Introduction
T-cell-expressed coinhibitory receptors act as essential immune checkpoints to prevent aberrant activation, thereby maintaining peripheral tolerance. However, they also impair productive immunity in response to pathogens and tumor cells. Blockade of the PD-1/PD-L axis and CTLA-4 has been shown to induce durable responses in patients suffering from different tumors including melanoma and lung cancer [
1‐
3]. T cells harbor additional inhibitory receptors that are considered as potential targets in cancer therapy. Studies in murine tumor models have demonstrated that blockade of lymphocyte-activation gene 3 (LAG-3) alone or in combination with PD-1 antibodies limits tumor growth and promotes clearance of malignant cells [
4‐
7]. Several LAG-3 antibodies and a bispecific agent that concomitantly binds to LAG-3 and PD-1 are currently in clinical development [
8]. Another promising target is B- and T-lymphocyte attenuator (BTLA), which is broadly expressed on human T cells and transduces strong inhibitory signals upon engagement by its ligand herpesvirus entry mediator (HVEM). Several studies including the work by our group have revealed that blocking antibodies to this molecule can enhance human T-cell responses when used alone or in combination with PD-1 antibodies [
9‐
11]. In addition, this receptor is robustly expressed in the tumor microenvironment and can function to inhibit tumor-specific human T cells [
12].
Studies on immune checkpoints have focused on CD8
+ T cells since they are the major effectors participating in anti-tumor immunity. However, these molecules are also expressed on CD4
+ effector T cells, which provide help to other immune cells, augmenting immunity at several levels. Importantly, CD4
+ T cells can also promote cytotoxicity, e.g., by killing target cells in a MHC class II-dependent or -independent fashion, or by licensing DC to effectively activate cytotoxic CD8
+ T cells [
13,
14].
Here, tetanus toxoid (TT) stimulation was used as a robust in vitro model for analyzing human CD4
+ T-cell responses to address the stimulatory capacity of immune checkpoint inhibitors targeting PD-L1, CTLA-4, LAG-3, and BTLA. We found that only the blockade of PD-L1 effectively enhanced the response to TT, while LAG-3 and BTLA antibodies had no effect. Surprisingly, addition of the therapeutic CTLA-4 antibody ipilimumab significantly reduced cytokine production and CD4
+ T-cell proliferation. Ipilimumab is an IgG1 antibody and can, therefore, efficiently interact with Fc receptors. Several recent studies have indicated that ipilimumab might function at least in part by depleting intra-tumoral CTLA-4
high Tregs via Fc receptor-dependent mechanisms [
15‐
18]. We observed reduced numbers of proliferated CD4
+ T cells in the presence of IgG1-ipilimumab but not with an IgG4 variant, indicating that impairment of CD4
+ T-cell responses by ipilimumab is mediated by its Fc part. Moreover, we demonstrate that depleting CD16
+ cells abrogated the inhibitory effects of ipilimumab.
Materials and methods
Donor collection and PBMC isolation
Thirty-five female and 30 male individuals with a mean age of 35.5 years (range from 19.6 to 61.2) participated in the study. Heparinized whole blood samples were collected to isolate peripheral blood mononuclear cells (PBMCs) by standard gradient density centrifugation with Lymphoprep® solution (Technoclone, Austria).
Proliferation assay
Carboxyfluorescein succinimidyl ester (CFSE) labeling was performed as described previously [
11]. CFSE-labeled PBMCs (1 × 10
5/well) were stimulated with TT (10 Lf/mL; Statens Serum Institut, Copenhagen, Denmark) in AIM V™ media (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1.5% human serum. Blocking antibodies to immune checkpoints were used at a final concentration of 8 µg/mL. After 6–7 days, percentage of CFSE
low CD4
+ T lymphocytes was analyzed by flow cytometry. A single data point represents the triplicate mean of a donor. Responses with a stimulation index of 1.5 (at least 1.5-fold increase in the percentage of CFSE
low CD4
+ T cells in TT-stimulated cultures with respect to the CFSE
low CD4
+ T cells in control cultures) were considered reactive and used for further analysis.
