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. 2015 Jan 2;179(2):265–276. doi: 10.1111/cei.12451

Modulation of heme oxygenase-1 by metalloporphyrins increases anti-viral T cell responses

C E Bunse *,†,1, V Fortmeier *,1, S Tischer *,†, E Zilian *,†, C Figueiredo *, T Witte ‡, R Blasczyk *,†, S Immenschuh *,†, B Eiz-Vesper *,†
PMCID: PMC4298404  PMID: 25196646

Abstract

Heme oxygenase (HO)-1, the inducible isoform of HO, has immunomodulatory functions and is considered a target for therapeutic interventions. In the present study, we investigated whether modulation of HO-1 might have regulatory effects on in-vitro T cell activation. The study examined whether: (i) HO-1 induction by cobalt-protoporphyrin (CoPP) or inhibition by tin-mesoporphyrin (SnMP) can affect expansion and function of virus-specific T cells, (ii) HO-1 modulation might have a functional effect on other cell populations mediating effects on proliferating T cells [e.g. dendritic cells (DCs), regulatory T cells (Tregs) and natural killer cells] and (iii) HO-1-modulated anti-viral T cells might be suitable for adoptive immunotherapy. Inhibition of HO-1 via SnMP in cytomegalovirus (CMV)pp65-peptide-pulsed peripheral blood mononuclear cells (PBMCs) led to increased anti-viral T cell activation and the generation of a higher proportion of effector memory T cells (CD45RA− CD62L−) with increased capability to secrete interferon (IFN)-γ and granzyme B. Treg depletion and SnMP exposure increased the number of anti-viral T cells 15-fold. To test the possibility that HO-1 modulation might be clinically applicable in conformity with good manufacturing practice (GMP), SnMP was tested in isolated anti-viral T cells using the cytokine secretion assay. Compared to control, SnMP treatment resulted in higher cell counts and purity without negative impact on quality and effector function [CD107a, IFN-γ and tumour necrosis factor (TNF)-α levels were stable]. These results suggest an important role of HO-1 in the modulation of adaptive immune responses. HO-1 inhibition resulted in markedly more effective generation of functionally active T cells suitable for adoptive T cell therapy.

Keywords: antigen-specific T cells, HO-1 modulation, immunotherapy, metalloporphyrins

Introduction

Heme oxygenase (HO)-1, the inducible isoform of the enzyme heme oxygenase, catalyzes the first and rate-limiting step of heme degradation into biliverdin, yielding carbon monoxide (CO) and free iron 1–4. HO-1, also known as heat shock protein 32 (HSP32), is a potent cytoprotective stress-response protein which is highly up-regulated by its substrate heme and various oxidative stress stimuli 5–10. HO-1 and its products have been found to mediate immunomodulatory effects by anti-oxidant, anti-proliferative, anti-inflammatory, anti-apoptotic and immunosuppressive mechanisms in various cell types 4,11–21.

The multi-functional role of HO-1 as an immunomodulator suggests that it might have potential as a therapeutic target for the modification of T cell responses and the clinical application of T cells. HO-1 is up-regulated upon T cell activation via the T cell receptor (TCR), resulting in increased CO production and further inhibition of T cell proliferation. This complex mechanism suggests that HO-1 might play a crucial role in the regulation and homeostatic control of T cell activation 13,22. HO-1–/– knock-out mice develop a progressive inflammatory state, and splenocytes isolated from these mice respond to TCR activation by producing inflammatory cytokines 23. Different organ transplant models have indicated a protective effect of carbon monoxide during transplantation 24. This effect was associated with increased survival as well as severe T cell-mediated graft-versus-host disease (GvHD) 25.

Interaction between antigen-presenting cells (APCs), effector T cells and regulatory T cells (Tregs) is strongly affected by the regulation of HO-1 activity in human cells. Modulation of HO-1 by tin-mesoporphyrin (SnMP) inhibits the maturation of dendritic cells (DCs) and the secretion of proinflammatory cytokines, resulting in decreased T cell proliferation 18,26. Burt et al. showed that pharmacological inhibition of HO-1 by SnMP in peripheral blood mononuclear cells (PBMCs) leads to the activation, proliferation and maturation of naive CD4+ and CD8+ T cells in an antigen-independent manner and in the presence of CD14+ monocytes 17.

Therefore, we aimed to identify the role of HO-1 in the modification of antigen-specific T cell responses and to estimate the application of the pharmacological modulators of HO-1 activity in a clinical-like setting for the isolation of clinical grade anti-viral T cells. The adoptive transfer of virus-specific cytotoxic T lymphocytes (CTLs) directed against cytomegalovirus (CMV) 27–29, Epstein–Barr virus (EBV) 30,31 and adenovirus (ADV) 32 can safely and effectively reduce or prevent the clinical manifestations of these viruses in immunosuppressed patients after haematopoietic stem cell transplantation and solid organ transplantation. Prophylactic anti-viral drug treatments are effective at preventing invasive viral infections post-transplant, but are typically administered for a limited time due to cumulative drug toxicity and cost 33,34. However, hosts with impaired T cell immunity remain susceptible to subsequent infections. The isolation of anti-viral T cells for adoptive T cell transfer requires the induction and expansion of specific T cells from seropositive donors. As the frequency of memory precursor T cells is often quite low, potent mediators of T cell proliferation are needed.

The present study analysed the influence of HO-1 modification by metalloporphyrins on anti-viral T cells and investigated the question of whether such modification could: (1) enhance the expansion of virus-specific CD8+ T cells in vitro, (2) influence other cell subsets within the PBMC population (e.g. DCs, Tregs and natural killer (NK) cells) in vitro, resulting in mediating effects on proliferating T cells and (3) make antigen-specific T cells suitable for adoptive immunotherapy.

