Regulatory T cells (Tregs): A major immune checkpoint to consider in combinatorial therapeutic HIV-1 vaccines

ABSTRACT

The field of immunotherapeutics is living an exceptional time as new antibodies that take brakes off T-cells and unleash them on tumours are being approved by the US-Food and Drug Administration (FDA). For the design and development of an HIV-1 therapeutic-vaccine, one would need preferably to shift the balance T-effectors/T-regulatory cells (Teff/Tregs) towards effectors to improve vaccine-specific immune-responses. Given the success with the new immune-checkpoint-blockers (ICB), it is an appropriate time for HIV-1 field to seize this opportunity and develop new therapeutic vaccine-strategies that take into consideration ICB and other immunomodulators such as cytokines. While the vaccine is important to stimulate HIV-1-specific T-cell responses, cytokines will support the expansion of the stimulated virus-specific T-cells and ICB will reverse exhaustion and unchain cytotoxic T-cells. In this commentary, we will spotlight Tregs as another major brake for T-cell immunity and address the main stumbling-blocks that often blurs HIV-1-specific-Tregs status with regards to their role (beneficial or detrimental) and we will recall some proof-of-concept studies where therapeutic immunization skewed the HIV-1-specific response from Tregs to Teffs which impacts on the magnitude of viral replication. We will also suggest some strategies to shift the balance towards Teffs and potentiate HIV-1-specific immune-responses.

KEYWORDS:

• Treg modulation
• Immune checkpoint blockers, cytokines
• combinatorial strategies
• HIV-1-therapeutic vaccine
• OX40
• CD39

Introduction

The main goal of therapeutic vaccines is to strengthen and help the immune system to fight an existing disease. Different strategies of immunotherapy have been described ranging from peptides therapeutic vaccine to targeted-cell therapies.

In HIV-1 infection, different therapeutic vaccine strategies have been developed and trialled, but none of these succeeded in eradicating the virus despite induction of strong specific immune responses (reviewed in¹).

CD4⁺ T-cell subsets including Teffs and Tregs play key roles during infections, and monitoring the development of such immune responses after vaccination is crucial. The role of Tregs has been extensively studied in cancer and HIV-1 vaccine trials.^2-4 In HIV-1 infection, these cells have been shown to be both beneficial and deleterious, creating thus some confusion in the field. This is due in part to the variable settings used when Tregs have been looked at and focused on at the time of the study. These include the stage of infection (acute versus chronic), the sampling (blood, lymph node or gut tissue) and/or the phenotypic markers used to identify and quantify Tregs (reviewed in⁵). Moreover, an additional important factor that has not been substantially discussed so far and may add a layer of confusion in data interpretation, is related to the nature of Tregs, i.e. “bulk” or antigen-specific subset. In this context, recent advances have been made in HIV-1 infected patients, where both subsets (bulk and antigen-specific) have been simultaneously measured and compared in the same patients.⁶ These aspects will be highlighted below.

For the design and development of an HIV-1 therapeutic vaccine, one would need preferably to shift the balance of Teff/Tregs toward effector cells in order to improve vaccine-specific immune responses. Given that we are living an exciting time where new therapeutic strategies based on ICB that target negative molecules such as protein-death 1 (PD-1, CD279) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4, CD152),⁷ it is time to acknowledge that Tregs are a full-fledged additional immune checkpoint that needs to be considered for depletion and/or modulation in combination with an anti-PD-1 antibody for example, in order to strengthen specific immune responses (particularly CD8 T cell response). In this commentary, we will highlight the main stumbling-blocks that often blurs HIV-1-specific-Tregs status in the field with regards to their role during anti-HIV responses and we will recall some proof-of concept studies where therapeutic immunization skewed the HIV-1-specific immune response from Tregs to Teffs which impacts on the magnitude of viral replication following treatment interruption. We will also suggest some strategies to shift the balance towards Teffs and potentiate HIV-1-specific immune responses (Figure 1).

