendered lymphopenic by irradiation (2, 3, 4). Because donor TCR-transgenic T cells were RAG deficient, it ensured that these T cells were monoclonal, expressing a single {alpha}beta TCR, and verified that this slow proliferation occurred in the absence of agonist peptides. Moreover, the product of this cell division possesses a memory T cell phenotype resembling the functional attributes and gene expression profiles of conventional memory T cells (2, 5). Despite these observations, recent work has suggested that foreign Ags may be responsible for some donor T cell proliferation observed following transplantation into congenitally T cell-deficient RAG–/–, TCR{alpha}–/–, or SCID hosts (6, 7). Moreover, because mice lacking any {alpha}beta T cells are severely immunocompromised, these hosts are highly susceptible to developing chronic infections and, as a result, may present a broad array of microbial Ags to donor T cells. In support of this hypothesis, the adoptive transfer of normal (polyclonal) T cells into a congenitally T cell-deficient host (chronically immunodeficient) results in a subset of T cells undergoing massive T cell expansion whereas only slow division is apparent when such T cells are transferred into wild-type hosts made lymphopenic by irradiation (acutely immunodeficient). The observation that donor TCR-transgenic (monoclonal) T cells undergo similar rates of division in these two disparate recipients demonstrates that rapid T cell proliferation only applies to a subset of T cells and suggests that it may be dependent on TCR specificity (6, 7). Because rapid T cell proliferation is lost when T cells are transferred into congenitally T cell-deficient, gnotobiotic (germfree) hosts (7), it argues that foreign Ags, likely derived from commensal microbes within the gut, are driving fast T cell proliferation in congenitally T cell-deficient animals.

The loss of T cell immunity is a common occurrence during chronic viral infections in both mice and humans (8). Recent evidence suggests that continual exposure to cognate Ag results in the overstimulation of viral-specific T cells and the development of an "exhausted" memory T cell phenotype. In contrast to acute infection, memory T cells derived from chronic infection exhibit Ag dependency, limited self-renewal capacity, diminished cytokine production, and reduced cytotoxicity (9). Therefore, these functional impairments displayed by chronically activated T cells may contribute to the failure to clear virus. Programmed death-1 (PD-1),4 a negative regulator of activated T cells (10), is strongly up-regulated on exhausted viral-specific CD8 T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice and HIV infection in humans (11, 12). Because blockade of PD-1 interaction with its ligand PD-L1 can restore function in exhausted CD8 T cells (11), it makes the case that PD-1 is not simply indicative of an exhausted state but also plays a key role in its maintenance.

In thymocytes, Sos (13) and RasGRP1 (14), two Ras-guanyl-nucleotide exchange factors, link Ras and MAPK activation to TCR signal transduction with their respective functions dependent on relocating to membranes by two distinct mechanisms (15). RasGRP1 mobilizes to membranes by binding the phospholipase C{gamma}1 product diacylglycerol through its C1 domain whereas Sos is recruited to the phosphorylated adaptor molecule linker for activated T cells by way of its association with the Src homology 2-domain-containing protein Grb2. RasGRP1–/– thymocytes show signs of reduced TCR signaling (14, 16, 17) and a selective impairment of positive but not negative selection (16). Therefore, mice lacking RasGRP1 serve as a model lacking a positive regulator of TCR signaling. RasGRP1–/– mice exhibit a marked T cell lymphopenia (14, 16), likely a consequence of decreased single-positive (SP) thymocyte maturation and T cell hyporesponsiveness (14, 16, 17). Paradoxically, a recently described novel mouse strain called RasGRP1lag (lymphoproliferation-autoimmunity glomerulonephritis), bearing a spontaneous mutation in RasGRP1, develops an autoimmune syndrome resembling systemic lupus erythematosus (SLE), exhibiting massive lymphoproliferation, high levels of serum autoantibodies and, eventually, advanced disease that required euthanasia (18). Therefore, RasGRP1 signaling may be critical for both thymocyte maturation and T cell tolerance.

In this study, we report that although RasGRP1–/– mice remain T cell lymphopenic and free of overt disease until at least 1 year of age, they possess a population of proliferating CD4 T cells that display an exhausted phenotype, characteristic of chronic infection (8). Adoptive transfer experiments suggest that chronic immunodeficiency and foreign Ags might be responsible for inducing RasGRP1–/– CD4 T cell proliferation rather than autoreactivity to self-Ags. Supporting the notion of a dysfunctional immune system, bacterial and viral challenge experiments revealed that RasGRP1–/– mice exhibited impaired T cell responses and a delay in pathogen clearance. Lastly, we find that chronic T cell immunodeficiency in RasGRP1–/– mice is likely a consequence of RasGRP1 protein loss in thymocytes and/or T cells rather than due to defects in innate immunity. In conclusion, these investigations highlight the roles of RasGRP1 in determining a normal immune status and as an essential regulator of adaptive T cell immunity toward experimental infection.


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

C57BL/6J (B6), B6.PL-Thy1a/Cy (Thy 1.1+), B6.SJL-Ptprca Pep3b/BoyJ (Ly 5.1+), B6.RAG-1–/–, and B6.TCR{alpha}–/– mice were acquired from The Jackson Laboratory. RasGRP1–/– breeder mice were provided by J. C. Stone (University of Alberta, Alberta, Canada) and bred onto a B6 background at least seven generations. To generate Thy1.1+ 2C TCR-transgenic animals, the 2C TCR transgene was bred onto the B6.PL-Thy1a/Cy background (H-2b, Thy1.1+). All studies followed guidelines set by the Animal Care Committee at the University of British Columbia in conjunction with the Canadian Council on Animal Care.

Flow cytometry

Abs against CD4 (GK1.5), CD8 (53-6.7), CD5 (53-7.3), TCRbeta (H57-597), CD62L (MEL-14), CD25 (PC61.5), CD69 (H1.2F3), CD45.1 (A20), Thy1.1 (HIS51), CD44 (IM7), CD62L (MEL-14), CD127 (AKR34), PD-1 (J43), PD-L1 (MIH5), PD-L2 (TY25), TNF-{alpha} (MP6-XT22), and IFN-{gamma} (XMG1.2) were purchased from eBioscience. Annexin VPE, anti-Fas (DX2), anti-FasL (MFL3), anti-Ki-67 Abs (B56), and anti-TCR Vbeta screening panel (no. 0143KK) reagent sets were purchased from BD Biosciences. For anti-Ki-67 staining, cells were fixed with 2% formaldehyde (Polysciences) for 10 min, permeabilized with 90% methanol, washed with 2% FCS/PBS and subsequently, incubated with anti-Ki-67 Ab for 30 min at room temperature. Isotype-control Ab (clone MOPC-21; BD Biosciences) staining was negligible (> 0.2%). Annexin V-PE staining was conducted as described previously (19). Data were acquired using either a FACScan or FACSCalibur and CellQuest software (BD Biosciences). Data were analyzed either with CellQuest, FCSPRESS, or FlowJo (Tree Star) software.