Cell culture, antibodies, and flow cytometry
For flow cytometry analysis, the monoclonal antibodies CD4-PE (OKT4), BTLA-PE (MIH26), PD-1-PE (EH12.2H7), and isotype control (MOPC-21) were obtained from Biolegend (San Diego, CA, USA). CTLA-4-PE (14D3) and LAG-3-PE (3DS223H) monoclonal antibodies were obtained from eBioscience (San Diego, CA, USA). Staining was performed in FACS buffer (1% BSA and 0.1% NaN3 in PBS) for 30 min and 10 mg/mL Beriglobin (CSL Behring, King of Prussia, PA, USA) was added to prevent non-specific binding to Fc receptors while staining of the immune checkpoints. 7-Aminoactinomycin (7-AAD; Biolegend) was used to exclude dead cells from analysis. To determine CTLA-4 expression, cells were permeabilized using the Cytofix/Cytoperm™ kit (BD Biosciences, Franklin Lakes, NJ, USA). For depletion of CD16-expressing cells, PBMCs were labeled with CD16-APC mAb (3G8, Biolegend). The CD16-negative population was isolated using an SH800S cell sorter (Sony Biotechnology, Japan). The purity of the isolated population was examined by flow cytometry.
The following monoclonal blocking antibodies (final concentration 8 μg/mL) were used: functional grade PD-L1 (29E.2A3; LEAF™) and PD-1 (EH12.2H7 LEAF™) from Biolegend, CTLA-4 (IgG1, Ipilimumab, Yervoy
®), CTLA-4 (Ipilimumab-IgG4, S228P, InvivoGen, San Diego, CA, USA), and a mouse IgG1 κ isotype control antibody (MOPC-21; LEAF™, Biolegend). BTLA and LAG-3 blocking antibodies were described previously [
10,
11]. FACSCalibur™ and LSRFortessa™ flow cytometers (BD Bioscience) were used for sample measurement, and the FlowJo software (version 10.4.1., Tree Star, Ashland, OR, USA) was used for flow cytometry data analysis.
LEGENDplex™ and Luminex-based cytokine analysis
At days 6–7 of the PBMC proliferation assays, culture supernatants were collected from the triplicates, pooled and stored at − 20 °C. Next, IL-5, IL-10, IL-13, IL-17F, IFN-γ, and TNF-α concentrations were measured using the LEGENDplex™ human T
H cytokine panel (13-plex, Biolegend). Cytokine data shown in Fig.
6 were obtained by measuring the IFN-γ and TNF-α concentrations in culture supernatants using the Luminex System 100 (Luminex, Texas, USA) as described [
19].
Statistics
Normalization of proliferation and cytokine data was performed using methods described previously [
20]. GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA) was used to perform statistical analyses. Red markings in the figures represent median values of proliferation data, cytokine concentrations, and expression levels. Wilcoxon–Mann–Whitney tests and non-parametric repeated measurement ANOVA (Friedman test) were performed to analyze receptor expression, proliferation, and cytokine data for two or more groups in relation to mock control conditions, respectively. Immune checkpoint conditions were compared with the control conditions without antibody using Dunn’s multiple comparison post hoc test. Two-tailed Student’s
t test was used to assess the significance for data summarized in Fig.
6. The
p values below 0.05 were considered significant (*),
p < 0.01 (**),
p < 0.001 (***), and
p < 0.0001 (****).
Discussion
In our study, we have compared blocking antibodies targeting four major inhibitory immune checkpoints (PD-1, BTLA, LAG-3, and CTLA-4) in terms of their capability to enhance CD4
+ T-cell responses to TT in vitro. Although we found that each of these receptors is expressed on CD4
+ T cells, which responded to antigen stimulation, a significant enhancement of proliferation and cytokine production was only seen during disruption of PD-1 inhibition using antibodies against PD-1 or PD-L1 (Figs.
3,
4 and Fig. S1). This is in line with numerous previous studies, which report a sturdy increase in the T-cell responses upon PD-1 blockade. Besides, this result highlights the unique potential of PD-1 as a therapeutic target to enhance T-cell responses against pathogen- or tumor-derived antigens [
10,
11,
23‐
26]. Anti-LAG-3 or anti-BTLA antibodies had limited effects on the T-cell responses in our study. The main potential of antibodies targeting emerging immune checkpoints like LAG-3 or BTLA may be realized upon coupling their use with PD-1 blockers. Several studies including work by our group have demonstrated that blocking multiple immune checkpoints can be more effective than blocking PD-1 alone [
9‐
11,
27,
28]. However, we did not observe an enhanced response of TT-specific CD4
+ T cells upon combined blocking of immune checkpoints compared to blocking PD-1 alone (Fig. S2).
