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International Immunology Advance Access originally published online on March 15, 2006
International Immunology 2006 18(4):565-572; doi:10.1093/intimm/dxh398
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Quantitative assessment concerning the contribution of IL-2Rß for superantigen-mediated T cell responses in vivo

Haoli Jin*, Dapeng Gong*, Dennis Adeegbe, Allison L. Bayer, Cleo Rolle, Aixin Yu and Thomas R. Malek

Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, PO Box 016960, Miami, FL 33101, USA
Present address: Division of Immunology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA

Correspondence to: T. Malek; E-mail: tmalek{at}med.miami.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
IL-2- and IL-2R-deficient mice readily develop T cell-dependent immune responses in vivo, but the relevance of this finding is complicated by severe concurrent autoimmunity. Furthermore, the detection of such responses does not address whether under normal circumstances IL-2 dominates T cell immunity. In the present report, we investigated the extent IL-2-independent T cell growth is mediated by other cytokines in the IL-2 family and compared such responses to those generated by IL-2/IL-2R-sufficient T cells. T cell expansion and contraction to the superantigen staphylococcal enterotoxin A (SEA) by autoimmune-free IL-2Rß–/– CD4 and CD8 T cells were comparable to normal control mice, although their CD8+ T cells did not optimally develop into IFN{gamma}-producing effector cells. The proliferation by these IL-2Rß-deficient T cells in vivo was independent of IL-2, IL-4 and IL-15 and not blocked by mAbs to the common {gamma} chain. However, in co-adoptive transfer experiments, wild-type T cells exhibited somewhat more extensive proliferation than IL-2Rß-deficient T cells to SEA and this difference was almost entirely accounted for by CD8+ T cells. Collectively, these data indicate that substantial T cell proliferation occurs in the absence of responsiveness to cytokines in the IL-2 family, although maximal T cell proliferation and development of IFN{gamma}-producing effector CD8+ T cells depend upon IL-2Rß.

Keywords: cytokines, cytokine receptors, cell proliferation, superantigen, T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Work over the last 20 years has established that IL-2 is a critical cytokine for the T cell growth and effector cell development in vitro (1, 2). Paradoxically, effective immune responses occur in vivo after challenging IL-2- or IL-2R-deficient mice in vivo (3–8). This finding suggests that IL-2 is not mandatory for T cell-dependent immune responses. However, attempts to ascertain the physiological role of IL-2 in vivo by direct analysis of IL-2/IL-2R-deficient mice is complicated by autoimmunity and lymphoproliferation leading to death by 4–12 weeks of age (9–12). This type of autoimmunity that leads to chronic T cell activation and cytokine production is an ideal environment to bypass a potential normal requirement for IL-2. To minimize this complication, some investigators have used young (4–8 weeks old) IL-2–/– mice and/or bred a TCR transgene onto IL-2/IL-2R-deficient animals (13–16). However, this may still not necessarily be sufficient as the onset of the activated phenotype and lymphoproliferation in IL-2Rß-deficient mice is already evident in the periphery by 2 weeks of age. Furthermore, breeding of the OT-I TCR transgene onto the IL-2Rß–/– genetic background only delayed the symptoms of autoimmunity (16).

The enigma concerning the lethal autoimmune disorder associated with IL-2 and IL-2R deficiency is accounted for by a failure in the production of CD4+CD25+ T regulatory (Treg) cells (17–21). Once this problem is corrected, such mice live a normal lifespan and develop T cell-dependent immune responses even though their peripheral T cells remain non-responsive to IL-2 (17). Thus, the immune system effectively compensates for the absence of IL-2/IL-2R for T cell-dependent immunity without an ongoing autoimmune response.

