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Keywords:

  • CD4;
  • CD25;
  • Co-stimulation;
  • IL-2;
  • Immunotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflicts of interest
  9. References
  10. Supporting Information

Surface expression of the IL-2 receptor α-chain (CD25) has been used to discriminate between CD4+CD25HIFOXP3+ regulatory T (Treg) cells and CD4+CD25NEGFOXP3 non-Treg cells. However, this study reports that the majority of resting human memory CD4+FOXP3 T cells expresses intermediate levels of CD25 and that CD25 expression can be used to delineate a functionally distinct memory subpopulation. The CD25NEG memory T-cell population contains the vast majority of late differentiated cells that respond to antigens associated with chronic immune responses and are increased in patients with systemic lupus erythematosus (SLE). In contrast, the CD25INT memory T cells respond to antigens associated with recall responses, produce a greater array of cytokines, and are less dependent on costimulation for effector responses due to their expression of CD25. Lastly, compared to the CD25NEG and Treg-cell populations, the CD25INT memory population is lost to a greater degree from the blood of cancer patients treated with IL-2. Collectively, these results show that in humans, a large proportion of CD4+ memory T cells express intermediate levels of CD25, and this CD25INTFOXP3 subset is a functionally distinct memory population that is uniquely affected by IL-2.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflicts of interest
  9. References
  10. Supporting Information

T-cell survival and effector function are sensitive to the availability of essential cytokines during development, homeostasis, and activation. Interleukin-2 (IL-2) is a 15.5 kDa α-helical protein discovered for its ability to culture T cells long term in vitro [1]. IL-2 has broad effects on T lymphocytes, including survival, proliferation, activation-induced cell death (AICD), T-cell differentiation, cytokine production, and immune tolerance [2-4]. The high-affinity receptor for IL-2 (IL-2R) is composed of three subunits, the α-subunit (CD25), β-subunit (CD122), and the common γ-chain (CD132). CD122 and CD132 are also subunits for other cytokine receptors, whereas CD25 is specific to the IL-2 receptor. IL-2 signaling occurs exclusively through the cytoplasmic tails of CD122 and CD132; CD25 has a short cytoplasmic tail and is not involved in IL-2 signaling. Instead, CD25 has the highest affinity for IL-2 among the individual subunits and acts as an affinity converter [2]. At high concentrations, IL-2 can signal in the absence of CD25 through CD122 and CD132, which form the intermediate-affinity IL-2R. However, CD25 in addition to CD122 and CD132 is required to respond to low concentrations of IL-2 by forming the high-affinity IL-2 receptor [2]. Once formed, the IL-2/CD25/CD122/CD132 quaternary complex is short-lived (t1/2 10–20 min) on the cell surface [5]. Upon internalization, IL-2, CD122, and CD132 are targeted for lysosomal degradation, whereas CD25 is recycled to the cell surface [6, 7].

Though CD25 has been shown to influence effector function of lymphocytes, CD25 is thought to play a greater role in immune tolerance in mice [2, 8]. Initially, it was found that depletion of CD4+CD25+ T cells from adoptive cell transfer experiments into nude mice resulted in systemic autoimmune disease [9]. These CD4+CD25+ cells were later shown to express the transcription factor Foxp3 (FOXP3 in humans) and are now termed regulatory T (Treg) cells that comprise 5–15% of CD4+ T cells in humans [10]. Treg cells depend on IL-2 signaling for their survival in vitro and in vivo [11-13]. Therefore, constitutive expression of CD25 on Treg cells is thought to be crucial to their survival and maintenance of immune homeostasis. This idea is supported by studies of mice deficient in CD25 or IL-2, which have low numbers of Treg cells and develop severe systemic autoimmune disease as they age [14, 15]. Despite the positive effects of IL-2 on effector and memory T cells, CD25/IL-2 deficiency in mice does not appear to greatly hinder T-cell immunity, reviewed elsewhere [8]. Therefore, it is thought that in mice, CD25/IL-2 plays a dominant role in immune tolerance and less for adaptive immunity, perhaps because CD25 is expressed only transiently on activated effector cells and constitutively on Treg cells. However, expression of CD25 and its role in immunology may be species dependent, since CD25 appears to play a larger role in T-cell effector responses in humans compared to mice, and may be somewhat dispensable for the maintenance of Treg cells as seen in patients treated with CD25-blocking antibodies [16-18]. This notion has been discussed elsewhere in the literature [19, 20] and is supported by the phenotype of CD25 deficiency in humans, who in contrast to mice, are severely immunocompromised and have a normal frequency of Treg cells [21-24].

This difference between mice and humans may be related to the presence of a large population of CD4+FOXP3 T cells in humans that express intermediate levels of CD25, a population that has not been found in mice [25]. Given the importance of IL-2 in the immune system and in the clinic, we sought to determine if resting CD4+FOXP3 T cells that expressed CD25 represent a functionally distinct human T-cell population that responds to IL-2 immunotherapy in cancer patients. We report that CD4+CD25INTFOXP3 cells comprised up to 65% of resting human CD4+ T cells and constituted the majority of the CD4+ memory compartment in healthy individuals. Further evaluation revealed that CD4+CD25NEG memory and CD4+CD25INT memory populations are composed of functionally distinct memory subsets. Also, CD25INT T cells exhibit enhanced effector function when activated in the absence of costimulation that is in large part due to IL-2 signaling. Lastly, we found that compared to the CD25NEG and Treg populations, the CD25INT population proliferated more vigorously to rhIL-2 in vitro and decreased in the peripheral blood of cancer patients undergoing IL-2 immu-notherapy. Together, these data show that in humans, there exist a larger proportion of resting CD4+FOXP3 T cells that express CD25 than previously thought, and that CD25INT cells are a functionally distinct memory population that appear to play a role in IL-2 immunotherapy in cancer patients. Ultimately, further studies of this population may help us understand and improve the efficacy of immunotherapies that influence IL-2 signaling.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflicts of interest
  9. References
  10. Supporting Information

CD25 expression on human CD4+FOXP3 memory T cells

The IL-2 receptor alpha chain (CD25) has been used as a marker for Treg cells (CD4+CD25HIFOXP3+) as well as activated T cells [2]. However, analysis of CD4+ cells using two different monoclonal antibodies to CD25 clearly revealed a population of resting FOXP3 human CD4+ T cells that expressed intermediate levels of CD25 [25]. We found that these two commercially available anti-human CD25 antibodies revealed a significant proportion of CD4+FOXP3 T cells expressed intermediate levels of CD25 (Supporting Information Fig. 1A). We subsequently used clone 4E3 for the remainder of this study and found that CD25INT CD4+ T cells were found in all individuals studied, comprising 35–65% of all CD4+ T cells in normal donors. Representative FACS plots from four individuals are shown in Fig. 1A.

