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Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20814
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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CTLA-4 blockade using an anti-CTLA-4 Ab has been shown to enhance antitumor responses in multiple murine models (12, 13, 14, 15, 16, 17, 18), but it can also induce autoimmune phenomena, including depigmentation, encephalomyelitis, colitis, and diabetes (15, 18, 19, 20, 21, 22). Similar phenomena have been seen in human studies. We have reported previously that treatment of patients with metastatic melanoma using a mAb against CTLA-4 can mediate both objective clinical regressions as well as autoimmunity, including colitis, dermatitis, hepatitis, hypophysitis, and uveitis (23, 24). The durability of these clinical responses has stimulated substantial interest in understanding the mechanism of action of CTLA-4 blockade.
CD4+CD25+ T regulatory cells constitutively express surface CTLA-4 (25, 26), and it has been suggested that this molecule is integral to their regulatory function (22, 25, 26). Therefore, this cell population may be most susceptible to the effects of CTLA-4 blockade. In both mice and humans, CD4+CD25+ T regulatory cells can inhibit the activation of other T cells and suppress proliferation and cytokine secretion of CD4+CD25– cells (27). Selective elimination of CD4+CD25+ cells in mice can result in severe autoimmune disease, while readministration of these cells could prevent autoimmunity (28, 29, 30). Moreover, depletion of CD25+ cells delayed growth of the B16 melanoma in C57BL/6 mice (18), and the adoptive transfer of CD25+-depleted CD4+ cells with tumor self-reactive CD8+ T cells and a vaccine induced regression of established melanoma in mice (31), implying that inhibition of T regulatory functions may release autoreactive T cells, including tumor reactive T cells, from peripheral suppressive mechanisms.
Thus, the constitutive expression of CTLA-4 on CD4+CD25+ T regulatory cells has raised the possibility that the clinical impact of CTLA-4 blockade was manifested by depletion or blockade of T regulatory cells. Alternatively, CTLA-4 blockade could act directly on CD4+ and/or CD8+ cells to release inhibitory influences and enhance effector functions. These hypotheses have been explored in humans in the current study by measuring cellular changes following the administration of anti-CTLA-4 Ab to patients with metastatic disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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All patients were treated on Investigational Review Board-approved protocols in the Surgery Branch, National Institutes of Health in Bethesda, Maryland. Patients A, E, and G had metastatic renal cell cancer. All other patients had metastatic melanoma. Patients eligible for treatment with anti-CTLA-4 Ab had an Eastern Cooperative Oncology Group performance status 
2, stage IV disease, no evidence of autoimmune or immunodeficiency disease, and 
3 wk had elapsed since any previous systemic cancer therapy. The human IgG1
anti-CTLA-4 mAb, MDX-010 (Medarex), was administered as an i.v. bolus over 90 min every 3 wk. Before Ab administration and 3 wk after each dose course, PBMC were obtained by apheresis, isolated by Ficoll-Hypaque separation, and cryopreserved at –180°C in heat-inactivated human AB serum with 10% DMSO. For suppression assays, fresh lymphocytes were used without any prior cryopreservation.
Response evaluation criteria in solid tumors (RECIST) criteria were used to determine radiographic response to treatment (32). The sum of the longest diameters of all tumors before and after therapy was calculated. A partial response was defined as a decrease of 30% (but not 100%) of the sum of the longest diameters of index lesions, lasting at least 1 mo, with no growth of lesions or the appearance of new lesions. A complete response was defined as the disappearance of all lesions for 1 mo. Patients not achieving either a partial or complete response were nonresponders. Treatment, autoimmunity, and response characteristics for patients treated with anti-CTLA-4 Ab are summarized in Table I. Patients F and G provided cells for study but were not enrolled in anti-CTLA-4 treatment protocols.
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CD4+ cells were isolated from whole PBMC by negative selection according to manufacturer’s instructions (CD4-negative isolation kit; Dynal Biotech). For suppression assays, CD4+ cells were further purified into CD25+ and CD25– subpopulations with the Dynal T regulatory kit, and accessory cells were T cell depleted from autologous PBMC using the Dynal CD3+ depletion kit (Dynal Biotech) according to the manufacturer’s instructions. For RT-PCR gene analysis of CD4+ subpopulations, CD4+-purified cells were sorted using a FACSVantage (BD Biosciences) into CD4+CD25+"high" and CD4+CD25–"low" cells, defined as the upper and lower 10% of cells staining for CD25 (CD25-PE; BD Biosciences).