Adoptive transfer experiments

Wild-type splenic and lymph node T cells were purified from Thy1.1+ animals and labeled with 1 µM CFSE (Molecular Probes) as previously described (20). Approximately 2 x 106 purified wild-type (polyclonal) Thy1.1+ T cells or 1 x 106 Thy1.1+ 2C TCR CD8 T cells were i.v. injected into Thy1.2+ recipients, either irradiated (600 rad) wild-type B6 or nonirradiated wild-type B6, B6.RasGRP1–/–, B6.RAG-1–/–, and B6.TCR{alpha}–/– mice. Conversely, ~2 x 106 CFSE-labeled RasGRP1–/– T cells (Thy1.2+) were transferred into either B6. Thy1.1+, irradiated B6.Thy1.1+, or B6.RAG-1–/– hosts. Spleens were recovered 1 wk posttransfer and proliferation of donor cells assessed by flow cytometry using a FACSCalibur (BD Biosciences).

Bacterial and viral infections

Mice were infected i.v. with ~10,000 CFU of a recombinant strain of Listeria monocytogenes engineered to express the 2C TCR agonist peptide SIYRYYGL (J. Priatel, L. Zenewicz, H. Shen, and H. Teh, manuscript in preparation). For viral infection, mice were injected i.p. with ~100,000 PFU of LCMV-Armstrong. Splenic viral titers were determined as described previously (21). For wild-type T cell infusion into RasGRP1–/– (Ly5.2+) animals, purified (10 million) Ly5.1+ T cells (~55:45% ratio of CD4 vs CD8 T cells) were i.v. injected into RasGRP1-deficient animals 1 day before infection with either rLM-SIY or LCMV.

Detection of IFN-{gamma} production by intracellular flow cytometry

Spleens were harvested from mice at either day 7 (rLM-SIY) or day 8 (LCMV) postinfection, pressed through metal mesh to generate single-cell suspensions, and subjected to RBC lysis by ammonium chloride treatment. Splenocytes were cultured for 5 h in 96-well, flat-bottom plates, at a concentration of 2–4 x 106 cells/well, in 0.2 ml of complete medium supplemented with 1 µl/ml Golgi Plug (contains brefeldin A; BD Biosciences) to block cytokine secretion. Cells were stimulated with a concentration of 1 µM for the MHC class I peptides (SIY, SIYRYYGL; GP33–41, KAVYNFATC; GP34–43, AVYNFATCGI; NP396–404, FQPQNGQFI; GP276–286, SGVENPGGYCL; NP205–212, YTVKYPNL; OVA257–264, SIINFEKL) and 10 µM for the I-Ab MHC class II peptides (LLO190–201, NEKYAQAYPNVS; GP61–80, GLKGPDIYKGVYQFKSVEFD). For anti-TCR stimulations, 2 x 106 splenocytes were incubated for 5 h in a 24-well plate that had been precoated with 10 µg/ml anti-CD3{epsilon} (145-2C11) Ab. After culture, cells were fixed for 15 min in 2% paraformaldehyde/PBS solution, permeabilized for 15 min with 0.2% Tween 20/PBS and stained with anti-CD4-allophycocyanin, anti-CD8-PE-Cy5, and anti-IFN-{gamma}-FITC Abs (eBioscience). Data were acquired on a FACSCalibur using CellQuest software (BD Biosciences) and analyzed with FCSPress (www.fcspress.com). All peptides were synthesized at the University of British Columbia’s Nucleic Acid Protein Service Unit.

Direct ex vivo CTL assays

After 7 days postinfection with rLM-SIY, splenic CD8 T cell effectors were isolated by staining total splenocytes with rat anti-mouse CD4 (GK1.5) Abs and subsequently depleted of CD4+ and surface Ig+ cells with anti-mouse (and rat-reactive) Ig-linked Dynabeads (catalog no. 110.02; Dynal Biotech). The target EL-4 cell line was labeled with 51Cr, pulsed with SIY peptide, washed, and incubated with various numbers of effectors as previously described (20).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
RasGRP1–/– CD4 T cells exhibit markers of acute activation, exhaustion, proliferation, and spontaneous apoptosis

The impaired T cell development results in a T cell lymphopenia, exhibiting a 10-fold decreased abundance of peripheral T cells, in 1-mo-old RasGRP1–/– mice (14). To account for the limited positive selection in RasGRP1–/– animals, it had been proposed that thymocytes capable of being selected without RasGRP1 must express strongly self-reactive TCRs to overcome their signaling deficits (16, 17, 18). Notably, autoreactive CD4 T cells were suspected to be the root cause of massive lymphoproliferation and an underlying autoimmune disorder in RasGRP1lag mice (18). However, although we observed some incidents of massive lymphoproliferation, splenomegaly, generalized lymphadenopathy, and a 20- to 30-fold increase in total cellularity, in mice homozygous for the targeted mutation on a mixed C57BL/6J:129 background (our unpublished observations), this phenotype seems to have vanished after sequential backcrossing of the RasGRP1 gene-knockout allele to the C57BL/6J (B6) genetic background. In this report, we focus on RasGRP1–/– mice that have been bred at least seven generations onto the B6 mouse background.

B6-backcrossed RasGRP1–/– mice appear healthy until at least 1 year of age and do not develop massive lymphoproliferation. Sampling of 2- to 4-mo-old RasGRP1–/– mice revealed that they remain T cell lymphopenic, the recovery of both CD4 and CD8 T cells from spleens and pooled lymph nodes (LNs) were reduced vs age-matched wild-type mice (Fig. 1A). Comparison of secondary lymphoid organs revealed that RasGRP1–/– mice have similar-sized spleens with respect to wild type (Fig. 1B). Curiously, RasGRP1–/– mice possess small peripheral LN such as axillary, brachial and inguinal LNs whereas mesenteric LNs (MLN) from the mutant mice were enlarged as compared with age-matched wild-type animals (Fig. 1B). Next, flow cytometric analyses revealed that RasGRP1–/– CD4 and CD8 T cells express very high levels of CD44 as compared with wild type regardless of whether they were isolated from the spleen, peripheral LN, or MLN (Fig. 1C and our unpublished observations). In addition, a large proportion of RasGRP1–/– CD4 T cells also display signs of acute activation (CD69high, CD127low, CD62Llow, Fashigh, FasLhigh). The activated and memory phenotype for RasGRP1–/– T cells is particularly conspicuous because our TCR-transgenic studies demonstrated that central tolerance was not affected by RasGRP1 deficiency and that RasGRP1–/– T cells displayed diminished capacities to undergo homeostatic expansion and respond to cognate Ag (16).