image

Figure 1. A new anti-human CD25 monoclonal antibody reveals a stable memory CD4+ T-cell population that expresses CD25 and FOXP3. (A) CD25 and FOXP3 staining of CD3+CD4+ T cells from four individuals. (B) Anti-CD25 antibody was incubated with different concentrations of CD25:Ig or OX40:Ig fusion protein for 30 min at room temperature. PBMCs were then added and evaluated for binding of anti-CD25 antibodies. (C) Enriched CD4+ cells from fresh PBMCs were stained for CD25 prior to incubation at 37°C with various concentrations of rhIL-2 (U/mL) in the presence or absence of anti-CD25-blocking antibodies for 15 min and evaluated for intracellular pSTAT5. (D) CD25 and FOXP3 staining of CD3+CD4+ and CD3+CD8+ T cells from the same individual. (E) The percentage of CD3+CD4+CD25NEG and CD3+CD4+CD25INT cells that express CD95 was determined in PBMCs from healthy donors (n = 15). (A, D) The flow cytometry plots are representative from ten individuals. (C) Flow cytometry plots that are representative of 1 of 3 independent experiments using PBMCs from different healthy individuals. (E) The flow cytometry plots show the data from one healthy individual. The graphs show the data from all 15 individuals; each symbol represents an individual, the wide horizontal bar represents the mean ± SD. Statistical significance was determined by paired student's t-test analysis. 0*p < 0.05

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To show that this new antibody recognized functional CD25, CD4+ T cells from fresh PBMCs were stimulated with various concentrations of rhIL-2 and then evaluated for upregulation of intra-cellular pSTAT5, as pSTAT5 is downstream of IL-2 signaling (Fig. 1B). Cells expressing higher levels of CD25 responded to lower concentrations of IL-2, while cells expressing little or no CD25 required higher concentrations of rhIL-2. When preincubated with an anti-CD25 blocking antibody that does not interfere with binding of the 4E3 anti-CD25 antibody, the cells expressing intermediate and high levels of CD25 were unable to respond to the lower concentrations of rhIL-2 but did respond to a higher dose of rhIL-2, presumably through the β and γ chains of the IL-2 receptor (Fig. 1B).

Although we found the CD25INTFOXP3 cells mainly among CD4+ T cells, a small proportion of resting CD8+ T cells also expressed CD25 (Fig. 1C). CD25INT CD4+ T cells were interrogated by flow cytometry for expression of markers of naïve and memory cells. The majority of CD25INT cells expressed the memory marker CD95 (Fig. 1D) [26]. This observation was reaffirmed by the expression of the naïve and memory markers CD45RA and CD45RO (Supporting Information Fig. 1B) [27]. In the normal individuals studied, CD25INT T cells comprise the majority (as much as 80%) of memory cells in the CD4+ T-cell compartment (data not shown). We were unable to find a significant relationship between the percent of CD4+ that were CD25INT as a function of age within the cohort of healthy individuals used in this study (data not shown).

We next evaluated whether CD95+CD25NEGFOXP3 and CD95+CD25INTFOXP3 CD4+ T cells maintain their respective CD25 phenotype over time. CD4+ T cells were enriched from fresh blood samples and then sorted into four groups: naïve (CD95), memory CD25NEG, memory CD25INT, and Treg cells (Supporting Information Fig. 1C and D) [28]. The sorted cells were cultured without stimulation and reevaluated for expression of CD25 2 and 5 days later. These sorted populations maintain their relative levels of CD25, suggesting the CD25INT memory cells were not recently activated cells with transient CD25 expression (Supporting Information Fig. 1E). These data imply that CD25INT and CD25NEG memory populations represent two distinct resting memory populations.

Phenotypic evaluation of CD25INT and CD25NEG memory T cells

Next, we tested the hypothesis that CD25INT memory cells were distinct from their memory CD25NEG counterparts by examining differences in differentiation/activation markers that are expressed by memory cells. The majority of CD4+ naïve and memory cells from normal donors express CD28. However, others have shown that individuals with ongoing chronic immune responses, such as autoimmune disease, have a higher proportion of late-differentiated memory CD4+ T cells that do not express CD28 [29, 30]. We found the majority of these memory CD4+CD28NEG cells were within the CD25NEG population (Fig. 2A). The memory CD4+CD28NEG population has been reported to produce cytolytic proteins such as granzyme B [31], which are typically expressed by CD8+ T-cell subsets. We found that memory CD4+ T cells that produce granzyme B were within the CD25NEG population and not found in the CD25INT population (Fig. 2A). We did not find clear differences in expression of the differentiation markers CCR7, CD62L, or CCR5 between CD95+CD25NEG and CD95+CD25INT CD4+ memory T cells (Supporting Information Fig. 2A) [32-34]. However, CCR7 for the most part was coexpressed on the CD25INT subpopulation.

image

Figure 2. CD25INT and CD25NEG memory CD4+ T cells contain distinct memory phenotypes. CD3+CD4+ T cells from fresh PBMCs obtained from healthy donors evaluated for (A) CD25, CD28, and Granzyme B expression, (B) CD25, EOMES, CD319, and CD134, and (C) EOMES, Granzyme B, CD28, and CD319. Flow cytometry plots are representative of 2 (A) or 4 (B) of eight healthy individuals. (C) Flow cytometry plots show that data are taken from one of three healthy individuals.

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To further assess the differences between the CD25NEG and CD25INT memory populations, we performed a microarray analysis with RNA from sorted CD95+ memory populations. Two genes whose expression levels were lower in the CD25INT cells were CD319, a member of the signaling lymphocyte activation molecule (SLAM) family receptors, and the T-box transcription factor Eomesodermin (EOMES), both of which are upregulated in activated CD8+ and NKT cells. Previous studies have shown that granzyme B is regulated in part by EOMES, while CD319 has activating properties on NKT cells, but little information regarding these two proteins is available for human CD4+ T cells [35-37]. Therefore, we evaluated intracellular and surface expression levels of EOMES and CD319 protein in CD4+ T cells from normal individuals. We found EOMES and CD319 were preferentially expressed within the CD4+CD25NEG population, confirming our microarray data (Fig. 2B). In contrast, the costimulatory TNF-receptor family member OX40 (CD134) was preferentially expressed on the surface of CD25INTFOXP3 population within normal individuals (Fig. 2B and Supporting Information Fig. 2B).