Figure 1. To potentiate antigen-specific immune responses in the context of HIV-1-therapeutic vaccine. T cell breath and magnitude will be improved by using peptides-loaded-DC (1). Immune response is regulated by inhibitory and co-stimulatory signals mediated by immune-checkpoint. These signals can be modulated using IC blockers (ICB) in order to amplify HIV-1-specific responses and to prevent cell exhaustion (2). T cell proliferation can be boosted using cytokines (IL-2, IL-7 or IL-15) in combination with therapeutic vaccines in order to promote antigen-specific T cell survival and expansion (3). It is important to block Tregs expansion and shift the balance toward effectors (4). To this end, cytokines, i.e IL-7 will modulate Tregs phenotype and function towards and potentiates Teffs differentiation (4a). Vaccines could be combined with other molecules that will suppress Treg-activity (c-Rel inhibitor, anti-CD39) (4b). Antibodies, i.e anti-CD25 (Daclizumab), Fc-optimized anti-CD25, anti-CD127 or anti-CD39 may be used to efficiently deplete Tregs (4c).

Monitoring vaccine impact: How to detect the full range of Th responses

Monitoring vaccine responses is crucial for predicting its impact on the immune system and improving therapeutic strategies. These responses are usually evaluated by assessing T-cell phenotype and proliferation as well as T-cell cytotoxicity and cytokine release following stimulation with peptides/proteins.⁸ However, these commonly used assays do not include Tregs responses. Indeed, the lack of tools that one can easily use in clinical trials setting has prevented the standartized measurement of Tregs specific responses. Therefore, Tregs frequency and/or function have often been studied on bulk CD4⁺CD25^hi CD127^lowFoxP3⁺ Tregs (reviewed in^5,9 and less often on antigen-specific Tregs. Angin et al., reported the presence of gag-specific Tregs in infected individuals by using MHC Class II tetramer loaded with gag peptides.⁹ This approach is interesting but very challenging when used in clinical trials due to the genetic variability of MHC Class II as well as the limited availability of Class II tetramers. Tregs could also have different affinity with MHC comparing to Teffs, which could lead to differential staining and probable under- or over- estimation of their frequencies. Therefore, more practical tools have been developped, and one of them is the “OX40 assay”¹⁰ that simultaneously detects a full range of Th responses including antigen-specific Tregs¹¹ and T follicular helper (Tfh) responses (Seddiki et al., unpublished). Moreover, it could be combined with other methods i.e, intracellular staining (ICS) or genomic and proteomic analyses. Importantly, this assay has served as a platform for the development of other assays such as the activation-induced marker (AIM) which is efficient at detecting antigen-specific CD4 T cells.^12,13 A newer version of the AIM assay based on the use of OX40/PD-L1 and OX40/4-1BB in human and rhesus macaque samples respectively, has been reported.¹⁴ However, these assays, contrary to the « OX40 assay», do not allow antigen-specific Tregs response detection, which are part of the immune response and should not be neglected.

Taking advantage of the reliability of the « OX40 assay », we demonstrated that HIV-1-infected individuals harbor high levels of HIV-1-specific Tregs at baseline.¹⁵ After vaccination with autologous dendritic cells (DC) loaded with HIV-1-derived peptides, patients who exhibited lower viral load after treatment interruption, displayed lower levels of HIV-1-specific CD4⁺CD134⁺CD25⁺CD39⁺FoxP3⁺ Tregs by shifting the HIV-1-specific Tregs:Teffs ratio towards polyfunctional effector responses.¹⁵ Of note, there was no difference in bulk CD4⁺CD25⁺CD127^lowFoxp3⁺Tregs levels before and after vaccination.

In a more recent work, we showed that therapeutic vaccination, of HIV-1-infected patients under therapy (cART), using ALVAC-HIV and Lipo-6T followed by 3 cycles of subcutaneous interleukin-2 (IL-2), decreased CD4⁺CD25⁺OX40⁺CD39⁺Foxp3⁺ HIV-1-specific Tregs thus shifting the balance towards effectors, while peripheral bulk CD4⁺CD25^hiCD127^lowFoxP3⁺ increased significantly.⁶ Moreover, we have been able to demonstrate that increased bulk Tregs frequency impacts on T-cell exhaustion while decreased HIV-specific Tregs impact on the balance towards effectors. This demonstrates the need for assessing different subsets of Tregs as each population is provided with its specific phenotype and/or function, playing thus different roles in HIV-1 infection.