Figure 1
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  		FIGURE 1. Peripheral CD4 T cells from RasGRP1–/– mice display signs of activation, exhaustion, proliferation, and spontaneous apoptosis. A, RasGRP1–/– animals possess a reduced frequency and numbers of peripheral CD4 and CD8 T cells. Decreased splenic CD4 and CD8 {alpha}beta TCR+ T cell numbers in mice lacking RasGRP1. The single (*) and double asterisk (**) represent p values, using an unpaired, two-tailed Student’s t test, calculated at p < 0.02 and p < 0.005, respectively. B, Comparison of spleens, PLNs (brachial LN is shown), and MLN between wild-type and age-matched RasGRP1–/– mice. C, RasGRP1–/– CD4 and CD8 {alpha}beta TCR+ T cells (bold line) possess markers of memory and acute activation. Thin line, shaded histograms indicate staining pattern of wild-type T cell counterparts. D, RasGRP1–/– CD4 T cells (bold line) express elevated levels of PD-1 and its ligand PD-L1 as compared with wild type (thin line). Shaded histograms represent background autofluorescence of unstained cells. E, An increased frequency of RasGRP1–/– CD4 T cells exhibit expression of the proliferation-associated nuclear Ag Ki-67. F, RasGRP1–/– CD4 T cells bind high levels of apoptotic marker annexin V.

 
The activated state of RasGRP1–/– CD4 T cells is reminiscent of the exhausted phenotype described in mice and humans suffering from chronic viral infections (8). Therefore, we sought to determine whether RasGRP1–/– T cells expressed PD-1, a molecule that is strongly expressed by exhausted but not normal T cells (10). Strikingly, a large fraction of RasGRP1–/– CD4 T cells possess high levels of PD-1 on their surface (Fig. 1D). In addition, PD-L1, a PD-1 ligand that is constitutively expressed by most splenocytes (10), is markedly up-regulated by RasGRP1–/– CD4 T cells whereas it is more modestly elevated by mutant CD8 T cells (Fig. 1D). By contrast, expression of PD-L2 does not appear to be altered in RasGRP1–/– mice (our unpublished observations). Next, we examined whether T cell activation in RasGRP1–/– mice was linked with cellular proliferation (Fig. 1E). Indeed, RasGRP1–/– CD4 T cells exhibit an elevated frequency of cells bearing the proliferation-associated nuclear Ag Ki-67 as compared with wild type (13.3 vs 5.0%). Because elevations in CD44 levels are intimately linked with peripheral T cell expansion (2), Ki-67 staining associates with increased CD44 expression in both wild-type and mutant mice (Fig. 1E). The difference in Ki-67 expression between wild-type and mutant CD4 T cells is significantly less pronounced when comparisons are based on the CD44high subsets (wild type = 13.4%; mutant = 15.7%). Notably, the CD44 expression profiles shown for wild-type and mutant T cells (Fig. 1E) is altered as compared with Fig. 1C because it was performed after formaldehyde fixation and permeabilization with methanol to facilitate the detection of the intracellular Ag Ki-67 (see Materials and Methods). Studies have shown that formaldehyde/methanol treatment of cells results in a reduction in the intensity of CD44 staining, as detected by the clone IM7 mAb, and an increase in background cell staining levels (22). Subsequently, we sought to determine whether RasGRP1–/– CD4 T cells show signs of accelerated cell death because elevated Fas/FasL levels and exhausted T cell phenotypes are associated with increased rates of T cell apoptosis (Fig. 1F). Staining with the apoptotic marker annexin V revealed that RasGRP1–/– CD4 T cells are strongly reactive toward this reagent (RasGRP1–/– = 76.0% vs RasGRP1+/+ = 24.1%). Collectively, these findings suggest that RasGRP1–/– CD4 T cells are highly activated, cycling, and apoptotic as compared with wild type.

RasGRP1–/– CD4 SP thymocytes display a naive cell surface phenotype

Mice deficient in RasGRP1 exhibit severely diminished numbers of mature SP thymocytes demonstrating that this molecule plays a critical role in thymopoiesis (14, 16, 18) (Fig. 2A). To examine whether RasGRP1–/– CD4 and CD8 T cells spontaneously acquire a memory phenotype from their development in the thymus, we stained thymocytes with Abs specific for CD4, CD8, and TCRbeta to identify mature (TCRbeta+) SP (CD4+CD8– or CD4–CD8+) thymocyte subpopulations. In stark contrast to peripheral RasGRP1–/– T cells, RasGRP1–/– SP thymocytes express abnormally low amounts of CD44 and CD69 as compared with their wild-type counterparts (Fig. 2B). However, in concordance with SP thymocyte maturation, RasGRP1–/– CD4 SP thymocytes bear equivalent expression of CD5, a marker of TCR signaling during positive selection (23), as well as similar levels of CD62L and Bcl-2 as compared with wild-type CD4 SP thymocytes (Fig. 2B and our unpublished observations). Strikingly, the CD44low phenotype of most RasGRP1–/– CD4 SP thymocytes contrasts with the elevated CD44 expression levels previously reported for RasGRP1lag CD4 SP thymocytes (18). To explain the contradiction between these findings, we hypothesize that the massive lymphoproliferation and lymphocytic tissue infiltration observed in RasGRP1lag animals (18) results in activated peripheral CD4 T cells also infiltrating the thymus. Because B6-backcrossed RasGRP1–/– mice remain lymphopenic, fewer RasGRP1–/– CD4 T cells likely traffic to the thymus and contaminate the CD4 SP electronic gate. Therefore, we argue that RasGRP1 deficiency supports the development of naive CD4 SP thymocytes and that their conversion to a CD44high phenotype in the periphery may result from homeostatic pressures.