EOMES partly controls granzyme B expression, therefore we addressed whether EOMES was expressed in granzyme B+ and/or CD28NEG cells. We observed that the majority of both the CD28NEG and the granzyme B+ cells coexpressed EOMES, but not all of the EOMES+ cells were CD28NEG or granzyme B+ (Fig. 2C). Lastly, since granzyme B, EOMES, and CD319 are expressed by cytolytic CD8+ T cells, we wanted to determine if a similar trend was found in CD8+ T cells. As mentioned, most of the human CD8+ T-cell populations are CD25NEG. However, we observed a high proportion of CD8+ T cells that express intermediate levels of CD25 in some cancer patients. The majority of the CD8+ T cells that express granzyme B, EOMES, CD319, and lack CD28 are within the CD8+CD25NEG subpopulation (Supporting Information Fig. 2C). Collectively, these results show that the CD25NEG and CD25INT memory cells are stable populations that contain distinct markers associated with known memory subsets.

CD25INT memory cells are not associated with chronic immune responses

Since late-differentiated memory cells were associated with the CD25NEG but not the CD25INT memory population (Fig. 2A and B), we hypothesized that CD25NEG memory cells would preferentially respond to antigens associated with chronic infections in humans. To test this hypothesis, we evaluated cytokine responses of memory CD4+ T cells after activation with antigens associated with a typical recall memory response (Influenza) and antigens associated with chronic immune responses (HCMV). CD4+ T cells stimulated with the superantigen Staphylococcal Enterotoxin B (SEB) served as a positive control for cytokine stimulation. CMV-specific T cells were skewed toward the CD25NEG population when compared to SEB, whereas responses to Influenza were skewed toward the CD25INT population (Fig. 3A and B).

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Figure 3. CD4+ memory T cells associated with chronic immune responses are CD25NEG. (A) CD3+CD4+ PBMCs from a healthy donor were cultured with either SEB (1 μg/mL), flu vaccine (3 μg/mL), or CMV lysate (10 μg/mL) in the presence of anti-CD28/49d (5 μg/mL) and Brefeldin A (5 μg/mL) for 18 h and stained for IFN-γ. Flow cytometry plots are representative of one individual's responses (B) CD25 MFI intensity of CD3+CD4+ IFN-γ and IL-2-producing cells isolated from the PBMCs of eight healthy donors. The bar graphs depict the cumulative mean ± SD from eight independent experiments using PBMCs from different healthy individuals. *p < 0.05 (C, D) Fresh CD3+CD4+ PBMCs from sex-matched healthy donors and SLE patients were evaluated for the proportion of CD95+ (C) and OX40+ (D) that are CD25NEG (N = 10). The flow cytometry plots show the data from one healthy individual and one SLE patient. The graphs show the data from all ten individuals per group; each symbol represents an individual, the wide horizontal bar represents the mean and the error bars represent mean ± SD. Statistical significance was determined by one-way ANOVA analysis (B) or by unpaired student's t-test (C, D).

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The production of cytokines by CD25NEG memory cells in response to HCMV suggests that they are involved in chronic inflammatory responses. Therefore, we hypothesized that patients with systemic lupus erythematosus (SLE), who suffer from chronic inflammation, would have a greater proportion of CD4+ memory T cells skewed toward the CD25NEG population. We compared CD4+ T cells from SLE patients and gender-matched healthy volunteers using CD95 and CD134 as markers of memory and ac-tivation, respectively. As reported by others, we observed a higher percentage of memory (CD4+CD95+) and activated memory cells (CD4+CD134+) in SLE patients compared to healthy donors (data not shown) [38, 39]. We also found that the memory/activated cells were skewed toward the CD25NEG compartment in SLE patients compared to normal donors (Fig. 3C and D). These data suggest that the late-differentiated CD4+ memory T cells are primarily within the CD25NEG memory population, which are expanded in SLE patients.

Functional characterization of CD25INT and CD25NEG memory CD4+ T cells

Next, we wanted to determine whether there were functional differences between CD95+CD25NEG and CD95+CD25INT memory cells upon activation with anti-CD3. We observed that sorted CD95+CD25INT memory cells (Supporting Information Fig. 1C and D) stimulated with anti-CD3 alone (in the absence of co-stimulation) formed larger clusters of cells compared to sorted CD95+CD25NEG cells (Fig. 4A). Expression of CD25 prior to activation may provide the CD95+CD25INT memory population with an advantage in the absence of added costimulation by allowing them to respond to lower levels of IL-2. CD25 is known to be greatly upregulated on T cells after activation and would negate any benefit of CD25 expression prior to activation [40, 41]. However, we found that only the CD95+CD25INT population upregulated CD25 in response to anti-CD3 alone (Fig. 4B). Since IL-2 signaling is known to augment CD25 expression on activated T cells [42], we evaluated IL-2 responses by intracellular pSTAT5 levels and found that only the CD95+CD25INT memory population increased pSTAT5 levels (Fig. 4C). Stimulation in the presence of high concentrations of exogenous IL-2 demonstrated that both populations are capable of upregulating both CD25 and pSTAT5 levels (Fig. 4B and Supporting Information Fig. 3A).

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Figure 4. CD25 expression enhances the effector function of the CD25INT population during TCR stimulation in the absence of costimulation. (A–D) CD95+CD25NEG and CD95+CD25INT cells were sorted from fresh PBMCs from three individuals and plated at 50,000 cells/well in triplicate (A) Image of sorted cells stimulated with plate-bound anti-CD3 in the presence or absence of soluble anti-CD25 for 48 h. (B) CD25 expression on sorted cells 72 h after culture with or with out anti-CD3 ± either anti-CD25-blocking antibody or rhIL-2 (5000 U/mL). (C) Seventy-two hours after stimulation as in (B), the cells were stained for CD25 in culture for 20 min and then fixed and evaluated for intracellular pSTAT5 by flow cytometry. (D) Total cell number after culture of the sorted cells with or without anti-CD3 ± anti-CD25-blocking antibody for 72 h and resting for 48 h without anti-CD3 ±. (E) Cytokine concentration in the supernatant of the cultures described in (D) taken 72 h after stimulation with anti-CD3 ± anti-CD25-blocking antibody. BLD: below level of detection. NS: not statistically significant (A, B, C) Representative images and flow cytometry plots are of one of three independent experiments using sorted cells from different healthy individuals. (D, E) Bar graphs depict the mean of triplicate wells ± SD for three independent experiments using sorted cells from different healthy individuals. Statistical significance was determined by one-way ANOVA analysis. *p < 0.05