Flow cytometry
Flow cytometry was used to evaluate surface expression of selected T cell markers. Whole PBMC were washed in ice-cold PBS with 0.5% BSA and incubated with appropriate fluorochrome-labeled Abs and relevant isotype controls (CD4-FITC (clone SK3), CD8-FITC (clone SK1), CD25-PE (clone 2A3), CD69-APC (clone L78), and HLA-DR-APC (clone L243); BD Biosciences). Sample fluorescence was acquired and analyzed with a FACSCalibur and CellQuest software (BD Biosciences).
RNA isolation, cDNA synthesis, and real-time PCR
RNA isolation and cDNA synthesis were performed in batches containing pre- and posttreatment samples to minimize variability. Total RNA was isolated using an RNAeasy minikit (Qiagen) and was reverse transcribed to cDNA using the ThermoScript RT-PCR system (Invitrogen Life Technologies) according to the manufacturers’ instructions.
Levels of 
-actin and Foxp3 gene expression were assessed with the ABI Prism 7700 Sequence Detection System (PerkinElmer). For -actin, the forward primer, 5'-GGCACCCAGCACAATGAAG-3', reverse primer, 5'-GCCGATCCACACGGAGTACT-3', and probe, 5'-6-FAM TCAAGATCATTGCTCCTCCTGAGCGC-TAMRA-3', were used. For Foxp3, the combined primer probe reagent (Assay-on-Demand gene expression assay; Applied Biosystems) was used. cDNA was analyzed in a 25-µl mixture containing TaqMan 2x Universal MasterMix (Applied Biosystems) and respective primers and probes at optimized concentrations. All samples were run in duplicate. Foxp3 copy numbers were calculated from a linear regression of known standards that were included in each RT-PCR run. All samples were run with the same set of standards, except samples from patients 5–10, where a different set of internal standards were used. Thermal cycler parameters were 2 min at 50°C, 10 min at 95°C, and 40 cycles of denaturation at 95°C for 15 s with annealing/extension at 60°C.
Suppression assay
The proliferative potential of CD4+CD25+ subpopulations were assessed in coculture assays. CD4+CD25+, CD4+CD25– (1.0 x 104), or both cell subpopulations (1:1; 1.0 x 104 each per well) were cultured with T cell-depleted irradiated accessory cells (5 x 104/well). For polyclonal activation, cells were cultured with 1.0 µg/ml soluble anti-CD3. One microcurie of [3H]thymidine per well was added during the final 18 h of a 5- or 6-day culture, and proliferation was measured using a scintillation counter. These culture conditions and reagent concentrations were optimized for sensitivity in prior experiments. Cultures were performed in 96-well round-bottom plates in sextuplets. Suppression of CD4+CD25– proliferation by CD4+CD25+ cells was calculated using the formula: percentage of suppression = (1 – (1:1 – CD25+/CD25–)) x 100%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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To test whether CTLA-4 blockade could abrogate the suppressive activity of CD4+CD25+ regulatory cells in vitro, coculture assays were performed using pretreatment cells in the presence and in the absence of anti-CTLA-4 Ab. Zero, 10, or 100 µg/ml anti-CTLA-4 Ab was added on day 0 to cultures for all patients, except patient G where Ab was added on days 0, 2, and 4. A typical example of the coculture suppression assay (patient F) is shown in Table II, and a summary of the suppression results on all five patients is shown in Fig. 1. The suppression of CD4+CD25– proliferation by coculture with CD4+CD25+ cells was not significantly affected by the addition of anti-CTLA-4 Ab in vitro.
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Pheresis samples from four patients treated in vivo with escalating doses of anti-CTLA-4 Ab every 3 wk were evaluated using flow cytometry for phenotype expression before treatment and after receiving two doses each at 3, 5, and 9 mg/kg. The percentage of PBMC expressing CD4+CD25+ was analyzed in the total CD4+ population by gating on the isotype control (Fig. 2, lower panel). In addition, the CD4+CD25+"high" population, thought to contain a higher proportion of regulatory cells (27, 33), was analyzed by gating the upper 10% of CD4+CD25+ cells in the pretreatment sample and using that gate to evaluate subsequent posttreatment samples (Fig. 2, upper panel). The administration of anti-CTLA-4 Ab in vivo did not appear to cause a consistent change in the percentage of CD4+ lymphocytes expressing CD4+CD25+ posttreatment compared with pretreatment when analyzed in both the total CD4+ and the "high" CD25+ subpopulations (Fig. 2).