Figure 2
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  		FIGURE 2. Aberrant-positive selection in RasGRP1-deficient mice results in a small population of SP thymocytes that bear a naive cell surface phenotype. A, Regions used for gating are indicated in the developmental profiles from wild-type and mutant mice. B, Expression of the CD5, CD44, CD62L, and CD69 differentiation markers are shown for wild-type (thin line) and RasGRP1–/– (bold line) DP, CD4 SP, and CD8 SP thymocyte subpopulations. Shaded histograms represent autofluorescence of unstained thymocyte subpopulations.

 
RasGRP1–/– host animals support rapid donor T cell proliferation

A hypothesis for the origin of memory phenotype (CD44high) RasGRP1-deficient T cells is that they are the product of slow homeostatic proliferation that result in the conversion of naive T cells into memory T cells. Because a substantially reduced number of SP thymocytes develop and are exported to the peripheral lymphoid organs in RasGRP1–/– mice (16), the few mature SP thymocytes that immigrate to the periphery are subjected to a T cell lymphopenic environment. Therefore, the availability of IL-7 and self-MHC interactions in RasGRP1–/– mice may be well-suited for inducing the peripheral T cell expansion. To test this hypothesis, we adoptively transferred equivalent numbers of wild-type T cells (Thy1.1+), labeled with the mitotic tracker CFSE, into RasGRP1–/–, wild-type B6, irradiated B6 (600 rad) and TCR{alpha}–/– host animals for a 1-wk period (Fig. 3A). As expected, the majority of CD4 and CD8 T cells recovered from normal (lymphoreplete) mice had failed to divide whereas those from irradiated recipients had undergone slow expansion that is characteristic of homeostatic proliferation, still retaining some fluorescence imparted by CFSE. In contrast, the outcome is substantially different when T cells are transferred into in congenitally T cell-deficient TCR{alpha}–/– or RAG-1–/– mice (6, 7). In these chronically immunodeficient animals, which are completely devoid of any {alpha}beta TCR+ cells, some donor T cells underwent "typical" homeostatic proliferation, cycling one to four times per week, while other donor T cells divide rapidly, greater than eight times within a week, and completely lost their CFSE fluorescence. The massive growth of these rapidly dividing cells results in their increased numbers and representation when looking at the distribution of donor cell CFSE fluorescence 1-wk posttransplantation (TCR{alpha} null; Fig. 3A). Interestingly, the donor cell division history in RasGRP1–/– recipients revealed a unique CFSE profile bearing similarities to both TCR{alpha}–/– and irradiated B6 hosts: a rapidly dividing population (52% of CD4+ and 37% of CD8+ T cells) and a slowly dividing population (21% of CD4+ and 50% of CD8+ T cells), respectively. These findings demonstrate that the lymphopenia present within RasGRP1–/– mice promotes spontaneous peripheral T cell expansion.


Figure 3
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  		FIGURE 3. The cellular environment within RasGRP1-deficient animals promotes T cell expansion. A, Purified wild-type T cells (Thy 1.1+) were labeled with CFSE and adoptively transferred into normal C57BL/6J (B6), irradiated B6, normal B6.RasGRP1–/–, and B6.TCR{alpha}–/– mice. Splenocytes were harvested 1-wk posttransfer and the proliferation of donor CD4 and CD8 T cells measured by flow cytometry. B, Same type of experiment as in A except that contour plots are presented, displaying CD62L expression as a function of CFSE fluorescence. C, Same experiment as in A except that 2C CD8 T cells (Thy 1.1+) were adoptively transferred into the indicated host animals and donor T cell proliferation tracked by gating on Thy1.1+CD8+ 2C TCR+ cells.

 
Foreign Ags, derived from commensal microbes, are thought to be responsible for the rapid naive T cell expansion observed after transfer of naive T cells into chronically immunodeficient mice RAG-1–/– and TCR{alpha}–/– mice (7). To support the idea that these rapidly dividing T cells in RasGRP1–/– recipients are undergoing an Ag-driven differentiation program, we charted CD62L (L-selectin), a marker that is down-modulated upon acute activation, vs CFSE fluorescence after 1-wk residence in vivo (Fig. 3B). Strikingly, a large proportion of the CFSElow CD4 (69%) and CD8 (47%) T cells recovered from RasGRP1–/– mice have low CD62L expression. By comparison, T cells residing in irradiated B6 recipients maintain high levels of CD62L while the majority of T cells recovered from RAG-1–/– hosts have down-regulated this marker (Fig. 3B). Next, we examined whether RasGRP1–/– mice could also promote expansion of TCR-transgenic T cells by infusing wild-type 2C TCR-transgenic CD8 T cells (Thy1.1+), labeled with CFSE, into normal B6, irradiated B6, RAG-1–/–, and RasGRP1–/– host animals and measuring cellular proliferation 1 wk later (Fig. 3C). Notably, RasGRP1–/– recipients induce slow 2C T cell proliferation but this amount is considerably less than both RAG-1–/– and irradiated B6 mice (97 and 87%, respectively). Moreover, the observation that 2C T cells do not undergo rapid proliferation in either RasGRP1–/– or RAG-1–/– hosts suggests that this type of T cell expansion may apply to only a subset of T cells with unique TCR specificity. Together, these results suggest that RasGRP1–/– mice are chronically immunodeficient and that forces driving wild-type T cell expansion in RasGRP1–/– hosts could be a combination of self- and foreign-Ags.

RasGRP1–/– CD4 T cells proliferate vigorously in chronically immunodeficient RAG-1–/– mice

The previous experiments examined how wild-type T cells respond following transplant into RasGRP1–/– hosts and therefore may not be reflective of how RasGRP1–/– T cells react to environmental cues. To define how mutant T cells respond in these different settings, CFSE-labeled RasGRP1–/– T cells (Thy1.2+) were i.v. injected into normal B6 (Thy1.1+), irradiated B6 (Thy1.1+), and RAG-1–/– animals (Fig. 4A). Strikingly, the majority of RasGRP1–/– CD4 T cells recovered from either normal or irradiated B6 animals were not recruited into cell cycle (63 and 65%, respectively) while the large fraction of RasGRP1–/– CD8 T cells isolated from irradiated recipients were proliferating slowly (87%). By contrast, RasGRP1–/– CD4 and CD8 T cells transplanted into RAG-1–/– hosts proliferated vigorously. Furthermore, RasGRP1–/– T cells, particularly the CD4 T cells, recovered from RAG-1–/– recipients strongly down-regulated CD62L expression whereas those placed in irradiated B6 hosts resembled the surface phenotype before adoptive transfer (Figs. 4B and 1C). Next, we addressed whether the residence of RasGRP1–/– T cells in different hosts influenced effector function (Fig. 4C). Upon TCR stimulation, RasGRP1–/– T cells recovered from RAG-1–/– hosts possessed an increased frequency of cells capable of producing the proinflammatory cytokines TNF-{alpha} and IFN-{gamma} vs those residing in irradiated wild-type recipients. In summation, these studies demonstrate that chronically immunodeficient hosts are capable of inducing RasGRP1–/– T cells to proliferate rapidly and boost effector function.