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To test the function of CD25 expression within the CD95+CD25INT population, we tested their ability to activate in the absence of costimulation. We found that anti-CD25-blocking antibodies interfered with the ability of CD25INT cells to form aggregates, upregulate CD25, and phosphorylate STAT5 (Fig. 4A–C). The decrease in CD25 staining was not due to blocking of the anti-CD25 detection antibodies, since the anti-CD25-blocking antibodies do not interfere with the anti-CD25 detection antibody (Fig. 1C and Supporting Information Fig. 3A). To further compare differences between CD95+CD25NEG and CD95+CD25INT memory cells and the role of CD25 during activation in the absence of costimulation, proliferative responses were determined. When stimulated with anti-CD3 alone, the CD95+CD25INT but not the CD95+CD25NEG cells proliferated robustly (Fig. 4D). However, blocking CD25 on the CD95+CD25INT cells interfered with their ability to proliferate (Fig. 4D). Conversely, when stimulated in the presence of anti-CD28 or exogenous rhIL-2, the CD95+CD25NEG population proliferated robustly, demonstrating that the CD95+CD25NEG cells are capable of proliferation. The CD95+CD25INT memory population consistently proliferated as well or better than the CD95+CD25NEG memory population under all conditions (data not shown).

Lastly, cytokine concentrations determined from supernatant showed that CD95+CD25INT cells produced more cytokines than the CD95+CD25NEG population and that blocking CD25 had a negative impact on these cytokine levels (Fig. 4E). Interestingly, blocking CD25 on the CD95+CD25INT population increased levels of detectable IL-2. This observation may be explained by a lack of IL-2 internalization and also a lack of negative feedback on IL-2 production. Collectively, these data suggest that CD95+CD25INT cells stimulated in the absence of costimulation are able to respond to lower concentrations of IL-2 due to their expression of CD25 prior to activation.

CD25INT memory cells differ in their sensitivity and functional responses to rhIL-2 in vitro

Since CD25 expression increased the ability of CD95+CD25INT memory cells to proliferate and produce cytokines (Fig. 4), we investigated their functional responses to rhIL-2 alone. Cells were sorted from fresh PBMCs (Supporting Information Fig. 1C and D) and stimulated with various concentrations of rhIL-2 (no anti-CD3). To determine their sensitivity to rhIL-2, cells were analyzed for intracellular pSTAT5 (Fig. 5A). The majority of cells in the Treg and CD95+ memory populations upregulated pSTAT5 following stimulation with high concentrations of rhIL-2 (1000 U/mL). However, each population differed in their response to lower concentrations of rhIL-2, showing an expected gradient of decreasing sensitivity to low concentrations of rhIL-2 from Treg cells to CD95+CD25INT to CD95+CD25NEG to naïve cells.

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Figure 5. The CD25INT population is differentially affected by rhIL-2 in vitro as compared with other memory CD4+ T cells. Sorted populations from fresh healthy donor PBMCs were cultured with the indicated concentrations of rhIL-2 for (A) 15 min and evaluated for intracellular pSTAT5. (Left) Flow cytometry plots of one of three healthy individuals at 10 U/mL and (Right) bar graphs of the cumulative mean ± SD from three independent experiments using PBMCs from different individuals (B) 7 days and evaluated for 7AAD and Annexin V staining. (Left) Flow cytometry plots of one of three healthy individuals at 10 U/mL and (Right) bar graphs of the cumulative mean ± SD from four independent experiments using PBMCs from different individuals or (C) intracellular Ki67 levels (Left) Flow cytometry plots of one of three healthy individuals at 1000 U/mL and (Right) bar graphs of the cumulative mean ± SD from four independent experiments using PBMCs from different individuals. (D) Sorted populations cultured for 48 h with or without rhIL-2 (5000 U/mL) and evaluated for expression levels of CD25 and FOXP3. The flow cytometry plots show the data from one of three independent experiments using PBMCs from different healthy individuals. Statistical significance was determined by one-way ANOVA analysis. *p < 0.05

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The effect of rhIL-2 on survival was evaluated in sorted populations cultured for 7 days with or without rhIL-2 (Fig. 5B). We found that the majority of the Treg populations were dead/dying when cultured alone and that exogenous rhIL-2 rescued the Treg cells from cell death (Fig. 5B). The CD95+CD25NEG cells were dependent on the addition of exogenous rhIL-2 for cell survival to a lesser extent than the Treg cells. In contrast, the CD95+CD25INT cells survived well without exogenous rhIL-2. We also found that compared to the CD95+CD25NEG population, the CD95+CD25INT population was better able to survive when stimulated with anti-CD3 in the absence of costimulation and had higher levels of the prosurvival protein BCL-2 ex vivo (data not shown).

Proliferative responses induced by rhIL-2 in the absence of TCR stimulation were evaluated by expression of intracellular Ki67. Coincubation with increasing concentrations of rhIL-2 induced proliferation by CD25INT cells and to a lesser extent CD25NEG cells (Fig. 5C). The Treg population did not proliferate in response to increasing concentrations of rhIL-2 alone, which has been reported by others [43]. Since IL-2 is known to regulate CD25 and FOXP3, we examined expression of these proteins in response to rhIL-2 (Fig. 5D) [42, 44]. Surprisingly, the CD95+CD25NEG population showed no change in CD25 expression, while the Treg-cell population greatly increased CD25 levels. In contrast, the CD95+CD25INT population displayed a bimodal expression of CD25 in response to rhIL-2, with some of the cells increasing and some decreasing expression of CD25. In addition, the Treg cells upregulated FOXP3 to a greater degree compared to the CD95+CD25NEG and CD95+CD25INT cells. These results were consistent among the three individuals tested. Together, these results show that these distinct populations differ in their sensitivity and functional responses to rhIL-2 in vitro.

IL-2 immunotherapy differentially affects CD25INT CD4+ T cells

Based on the differential responses by the CD25INT subset to rhIL-2 in vitro, we evaluated CD25 expression on CD4+ T cells isolated from cancer patients receiving immunotherapy with high-dose IL-2. Analysis of CD4+ T cells before and after the first infusions revealed that the CD25INT population as a percentage of total CD4+ T cells was significantly reduced, while the percentage of CD25NEG increased (Fig. 6A). The decrease in proportion of CD25INT cells with a concomitant increase of CD25NEG cells was a trend observed in ten patients (Fig. 6B). In contrast, no significant change was found in the proportion of FOXP3+ Treg cells (Fig. 6B). These changes began within 30 min of IL-2 infusion, suggesting that the effect is due to direct rhIL-2 stimulation and not downstream effects (Fig. 6C).