Our laboratory has developed two model systems that indicate that the main role for IL-2 in vivo is the production of Treg cells (17, 22, 23). In one model, IL-2Rß is selectively expressed as a transgene within the thymus of IL-2Rß-deficient mice (designated Tg–/– in this report). In the other, wild-type CD4+CD25+ Treg cells are adoptively transferred into neonatal IL-2Rß–/– mice. In both cases, the mice lived a normal autoimmune-free life with a largely normal peripheral immune compartment, including a lack of activated lymphocytes. When conventional peripheral T cells are isolated from these mice, they are essentially non-responsive to IL-2 in vitro. More detailed studies using peripheral Tg–/– T cells in vitro indicate that they undergo three to four cell divisions after engaging the TCR and co-stimulatory molecules, but after this limited response, Tg–/– T cells do not develop into CTL or IFN{gamma}-producing cells (2). Such activated Tg–/– T cells are not only unable to proliferate to IL-2 or IL-15 but do not undergo normal cytokine-dependent expansion to other {gamma}c-dependent cytokines (2). Thus, the behavior of Tg–/– T cells is in line with the notion that IL-2 is a required cytokine for development and clonal expansion of effector T cells in vitro. However, when Tg–/– mice were challenged in vivo, they readily developed primary and secondary antibody responses, mounted first and second set allogeneic skin graft rejection responses, developed primary and secondary antiviral T cell responses to vaccinia virus infection and their CD8+ T cells developed proliferative and CTL response to nominal antigen (16, 24). These findings indicate that the strict requirement for IL-2 for effector T cell responses in vitro does not apply for the generation of T cell immunity in vivo.

The capacity of Tg–/– mice to develop effective immunity implies that there must be redundant pathways that are effective in vivo that compensates for the lack of IL-2Rß function. In this regard, when Tg–/– T cells were stimulated with anti-CD3 in the presence of IL-4, the Tg–/– T cells mounted near normal CTL responses and subsequently expanded upon further culture with IL-4, suggesting that IL-4 in particular, or perhaps other {gamma}c-dependent cytokines, might effectively substitute for the lack of IL-2Rß in vivo (2). Even though there is IL-2-independent immunity in vivo, there are limited data concerning the magnitude of such responses in the absence of autoimmunity. Moreover, although IL-2-independent T cell immunity readily develops, under normal circumstances IL-2 may remain as the predominant means to drive T cell expansion in vivo. These points also represent critical practical concerns because IL-2 and its receptor have been favorite targets in immunotherapy, primarily to augment or suppress effector T cell responses. Therefore, to address these issues, we examined the responses by Tg–/– T cells in vivo either directly or in competition with wild-type cells after challenge with the bacterial superantigen (Sag) staphylococcal enterotoxin A (SEA). Unlike immune responses to infectious agents or antigens emulsified in adjuvants, the response to Sag results in T cells to undergo well-characterized expansion and contraction without an extensive or prolonged inflammatory response. We viewed, therefore, that the response to SEA provided a more direct analysis of the potential requirement for IL-2Rß for T cell growth in vivo by minimizing the contribution of signaling induced by the large array of cytokines and other interactions that occur during most other immune responses.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
C57BL/6J, Thy-1.1-congenic C57BL/6 mice and IL-4–/– mice on the C57BL/6 genetic background were obtained from Jackson Laboratory (Bar Harbor, ME, USA). CD45.1-congenic C57BL/6 mice were obtained from Taconic (Germantown, NY, USA) and bred in our animal colony. IL-2Rß–/– mice on the C57BL/6 genetic background (10) were bred in our animal colony after preventing autoimmunity by the adoptive transfer of CD4+CD25+ Treg cells into new born IL-2Rß–/– mice (17), as previously described. Tg–/– mice, i.e. thymic transgenic IL-2Rß expression in C57BL/6 IL-2Rß-deficient mice, were previously described (22). These mice and Tg–/– x IL-4–/– mice were maintained in our animal colony. SEA (10 µg; Toxin Technology, Inc., Sarasota, FL, USA) was administered intra-peritoneally (i.p.) in 200 µl of sterile HBSS. In competitive adoptive transfer experiments, all recipients were normal unmanipulated Thy-1.1-congenic C57BL/6 mice. Donor spleen cells were treated with CFSE as previously described (23). These mice were injected with SEA 24 h after adoptive transfer to permit homing and engraftment of the donor cells before in vivo activation with SEA. For anti-{gamma}c treatment (25), mice received 0.5 mg each of the 4G3 and 3E12 mAbs i.p. in PBS 12 h before and 12 h after injection of SEA. Control mice received PBS instead of anti-{gamma}c mAb.