Figure 4
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  		FIGURE 4. RasGRP1–/– T cells expand vigorously after transfer into chronically immunodeficient RAG-1–/– hosts. A, CFSE-labeled RasGRP1–/– T cells (Thy 1.2+) were adoptively transferred into normal B6 (Thy 1.1+), irradiated B6 (Thy 1.1+), or B6.RAG-1–/– host animals. After residing within recipient animals for 1 wk, donor T cell proliferation was measured by staining splenocytes with specific Abs to Thy1.2, CD4, CD8, and TCRbeta and electronic gating. B, Same type of experiment as in A except that the relationship between cellular proliferation and CD62L expression is presented as contour plots. C, RasGRP1–/– T cells were recovered from either B6.RAG-1–/– or irradiated wild-type B6 recipients after 1-wk residence, stimulated on anti-TCR Ab-coated plates, and assessed for cytokine production.

 
RasGRP1–/– mice generate poor pathogen-specific T cell responses

To test the hypothesis that RasGRP1–/– mice are immunodeficient, wild-type and mutant mice were infected with a novel recombinant strain of L. monocytogenes (rLM-SIY) expressing a MHC class I Kb-restricted peptide SIYRYYGL (SIY), an agonist for the 2C TCR (24). One week postinfection, splenocytes were stimulated with either the endogenous MHC class II-restricted peptide LLO190–201 (listeriolysin O (LLO); Fig. 5A) or SIY (Fig. 5B) and Ag-specific T cell responses were monitored by IFN-{gamma} production using intracellular flow cytometry. As an additional control, splenocytes were also cultured on anti-TCR Ab-coated plates to test for the capacity to produce IFN-{gamma}. Strikingly, RasGRP1–/– mice mounted a barely detectable immune response toward the LLO peptide (Fig. 5C). The fact that RasGRP1–/– CD4 T cells can respond to anti-TCR Abs suggests that the weak response by RasGRP1–/– mice is the result of a failure to generate LLO-reactive T cells rather than to secrete IFN-{gamma}. By contrast, RasGRP1–/– CD8 T cells generate a strong anti-SIY response that is modestly reduced in Ag-specific T cell numbers as compared with wild type (Fig. 5C). In addition, we tested the function of RasGRP1–/– CD8 T cell effectors in a standard 51Cr-release assay and found that these cells displayed cytotoxicity similar to their wild-type counterparts (Fig. 5D). These rLM-SIY infection studies suggest that RasGRP1 is particularly critical for generating MHC class II-restricted immune responses.


Figure 5
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  		FIGURE 5. RasGRP1–/– mice mount a poor MHC class II-restricted LLO response upon infection with L. monocytogenes. Wild-type and RasGRP1–/– animals were infected i.v. with rLM-SIY. Spleens of infected mice were harvested 7 days postinfection and assayed for immune responses toward the immunodominant MHC class II-restricted peptide LLO190–201 (A) or the MHC class I-restricted peptide SIY (B) by measuring IFN-{gamma} production using intracellular flow cytometry. Numbers within the plot reflects the frequency of CD8 or CD4 T cells responding to a particular condition. C, RasGRP1–/– mice possess reduced numbers of anti-LLO-reactive CD4 T cells. Error bars, SD. D, RasGRP1–/– CD8 T cell effectors display potent cytotoxic activity. Various numbers of splenic CD8 T cells from wild-type and mutant mice were incubated with a 51Cr-labeled EL-4 target.

 
To address the possibility that the importance of RasGRP1 on mounting MHC class II-restricted immune responses may be restricted to the LLO peptide or bacterial infection, we used LCMV to infect wild-type and RasGRP1–/– mice. Eight days postinfection with a sublethal dose of LCMV, Ag-specific T cells were enumerated to viral immunodominant MHC class II-restricted GP61–80 (Fig. 6A) and MHC class I-restricted peptides (GP33–41, GP34–43, GP276–286, NP205–212 and NP396–404; Fig. 6B). Similar to the bacterial infection, RasGRP1–/– mice generated a weak MHC class II-restricted response although this time a reduced fraction of mutant CD4 T cells produced IFN-{gamma} upon TCR stimulation (3.6 vs 13.0%). However, in contrast to rLM-SIY results, RasGRP1–/– mice mounted weak immune responses toward all five MHC class I-restricted viral peptides analyzed despite the fact that a similar proportion of wild-type vs mutant CD8 T cells can secrete IFN-{gamma} upon TCR stimulation (48%). The fact that total T cell numbers were also sharply reduced in RasGRP1–/– animals makes the drop in Ag-specific T cell numbers even more dramatic (Fig. 6C). To evaluate whether the diminished T cell responses were physiologically relevant, splenic tissue, harvested 8 days postinfection, was screened for virus using plaque-forming assays (Fig. 6D). Indeed, 3 of 4 RasGRP1–/– spleens in this experiment (9 of 13 total) contained readily detectable virus at this late period postinfection, at a time point when virus is undetectable among wild-type mice. In conclusion, the weakened immune responses by RasGRP1–/– mice is associated with delayed viral clearance.


Figure 6
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  		FIGURE 6. RasGRP1–/– animals generate a weak T cell response toward LCMV and exhibit delayed viral clearance. Wild-type and mutant mice were infected i.p. with the LCMV. At day 8 postinfection, splenocytes were cultured in either medium alone (no peptide), stimulated with the indicated immunodominant viral peptide or placed in a well coated with anti-TCR Ab ({alpha}TCR). Anti-LCMV T cells were enumerated by IFN-{gamma} production by gating on either CD4 (A) or CD8 (B) T cells. Numbers within the plot reflects the frequency of CD8 or CD4 T cells responding to a particular condition. C, RasGRP1–/– animals possess greatly reduced numbers of Ag-specific T cells. Error bars represent the SD. D, At day 8 postinfection, LCMV can still be detected in the spleens of some RasGRP1–/– animals. Dashed line indicates the approximate detection level of the assay.