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Figure 6. The CD25INT population is differentially affected by rhIL-2 in cancer patients undergoing IL-2 immunotherapy. CD3+CD4+ T cells from PBMCs collected from cancer patients with metastatic melanoma or renal cell carcinoma over the course of IL-2 immunotherapy and subsequently frozen were analyzed. (A) Representative flow cytometry plots of CD3+CD4+ cells collected from two patients (n = 10 in total) immediately before and 18 h after the start of IL-2 immunotherapy and evaluated for CD25 and FOXP3 expression. (B) Cumulative results from all patients (n = 10) following analysis as detailed in (A) (C) CD25 and FOXP3 expression on CD3+CD4+ T cells from one patient during the first 3 h of IL-2 immunotherapy. Flow cytometry plots are from one of three experiments using different patients. (D) Evaluation of CD3+CD4+ T cell subsets from four patients’ PBMCs before, 18 h, 1 week, and 2 weeks after the start of IL-2 immunotherapy. Each line represents data from one individual from four independent experiments. Statistical significance was determined by paired student's t-test. *p < 0.05

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Since rhIL-2 binds to CD25, we wanted to confirm that the disappearance of the CD25INT cells was not due to blocking of the anti-CD25 detection antibody by rhIL-2. We noted that preincubation with rhIL-2 does not interfere with binding of the CD25 antibody used in these studies (Supporting Information Fig. 4A). Moreover, if rhIL-2 did block the CD25 detection antibody, we would not expect to observe CD25 staining on the Treg cells after IL-2 treatment. Instead, we observed an overall increase in CD25 expression on the Treg cells (Supporting Information Fig. 4B). This is consistent with our in vitro finding (Fig. 5D) and was confirmed with sorted cells (Supporting Information Fig. 4C).

Lastly, we wanted to determine whether IL-2 immunotherapy modulated the CD4+ T-cell compartment in a transient or lasting fashion. Therefore, patients were evaluated over time after the start of IL-2 therapy, which was between 4 and 11 days after the final infusion. We observed that within a few days after the last IL-2 infusion, the CD25INT population returned and remained at near pretreatment levels in four individual patients (Fig. 6D). In contrast, the Treg data were not consistent between patients. Taken together, it is apparent that the CD25INT population is differentially affected by IL-2 and could potentially be playing an integral role in antitumor immunity in cancer patients undergoing IL-2 immunotherapy.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflicts of interest
  9. References
  10. Supporting Information

Previous studies in mice and humans have shown that CD25 is expressed primarily on resting FOXP3+ Treg cells and transiently on activated T cells. Here, we have shown that a large proportion of resting CD4+ T cells in humans express intermediate levels of CD25 and are FOXP3. We have found no mouse equivalent for this population when staining CD4+ T cells for CD25 and FOXP3 in our mouse colony in either young, old or tumor-bearing C57BL/6 male and female mice. In addition, when enriched resting CD4+ cells from mice are stimulated ex vivo with low concentrations of IL-2, much fewer cells from mice upregulated pSTAT5 compared to human cells (7% versus 40%) (data not shown). However, there have been some reports of variable levels of CD4+CD25+FOXP3 cells in mice under certain inflammatory conditions, though it is unclear if these are activated cells that have transiently upregulated CD25 or represent a resting memory population similar to what we have found in humans [45-48]. Therefore, there may be differences in the expression and role of IL-2/CD25 in cellular immunology between laboratory mice and humans. This notion has been discussed by other groups and is supported by studies of humans who lack functional CD25, and appear to have functional and phenotypic differences when compared to CD25−/− mice [2, 16, 19-24]. In contrast to mice, CD25 deficiency in humans is accompanied by severe immunodeficiency that is characterized by susceptibility to opportunistic pathogens and a normal Treg frequency [9, 14, 15, 21-24]. In addition, IL-2-deficient mice are fully capable of rejecting allografts, whereas CD25-deficient humans are not [24, 49, 50]. Therefore, CD25 may be more important for effector function in humans and more important for tolerance in mice since only Treg cells constitutively express CD25 in mice. This may explain why blocking CD25 during tumor immuno-therapy has not translated well from mice to humans [51]. Discrepancies between mouse and human immunology have been described elsewhere and is not unexpected since the species diverged 65–75 million years ago [52]. Therefore, studies conducted in mice on the role of IL-2 in T-cell function may not exactly translate to humans, and this study may offer one possible explanation for these differences.

We believe that the discovery of this CD4+CD25INT population is particularly important for therapies that target CD25/IL-2 and that hopefully by studying the response of this population we can better understand the mechanism of these therapies and improve their clinical efficacy. We evaluated the response of the CD4+CD25INTFOXP3 population to IL-2 immunotherapy. Over the course of IL-2 immunotherapy in cancer patients, the percentage of CD4+ T cells that were CD25INT population decreased, while the CD25NEG increased and Treg populations stayed relatively stable, suggesting these populations were differentially affected by the therapy. From these studies, it was clear that the CD25INT population was affected by the IL-2 therapy, however, it is currently not known exactly how the CD25INT population responded to the therapy. One possibility is that the CD25INT cells may have downregulated or shed CD25 [53]. However, we did not see diminution of CD25 on the Treg cells, and we demonstrated that not all of the CD25INT population downregulated expression of CD25 in response to rhIL-2 in vitro and that some even increased CD25 expression. In addition, in vitro stimulation with rhIL-2 also suggested that the CD25INT cells are differentially responsive to rhIL-2, as shown by Ki67 staining, and could therefore be act-ivated to a greater degree than the CD25NEG and Treg populations. Therefore, we believe that the disappearance of the CD25INT population observed in IL-2 cancer patients is most likely a combination of events, including decreased surface expression of CD25 and increased activation, which might have led to AICD and/or egress from the blood to tissue. Nevertheless, it is clear that the CD25INT population is greatly affected by IL-2 immunotherapy and may be integral to the antitumor immune response.

In addition to studying the CD25INT population in the context of IL-2/CD25 therapies, these different memory subsets may provide insight into the severity of disease or responses to treatments, as well as the role of these populations in different diseases. For instance, we found that the memory CD25NEG, but not the memory CD25INT cells, were associated with chronic immune responses and were expanded on SLE patients (Fig. 2 and 3). This suggests that the CD25NEG memory population may play a role in auto-immune disease.