Cell purification and culture
The lymphocytes from the liver and lung were isolated as previously described (26). Briefly, the liver tissue was dispersed through 70-µm wire mesh in HBSS containing 2% FCS and 10 mM HEPES. After centrifugation, the cell pellet was re-suspended in 35% Percoll (Amersham Biosciences, Piscataway, NJ, USA) containing 200 U ml–1 heparin and centrifuged at 600 g for 30 min. The cell pellet was re-suspended in Tris (17 mM) ammonium chloride (140 mM) pH 7.6 for 2 min and washed prior to FACS analysis. Lung tissue was minced in HBSS containing 1.3 mM EDTA and incubated for 30 min in a 37°C shaking water bath. The tissue was then incubated for 1 h at 37°C with 150 U ml–1 collagenase (Sigma, St Louis, MO, USA) in RPMI 1640 containing 5% FCS and calcium chloride (5 mM). After centrifugation, the cell pellet was re-suspended in 44% Percoll and then layered on 67.5% Percoll. After centrifugation at 600 g for 30 min, the cells at the interface were harvested, and washed prior to FACS analysis.

To assess development of IFN{gamma}-producing T cells after anti-CD3 activation in vitro, spleen cells (2 x 106 per well) were cultured in 24-well flat bottom culture plates with anti-CD3 (5% 145-2C11 supernatant) for 48 h in the absence or presence of IL-12 (20 ng ml–1; PeproTech, Rocky Hill, NJ, USA). Cultured cells were harvested, washed and re-cultured for 6 h in the presence of brefeldin A (1 µg ml–1) prior to staining for T cell surface markers and intracellular IFN{gamma}. To assess IFN{gamma} production directly ex vivo, spleen cells were cultured with phorbol myristate acetate (PMA) (50 ng ml–1), ionomycin (1 µM) and brefeldin A (1 µg ml–1) for 4 h prior to staining for T cell surface markers and intracellular IFN{gamma}.

mAbs and FACS analysis
Fluorescent-conjugated mAbs to CD4, CD8, Vß3, Thy-1.2, Thy-1.1, CD45.1 and IFN{gamma} were obtained from Pharmingen BD Biosciences (San Jose, CA, USA). Unconjugated anti-{gamma}c 4G3 was obtained from Pharmingen BD Biosciences and 3E12 was prepared in our laboratory. Cell surface FACS analysis was performed as previously described (23). Intracellular staining for IFN{gamma} was performed after surface staining by permeabilization in Cytofix/Cytoperm solution and washing in Perm/Wash solution according to the manufacturer's instruction (Pharmingen BD Biosciences). Cells were analyzed using a Becton Dickinson LSR1 and CellQuest software. Typically, FACS analysis was performed on 50 000–100 000 cells per sample except for the competitive adoptive transfer experiments where 1 x 106 cells per sample were analyzed.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Role of IL-2Rß for T cell expansion and contraction to SEA
SEA activates T cell proliferation for both CD4+ and CD8+ C57BL/6 T cells bearing Vß1, Vß3, Vß10, Vß11 and Vß12 TCR leading to expansion, effector cell activity and then contraction (27). This provides a means to investigate the role of IL-2 in these responses where T cell activation is not limiting while the inflammatory response is minimized. When normal C57BL/6 or Tg–/– mice were injected with 10 (Fig. 1) or 1 µg (not shown) of SEA, both expansion, which peaks 2–3 days after SEA challenge, and subsequent contraction which is readily observed by day 5 were similar for CD4+ and CD8+ T cells when monitoring Vß3+ T cells (Fig. 1A).