 
The ability of RasGRP1–/– mice to respond strongly to the SIY peptide but only weakly to immunodominant LCMV peptides led us to speculate whether the differential responses were intrinsic to the peptides or the pathogen. To resolve this issue, wild-type and RasGRP1–/– mice were infected with two other rLMs, either rLM-OVA or rLM-GP33, and the frequency of MHC class I-restricted anti-OVA257–264- and anti-GP33–41-specific T cells assessed 7 days postinfection. In stark contrast to the SIY peptide, RasGRP1–/– mice mounted weak, barely detectable responses toward both OVA257–264 and GP33–41 MHC class I-restricted peptides (our unpublished observations). These findings that RasGRP1–/– mice can respond to a limited set of immunodominant peptides suggest that its resident T cells may possess an altered TCR repertoire. To look for differences in the TCR repertoire between wild-type and RasGRP1–/– mice, Vbeta TCR chain usages were determined by staining splenocytes with various anti-Vbeta TCR chain-specific Abs in conjunction with Abs specific for CD4, CD8, and the constant region of the TCRbeta chain (Fig. 7). Although we observed differences among the Vbeta TCRs used by both RasGRP1–/– CD4 and CD8 T cells, the CD4 T cell population exhibited fewer statistically significant changes, due in part to a large deviation between RasGRP1–/– mice. It is not clear whether the TCR repertoire changes in RasGRP1–/– mice results from aberrant T cell development, homeostatic strain, or a mixture of both processes. Moreover, the possibility exists that CD4 T cell immune activation, resulting in persistent rounds of cellular proliferation and death, could contribute to great variability exhibited by RasGRP1–/– CD4 T cell TCR repertoire. Collectively, these studies raise the prospect that changes in the TCR repertoire in RasGRP1–/– mice could be responsible for the impaired generation of pathogen-specific T cells.


Figure 7
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  		FIGURE 7. Peripheral RasGRP1–/– T cells possess an altered TCR repertoire. The TCR Vbeta usage of wild-type (n = 5) and mutant T cells (n = 7) was analyzed by using a panel of anti-TCR Vbeta Abs, flow cytometry and electronic gating on either CD4+ TCRbeta+ (A) or CD8+ TCRbeta+ cells (B). To determine the significant differences observed between the two sets of animals, a two-tailed Student’s t test (two-sample, unequal variance; heteroscedastic) was performed. The single asterisk (*) and double asterisk (**) represent values of p < 0.02 and p < 0.005, respectively.

 
Failure of RasGRP1–/– mice to generate pathogen-specific T cells is the result of RasGRP1 loss in thymocytes and/or T cells

Because the engineered mutation in RasGRP1 results in a systemic loss of RasGRP1 function (14), it is possible that RasGRP1 deficiency in another cell type, besides T cells, may contribute to defective pathogen-specific responses. To investigate whether the innate immune system within RasGRP1–/– mice is capable of nurturing T cell responses, mutant mice (Ly5.2+) were infused with purified wild-type T cells (Ly5.1+) and infected the next day with either rLM-SIY or LCMV (Fig. 8A). Seven days post-rLM-SIY infection, a sizable proportion (7.1%) of the wild-type CD4 T cells were LLO reactive whereas few RasGRP1–/– CD4 T cells produced IFN-{gamma} upon peptide stimulation (Fig. 8B). Interestingly, RasGRP1–/– CD8 T cells (Ly5.1–) mounted a weaker anti-SIY response in the presence of wild-type T cells (Figs. 8B and 5B), implying that they may not compete well for Ag. These findings suggest that the innate immune system in RasGRP1–/– mice is not to blame for the impaired generation of rLM-specific T cells.


Figure 8
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  		FIGURE 8. Failure of RasGRP1–/– mice to generate pathogen-specific T cells is the result of RasGRP1 loss in thymocytes and/or T cells. A, RasGRP1–/– mice (Ly 5.2+) were infused with ~10 million wild-type (Ly 5.1+) T cells and infected 1 day later with either rLM-SIY or LCMV. Splenocytes were recovered from animals after infection with either rLM-SIY (B) or LCMV (C), stimulated with the indicated peptides in vitro, and IFN-{gamma} production measured by flow cytometry. Data were electronically gated on either CD4 or CD8 and the frequency of responding wild-type (Ly5.1+; right) or RasGRP1–/– T cells (Ly5.1–; left) is indicated within the density plot.

 
To determine whether the wild-type T cells could also respond in mutant mice after viral infection, we performed an analogous wild-type T cell infusion experiment except this time we infected with LCMV. Consistent with our rLM-SIY observations, wild-type CD4 and CD8 T cells generate strong anti-LCMV responses toward immunodominant viral peptides (Fig. 8C). Interestingly, RasGPR1–/– mice seem to mount a better IFN-{gamma} response to the NP396 peptide in the presence of wild-type donor T cells (12 vs 0.8%; Figs. 8C and 6B). A plausible explanation for this observation is that wild-type CD4 T cells help the mutant CD8 T cell response by producing cytokines or providing costimulation. Notably, we have previously shown that the addition of the cytokine IL-2 can restore Ag-driven proliferation by RasGPR1–/– 2C CD8 T cells in vitro (16). These experiments argue that poor T cell responses observed in RasGRP1–/– mice are the consequence of RasGRP1 deficiency in thymocytes and/or T cells.

The finding that the "empty" lymphoid compartment in RasGRP1–/– mice can induce spontaneous T cell expansion suggests the possibility that it could initiate the differentiation of donor T cells into pathogen-specific T cell effectors without infection. To explore the likelihood of this possibility, we compared cohorts of RasGRP1–/– mice receiving wild-type T cell infusions that were left untreated with those infected with LCMV. Nine days postdonor T cell infusion, mice that were left untreated did not exhibit significant numbers of viral-specific T cells (our unpublished observations) and the donor cell recovery was substantially lower than those infected with LCMV (3.4- ± 0.7-fold-decrease for CD4 T cells; 7.3- ± 1.2-fold-decrease for CD8 T cells). These experiments demonstrate that the adoptive transfer of wild-type T cells into RasGRP1–/– mice does not induce their spontaneous differentiation into anti-LCMV T cell effectors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The prime directives of thymocyte development are to generate a TCR repertoire that is self-restricted, self-tolerant, and diverse, enabling responses toward a vast array of foreign peptides associated self-MHC molecules. Because the generation of the TCR repertoire is dependent on TCR signaling, mutations affecting signaling molecules downstream of the TCR may have deleterious effects on both T cell function and TCR repertoire. In this study, we report the consequences of RasGRP1 deficiency and reduced TCR-induced Ras signaling on peripheral T cell homeostasis and T cell immunity.