In summary, we report in this study that a large percentage of memory CD4+ T cells in humans express intermediate levels of CD25. CD25 expression on the CD25INT memory population appears to be important biologically and that the CD25INT population is greatly affected by IL-2 immunotherapy in cancer patients. These findings not only improve our understanding of the role of CD25 in human immunology, but may also have clinical implications by helping to illuminate the mechanisms and potentially improve the efficacy of therapies that target IL-2 and CD25.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflicts of interest
  9. References
  10. Supporting Information

Lymphocyte isolation and phenotypic analysis

Human PBMCs were isolated by centrifugation of heparinized blood over Ficoll-Plaque™ PLUS (GE Healthcare). Isolated PBMCs were either analyzed fresh or were frozen in 45% RPMI/45% FBS/10% DMSO and then thawed for analysis. Staining for flow cytometry was done at either 4°C or room temperature for 30 min with: CD3 (UCHT1), CD4 (SK3), CD8 (SK2), CD25 (Miltenyi, 4E3), CD25 (BD, M-A251), CD95 (DX2), CD45RA (HI100), CD45RO (UCHL1), CD127 (eBioRDR5), CD28 (CD28.2), CD134 (ACT35), CCR5 (2D7/CCR5), or CD319 (162.1). For intracellular staining, cells prepared with Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer's instructions and incubated at either 4°C or room temperature for 30 min with: EOMES allophycocyanin (WD1928), FOXP3 (236A/E7), Ki67 (B56), pSTAT5 (47), IL-17A (BL168), Granzyme B (GB11), BCL-2 (100), IL-2 (MQ1-17H12), or IFN-γ (B27). Antibodies were acquired from Miltenyi, eBioscience, BD Biosciences, BioLegend, Invitrogen, and Beckman Coulter. All samples were run on an LSR II flow cytometer or FACSAria II and analyzed by FlowJo or Winlist. Sorting experiments were done using CD4+ cells enriched by Miltenyi LS columns from fresh PBMCs that were stained and sorted using a BD FACSAria II Cell Sorter.

Healthy donors and patients

PBMCs from individuals (ten females, five males; mean age, 36; age range, 27–61) without known autoimmune disease or cancer were used as healthy donors in this study. Patients with SLE (ten females; mean age, 40; age range, 20–49) that took part in the study fulfilled the American College of Rheumatology revised classification criteria for lupus [54]. Patients had active (n = 7) or inactive (n = 3) renal nephritis and were being treated with a variety of drugs (hydroxychloroquine n = 9, mycophenolate n = 4, prednisone n = 7). Cancer patients undergoing IL-2 immuno-therapy (three females, seven males; mean age, 53; age range, 24–68) had either metastatic melanoma or renal metastatic carcinoma. Analysis of PBMCs from healthy donors and SLE patients was done on fresh samples. Samples from IL-2-treated patients were frozen PBMCs that had been collected immediately before treatment and 18 h, 1 week, and 2 weeks after the first infusion. All IL-2 patients received 600,000 IU/kg of rhIL-2 (Proleukin) every 8 h by intravenous bolus for up to 14 doses. Two cycles of IL-2 immunotherapy were given at 2-week intervals following which clinical response was determined and further IL-2 was administered at the discretion of their physician for patients with stable or responding disease.

IL-2 in vitro stimulations

Enriched CD4+ or sorted cells from fresh PBMCs were cultured in 10% complete RPMI and incubated at a concentration of 100,000 cells/100 μL in 96 well plates. For pSTAT5 analysis, cells were incubated for 1 h at 37°C with or without 2 μg/mL of anti-CD25-blocking antibody (R&D Systems, clone no. 22722) and stimulated with rhIL-2 (Proleukin) for 15 min. The cells were then fixed and permeabilized with the Fix & Perm Cell Permeabilization Reagents from Invitrogen following the methanol-modified protocol and stained for pSTAT5. For survival and proliferation assay, sorted cells were cultured for 7 days with or without rhIL-2 and evaluated for survival by Annexin V/7AAD staining (BD Biosciences) and proliferation by intracellular Ki67.

Antigen stimulation and intracellular cytokine staining

Frozen PBMCs from healthy individuals were thawed and cultured at 37°C in 10% complete RPMI at a concentration of 1 × 106 cells/100 μL in 96 well plates. Cells were cultured with 5 μg/mL of anti-CD28/49d alone or with Flu Vaccine (afluria®, 3 μg/mL), SEB (Toxin Techonology Inc., 1μg/mL), or CMV lysate (Advanced Biotechnologies Inc., 10 μg/mL) for 1 h, after which brefeldin A (5 μg/mL) was added. After 18 h, cells were stained for extracellular CD3, CD4, CD95, and CD25 and then stained for the intracellular cytokines IFN-γ and IL-2 after permeabilization. CD25 MFI background was determined by staining for all markers except CD25 in each assay.

Anti-CD3 stimulation assays

Fresh PBMCs were sorted, suspended in 10% RPMI at a concentration of 50,000 cells/100 μL in 96 well plates that were uncoated or precoated with 5 μg/mL anti-CD3 (OKT3). All samples were done in triplicate with and without 2 μg/mL of anti-CD25-blocking antibody (R&D Systems, clone no. 22722). Cells were cultured for 3 days, after which 100 μL of supernatant was collected and the cells were transferred to uncoated 96 well plates and given 100 μL of fresh media with and without anti-CD25 (2 μg/mL). Two days after replating, proliferation was analyzed by counting cells with a hemocytometer and survival was determined by Annexin V/7AAD staining (Invitrogen) analyzed by flow cytometry.

Statistical analysis

Statistical significance was determined by paired or unpaired student's t-test (for comparison between two groups) or one-way ANOVA (for comparison among more than two groups) using Prism software (GraphPad, San Diego, CA, USA); a p-value of <0.05 was considered significant.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflicts of interest
  9. References
  10. Supporting Information