Figure 1
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  		Fig. 1. IL-2Rß is not required for T cell expansion and contraction to SEA. (A) The indicated mice were injected with SEA (10 µg i.p.) and the percentage of Vß3+ CD4+ or CD8+ T cells in the spleen was determined by FACS analysis. Data are the mean ± SD of four to six mice per group. (B) Normal C57BL/6 and autoimmune-free cured IL-2Rß–/– mice, which received CD4+CD25+ Treg cells at birth, were challenged with SEA at 8 weeks of age. T cell expansion to SEA was assessed by FACS analysis and then determining the percentage of Vß3+ CD4+ or CD8+ T cells in the spleen. Data are the mean ± SD of two to four mice per group.

 
We have previously extensively documented that conventionally activated peripheral Tg–/– T cells do not express IL-2Rß and are non-responsive to IL-2 or IL-15 in vitro and in vivo (2, 17, 22, 24, 28). Nevertheless, to further address that the response to SEA was IL-2Rß independent, we also examined Vß3+ T cell expansion and contraction by non-transgenic IL-2Rß–/– mice that were ‘cured’ of autoimmunity by the adoptive transfer of wild-type Treg cells (17, 23). Since we used CD45.1 congenic Treg cells, the response by the recipient IL-2Rß–/– T cells is directly examined by staining for CD45.2 and cannot be construed to be due to a response by donor cells. In this situation, the level of expansion of recipient Vß3+ IL-2Rß–/– CD4+ and CD8+ T cells was also largely comparable to that detected for normal C57BL/6 T cells (Fig. 1B). Collectively, these data indicate that IL-2Rß function is not required for T cell growth and contraction in response to SEA.

SEA responses by Tg–/– T cells in competitive adoptively transferred mice
The response to SEA shown above by Tg–/– T cells demonstrates that expansion and contraction occur efficiently in the absence of IL-2Rß. However, under normal circumstance, IL-2-dependent T cell growth may be much more effective than this IL-2Rß-independent pathway. To investigate this possibility, T cell expansion and contraction were evaluated for a 1:1 mixture of CFSE-labeled normal C57BL/6 CD45.1 congenic and CD45.2 Tg–/– T cells that were adoptively transferred to Thy-1.1 normal C57BL/6 mice and then were injected with SEA 24 h later. We reasoned that in any case where IL-2Rß signaling dominated the response to SEA, a preference would be noted in the number, the level of CFSE staining or anatomical location of the donor wild-type T cells. By the use of congenic mice, recipient T cells were distinguished from donor cells by expression of Thy-1.1 and Thy-1.2, respectively. The wild-type donor T cells were distinguished from Tg–/– T cells by expression of CD45.1 and CD45.2, respectively.

Three days after injection of SEA, two populations of CFSE-labeled cells were noted for wild-type and Tg–/– T cells (Fig. 2A), or after gating on CD4 (Fig. 2B) and CD8 (Fig. 2C) T cell subsets. One population of cells was homogeneously and brightly CFSE labeled that reflected undivided T cells. The other population was heterogeneously staining with CFSE and represented T cells that divided in response to SEA. Importantly, after gating on Vß3+ T cells (Fig. 2D), virtually all the donor wild-type and Tg–/– T cells diluted CFSE, indicating that all potential SEA-responsive T cells proliferated to this Sag. Thus, even in a competitive environment Tg–/– T cells readily proliferated to SEA.


Figure 2
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  		Fig. 2. Competitive proliferative responses by normal and Tg–/– T cells to SEA. Spleen cells from CD45.1-congenic C57BL/6 mice (Thy-1.2) and Tg–/– (CD45.2, Thy-1.2) mice were mixed 1:1, labeled with CFSE and adoptively transferred (20 x 106) into normal Thy-1.1 congenic C57BL/6 mice. Twenty-four hours later mice were challenged with SEA, and 3 days later, the histograms for donor CFSE staining in the spleen (SP), lymph nodes (LN), lung and liver, as indicated, were determined after gating on all T cells (A), CD4+ (B), CD8+ (C) or Vß3+ (D) T cells. The cells to the right of the gate in each histogram are those cells that did not respond to SEA and did not dilute CFSE (undivided cells). This gating was used in the calculations to derive the data in Figs 3 and 4.