We found that the presence of the therapeutic CTLA-4 antibody ipilimumab substantially decreased the number of proliferated CD4
+ T cells and lowered the cytokine production in response to TT. This finding was coherent with an earlier study performed by us that demonstrated an impaired in vitro proliferation and cytokine production of allergen-specific CD4
+ T cells in the presence of ipilimumab [
20]. Although CTLA-4 was the first immune checkpoint successfully targeted clinically and its inhibitory role in T-cell responses is clearly established, the mechanisms of CTLA-4-mediated T-cell suppression are still not well understood [
29]. Initially, the inhibitory signaling mediated via the cytoplasmic tail of CTLA-4 was examined [
30‐
34], but there is also evidence that this receptor limits T-cell responses in an extrinsic manner [
35,
36]. It can prevent costimulatory signaling by outcompeting the binding of CD28 to the B7 molecules and by depleting B7 molecules from APCs through the process of transendocytosis [
37,
38]. We have previously used CTLA-4 variants lacking a cytoplasmic domain in a T-cell reporter system and found that they are fully competent to inhibit T-cell responses, which corroborates the vital role of extrinsic effects on CTLA-4 function [
39]. CTLA-4 is strongly expressed in Treg cells and it possibly inhibits T-cell responses via them. Importantly, several recent reports have otherwise suggested that, in vivo, CTLA-4 antibodies function by depleting intra-tumoral Tregs rather than by blocking CTLA-4 [
16,
21]. Ipilimumab has been shown to engage FcγRIIIA (CD16)-expressing non-classical monocytes resulting in ADCC-mediated lysis of Tregs [
18]. Based on this observation, the response to ipilimumab treatment was seen to be correlated with the levels of CD16
+ monocytes and the CD16a-V158F high-affinity polymorphism, implying that depletion of Tregs by CD16
+ cells might indeed contribute to the efficacy of ipilimumab treatment [
15,
18].
There is a surprising lack of in vitro studies reporting robust stimulatory effects of ipilimumab on primary human T cells, whereas numerous reports have documented that PD-1 blockade strongly enhances human CD4
+ and CD8
+ T-cell responses in vitro. Previous studies of our group show a tendency of reduced CD4
+, but not CD8
+, T-cell responses to allogeneic DC or HIV peptides using ipilimumab, although this effect was not statistically significant in the tested cohorts [
10,
11]. Moreover, we have found that ipilimumab induced a profound reduction in CD4
+ T-cell proliferation and cytokine production in PBMC cultures stimulated with allergen-containing extracts [
20]. In the current study, we demonstrate that ipilimumab significantly reduced CD4
+ T-cell responses to TT in a large cohort of donors. Since this effect was specifically observed with ipilimumab–IgG1, but not with an IgG4 variant of this antibody, we hypothesized that cytotoxic effects might have resulted in depletion of CTLA-4
high CD4
+ responder T cells in our PBMC cultures. To support our hypothesis, we used PBMC cultures depleted of CD16
+ cells and demonstrated that the inhibitory effect of ipilimumab was abrogated in such cultures. Increasing evidence suggests that Tregs, which express high levels of CTLA-4, are subject to ADCC mediated by CTLA-4 antibodies including ipilimumab [
15,
16,
18,
21]. To our knowledge, this is the first study to present data indicating that ipilimumab might also target CD4
+ effector T cells. Romano et al. co-cultured flow sorted CD4
+ T cells with CD16
+ monocytes. They showed that CD25
high CD4
+ T cells had the highest expression of CTLA-4 as well as FOXP3 and revealed that these cells, which were most likely Tregs, were killed in the presence of ipilimumab [
18]. In line, our experiments illustrate that upon strong upregulation of CTLA-4, stimulated CD4
+ effector T cells can also become targets of ipilimumab-mediated ADCC. We assume that ipilimumab will also deplete Tregs in our assay, but their low numbers in PBMC fractions will not significantly affect the CD4
+ T-cell response and the net effect of this antibody might be inhibitory in vitro. Further efforts are needed to address whether ipilimumab can deplete CTLA-4-expressing CD4
+ effector T cells in vivo. CTLA-4 expression is lower on CD8
+ than on CD4
+ T cells and our previous studies did not indicate impairment of CD8
+ T-cell response by ipilimumab [
10,
11,
40]. This outcome leads us to believe that the depletion of activated CD8
+ effector T cells in vivo by this antibody seems less likely.
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