Todd Triplett is a Ph.D. candidate at Oregon Health and Science University and this work is submitted in partial fulfillment of the requirement for the Ph.D. We thank Dr. Walter Urba, Dr. David Parker, Dr. William Redmond, Dr. Nick Morris, Dr. Amy Moran, Dr. Stephanie Lynch, Kendra Garrison, and Sarah Church for helpful discussions and critical reading of the manuscript, and Mr. Dan Haley for his expertise with flow cytometry.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflicts of interest
  9. References
  10. Supporting Information
  • 1
    Morgan, D. A., Ruscetti, F. W. and Gallo, R., Selective in vitro growth of T lymphocytes from normal human bone marrows. Science 1976. 193: 10071008.
  • 2
    Malek, T. R., The biology of interleukin-2. Annu. Rev. Immunol. 2008. 26: 453479.
  • 3
    Dooms, H. and Abbas, A. K., Control of CD4+ T-cell memory by cytokines and costimulators. Immunol. Rev. 2006. 211: 2338.
  • 4
    Refaeli, Y., Van Parijs, L., London, C. A., Tschopp, J. and Abbas, A. K., Biochemical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis. Immunity 1998. 8: 615623.
  • 5
    Yu, A., Olosz, F., Choi, C. Y. and Malek, T. R., Efficient internalization of IL-2 depends on the distal portion of the cytoplasmic tail of the IL-2R common gamma-chain and a lymphoid cell environment. J. Immunol. 2000. 165: 25562562.
  • 6
    Hemar, A., Subtil, A., Lieb, M., Morelon, E., Hellio, R. and Dautry-Varsat, A., Endocytosis of interleukin 2 receptors in human T lymphocytes: distinct intracellular localization and fate of the receptor alpha, beta, and gamma chains. J. Cell Biol. 1995. 129: 5564.
  • 7
    Yu, A. and Malek, T. R., The proteasome regulates receptor-mediated endocytosis of interleukin-2. J. Biol. Chem. 2001. 276: 381385.
  • 8
    Malek, T. R. and Bayer, A. L., Tolerance, not immunity, crucially depends on IL-2. Nat. Rev. Immunol. 2004. 4: 665674.
  • 9
    Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M., Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 1995. 155: 11511164.
  • 10
    Antony, P. A. and Restifo, N. P., CD4+CD25 +T regulatory cells, immunotherapy of cancer, and interleukin-2. J. Immunother. 2005. 28: 120128.
  • 11
    Papiernik, M., de Moraes, M. L., Pontoux, C., Vasseur, F. and Penit, C., Regulatory CD4 T cells: expression of IL-2R alpha chain, resistance to clonal deletion and IL-2 dependency. Int. Immunol. 1998. 10: 371378.
  • 12
    Fontenot, J. D., Rasmussen, J. P., Gavin, M. A. and Rudensky, A. Y., A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. 2005. 6: 11421151.
  • 13
    Setoguchi, R., Hori, S., Takahashi, T. and Sakaguchi, S., Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J. Exp. Med. 2005. 201: 723735.
  • 14
    Willerford, D. M., Chen, J., Ferry, J. A., Davidson, L., Ma, A. and Alt, F. W., Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity 1995. 3: 521530.
  • 15
    Furtado, G. C., Curotto de Lafaille, M. A., Kutchukhidze, N. and Lafaille, J. J., Interleukin 2 signaling is required for CD4(+) regulatory T cell function. J. Exp. Med. 2002. 196: 851857.
  • 16
    de Goer de Herve, M. G., Gonzales, E., Hendel-Chavez, H., Decline, J. L., Mourier, O., Abbed, K., Jacquemin, E. et al., CD25 appears non essential for human peripheral T(reg) maintenance in vivo. PloS one 2010. 5: e11784.
  • 17
    Wang, Z., Shi, B. Y., Qian, Y. Y., Cai, M. and Wang, Q., Short-term anti-CD25 monoclonal antibody administration down-regulated CD25 expression without eliminating the neogenetic functional regulatory T cells in kidney transplantation. Clin. Exp. Immunol. 2009. 155: 496503.
  • 18
    Vondran, F. W., Timrott, K., Tross, J., Kollrich, S., Schwarz, A., Lehner, F., Klempnauer, J. et al., Impact of Basiliximab on regulatory T-cells early after kidney transplantation: down-regulation of CD25 by receptor modulation. Transplant Int. 2010. 23: 514523.
  • 19
    Wuest, S. C., Edwan, J. H., Martin, J. F., Han, S., Perry, J. S., Cartagena, C. M., Matsuura, E. et al., A role for interleukin-2 trans-presentation in dendritic cell-mediated T cell activation in humans, as revealed by daclizumab therapy. Nat. Med. 2011. 17: 604609.
  • 20
    Cohen, A. C., Nadeau, K. C., Tu, W., Hwa, V., Dionis, K., Bezrodnik, L., Teper, A. et al., Cutting edge: decreased accumulation and regulatory function of CD4+ CD25(high) T cells in human STAT5b deficiency. J. Immunol. 2006. 177: 27702774.
  • 21
    Sharfe, N., Dadi, H. K., Shahar, M. and Roifman, C. M., Human immune disorder arising from mutation of the alpha chain of the interleukin-2 receptor. Proc. Natl. Acad. Sci. USA 1997. 94: 31683171.
  • 22
    Caudy, A. A., Reddy, S. T., Chatila, T., Atkinson, J. P. and Verbsky, J. W., CD25 deficiency causes an immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome, and defective IL-10 expression from CD4 lymphocytes. J. allergy Clin. Immunol. 2007. 119: 482487.
  • 23
    Aoki, C. A., Roifman, C. M., Lian, Z. X., Bowlus, C. L., Norman, G. L., Shoenfeld, Y., Mackay, I. R. et al., IL-2 receptor alpha deficiency and features of primary biliary cirrhosis. J. Autoimmun. 2006. 27: 5053.
  • 24
    Roifman, C. M., Human IL-2 receptor alpha chain deficiency. Pediatr. Res. 2000. 48: 611.
  • 25
    Lee, R. W., Creed, T. J., Schewitz, L. P., Newcomb, P. V., Nicholson, L. B., Dick, A. D. and Dayan, C. M., CD4+CD25(int) T cells in inflammatory diseases refractory to treatment with glucocorticoids. J. Immunol. 2007. 179: 79417948.
  • 26
    Pitcher, C. J., Hagen, S. I., Walker, J. M., Lum, R., Mitchell, B. L., Maino, V. C., Axthelm, M. K. et al., Development and homeostasis of T cell memory in rhesus macaque. J. Immunol. 2002. 168: 2943.
  • 27
    Merkenschlager, M., Terry, L., Edwards, R. and Beverley, P. C., Limiting dilution analysis of proliferative responses in human lymphocyte populations defined by the monoclonal antibody UCHL1: implications for differential CD45 expression in T cell memory formation. Eur. J. Immunol. 1988. 18: 16531661.
  • 28
    Liu, W., Putnam, A. L., Xu-Yu, Z., Szot, G. L., Lee, M. R., Zhu, S., Gottlieb, P. A. et al., CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J. Exp. Med. 2006. 203: 17011711.