A recently described mouse strain called RasGRP1lag suffers from massive lymphoproliferation and an autoimmune syndrome sharing similarities with SLE (18). Although young mice appeared normal, older RasGRP1lag mice developed massive lymphoproliferation, displaying splenomegaly and lymphadenopathy, with an excess of 10-fold larger lymph node size and cell numbers as compared with age-matched controls (18). By 5–8 mo of age, RasGRP1lag mice were found to be so anorexic and lethargic that it necessitated euthanasia (18). Although we observed RasGRP1–/– mice that developed substantial splenomegaly and lymphadenopathy (our unpublished observations), the penetrance of this phenotype disappeared after successive backcrossing of the targeted RasGRP1 mutation to the B6 background. Our B6 backcrossed RasGRP1–/– mice remain T cell lymphopenic and appear healthy until at least 1 year of age. However, despite the absence of massive lymphoproliferation, these RasGRP1–/– mice do possess elevated levels of serum autoantibodies (our unpublished observations). Because autoimmune disease often requires a complex mixture of genetics and environmental factors (25, 26), it is perhaps not surprising that a change in genetic background may be responsible for the contradictions between our findings and those previously reported (18). Moreover, a recent study has found that SLE can simply develop from a hybrid 129:B6 background rather than targeted gene disruption (27). Therefore, we suspect that genetic modifiers from the 129/SvJ mouse strain may synergize with RasGRP1 deficiency to cause massive lymphoproliferation and exacerbate autoimmune disease.

It has been proposed that RasGRP1-deficient thymocytes capable of maturing into SP thymocytes need to express more strongly self-reactive TCRs to overcome their signaling deficits (16, 17, 18). Because central tolerance does not appear to be affected by RasGRP1 deficiency (16), it has led us to hypothesize that self-reactivity of TCRs mediating positive selection of RasGRP1–/– double-positive thymocytes (DP) must bridge the boundary between positive and negative selection. The question of why immune activation selectively affects the CD4 T cell lineage in RasGRP1–/– mice is unknown. Because CD4 T cell development has been proposed to be more highly dependent on RasGRP1/ERK signaling (17, 28), RasGRP1 deficiency may affect thymic ontogeny by selecting more strongly self-reactive CD4 than CD8 SP thymocytes. Alternatively, RasGRP1-dependent mechanisms preserving peripheral tolerance or a relentless homeostatic strain may preferentially induce the activation of RasGRP1–/– CD4 T cells. However, because RasGRP1–/– T cells are severely hyporesponsive as compared with wild-type cells (16), their autoimmune potential may be counterbalanced by their inefficient TCR signaling and reduced proliferation upon Ag encounter.

Homeostatic mechanisms that function to regulate peripheral T cell numbers may be basis for the association between autoimmunity and T cell lymphopenia observed in both animals and humans (4, 25). Our studies of the homeostatic mechanisms operating in RasGRP1–/– mice suggest that both self- and foreign-Ags could be driving T cell proliferation (Fig. 3). However, because the RasGRP1 mutation was made in 129/SvJ embryonic stem cells (14), it is plausible that 129/SvJ-derived alloantigens may be responsible for some donor wild-type B6 T cell proliferation observed after their transfer into RasGRP1–/– mice (Fig. 3). Although the RasGRP1–/– mice used in this study have been bred at least seven generations onto the B6 background, this mouse line may still contain a significant amount of 129/SvJ DNA that is likely closely linked to the targeted locus. To identify the forces driving the cell cycling of RasGRP1–/– CD4 T cells in vivo (Fig. 1E), we initiated a series of adoptive transfer experiments using RasGRP1–/– donor T cells. Notably, it had been hypothesized that RasGRP1 deficiency allows autoreactive T cells to escape the thymus, proliferate upon encounter with peripheral self-Ags, and initiate autoimmunity (18). However, the failure of the majority of RasGRP1–/– CD4 T cells to proliferate after placement in wild-type hosts, either unmanipulated recipients or ones made lymphopenic through irradiation, suggests that self-Ags may not be responsible for their expansion (Fig. 4). By contrast, the observation that a subset of RasGRP1–/– CD4 T cells can undergo massive expansion in RAG-deficient hosts suggests that foreign Ags could be stimulating CD4 T cell proliferation in RasGRP1–/– animals (Fig. 4). Moreover, foreign Ags may be ideally suited to provoke weakly responsive RasGRP1-deficient CD4 T cells to proliferate vigorously because they can simultaneously act as a direct TCR stimulus, an activator of APCs and an inducer of inflammatory cytokine production. Therefore, these studies demonstrate that the environment within irradiated wild-type mice, possessing increased availability to both self-Ags and cytokines, is insufficient to recruit most RasGRP1–/– CD4 T cells into cell cycle.

The observation that RasGRP1–/– mice exhibit diminished T cell responses and delayed pathogen clearance suggests that they could be prone to developing chronic infections. Notably, RasGRP1–/– CD4 T cells share some attributes with functionally exhausted memory T cells found in both mice and humans during chronic infections (8, 9). First, RasGRP1–/– CD4 T cells possess markers of acute activation and T cell memory like exhausted T cells (Fig. 1C). Second, RasGRP1–/– CD4 T cells seem to have limited self-renewal capacity because they expand poorly in irradiated wild-type recipients, a cellular environment where the availability of the common {gamma}-chain-linked cytokines IL-7 and IL-15 is increased (Fig. 4). RasGRP1–/– CD4 T cells also have reduced IL-7R expression (Fig. 1C). Third, RasGRP1–/– CD4 T cells strongly express PD-1, an inhibitory receptor that is coupled with exhausted viral-specific CD8 T cells in both mice and humans (11, 12), as well as its ligand PD-L1 (Fig. 1D). In contrast to the CD4 T cell phenotype, RasGRP1–/– CD8 T cells do not possess an exhausted phenotype because they do not exhibit signs of acute activation (CD69low, CD62Lhigh, CD127high; Fig. 1C), fail to express elevated levels of PD-1 (Fig. 1E) and can mount a significant anti-SIY T cell response (Fig. 5, B–D). A complication of housing chronically activated CD4 T cells is that it could promote autoimmunity in RasGRP1–/– mice, perhaps through elevated FasL expression, inducing nonspecific cell death, or proinflammatory TNF-{alpha} production (Figs. 1C and 4C). Therefore, T cell immunodeficiency could predispose RasGRP1–/– mice to both chronic infections and autoimmunity.