Material and methods

Collection of PBMCs

Written informed consent was obtained from all donors, as approved by the Ethics Committee of Hannover Medical School. In total, cells from 17 donors were included in this study. PBMCs form healthy human leucocyte antigen (HLA)-A*02:01-positive, CMV-seropositive blood donors with no prior history of blood transfusion and no signs of acute infection were isolated by discontinuous gradient centrifugation. Cells were resuspended at a concentration of 1 × 107 cells/ml in culture medium consisting of RPMI-1640 (Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated human AB serum (C.C.pro, Neustadt, Germany) and 50 U/ml IL-2 (PeproTech GmbH, Hamburg, Germany) and allowed to rest overnight.

Metalloporphyrins for pharmacological modulation of HO-1 activity and peptide stimulation

SnMP and cobalt-protoporphyrin (CoPP) were purchased in powdered form from Frontier Scientific (Logan, UT, USA), dissolved in dimethylsulphoxide (DMSO) and kept protected from light. A 25-mM stock solution was stored at −20°C. Before use in cell culture, the stock solution was diluted in culture medium. Total concentration of DMSO did not exceed 2·8 μM in all experiments.

The HLA*02:01-restricted CMVpp65495–503 peptide (A02pp65P, NLVPMVATV; ProImmune, Oxford, UK) was used to stimulate antigen-specific T cells at a concentration of 10 μg/ml. To ensure equal DMSO concentration in all set-ups, DMSO was additionally added to all peptide-stimulated but SnMP- or CoPP-untreated samples.

Determination of optimal working concentrations of metalloporphyrins in T cell proliferation assays

Carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Darmstadt, Germany) dilution experiments were performed to investigate whether SnMP- or CoPP-induced proliferation of CMV-specific T cells is dose-dependent 35. PBMCs (n = 3) were labelled with CFSE (final concentration 1 μM) and plated at a concentration of 5 × 106 cells per ml in 24-well plates (Sarstedt, Inc., Newton, NC, USA). Cells were stimulated with A02pp65P in the presence of 1, 5, 10, 15 or 20 μM of SnMP and CoPP, respectively. After 4, 24 and 72 h, mRNA levels of HO-1 were assessed by quantitative real-time polymerase chain reaction (PCR) 36. On day 7, cells were counted and analysed by flow cytometry (purity panel). In all further experiments, 10 μM SnMP or 10 μM CoPP were used.

Evaluation of cell frequencies and phenotypes by flow cytometric analysis

Flow cytometric analyses were performed using fluorescence activated cell sorter (FACS)Canto II (BD Biosciences, Heidelberg, Germany) and BD FACSDiva Software version 6·1.2. At least 100 000 events were acquired in the live gate, or at least 30 000 in the CD3+ gate. Gates were set based on the light-scatter properties of lymphocytes and DCs, respectively. CMV-specific T cell frequencies were assessed by peptide major histocompatibility complex (pMHC) multimer staining using R-phycoerythrin (R-PE)-conjugated pentamer HLA-A*02:01/CMVpp65495–503 (A02pp65M; ProImmune) according to the manufacturer's instructions. Additionally, cells were stained with the following monoclonal antibodies (mAb): anti-CD8 allophycocyanin (APC) [Becton Dickinson (BD), Heidelberg, Germany], anti-CD45RA peridinin chlorophyll (PerCP)-cyanin (Cy)5·5 and anti-CD62L APC/Cy7 (all BioLegend, San Diego, CA, USA) (T cell phenotype panel). CMV-specific T cells were classified as naive T cells (TN; CD62L+ CD45RA+), central memory T cells (TCM; CD62L+ CD45RA−), effector memory T cells (TEM; CD62L− CD45RA−) or terminally differentiated effector memory T cells (TEMRA; CD62L− CD45RA+).

Resting PBMC populations were determined by staining with anti-CD3 PerCP, anti-CD14 APC-H7, anti-CD19 PE and anti-NKp46 APC (all Becton Dickinson), anti-CD8 FITC (Beckman Coulter, Brea, CA, USA) and anti-CD45RA PE/Cy7 (BioLegend) (purity panel).

Early activation of T cells was assessed by staining with anti-CD25 PE, anti-CD69 fluorescein isothiocyanate (FITC) and anti-CD137 PE/Cy7 (all BioLegend) 5 h after stimulation (early activation panel) (n = 6).

Phenotypical analysis of DCs was performed with anti-CD206 FITC, anti-CD209 PE-Cy7 (both BioLegend), anti-CD14 PerCP, anti-HLA-DR APC-Cy7, anti-CD83 PE and anti-CD86 APC (all from BD) (DC panel).

CMVpp65 peptide-specific expansion of CD8+ T cells through modification of HO-1 activity

To test whether the expansion of A02pp65P-restricted CD8+ T cells is altered by modified HO-1 activity, 5 × 106/ml freshly isolated PBMCs (n = 6) were stimulated with 10 μg/ml A02pp65P alone and in the presence of 10 μM SnMP or 10 μM CoPP, respectively. On day 7, supernatants were harvested and analysed for the effector molecules interferon (IFN)-γ and granzyme B secretion by enzyme-linked immunosorbent assay (ELISA). Secretion of multiple cytokines was assessed using a bead-based multiplexed assay. Stimulated cells were counted, stained with A02pp65M and analysed by flow cytometry using the T cell phenotype panel.