How could we potentiate HIV-1-specific responses with therapeutic vaccines?

An immune response is initiated following recognition of antigen by T cell receptor (TCR) and is regulated by inhibitory and co-stimulatory signals mediated by immune checkpoint (IC) molecules. In physiological conditions, IC protect from autoimmunity and immune system over-reaction following pathogenic infection. PD-1 and CTLA-4 constitute the most targeted molecules in immunotherapy as they have proven to be good candidates in cancer (reviewed in^7,16). In cART HIV-1 individuals, the first clinical trial with anti-PDL1 antibody has been completed and only two patients showed increased percentage of Gag-specific CD8⁺ T cells at day 28 after infusion.¹⁷ In SIV (simian immunodeficiency virus) infection, anti-PD-1 infusion resulted in significant enhancement of specific-immune responses and decrease in plasma RNA level.¹⁸ However, none of these studies has examined the impact of IC inhibitors on Tregs subsets dynamics. A recent study analyzed the effect of pembrolizumab (anti-PD1) on different populations of Tregs. Authors showed that PD-1 was mainly expressed on CD4⁺CD25⁺ cells of healthy donors and patients with primary breast cancer. However, pembrolizumab did not affect the frequency of FoxP3⁺ Tregs subsets but rather blocked signalling cascades via the PD-1/PD-L1 axis in activated T cells, suggesting that pembrolizumab may reverse immune escape through blockage of PD-1/PD-L1 interaction and not by altering Tregs phenotype or function.¹⁹

Given that therapeutic vaccines and cytokines have been commonly used to enhance HIV-1-specific cell-mediated immune responses and to suppress virus replication, combined immunotherapy including ICB, cytokines and vaccines represents potent combinatorial interventions in HIV-1-infected patients. While the vaccine is important to stimulate HIV-specific T-cell responses, cytokines and ICB may support the expansion of the stimulated virus-specific T cells.

The common γ-chain cytokines IL-2, IL-7 and IL-15 are crucial for T-lymphocyte development and survival, therefore their use in immunotherapy was proposed to improve quantitative and qualitative CD4⁺ T cell restoration.

Studies using a mouse model for LCMV indicated that combined IL-2 and PD-1 blockade therapy represents a promising strategy for increasing CD8 T cell function and reducing viral load during chronic infection despite an increase in Tregs.²⁰ In cancer, AIPV combination, i.e. tumor-antigen specific antibody (A), IL-2 (I), anti-PD1 (P) and amphiphile-vaccine (V, specific tumor peptides or CpG), induced tumor regression, tumor infiltration of CD8⁺ T cells and cross-presentation promotion in mice treated with subcutaneous syngeneic tumor models (melanoma, mouse breast cancer and tumors expressing the human papilloma virus (HPV) onco-antigens E6 and E7).²¹ In HIV-1 infection, recombinant IL-2 administration to patients on cART, showed sustained increase in CD4 T cell count²² but no clinical benefits were noted.²³ These results were explained by the expansion of CD4⁺CD25⁺FoxP3⁺ Tregs.²⁴ However, unlike IL-2 therapy alone, the use of IL-2 as an adjuvant to vaccines highlighted more positive effects by decreasing HIV-1-specific CD4⁺ CD39⁺FOXP3⁺CD25⁺CD134⁺ Tregs as mentioned above,⁶ emphasizing thus the promising character of this cytokine in combinatorial therapeutic strategies for HIV-1⁺ patients.

Il-7 therapy was seen as an alternative strategy to IL-2 therapy. Clinical trials performed in HIV-1 infected patients under cART revealed that IL-7 administration induced immunological improvement, particularly on T cells^25-27 with a relative decrease of Tregs.^28,29 Indeed, IL-7 treatment on Tregs led to reduced expression of CD39 and reduced suppressive activity with a shift toward Th17 phenotype.³⁰ However, IL-7 therapy failed to reduce viral reservoir^31,32 probably due to central memory CD4 T cell expansion.³³