  • 29
    Duftner, C., Goldberger, C., Falkenbach, A., Wurzner, R., Falkensammer, B., Pfeiffer, K. P., Maerker-Hermann, E. et al., Prevalence, clinical relevance and characterization of circulating cytotoxic CD4+CD28- T cells in ankylosing spondylitis. Arthritis Res. Ther. 2003. 5: R292R300.
  • 30
    Thewissen, M., Somers, V., Hellings, N., Fraussen, J., Damoiseaux, J. and Stinissen, P., CD4+CD28null T cells in autoimmune disease: pathogenic features and decreased susceptibility to immunoregulation. J. Immunol. 2007. 179: 65146523.
  • 31
    van Leeuwen, E. M., Remmerswaal, E. B., Vossen, M. T., Rowshani, A. T., Wertheim-van Dillen, P. M., van Lier, R. A. and ten Berge, I. J., Emergence of a CD4+CD28- granzyme B+, cytomegalovirus-specific T cell subset after recovery of primary cytomegalovirus infection. J. Immunol. 2004. 173: 18341841.
  • 32
    Sallusto, F., Lenig, D., Forster, R., Lipp, M. and Lanzavecchia, A., Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999. 401: 708712.
  • 33
    Sallusto, F., Geginat, J. and Lanzavecchia, A., Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu. Rev. Immunol. 2004. 22: 745763.
  • 34
    Okoye, A., Meier-Schellersheim, M., Brenchley, J. M., Hagen, S. I., Walker, J. M., Rohankhedkar, M., Lum, R. et al., Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection. J. Exp. Med. 2007. 204: 21712185.
  • 35
    Pearce, E. L., Mullen, A. C., Martins, G. A., Krawczyk, C. M., Hutchins, A. S., Zediak, V. P., Banica, M. et al., Control of effector CD8+ T cell function by the transcription factor Eomesodermin. Science 2003. 302: 10411043.
  • 36
    Intlekofer, A. M., Takemoto, N., Wherry, E. J., Longworth, S. A., Northrup, J. T., Palanivel, V. R., Mullen, A. C. et al., Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat. Immunol. 2005. 6: 12361244.
  • 37
    Stark, S. and Watzl, C., 2B4 (CD244), NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells. Int. Immunol. 2006. 18: 241247.
  • 38
    Watanabe, T., Suzuki, J., Mitsuo, A., Nakano, S., Tamayama, Y., Katagiri, A., Amano, H. et al., Striking alteration of some populations of T/B cells in systemic lupus erythematosus: relationship to expression of CD62L or some chemokine receptors. Lupus 2008. 17: 2633.
  • 39
    Patschan, S., Dolff, S., Kribben, A., Durig, J., Patschan, D., Wilde, B., Specker, C. et al., CD134 expression on CD4+ T cells is associated with nephritis and disease activity in patients with systemic lupus erythematosus. Clin. Exp. Immunol. 2006. 145: 235242.
  • 40
    Uchiyama, T., Broder, S. and Waldmann, T. A., A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells. J. Immunol. 1981. 126: 13931397.
  • 41
    Kalia, V., Sarkar, S., Subramaniam, S., Haining, W. N., Smith, K. A. and Ahmed, R., Prolonged interleukin-2Ralpha expression on virus-specific CD8+ T cells favors terminal-effector differentiation in vivo. Immunity 2010. 32: 91103.
  • 42
    Depper, J. M., Leonard, W. J., Drogula, C., Kronke, M., Waldmann, T. A. and Greene, W. C., Interleukin 2 (IL-2) augments transcription of the IL-2 receptor gene. Proc. Natl. Acad. Sci. USA 1985. 82: 42304234.
  • 43
    Thornton, A. M. and Shevach, E. M., CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 1998. 188: 287296.
  • 44
    Yao, Z., Kanno, Y., Kerenyi, M., Stephens, G., Durant, L., Watford, W. T., Laurence, A. et al., Nonredundant roles for Stat5a/b in directly regulating Foxp3. Blood 2007. 109: 43684375.
  • 45
    Berretta, F., St-Pierre, J., Piccirillo, C. A. and Stevenson, M. M., IL-2 contributes to maintaining a balance between CD4+Foxp3+ regulatory T cells and effector CD4+ T cells required for immune control of blood-stage malaria infection. J. Immunol. 2011. 186: 48624871.
  • 46
    Brinster, C. and Shevach, E. M., Costimulatory effects of IL-1 on the expansion/differentiation of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells. J. Leukoc. Biol. 2008. 84: 480487.
  • 47
    Yarkoni, S., Sagiv, Y., Kaminitz, A., Farkas, D. L. and Askenasy, N., Targeted therapy to the IL-2R using diphtheria toxin and caspase-3 fusion proteins modulates Treg and ameliorates inflammatory colitis. Eur. J. Immunol. 2009. 39: 28502864.
  • 48
    Lee, Y. K., Menezes, J. S., Umesaki, Y. and Mazmanian, S. K., Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. USA 2011. 108(Suppl 1): 46154622.
  • 49
    Steiger, J., Nickerson, P. W., Steurer, W., Moscovitch-Lopatin, M. and Strom, T. B., IL-2 knockout recipient mice reject islet cell allografts. J. Immunol. 1995. 155: 489498.
  • 50
    Dai, Z., Konieczny, B. T., Baddoura, F. K. and Lakkis, F. G., Impaired alloantigen-mediated T cell apoptosis and failure to induce long-term allograft survival in IL-2-deficient mice. J. Immunol. 1998. 161: 16591663.
  • 51
    Jacobs, J. F., Punt, C. J., Lesterhuis, W. J., Sutmuller, R. P., Brouwer, H. M., Scharenborg, N. M., Klasen, I. S. et al., Dendritic cell vaccination in combination with anti-CD25 monoclonal antibody treatment: a phase I/II study in metastatic melanoma patients. Clin. Cancer Res. 2010. 16: 50675078.
  • 52
    Mestas, J. and Hughes, C. C., Of mice and not men: differences between mouse and human immunology. J. Immunol. 2004. 172: 27312738.
  • 53
    Brusko, T. M., Wasserfall, C. H., Hulme, M. A., Cabrera, R., Schatz, D. and Atkinson, M. A., Influence of membrane CD25 stability on T lymphocyte activity: implications for immunoregulation. PloS one 2009. 4: e7980.
  • 54
    Hochberg, M. C., Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997. 40: 1725.
Abbreviations
SEB

Staphylococcal Enterotoxin B

SLE

systemic lupus erythematosus

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflicts of interest
  9. References
  10. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
eji2311-sup-0001-figures1.pdf3500K

Fig.1. Evaluation of CD4+CD25INT memory cells.

Fig.2. CD25 expression in relation to differentiation markers

Fig.3. CD25INT cells respond robustly to stimulation in the absence of co-stimulation.

Fig.4. Determining influence of rhIL-2 on CD25 expression.

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