Effects of HO-1 modification on DC maturation and their T cell stimulatory capacity

DCs were generated using a slightly modified protocol that accelerates the generation of mature DCs (mDCs) within 48 h 37. Briefly, monocytes were isolated from freshly isolated PBMCs (n = 3) by magnetic cell sorting using the Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes were suspended in RPMI-1640 supplemented with 2% heat-inactivated human AB serum and 500 U/ml IL-4 and 1000 U/ml granulocyte macrophage-colony stimulating factor (GM-CSF) (PeproTech) at a concentration of 5 × 105/ml and exposed to 10 μM SnMP and 10 μM CoPP, respectively. In order to eliminate any effect of DMSO, which was used to dissolve SnMP and CoPP, 2·8 μM DMSO was added to all metalloporhyrin-untreated samples. Immature dendritic cells (iDCs) were harvested, their phenotype was assessed by flow cytometry and loaded with A02pp65P (10 μg/ml). Maturation was further initiated by adding proinflammatory stimuli (10 ng/ml tumour necrosis factor (TNF)-α, 10 ng/ml interleukin (IL)-1β (all from PeproTech) and 1 U/ml PG-E2 (Sigma Aldrich, Munich, Germany). On day 2, mDCs were collected and their maturation status was analysed by flow cytometry.

Peptide-loaded mDCs treated with SnMP, CoPP or DMSO were co-cultured with autologous monocyte-depleted PBMCs, previously stored for 2 days in culture media (RESTORE protocol, RESetting T cells to Original Reactivity) 38 at an effector : target (E : T) ratio of 1 : 10 in 48-well round-bottomed plates (Nunc, Thermo Scientific, Waltham, MA, USA) in culture medium for another 7 days. Flow cytometry was used to analyse the expansion of CMV-specific T cells (T cell phenotype panel) and mRNA levels of Ki-67, IFN-γ and granzyme B were determined.

Depletion of CD25+ Tregs and NKp46+ NK cells

Freshly isolated PBMCs (i), CD4+ CD25+ Treg-depleted PBMCs (ii) and NKp46+ NK cell-depleted PBMCs (iii) were used to evaluate the role of Tregs and NK cells. Depletion was performed with MACS technology (Miltenyi Biotec) using CD25-APC and NKp46-APC mAbs and biotin-conjugated secondary antibodies. Purity was determined by flow cytometry. Post-depletion contamination of CD25+ cells was usually less than 0·5%, and that of NKp46 less than 2%. Non-depleted and CD25- and NKp46-depleted PBMCs were stimulated with A02pp65P with and without SnMP (10 μM) or CoPP (10 μM) and cultured at a concentration of 5 × 106 cells/ml for 1 week. On day 7, cells were counted and analysed (T cell phenotype panel).

Evaluation of HO-1, IFN-γ and granzyme B mRNA levels by quantitative real-time PCR and detection of granzyme B, and IFN-γ secretion by ELISA

To assess the mRNA levels of HO-1, Ki-67, IFN-γ and granzyme B, total cellular RNA was isolated (RNeasy Mini Kit; Qiagen, Hilden, Germany) and cDNA was amplified using the high capacity cDNA reverse transcription kit (Applied Biosystems, Darmstadt, Germany). Inventoried mixes (Applied Biosystems) were used for quantification of Ki-67, HO-1, IFN-γ and granzyme B mRNA levels. Amplification was performed using TaqMan Gene Expression Master Mix (Applied Biosystems). The constitutively expressed glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as reference gene. Granzyme B and IFN-γ secretion in the culture supernatant was analysed by ELISA (eBioscience, San Diego, CA, USA) on day 7, according to the manufacturer's instructions.

Multiple cytokine detection

A bead-based multiplexed assay (Luminex Cytokine Human Panel; Invitrogen) that quantifies multiple cytokines (IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-8, IL-12p70, TNF-α, GM-CSF) in single-sample supernatant was used to analyse the cytokine expression patterns of T cells after 7 days of stimulation with A02pp65P in the presence or absence of SnMP and CoPP, respectively (n = 5). Cytokine detection was performed according to the manufacturer's instructions on a Luminex-200 instrument (Invitrogen).

Applicability of SnMP in the isolation of anti-viral T cells in a clinical-like setting

The clinical applicability of SnMP for pharmacological inhibition of HO-1 was proved using the IFN-γ cytokine secretion and enrichment assay (CSA; Miltenyi Biotec). After short 4-h in-vitro stimulation with A02pp65P and the overlapping CMVpp65 peptide pool (PepTivator CMV pp65, pp65PP; Miltenyi Biotech) in the presence or absence of SnMP, CMV-specific IFN-γ-secreting T cells from six donors were detected and enriched according to the manufacturer's instructions. The pp65PP is a peptide pool covering the complete sequence of the pp65 protein of human cytomegalovirus. These peptides are mainly 15-mer peptides overlapping by 11 amino acid residues (aa) and represent an optimized solution for stimulating both CD4+ and CD8+ T cells in various applications 39. Cells were stained with anti-IFN-γ-PE (Miltenyi Biotec), anti-CD8 APC and anti-CD4 PerCP mAbs and analysed by flow cytometry. A total of 50 000 events were acquired in the live gate, or at least 20 000 in the CD3+ population. Gates were set based on the scatter properties of lymphocytes and on CD3+/IFN-γ+ T cell populations.