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Foxp3 is highly expressed in CD4+CD25+ T regulatory cells and has been described as a lineage specification factor for these cells (34). Thus, CD4+ cells were purified from the PBMC of the four patients from whom data were obtained for Fig. 2, and Foxp3 gene expression was determined relative to the expression of -actin. There was no consistent difference in Foxp3 expression in CD4+ cells as a function of Ab dose in patients treated with 3, 5, or 9 mg/kg anti-CTLA-4 Ab, although there was a trend toward higher relative Foxp3 levels at some anti-CTLA-4 doses compared with pretreatment levels in each patient (relative Foxp3 levels at 3, 5, and 9 mg/kg: +2.96, p = 0.24; +3.26, p = 0.18; +4.61, p = 0.05; not corrected for multiple analyses). (Fig. 3).
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-actin in PBMC from all 10 patients before and after receiving anti-CTLA-4 Ab. Eight of these 10 patients revealed an increase in relative Foxp3 gene expression posttreatment, again suggesting that this Ab may have an agonistic effect in vivo on Foxp3-expressing cells.
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-actin in these highly purified CD25+ and CD25– subpopulations was determined. As expected, very low levels of Foxp3 gene expression were seen in CD25– low populations compared with the CD25+ high populations. There was a significant increase in relative Foxp3 expression in PBMC obtained posttreatment compared with pretreatment in the CD4+CD25+ high population (p = 0.05, paired t test) (Fig. 4).
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Effects of in vivo CTLA-4 blockade on the function of T regulatory cells
To test whether CD4+CD25+ cells from patients treated with CTLA-4 blockade maintained suppressive function posttreatment, lymphocytes from patients treated with anti-CTLA-4 Ab in vivo were evaluated in coculture suppression assays. Lymphocytes from five patients for whom fresh pheresis samples were available were bead purified into CD4+CD25+ and CD4+CD25– cell populations. All patients had received multiple doses of anti-CTLA-4 Ab and were apheresed after the last dose (Table I). These posttreatment CD4+CD25+ cells displayed a persistent ability to suppress proliferation of CD4+CD25– cells, ranging from 49 to 84%, thus indicating that CTLA-4 blockade in vivo did not eliminate circulating lymphocytes with suppressive function (Fig. 5).
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To evaluate the influence of CTLA-4 blockade on the activation state of lymphocytes, whole PBMC from four patients who received the highest doses of anti-CTLA-4 were evaluated using flow cytometry to evaluate cell surface markers characteristic of lymphocyte activation. An example of HLA-DR expression on CD4+ cells is shown in Fig. 6. A trend toward an increase in HLA-DR expression was seen on CD4+ lymphocytes in patients tested after receiving 3 mg/kg (+8.55, p = 0.07, paired t test), 5 mg/kg (+12.78, p = 0.08, paired t test), and 9 mg/kg (+13.12, p = 0.11, paired t test) anti-CTLA-4 Ab. A trend toward increase in HLA-DR expression was also seen on CD8+ lymphocytes in patients tested after receiving 3 mg/kg (+9.69, p = 0.12, paired t test), 5 mg/kg (+14.35, p = 0.02, paired t test), and 9 mg/kg (+15.46, p = 0.03, paired t test) anti-CTLA-4 Ab, with the exception of patient 1, in whom CD8+HLA-DR+ levels remained stable at 3 mg/kg before increasing (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We first addressed the impact of anti-CTLA-4 Ab on CD4+CD25+ T cell regulatory cell function in vitro using a coculture suppression assay. This proliferation assay was developed to functionally identify murine and human CD4+CD25+ T regulatory cells by their ability to suppress the proliferation of CD4+CD25– cells (26, 27, 33, 35, 36). Physiologic concentrations of anti-CTLA-4 Ab cocultured with purified populations of CD4+CD25+ lymphocytes did not abolish their ability to suppress the proliferation of CD4+CD25+ human lymphocytes (Table II and Fig. 1).