IL-15 has emerged as another highly-promising candidates in immunotherapy. This cytokine shares many of the biological properties of IL-2.³⁴ Interestingly, IL-15Rα/IL-15 complex emerged as a potent adjuvant for dendritic cell immunotherapy or combinatorial therapy for the developpement of sustained memory-CD8 T cell responses.^35,36 In HIV-1-infected humanized mice, IL-15 superagonist (ALT-803) treatment inhibited HIV-1 infection through NK cell activation which highlights the pluricellular effect of IL-15.^37,38 ALT-803 molecule is moving into a clinical trial in cART HIV-1 infected subjects (ClinicalTrials.gov identifier: NCT02191098). It is noteworthy to mention that combined therapy with anti-PD-1, anti-PD-L1 or anti-CTLA-4, greatly enhanced the efficacy of ALT-803, in remission of primary and advanced tumors in different preclinical studies.^39,40

Treg depletion or modulation: An additional therapeutic strategy to potentiate vaccines

Meta-analysis work reviewing various cancer research studies, found a negative prognosis associated with tumor-infiltrating FoxP3⁺ Tregs.⁴¹

In HIV-1 and SIV infections, Tregs were associated with lower specific immune responses^42-44 and contributed to HIV-1 reservoir persistence.⁴⁵ Moreover, cART-patients who received a DC-based vaccine showed a better anti-HIV response that was correlated with lower frequency of HIV-specific CD4⁺CD134⁺CD25⁺CD39⁺FoxP3⁺ Tregs.¹⁵ Indeed, Tregs negatively impact anti-HIV-1 specific responses, it is thus necessary to pay attention to them and they should not be neglected.

Depletion of Tregs using anti-CD25 has been used therapeutically in combination with vaccines in cancer studies.⁴⁶ In a promising study, patients with breast cancer receiving Daclizumab (anti-CD25 mAb) followed by peptide-vaccination, displayed a prolonged depletion of CD25^highCD45RA⁻ Tregs with an increase in Teffs responses .⁴⁷ Moreover, in a recent study, Treg depletion was shown to synergize with PD-1 blockade to eradicate tumor-infiltrating Tregs.⁴⁸ Indeed, the use of an anti-CD25 antibody with enhanced binding to activating Fcγ-Receptors led to effective depletion of tumor-infiltrating Tregs and improved control of established tumors. More importantly, combination of anti-CD25 with anti-PD-1 promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches for immunotherapy.⁴⁸ In another recent study it has been shown that melanoma cell growth is drastically reduced in mice lacking NF-κB c-Rel subunit, but not p65, in Tregs. Interestingly, chemical inhibition of c-Rel function delayed melanoma growth by impairing Tregs-mediated immunosuppression and potentiated the effects of anti-PD-1 immunotherapy. These results therefore establish inhibition of NF-κB c-Rel as a viable therapeutic approach for enhancing checkpoint-targeting immunotherapy protocols.⁴⁹ Altogether, these studies open the field to novel molecules and possibilities of combinatorial strategies, i.e simultaneous Tregs inhibition and ICB use. Other strategies of Treg-depletion were developed via the targeting of CTLA-4,^50,51 CD27,⁵² ICOS⁵³ or OX40 (CD134).⁵⁴ These major advances are very attractive and provide the scientific basis for developing potent anti-HIV-1 combinatorial therapeutics to enhance immune responses and gain more efficient control of virus replication.

Conclusion

Despite major advances in the field of HIV-1 therapeutic vaccination, it became apparent that HIV cure will need to combine cytokines, i.e IL-2, IL-7 or IL-15, which are effective at increasing expansion, differenciation and maintenance of virus-specific memory T cells, in addition to ICB that have proven to be effective in cancer immunotherapy. Furthermore, the recent concepts of Tregs depletion based on NF-kB c-Rel inhibition⁴⁹ or the use of anti-CD25 antibody with enhanced binding to activating Fcγ-Receptors leading to effective Tregs depletion,⁴⁸ offer excellent opportunities to block Tregs which represent a major immune checkpoint that should not be neglected in future clinical studies.

Disclosure of potential conflict of interest

There are no conflicts of interests.

Additional information

Funding

This work was supported by the Investissement d'Avenir program managed by the ANR under reference ANR-10-LABX-77, the Agence Nationale pour la Recherche sur le SIDA et les hepatites virales (ANRS), and the Vaccine Research Institute (VRI).