In order to determine the influence of SnMP-treatment on the functionality of the anti-viral T cells, enriched CD3+/IFN-γ+ T cells (2·5 × 104/ml) isolated from three donors after pp65PP stimulation were further cultivated on an autologous IFN-γ-negative feeder layer (2·5 × 106/ml) for 10 days at an E : T ratio of 1 : 100. On day 10, intracellular levels of IFN-γ, TNF-α and granzyme B were detected. Cells were incubated with pp65PP for a total of 5 h. Brefeldin A (BioLegend) was added at a dilution of 1 : 1000 after 1 h. Intracellular staining was performed using the IntraPrep Kit (Beckmann Coulter, Krefeld, Germany), according to the manufacturer's instructions. Staining for IFN-γ (PE; Beckmann Coulter), TNF-α (PE-Cy7; BioLegend) and granzyme B (AlexaFluor467; BioLegend) was performed in combination with anti-CD3 PerCP (BD) and anti-CD8 APC (BD) or anti-CD8 FITC (BD, in case of granzyme B) staining.

In addition, anti-viral T cell degranulation was assessed as a surrogate marker of cytotoxicity 40–42 by detecting the expression of CD107a on the cell surface 43. Cells were restimulated with pp65PP and incubated with a PE-Cy7-conjugated anti-CD107a antibody (2·5 μl/1 × 106 cells; BioLegend) at 37°C and 5% CO2. After 1 h of incubation, a 1 : 1000 dilution of monensin (BioLegend) was added and the cells were further incubated for 4 h before staining with anti-CD3 PerCP and anti-CD8 APC.

Statistics

Statistical analyses were performed using paired or unpaired t-testa run on GraphPad Prism version 5·02 software (GraphPad Software, San Diego, CA, USA). Levels of significance are expressed as P-values (*P < 0·05, **P < 0·01, ***P < 0·001).

Results

Metalloporphyrins affect HO-1 expression and proliferation of human T cells

To assess the influence of the metalloporphyrins, CoPP and SnMP, on HO-1 expression levels, PBMCs were treated with either CoPP or SnMP and HO-1 mRNA levels were determined after 4, 24 and 72 h. SnMP did not affect HO-1 mRNA levels, whereas CoPP increased HO-1 expression significantly and in a dose-dependent manner, as reflected by an up to 225-fold increase at 4 h followed by weaker but still marked increases (61-fold at 24 h and 44-fold at 72 h) at later time-points (Supporting information, Fig. S1).

Next, we investigated the effects of treatment with CoPP or SnMP in combination with A02pp65P on the proliferative capacity of anti-viral T cells (Fig. 1a). Compared to levels in untreated cells, SnMP enhanced T cell proliferation in a dose-dependent manner; the most significant increase was observed at a concentration of 10 μM (61·35% CFSElow cells). Higher concentrations of SnMP did not lead to further increases in proliferation capacity. In contrast, CoPP reduced T cell proliferation in a dose-dependent manner up to a concentration of 10 μM (21·82% CFSElow cells). Thus, the optimal working concentrations were determined to be 10 μM of SnMP for HO-1 inhibition and 10 μM of CoPP for HO-1 induction.

Figure 1.

Figure 1

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Metalloporphyrin treatment increases proliferation and phenotype of T cells. Peripheral blood mononuclear cells (PBMCs) (n = 3) were treated with tin-mesoporphyrin (SnMP) or cobalt-protoporphyrin (CoPP) and stimulated with A02pp65P for 7 days. (a) Treatment with SnMP increases proliferation of CD3+ T cells after 7 days of A02pp65P stimulation dose-dependently up to 61·35% (10 μM) and CoPP treatment decreases CD3+ T cell proliferation to 21·82% (10 μM). (b) The frequency of A02pp65M+ CD8+ T cells increases under SnMP treatment up to 16·92%. Treatment with CoPP does not enhance the expansion of specific T cells (8·90% versus 7·20% of A02pp65P). (c) The composition of T cell subsets of A02pp65M-specific T cells is altered by metalloporphyrin treatment. Effector memory T cells (TEM; CD45RA−CD62L−) are increased, naive T cells (TN; CD45RA+CD62L+), central memory T cells (TCM; CD45RA−CD62L+) and terminally differentiated effector memory T cells (TEMRA; CD45RA+CD62L−) are reciprocally decreased. Shown are means of three donors. (d) Interferon (IFN)-γ and granzyme B secretion is increased under SnMP treatment as detected by enzyme-linked immunosorbent assay (ELISA) after 7 days of A02pp65P stimulation. (e) The secretion of additional cytokines was assessed by Luminex technology after 7 days of A02pp65P stimulation (n = 5).

Inhibition of HO-1 by SnMP increases expansion of CMVpp65-specific T cells and promotes development into effector memory T cells

Besides an increased proliferation rate of CD3+ T cells, HO-1 inhibition by SnMP also significantly up-regulated the percentages of CMVpp65-specific CD8+ T cells after 7 days of stimulation (mean A02pp65P: 7·20% versus mean 10 μM SnMP/A02pp65P 16·92%, Fig. 1b). As expected, HO-1 activation by CoPP did not significantly alter the percentage of CMVpp65-specific T cells (8·90%). Exposure to metalloporphyrins without stimulatory peptides did not have an effect on virus-specific T cell proliferation (data not shown).

We further determined the effects of HO-1 modulation on the phenotype of A02pp65M+ CD8+ T cells (Fig. 1c). HO-1 inhibition with 10 μM SnMP resulted in higher frequencies of A02pp65M+ TEM cells (CD62L−CD45RA−) than stimulation with A02pp65p alone or 10 μM CoPP (A02pp65p: mean 26·74%, SnMP/A02pp65p: mean 50·39%, CoPP/A02pp65p: mean 27·59%). Compared to A02pp65p alone, SnMP/A02pp65p resulted in a reciprocal decrease in TN cells (mean 10·36% versus 14·81%), TEMRA cells (mean 53·53% versus 37·11%) and TCM cells (mean 4·92% versus 2·14%). Regarding the activation of CMV-specific T cells, SnMP/A02pp65P treatment was associated with increased expression of the activation markers CD25, CD38 and CD69 (data not shown), indicating that an additional SnMP treatment induced a more active phenotype than A02pp65P alone.