We next studied the phenotype and function of PBMC obtained from patients who received multiple doses of anti-CTLA-4 Ab in vivo. There was no consistent dose-response effect in the percentage of cells expressing CD4+CD25+ posttreatment compared with pretreatment in either the total CD4+ fraction or the CD25+"high" fraction, which may be more representative of CD4+ cells with regulatory function (27, 33). These studies suggested that the administration of anti-CTLA-4 Ab did not deplete circulating CD4+CD25+ cells. This was further evaluated by assessing the effect of CTLA-4 blockade on a specific genetic marker of regulatory T cells. Foxp3 expression is restricted to CD4+CD25+ regulatory cells in mice, and forced gene expression of Foxp3 by viral transduction can impart regulatory activity to normal T cells (37, 38, 39). Furthermore, mutations in the murine foxp3 gene causes a fatal autoimmune disorder, termed scurfy, and a similar disease, immune dysregulation, polyendocrinopathy, X-linked syndrome, occurs in humans carrying a mutation in the human Foxp3 gene, which shares 86% similarity to the murine homologue (40, 41, 42, 43, 44, 45). Although the role of Foxp3 in defining T regulatory cells in mice is clearer than in humans (34), its gene expression is a surrogate for identification of human T regulatory cells. In CD4+ cells, relative Foxp3 expression did not consistently change in patients treated with anti-CTLA-4 Ab at multiple dose levels and, in fact, increased at some dose levels. In patients evaluated after administration of 3 mg/kg Ab, there appeared to be an increase in Foxp3 gene expression that was evident in patients that showed a clinical antitumor response and in those that did not, regardless of whether the patient experienced grade III/IV autoimmunity. We then further purified this CD4+ population into CD4+CD25+ and CD4+CD25– populations by flow sorting for the highest and lowest expressors of CD25 and evaluated Foxp3 gene expression in these subpopulations. Relative Foxp3 expression in the CD4+CD25+"high" population significantly increased in posttreatment compared with pretreatment CD4+ cells independent of patient clinical antitumor response or autoimmunity status. Thus, assessment of Foxp3 expression in either the CD4+ or the CD4+CD25+ lymphocyte populations suggested that administration of Ab to CTLA-4 may be agonistic to T regulatory cells, rather than antagonistic. It should be mentioned that all measurements were made on cells taken 3 wk after the dose of anti-CTLA4 Ab.
Functional assays represent the most direct method for assessing the presence of T regulatory cell-suppressive ability. Therefore, we next analyzed lymphocytes from patients treated with in vivo anti-CTLA-4 Ab, for whom fresh pheresis samples were available, using in vitro suppression assays. These studies revealed that circulating CD4+CD25+ cells obtained after in vivo treatment with anti-CTLA-4 Ab maintained the ability to suppress CD4+CD25– cells by 49–84%. This suppression was evident in PBMC obtained from patients whether or not they experienced clinical response to treatment. Therefore, we concluded that in vivo CTLA-4 blockade did not abolish the suppressive function of T regulatory cells. However, it should be emphasized that the T regulatory subset is incompletely defined at this time, and expression of the Foxp3 gene may not be an adequate marker. It remains possible that a subset of tumor-specific T regulatory cells were depleted.
Because CTLA-4 blockade did not inhibit the expression or function of CD4+CD25+ cells, it seemed very unlikely that the antitumor and autoimmune effects seen in patients were due to enhanced activity of effector T cells secondary to the absence of T regulatory-mediated suppression. Therefore, we turned our attention to an examination of the activation status of T cells from patients that responded to Ab treatment and/or experienced autoimmunity. HLA-DR expression, a T cell activation marker (46, 47), appeared to increase in CD4+ and CD8+ cells obtained from patients after treatment with escalating doses of anti-CTLA-4 Ab. HLA-DR expression also trended to increase in both the CD4+CD25+ and CD4+CD25– cell populations, implying a pan-lymphocytic activation. CD25 and CD69 expression did not change significantly in these patients, although the window in which these phenotypic markers of activation would be up-regulated may have been missed at the time points measured (46, 47). This data was consistent with our prior analyses of patients treated with anti-CTLA-4 Ab and tested at similar time points, where we observed an increase in HLA-DR expression on CD4+ and CD8+ cells and increased CD45RO expression on CD4+ cells after Ab administration in both responders and nonresponders with or without autoimmunity (23, 24).
It should be emphasized that the measurements of T regulatory function, Foxp3 expression, and phenotypic analyses reported here were performed on circulating lymphocytes and not specifically on lymphocytes with antitumor activity. The low levels of antitumor T cells in these patients precluded our ability to measure DR expression specifically on these cells. As reported earlier there was no apparent impact of anti-CTLA-4 Ab administration on the ability to generate antitumor precursors following peptide immunization (23).
Thus, the present data suggest that the antitumor and autoimmune effects seen after CTLA-4 blockade using an anti-CTLA-4 Ab are not due to inhibition or depletion of T regulatory cells but rather appear to act through direct activation of CD4+ and CD8+ effector cells.
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 Address correspondence and reprint requests to Dr. Steven A. Rosenberg, Surgery Branch, National Cancer Institute, National Institutes of Health, CRC Room 3-3888, 10 Center Drive, MSC 1201, Bethesda, MD 20814. E-mail address: sar{at}nih.gov 
Received for publication May 3, 2005. Accepted for publication September 19, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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AAUGAA) leads to the IPEX syndrome. Immunogenetics 53: 435-439. [Medline]
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, A total of 5000 cells/population was cultured. All other cultures were plated with 10,000 cells/population.