 

Figure 3
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  		Fig. 3. Expansion and contraction of donor T cells during competitive responses by normal and Tg–/– T cells to SEA. Data as generated in Fig. 2 were evaluated for the absolute number of donor T cells that responded, i.e. diluted CFSE (A), or did not respond, i.e. did not dilute CFSE (B), in the spleen and lymph nodes 3 and 5 days after challenge with SEA. Numbers are based on the percentage of all donor cells (Thy-1.2+ T cells) after acquiring 1 x 106 events during FACS analysis. Data are the mean ± SD of four to eight mice per group.

 

Figure 4
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  		Fig. 4. Wild-type CD8+ T cells preferentially expand during competitive responses by normal and Tg–/– T cells to SEA. Data as generated in Fig. 2 were evaluated for the fraction of wild-type (CD45.1) and Tg–/– (CD45.2) donor (Thy-1.2) T cells in the indicated tissues. Shown are (A) total T cells or (B) CD4+ or CD8+ T cells that responded (divided) or did not respond (undivided) to SEA 3 days later as measured by dilution of CFSE. The percentage of wild-type Tg–/– T cells were expressed as a ratio after collecting 1 x 106 events during FACS analysis and this ratio was corrected for input ratio of these cells when injected into recipient mice. Data are the mean ± SD of three to eight mice per group. The ratio of the divided versus undivided cells in each tissue was compared by t-test (*P < 0.05, **P < 0.01, and ***P < 0.001).

 
As all Vß3+ T cells diluted CFSE, it is highly likely that the T cells that divided represent those T cells that express TCRs bearing a Vß subgroup that is responsive to SEA while the undivided T cells expressed TCRs bearing a SEA non-responsive Vß subgroup. Consistent with this notion, when the number of donor cells within the spleen and lymph nodes were quantified, the number of divided (CFSE diluted) donor T cells decreased between days 3 and 5 (Fig 3A), representing the contraction phases of the SEA response. There was also not an increase, but rather a decline, in donor CFSE-diluted T cells in the liver and lungs on day 5, indicating that the decrease in proliferating T cells in the lymphoid compartment was not due to trafficking of SEA-responsive cells into non-lymphoid tissue (data not shown). In contrast, the number of undivided (CFSE bright) donor T cells in the spleen and lymph nodes remained constant and equivalent between both groups over this time (Fig. 3B). When the ratio of wild-type and donor T cells that diluted the CFSE were determined on day 3 and was compared with the SEA non-responsive population of T cells that did not dilute CFSE, a statistically significant preference (approximately 2:1) for wild-type cells was noted in the spleen, which was even greater (approximately 3:1) for the lung and liver (Fig. 4A). A similar degree of preference for wild-type donor T cells was seen on day 5 (data not shown) after the contraction phase of the response, suggesting that both types of T cells similarly underwent apoptosis. When the donor CD4+ and CD8+ T cells in the spleen and lymph nodes were similarly analyzed, the preference for wild-type cells was primarily accounted by an increased within the wild-type CD8+ T cell subset (Fig. 4B). Thus, although Tg–/– T cells readily respond to SEA, the response is somewhat more robust for T cells that express a functional IL-2R, especially CD8+ T cells.

Role of {gamma}c-dependent cytokines for T cell expansion and contraction to SEA
IL-4 has been shown to function as a redundant cytokine for T cell growth and effector cell development by Tg–/– T cells in vitro (2). Therefore, we were especially interested in testing whether IL-4 played an analogous role for the in vivo response to SEA by Tg–/– mice. Therefore, C57BL/6 IL-4–/– mice were bred to Tg–/– mice. The resulting Tg–/– IL-4–/– progeny and control IL-4–/– mice also showed largely normal expansion and contraction to SEA, although somewhat greater variability was noted for contraction by CD8+ Vß3+ Tg–/– IL-4–/– T cells on day 5 (Fig. 5A).