These findings demonstrate that HO-1 inhibition leads to a higher proportion of functional effective anti-viral T cells, as underlined by the increased levels of secreted IFN-γ and granzyme B detected in the cell culture supernatants of SnMP/A02pp65p-treated cells (Fig. 1d). These cells showed a 5·4-fold increase of IFN-γ secretion and a 2·22-fold increase of granzyme B secretion compared to stimulation with A02pp65p alone. CoPP/A02pp65p led to a negligible increase of IFN-γ and granzyme B secretion.

To further assess whether HO-1 inhibition might have an effect on the development of a proinflammatory cytokine milieu that might support antigen-specific T cell proliferation, cytokine detection was carried out using a multiplex assay for IL-1β, IL-6, IL-8, IL-10, IL-12p70, IL-17a, TNF-α and GM-CSF (Fig. 1e). IL-1β and TNF-α secretion increased in response to CoPP independently of A02pp65P. In contrast, GM-CSF, IL-6, IL-10 and IL-17A were secreted more efficiently in response to SnMP. Interestingly, the secretion of these cytokines was inhibited by combined stimulation with A02pp65P. There was no difference in IL-4 and IL-12p70 secretion between the two metalloporphyrins.

Modification of HO-1 activity during DC maturation does not affect DC maturation status or results in higher T cell stimulatory capacities

The effect of HO-1 activity modulation on DC maturation and the resulting consequences on T cell stimulation were evaluated (Fig. 2; Supporting information, Fig. S2). Inhibition or induction of HO-1 via SnMP and CoPP, respectively, did not alter the levels of CD83, CD86, CD206 and HLA-DR surface molecule expression (Supporting information, Fig. S2), while induction of HO-1 by CoPP decreased CD209 expression on both immature and mature DCs significantly (Supporting information, Fig. S2). Compared to SnMP-untreated but A02pp65P-stimulated cells, inhibition of HO-1 by SnMP led to a slight increase in the number of CMVpp65-specific T cells (1·46-fold) (Fig. 2a). No increase in CMVpp65-specific T cells was observed when CoPP-treated DCs were used (1·01-fold). Additionally, analysis of mRNA revealed no significant change in Ki-67, IFN-γ or granzyme B levels in response to SnMP or CoPP (Fig. 2b). In summary, we conclude that modulation of HO-1 in DCs by metalloporphyrins does not alter the maturation status, functionality or T cell stimulatory capacity of professional APCs.

Figure 2.

Figure 2

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Treatment of dendritic cells (DCs) with metalloporphyrins does not influence maturation and antigen presentation. DCs (n = 3) were treated with tin-mesoporphyrin (SnMP) or cobalt-protoporphyrin (CoPP) during maturation and mature and peptide-loaded DCs were used to stimulate autologous peripheral blood mononuclear cells (PBMCs) for 7 days. (a) Expansion of A02pp65M+ CD8+ T cells is not affected significantly by DCs matured under the influence of SnMP or CoPP. (b) Proliferation as detected by Ki-67 and effector molecule expression [interferon (IFN)-γ and granzyme B] is not affected when DCs were treated with metalloporphyrins.

HO-1 inhibition in Tregs indirectly affects expansion of antigen-specific T cells

Recently, it was found that SnMP inhibits the regulatory capacity of Tregs 17. Therefore, we investigated whether HO-1 inhibition in Tregs affects the proliferation of virus-specific T cells (Fig. 3a). SnMP/A02pp65P stimulation led to a 3·29-fold increase in CMV-specific CD8+ T cells compared to A02pp65P alone, which was further increased 5·69-fold by depletion of CD4+ CD25+ cells. HO-1 inhibition by SnMP in combination with Treg depletion led to a 13·23-fold increase in CMV-specific T cell frequencies. These results indicate that Treg depletion in combination with SnMP multiplies CMV-specific T cell frequencies.

Figure 3.

Figure 3

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Tin-mesoporphyrin (SnMP) treatment and depletion of regulatory T cells (Tregs) but not natural killer (NK) cells amplify the expansion of antigen-specific T cells. Peripheral blood mononuclear cells (PBMCs) (n = 3) were left untouched or either depleted from Tregs or NK cells and then stimulated with A02pp65P and treated with SnMP for 7 days. (a) Depletion of CD4+CD25+ T cells (Tregs) in combination with SnMP improves enhanced the expansion of A02pp65M+ CD8+ T cells. Depletion of NKp46+ NK cells does not affect A02pp65M+ CD8+ T cell expansion regardless of SnMP treatment. (b) Treatment with SnMP up-regulates the expression of NKp46 on NK cells.

Analysis of the influence of modified HO-1 activation on further cell subsets within the PBMC population showed that treatment with metalloporphyrins did not have any effects on CD14+ and CD19+ cells (Supporting information, Fig. S3). Interestingly, significant expansion of NK cells was observed, as reflected by increased expression of the NKp46 receptor in response to SnMP (3·44% with A02pp65P alone versus 17·79% with SnMP/A02pp65P, Fig. 3b).

To investigate whether SnMP alters the functionality of NK cells that affect T cell proliferation, non-depleted PBMCs and NKp46+-depleted PBMCs were stimulated with A02pp65P with or without SnMP (Fig. 3a). Compared to unmanipulated PBMCs, NK depletion had no effect on the expansion of CMVpp65-specific T cells independently of the addition of SnMP (SnMP/A02pp65P) (3·29-fold versus 3·09-fold increase).