The failure of RasGRP1–/– mice to generate normal numbers of Ag-specific CD4 and CD8 T cells after bacterial and viral infection likely results from a mixture of direct (T cell hyporesponsiveness) and indirect (altered thymic development and T cell homeostasis) influences of RasGRP1 deficiency. Decreased T cell responsiveness would be predicted to reduce the probability that a given T cell undergoes Ag-induced developmental programming whereas changes to T cell development and peripheral T cell homeostasis could alter the TCR repertoire and T cell differentiation. Notably, studies on RasGPR1–/– 2C CD8 T cells have found that RasGRP1 regulates homeostatic proliferation (16), TCR-signaling thresholds, and augments cytokine production (J. Priatel, X. Chen, L. Zenewicz, H. Shen, J. Coughlin, J. Stone, and H. Teh. manuscript in preparation). Because the precursor frequency of Ag-specific T cells is a critical parameter for the generation of effector and memory T cells (29, 30), we assessed Vbeta TCR usage among splenic T cells from naive (uninfected) mice to look for alterations in the TCR repertoire between normal and mutant mice. Although our results demonstrate that the TCR repertoire of RasGRP1–/– mice is significantly different, they do not provide a measure of TCR diversity. Moreover, the CD44high surface phenotype of RasGRP1–/– T cells (Fig. 1C) suggests the possibility that these cells may be derived from a considerable amount of peripheral expansion, a phenomena thought to restrict the TCR repertoire because it could result from the selective outgrowth of cells expressing a given TCR. In addition, the exhausted phenotype of RasGRP1–/– CD4 T cells suggests that their state of T cell differentiation could also contribute to their inaction. Therefore, reduced generation of Ag-specific T cells by RasGRP1–/– mice could result from changes in T cell differentiation, T cell function, and/or T cell TCR repertoire.

The fact that numerous T cell lymphopenic animals and humans exhibit T cell activation argues that an "empty" T cell compartment and changes to T cell homeostasis play a major role in the phenotype observed in RasGRP1–/– mice rather than being solely attributable to RasGRP1 loss in peripheral T cells. Interestingly, T cell lymphopenia is also often associated with T cell hyporesponsiveness and autoimmunity (25) and as a consequence, it raises the question of what are common denominators between these phenomena. The knowledge that TCR signaling is critically important for both T cell development and T cell survival has provided insight into the pairing of these occurrences (1, 31). Moreover, mutations affecting TCR signaling may cause T cell activation by impacting central and/or peripheral tolerance (32). In addition, immunodeficiency may lead to disruptions in T cell homeostasis, development of chronic infections, persistent T cell activation, and T cell exhaustion (8, 10). Additionally, studies on two models of spontaneous autoimmune diabetes, the NOD mouse and BioBreeding diabetic rat, suggest that T cell lymphopenia may initiate disease by altering T cell homeostasis (33, 34).

In conclusion, our study highlights the dangers associated with RasGRP1 loss, including changes to T cell development, peripheral T cell homeostasis and T cell immunity. Because our findings suggest that chronic immunodeficiency promotes persistent CD4 T cell activation and constitutive proinflammatory cytokine production, they provide insight into why the conditions of T cell lymphopenia and T cell hyporesponsiveness are linked to autoimmunity in humans (25). An added complication of persistent T cell activation is that it may result in functional impairments by inducing a state of T cell exhaustion, a phenomenon commonly observed during chronic infection in mice and humans (8). Recently, PD-1, an inhibitory receptor that functions to put a brake on TCR signaling (10), has been shown to impair function of exhausted viral-specific CD8 T cells during chronic LCMV infection (11). Because CD4 T cells in HIV-infected individuals (12) and RasGRP1–/– mice express high levels of PD-1 (Fig. 1D), it leads us to speculate whether these exhausted states arise from similar forces despite the fact that CD4 T cell lymphopenia in these scenarios has disparate origins. In HIV-infected individuals, it has been hypothesized that CD4 T cell immune activation results from a combination of homeostatic strain and immunodeficiency at mucosal sites, facilitating infections by opportunistic pathogens (35, 36). Thus, it is possible that chronic immunodeficiency in RasGRP1–/– mice allows for translocation of commensal microflora across intestinal epithelium, causing local or systemic infections and CD4 T cell immune activation. Further study of RasGRP1–/– mice will provide additional mechanistic insights into consequences of T cell immunodeficiency and contribute to understanding how such alterations predispose to autoimmune disease.


   Acknowledgments
 
We thank Salim Dhanji and Michael Chow for helpful discussions, Soo-Jeet Teh for technical assistance, Iryna Shanina and Diane Fang for LCMV plaque titer determinations, James C. Stone for RasGRP1–/– breeder mice, Ninan Abraham for providing B6.SJL-Ptprca Pep3b/BoyJ (Ly5.1+) mice, Hermann J. Ziltener for providing additional B6.RAG-1–/– mice, the Wesbrook Animal Unit for animal husbandry, and the Life Sciences Flow Cytometry Facility for cell sorting.


   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 Grant MOP-77547 from the Canadian Institutes of Health Research (CIHR; to H.-S.T.). J.J.P. was funded in part by a CIHR fellowship. Back

2 Current address: Department of Pathology and Laboratory Medicine, British Columbia Children’s Hospital, Vancouver, British Columbia, Canada Back

3 Address correspondence and reprint requests to Dr. Hung-Sia Teh, Department of Microbiology and Immunology, Life Sciences Centre, University of British Columbia, Room 3509, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3. E-mail address: teh{at}interchange.ubc.ca Back

4 Abbreviations used in this paper: PD-1, programmed cell death 1; PD-L1, PD-1 ligand 1; DP, double-positive thymocyte; SP, single-positive thymocyte; LCMV, lymphocytic choriomeningitis virus; LN, lymph node; MLN, mesenteric LN; SLE, systemic lupus erythematosus; LLO, listeriolysin O. Back

Received for publication December 19, 2006. Accepted for publication June 4, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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X. Chen, J. J. Priatel, M. T. Chow, and H.-S. Teh
Preferential Development of CD4 and CD8 T Regulatory Cells in RasGRP1-Deficient Mice
J. Immunol., May 1, 2008; 180(9): 5973 - 5982.
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