Figure 5
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  		Fig. 5. {gamma}c-dependent cytokines are not required for the T cell response to SEA. (A) The indicated mice were injected with SEA (10 µg i.p.) and the percentage of Vß3+ CD4+ or CD8+ T cells in the spleen was determined by FACS analysis. Data are the mean ± SD of three to five mice per group. (B) Twelve hours before and after challenge with SEA, Tg–/– mice received PBS or anti-{gamma}c mAbs and percentage of Vß3+ CD4+ or CD8+ T cells in the spleen was determined 2 days after SEA challenge. Data are the mean ± SD of three mice per group.

 
To further explore the role of {gamma}c-dependent cytokines in T cell expansion to SEA, Tg–/– mice also received a mixture of anti-{gamma}c mAbs prior to injection of SEA. We used a regimen of anti-{gamma}c that was previously shown to very effectively inhibit T cell development in bone marrow chimeric mice (25) after confirming that this batch of anti-{gamma}c similarly inhibited cytokine-induced proliferation in vitro (data not shown). Even with a genetic block in responsiveness to IL-2 and IL-15 and the presence of inhibitory mAbs to anti-{gamma}c, there was no impairment in the proliferation of Vß3+ CD4+ or CD8+ T cells to SEA (Fig. 5B). Collectively, these data indicate that the response to SEA appears to be independent of IL-4 and suggest that it is also independent of other {gamma}c-dependent cytokines.

IFN{gamma} production by Tg–/– T cells in vitro and in vivo
Past studies demonstrated that in the absence of IL-2R signaling, Tg–/– T cells failed to produce IFN{gamma} upon anti-CD3 stimulation in vitro or vaccinia virus infection in vivo (2, 24), even though these T cells proliferated to both stimuli. We were interested in determining whether this represented a strict requirement for functional IL-2Rß to generate IFN{gamma}-producing T cells or whether there might be redundant pathways to support the development of such effector cells. Since IL-12 is an important cytokine that promotes cell-mediated immune responses (29), this cytokine was added to cultures of Tg–/– spleen cells during their stimulation with anti-CD3 in vitro. Two days later the proportion of IFN{gamma}-producing CD4+ and CD8+ T cells was enumerated by FACS analysis. As noted previously, Tg–/– CD4+ or CD8+ T cells did not produce IFN{gamma} upon stimulation with only anti-CD3, while control wild-type CD57BL/6 T cells developed into IFN{gamma}-producing effector cells with a higher proportion of CD8+ T cells producing this cytokine. The inclusion of exogenous IL-12 increased the number of control T cells that produced IFN{gamma} and led to a measurable number of Tg–/– CD4+ and CD8+ T cells that also produced IFN{gamma}. Quantitative evaluation of these results indicted that a lower fraction of Tg–/– T cells produced IFN{gamma} in the presence of IL-12 in vitro (Fig. 6B).


Figure 6
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  		Fig. 6. IL-12 promotes the development of IFN{gamma}-producing effector T cells in vitro in the absence of IL-2Rß. Spleen cells from normal C57BL/6 or Tg–/– mice were stimulated with anti-CD3 in the absence or presence of exogenous IL-12 for 48 h. Representative (A) and the percentage of (B) CD4+ and CD8+ T cells that produced IFN{gamma} by intracellular FACS analysis. Data in (B) are the mean ± SD of three mice per group.

 
The percentage of IFN{gamma}-producing T cells was also enumerated 3 days after control and Tg–/– mice were injected with SEA, as this time represents the peak proliferative response to this Sag. For these experiments, it was necessary to briefly re-stimulate the T cells in vitro with PMA and ionomycin to observe IFN{gamma} production. When compared with uninjected mice, a small and comparable percentage of CD4+ wild-type and Tg–/– T cells produced IFN{gamma} (Fig. 7A). A more substantial fraction of both wild-type and Tg–/– CD8+ T cells also produced IFN{gamma} (Fig. 7A), and the level of IFN{gamma} per cell was equivalent based on the mean fluorescent intensity of the positive cells (data not shown). After subtracting the background percentage of IFN{gamma}+ CD8+ T cells from naive wild-type and Tg–/– mice, normal C57BL/6 mice that were injected with SEA developed approximately a 4-fold greater fraction of CD8+ T cells that produced IFN{gamma} than SEA-injected Tg–/– mice. Even without subtracting the background response, there was 1.9-fold greater number of wild-type CD8+ T cells that produced IFN{gamma}. Collectively, these data indicated that there are redundant mechanisms for T cell IFN{gamma} production, but optimal production of this cytokine, especially for CD8+ T cells, depends upon IL-2Rß expression by peripheral T lymphocytes.