SnMP results in greater enrichment of IFN-y-secreting cytotoxic T cells with equal cytolytic activity and cytokine expression

To answer the question of whether or not metalloporphyrin-treated antigen-specific T cells are suitable for clinical use, we examined if the expansion of virus-specific T cells via SnMP might be applicable in adoptive immunotherapy. Therefore, the enrichment of IFN-γ secreting anti-viral T cells was performed using CSA (Fig. 4). CSA is a method comparable to the cytokine capture system (CCS), which enables the isolation of clinical grade T cells for adoptive T cell transfer. Prior to enrichment of the cells via magnetic-activated cell sorting (MACS) technology, flow cytometric analysis revealed significantly more CD3+/IFN-γ+ T cells after stimulation with SnMP/A02pp65P and SnMP/pp65PP (Fig. 4a) than with A02pp65P and pp65PP. To assess the functionality and viability of enriched CD3+/IFN-γ+ cells, T cells from three donors were cultured in the presence of the IFN-γ-negative feeder cell population for 10 days. Treatment with SnMP did not alter the functional properties of virus-specific T cells during isolation and enrichment in a near-clinical setting, because no significant differences in the viability, degranulation capacity or secretion of the effector molecules IFN-γ, TNF-α and granzyme B were observed (Fig. 4b). Treatment with SnMP increased the number of IFN-γ-secreting cells significantly, but did not affect purification performance or cell properties after enrichment, such as T cell subset composition of naive, central memory, effector memory and terminally differentiated effector memory T cells and the ratio of CD4+ and CD8+ T cells. Therefore, we conclude that the application of SnMP for increasing the number of disposable anti-viral T cells for adoptive T cell immunotherapy is feasible.

Figure 4.

Figure 4

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Tin-mesoporphyrin (SnMP)-treated cells are suited for cell therapies. Peripheral blood mononuclear cells (PBMCs) were stimulated with the cytomegalovirus (CMV)pp65 peptide pool (pp65PP) for 4 h and interferon (IFN)-γ secreting cells were enriched in a clinical-like setting using the cytokine secretion assay (CSA, n = 6). Cells from three donors were cultivated for additional 10 days and their effector function was determined by intracellular staining and the degranulation assay. (a) CD3+/IFN-γ+ T cells are significantly augmented after pp65PP stimulation under SnMP treatment. (b) The composition of T cell subsets is not altered by SnMP treatment in CD3+, CD8+ or CD4+ cells after enrichment for IFN-γ-secreting cells. Shown are means of three donors. (c) The percentage of CD4+ and CD8+ T cells after enrichment for IFN-γ secreting cells is not affected by SnMP treatment. (d) SnMP-treated CD3+/IFN-γ+ T cells enriched in the CSA and cultivated for 10 days are equally functional to non-SnMP-treated cells, as determined by intracellular staining for IFN-γ, tumour necrosis factor (TNF)-α and granzyme B as well as by CD107a degranulation assay.

Discussion

Recently, it was shown that inhibition of HO-1 activity by the metalloporphyrin SnMP induces antigen-independent activation, proliferation and maturation of naive CD4+ and CD8+ T cells in vitro 17. To investigate the role of HO-1 in the modulation of adoptive immune responses, we examined the effects of HO-1 inhibition and activation on phenotypical and functional properties of virus-specific T lymphocytes. The three major findings of this study are that: (1) treatment of PBMCs with the HO-1 inhibitor SnMP is associated with increased anti-viral CD8+ T cell activation and a more active phenotype; (2) depletion of regulatory T cells in combination with SnMP treatment significantly increases virus-specific T cell frequencies; and (3) inhibition of HO-1 activity increases the number of anti-viral IFN-γ secreting cells, which may afford improvement of adoptive immunotherapeutic protocols for clinical T cell applications in conformity with GMP.

SnMP inhibits HO-1 activity in PBMCs effectively and increases the number of virus-specific T cells

HO-1, which is up-regulated during T cell activation, and its catalytic product CO inhibit T cell proliferation in response to activation via the TCR 13,22. Increased HO-1 activity in APCs, such as DCs and monocytes, inhibits the immunogenicity of these cells and thereby negatively influences their ability to stimulate allogeneic T cell responses 18,26,44. Burt and colleagues 17 have demonstrated that pharmacological inhibition of HO-1 activity in PBMCs by SnMP inverts the limiting effect of HO-1 on T cell activation, proliferation and maturation of naive CD4+ and CD8+ T cells in an antigen-independent manner in cultures where CD14+ monocytes are present. Given this, we asked whether the modulation of HO-1 activity would result in the increased proliferation of antigen-specific T cells and if the observed effects could be translated into clinical practice for the generation of clinical-grade T cells.

To investigate these questions, we used the immunodominant HLA-A*02:01-restricted CMV peptide (CMVpp65495–503) 45 and the CMVpp65 overlapping peptide pool as antigenic stimuli. The CMVpp65 overlapping peptide pool (PepTivator CMVpp65) is commercially available in GMP quality and was used to consider the use of the antigen and the technology for future manufacturing of clinical-grade T cells.