Figure 7
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  		Fig. 7. The development of IFN{gamma}-producing CD8+ T cells in response to SEA in vivo is impaired in Tg–/– mice. Three days after challenge with SEA, spleen cells were stimulated with PMA and ionomycin for 4 h in the presence of brefeldin A. FACS analysis for intracellular IFN{gamma} production was performed after gating on CD4+ or CD8+ T cells. Normal and Tg–/– mice that did not receive SEA served as a control for production of IFN{gamma} by naive T cells. Data are the mean ± SD of three to five mice per group.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the present study, we showed that the T cell expansion and contraction by both CD4+ and CD8+ T cells to the bacterial Sag SEA occurred normally when autoimmune-free IL-2Rß-deficient mice were challenged in vivo. This included an evaluation of the response by recipient IL-2Rß-deficient T cells that were rendered autoimmune free by the adoptive transfer of CD4+CD25+ Treg cells or by direct analysis of Tg–/– mice. These data demonstrate that there is not an essential requirement for IL-2/IL-2R for T cell proliferation to SEA in vivo. Furthermore, this T cell proliferation cannot be attributed to severe autoimmunity that is associated with unmanipulated IL-2- or IL-2R-deficient mice. Perhaps more importantly, it is highly unlikely that the IL-2Rß-indepenent response to SEA by Tg–/– mice is simply due to the high frequency of responding T cells because Tg–/– mice also generate effective immunity to other immunogens where the frequency of antigen-specific T cells is initially low (24).

When we consider the results of the competitive adoptive transfer experiments where a mixture of wild-type and Tg–/– CFSE-labeled T cells to SEA was transferred into a single recipient, two important points emerge concerning the requirements for IL-2 for T cell growth in vivo. First, all donor Vß3+ T cells of wild-type and Tg–/– origin diluted CFSE, indicating that substantial T cell proliferation occurred without a requirement for IL-2. Thus, the expansion and contraction to SEA by Tg–/– mice accurately demonstrate that this response is typically IL-2Rß-independent rather than reflecting some extraordinary compensatory mechanism. Second, a preference was noted for proliferation by wild-type donor T cells that was accounted for by an increased number of wild-type donor CD8+ T cells in the spleen and lymph nodes. Thus, signaling through IL-2Rß appears to predominantly aid the CD8+ T cell response to SEA in peripheral immune tissue. This finding is consistent with several other studies that indicate that IL-2 contributes to CD8+ T cell responses in vivo (13, 30–33). Thus, it is not surprising that even though aspects of T cell immunity might be somewhat impaired in the absence of IL-2, this is not sufficient to prevent the development of substantial and protective immune responses. Thus, blockade of IL-2 or IL-2R somewhat inhibits T cell immune response in vivo but more effective inhibition requires other immune suppressants that more broadly target T cell activation. In fact, this has often been observed in experimental and clinical immune therapy.

Our results in the competitive adoptive transfer experiments slightly differ from one other study that has also carefully compared the proliferation of IL-2- and IL-2R-deficient T cells, in which they examined only CD8+ OT-I TCR transgenic T cells in response to ovalbumin peptide in the context of a viral infection. In that study (14), the proliferation by wild-type and IL-2/IL-2R-deficient CD8+ OT-I T cells in secondary lymphoid tissue was comparable. However, we found that wild-type CD8+ T cells underwent more extensive proliferation. It is likely that this difference is due to optimal T cell help after SEA challenge as this Sag readily activates a large fraction of CD4+ T cells, the major source of IL-2. Analogous to the findings for OT-I T cells (14), this preference for donor wild-type T cells was even greater when examining non-lymphoid lung and liver tissues. We cannot distinguish whether this result is due to preferential homing of wild-type T cells into non-lymphoid tissue or whether this reflects more extensive IL-2-dependent T cell expansion within these non-lymphoid sites, as proposed by others (14).

IL-2 has been implicated as a key cytokine that promotes the development of IFN{gamma}-secreting effector T lymphocytes (34, 35). The inability of Tg–/– T cells to develop into IFN{gamma}+ effector T cells upon polyclonal stimulation in vitro supports this view (2). However, there also appears to be redundant pathways to elicit IFN{gamma}-producing effector cells because the inclusion of IL-12 during in vitro priming resulted in the generation of CD4+ and CD8+ Tg–/– T cells that readily secreted IFN{gamma}. Injection of SEA into Tg–/– mice also resulted in the production of both CD4+ and CD8+ IFN{gamma}-secreting effector T cells. Thus, there is not a mandatory requirement for IL-2 to elicit an IFN{gamma} response in vivo. However, even with a normal proliferative response to SEA by Tg–/– T cell in vivo, IFN{gamma} production was impaired, but not absent, for CD8+ Tg–/– T cells. As SEA broadly activates both CD4+ and CD8+ T cells, it is highly unlikely that this impaired IFN{gamma} response by CD8+ T cells is the result of a lack of CD4+ T cell help. A requirement for IL-2 for IFN{gamma} production in vivo has also been noted after challenge with vaccinia or lymphocytic choriomeningitis virus (24, 35). These data indicate that IL-2 is an important cytokine for the full development of IFN{gamma}-secreting CD8+ T cells. This finding in conjunction with the competitive adoptive transfer experiments indicates that IL-2 contributes to maximal CD8 T cell proliferation and effector function in vivo.

There is a very striking dichotomy concerning the strict requirement for IL-2 for T cell responses in vitro versus the capacity to induce IL-2-independent immunity in vivo. (2–8, 24, 36–40). Thus, there must be sufficient redundancy in vivo mediated by other cytokines and/or cell–cell molecular interactions to compensate for the lack of IL-2Rß signaling. IL-4 represented one attractive candidate for such a redundant cytokine because the provision of IL-4 to Tg–/– T cells during their in vitro priming partially restored their ability to undergo T cell clonal expansion to subsequent culture with IL-4 and to differentiate into CTL (2). Moreover, since SEA does not induce a substantial inflammatory response, a T cell-derived factor might be expected to provide this redundant function. However, IL-4 is not a major redundant cytokine for T cell expansion by Tg–/– T cells in vivo because normal expansion and near normal contraction were observed for CD4+ and CD8+ T cells after SEA challenge of Tg–/– IL-4–/– mice, which are genetically non-responsive to IL-2, IL-4 and IL-15. Moreover, T cell expansion to SEA was also normal after treatment of Tg–/– mice with potent inhibitory mAbs to {gamma}c. It is highly unlikely, therefore, that any of the {gamma}c-dependent cytokines are responsible for IL-2-independent proliferation in vivo. Consistent with this view, even though thymic T cell development is severely blocked in {gamma}c-deficient mice, when an MHC class II-restricted transgenic TCR is bred to {gamma}c-deficient mice, the few mature CD4+ TCR transgenic T cells that develop readily expanded when these lymphopenic mice were stimulated with antigen (41). Thus, the key redundant mechanism that compensates for the lack of IL-2Rß function in vivo remains to be identified.


   Acknowledgements
 
This work was supported by National Institutes of Health Grant R01 AI40114.


   Abbreviations
 
i.p.   intra-peritoneally
PMA   phorbol myristate acetate
Sag   superantigen
SEA   staphylococcal enterotoxin A
Treg   T regulatory

   Notes
 
* These authors contributed equally to this work. Back

Transmitting editor: M. Bevan

Received 31 October 2005, accepted 12 January 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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