Exposure of PBMCs to SnMP, which inhibits HO-1 activity, resulted in the activation and proliferation of anti-viral CD8+ T cells, as determined by multimer staining and functional assays. Two possible explanations based on the physiological roles of HO-1 mediated by the enzymatic product CO are detailed below:

  1. Caspase activity is up-regulated during T lymphocyte proliferation and is strongly related to proliferation, as shown by bromodeoxyuridine (BrdU) proliferation assay 13. The study demonstrated that CO inhibits caspase-3 and -8 activity, which suggests that the anti-proliferative effect of HO-1 on activated CD3+ T cells may be partially associated with increased expression of the cell cycle inhibitor p21Cip1 through p21Cip1-dependent activation of caspases. Therefore, inhibition of the enzymatic properties of HO-1 resulted in decreased CO generation and diminished CO-mediated inhibition of caspase activity, which further abolished the inhibition of T cell proliferation.

  2. Limitation of T cell activation in response to anti-CD3 is based on the suppressive effects of HO-1 and its enzymatic product CO on IL-2 secretion due to inhibition of the extracellular signal-regulated kinase (ERK) pathway in CD4+ T cells. Hence, if IL-2 secretion in CD4+ T cells is no longer limited after HO-1 inhibition, T cell help can be provided and antigen-specific CD8+ T cells can therefore proliferate strongly.

The results of the multiple cytokine expression profiling analysis revealed an enhanced secretion of GM-CSF, IL-6, IL-10 and IL-17A in in-vitro SnMP-treated PBMCs (Fig. 1e). This increase was found not only in human cells of our study, but also in splenocytes isolated from HO-1 knock-out mice in response to TCR activation 23. In contrast, exposure of T cells to CoPP resulted in an increase in IL-1β and TNF-α secretion. Both cytokines participate in the establishment of inflammatory lesions in acute and chronic inflammation act synergistically in many of these activities 46. Consequently, increased expression and the role of these two cytokines under CoPP treatment are not yet conclusive. IL-1β is secreted by myeloid cells (e.g. monocytes, DCs) and plays an important role via triggering the secretion of further proinflammatory cytokines in T helper target cells. Therefore, T cell proliferation was likely but was not determined (Fig. 1a). Conversely, secretion of IL-1β indicates stress conditions, which might be induced by CoPP treatment. TNF-α is also known to induce cellular proliferation and differentiation and the activation of cells, but has no effect on the expansion on CD3+ T cells in these cell culture conditions.

Our findings underline the role of HO-1 in the regulation of T cell activation and the maintenance of immune homeostasis. In order to gain insight into the regulatory mechanism of HO-1-mediated impairment of T cell function its inhibitor SnMP and inducer CoPP, subpopulations of CD4+ and CD8+ T cells should be investigated separately.

Modulation of HO-1 in the maturation process of DCs

Recent studies have shown that modulation of HO-1 activity in APCs significantly influences the outcome of their interaction with allogeneic T cells 17,18,26,44,47. Modulation of HO-1 has been shown to affect the secretion of cytokines, which are critical for DC generation and for effector T cell responses 18. Nevertheless, in all these studies, newly generated DCs were not used for antigen-specific expansion of memory T cells, and the antigen-presenting capacities of the cells were not proved. Metalloporphyrin-treated monocytes did not show significantly different expression of the DC maturation markers than untreated monocytes (Supporting information, Fig. S2). Treatment consistently had no significant impact on the number or functionality of CMV-specific CD8+ T cells (Fig. 2a).

Our findings are in concordance with those of Chauveau et al. 18, who observed a strong down-regulation of HO-1 expression during DC maturation, indicating that the modulatory role of HO-1 might be restricted to iDCs. These authors used a protocol in which iDCs were generated over a 6-day period followed by 2 days of incubation for maturation (RESTORE protocol) 38. Consequently, the duration of the iDC state in our study was probably too short to induce HO-1-restricted modulation of DC maturation or functionality. While the study by Chauveau and colleagues 18 showed that HO-1 can block the maturation of DCs, our study demonstrated that SnMP-mediated inhibition of HO-1 does not influence DC maturation and does not lead to improved T cell stimulation. In combination with previous findings, these new insights into the potential role of HO-1 in APC biology might be useful in the development of new immunosuppressive strategies in transplantation or autoimmune diseases.

The negative impact of regulatory T cells on expansion of virus-specific T cells can be enhanced synergistically by Treg depletion and SnMP treatment

The results of our cytokine multiplex assays revealed that IL-10 secretion increased after inhibition of HO-1 by SnMP (Fig. 1e). This result can be attributed to the presence of Tregs cells and monocytes within the SnMP-treated PBMC population 48. In our study, Treg depletion increased the expansion rate of CMV-specific anti-viral T cells approximately sixfold, as shown in an antigen-dependent stimulation assay (Fig. 3a). Even more impressively, Treg depletion combined with HO-1 inhibition in resting PBMCs resulted in a 13-fold increase. These results suggest that inhibition of the enzymatic activity of HO-1 by SnMP occurs in Tregs as well as in the resting PBMC population.

Burt et al. 17 postulated that SnMP-treated monocytes undergo several phenotypical changes leading to more effective T cell interactions. Furthermore, it is assumed that HO-1 inhibition renders monocytes unable to support Treg function and actively inhibit Treg activity. We found that inhibition of HO-1 in PBMCs increases the generation of anti-viral T cells, and that this is due most probably to a modified monocyte phenotype and monocyte-mediated impairment of Treg function (Fig. 3a).

The combination of Treg depletion and SnMP treatment has a synergistic effect on anti-viral T cell expansion that makes this strategy useful for the generation of antigen-specific T cells from naive T cells or from low precursor frequencies for adoptive T cell therapy.

Modulation of HO-1 increases the number of NKp46+ cells

It has been reported that HO-1 expression has a negative influence on both T cells and NK cells 49. We observed increased expression of the receptor NKp46 in cells where HO-1 activity was suppressed by SnMP treatment (Fig. 3b). As shown in a recent review, NK cells can positively influence CD4+ and CD8+ T cell responses to a variety of antigens by cytokine and chemokine production, but they can also inhibit T cell immunity 50. However, we found that depletion of NKp46+ NK cells and HO-1 inhibition by SnMP in resting PBMCs had no impact on anti-viral T cell proliferation. It is likely that the effects of NK cell depletion with respect to HO-1 modulation are hard to detect in this setting. The results are not yet conclusive and should be investigated in further studies.

Applicability and translation of the described strategies in clinical settings

Taken together, we found that HO-1 inhibition results in a significant increase in the expansion of peptide-specific cells. By increasing the number and functionality of specific T cells, SnMP treatment provides therapeutic advantages over conventional strategies. We proved the clinical applicability of this approach by isolating anti-viral T cells using the cytokine secretion assay – a method comparable to the cytokine capture system, which enables the isolation of clinical grade T cells for adoptive T cell transfer. In-vitro SnMP treatment increased the number of IFN-γ-secreting cells, the purity of which was equal to that of untreated cells (Fig. 4a). Moreover, the quality (Fig. 4b,c) and effector functions of these T cells were not impaired by treatment (Fig. 4d). Based on the safety of SnMP, as observed in the treatment of patients with jaundice and hyperbilirubinaemia for more than a decade 51,52, toxicity appears not to be of major importance. The proposed approach appears promising and may help to generate stronger T cell responses and thus improve the cell isolation efficiency of antigen-specific T cell therapies, especially when expanding these cells from low precursor frequencies. Moreover, the treatment is easily combined with the existing protocol, as SnMP treatment would be performed during the 4-h incubation time before the enrichment for IFN-γ cells.

In first experiments we have now investigated whether SnMP treatment is suitable for enrichment of further clinical relevant antigens such as tumor-associated antigens [e.g. melan-A/melanoma antigen recognized by T cells 1 (MART-1), Wilms' tumour (WT1)] (Supporting information, Fig. S4a). Melanoma antigens (Melan-A/MART-1) belong to the differentiation antigens and were identified on melanoma cells. The WT1 is found in a variety of haematological diseases and solid tumours and is involved in tumour proliferation. Both antigens represent a promising target for immunotherapy. Preliminary results from experiments in which SnMP treatment was applied in combination with Melan-A/MART-1 or WT1 for the enrichment of IFN-γ-positive specific T cells showed that SnMP increases the number of IFN-γ-secreting cells (Supporting information, Fig. S4a). The properties of T cells after enrichment are not affected (Supporting information, Fig. S4b). These results are promising, and indicate that this technology might also be suitable for the enrichment of sufficient numbers of tumour-specific T cells without need of further expansion.

In summary, this study demonstrates for the first time that modulation of the enzymatic activity of HO-1 can induce antigen-specific T cell activation. SnMP treatment results in the generation of functionally active T cells suitable for adoptive T cell therapy. These results underline the important role of HO-1 modulation in the adoptive immune response.

Acknowledgments

The authors would like to thank Sarina Lukis and Marina Kramer for their excellent technical assistance. This work is supported in part by funding from the Integrated Research and Treatment Center Transplantation (IFB-Tx) funded by the German Federal Ministry of Education and Research (reference number: 01EO0802), the Hannover Biomedical Research School (HBRS) and the PhD program ‘Molecular Medicine’.

Disclosures

The authors declare no conflicts of interest.

Author contributions

C. E. B. designed data analysis procedures, performed data acquisition and statistical analysis and co-wrote the manuscript. V. F. helped to design the study and carried out the T cell stimulation assays and immunoassays and co-wrote the manuscript. S. T. supported multimer staining in donor material. E. Z. provided support in the conduct of titration assays. C. F. provided support in assays to evaluate the effect of NK cells. T. W. critically discussed the data. R. B., Head of Hannover Medical School's Institute for Transfusion Medicine, contributed helpful discussions and helped to draft the manuscript. S. I. provided support in the assay design, critically discussed the data and co-wrote the manuscript. B. E.-V. conceived the study, participated in its design and coordination, designed the T cell stimulation assays, immunoassays and data analysis procedures and co-wrote the manuscript.

Supporting Information

Fig. S1. Metalloporphyrin treatment induces heme oxygenase-1 (HO-1) expression in peripheral blood mononuclear cells (PBMCs).

Fig. S2. Metalloporphyrin treatment does not affect maturation of dendritic cells.

Fig. S3. Tin-mesoporphyrin (SnMP) treatment significantly increases the proportion of natural killer (NK)p46+ but not CD14+ and CD19+ cells.

Fig. S4. Tin-mesoporphyrin (SnMP) treatment increases the number of interferon (IFN)-γ+ cells after Melan-A/melanoma antigen recognized by T cells 1 (MART-1PP) and Wilms' tumour protein (WT1PP) stimulation, but does not affect the phenotype.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Metalloporphyrin treatment induces heme oxygenase-1 (HO-1) expression in peripheral blood mononuclear cells (PBMCs).

Fig. S2. Metalloporphyrin treatment does not affect maturation of dendritic cells.

Fig. S3. Tin-mesoporphyrin (SnMP) treatment significantly increases the proportion of natural killer (NK)p46+ but not CD14+ and CD19+ cells.

Fig. S4. Tin-mesoporphyrin (SnMP) treatment increases the number of interferon (IFN)-γ+ cells after Melan-A/melanoma antigen recognized by T cells 1 (MART-1PP) and Wilms' tumour protein (WT1PP) stimulation, but does not affect the phenotype.

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