Diversity and Recognition Efficiency of T Cell Responses to Cancer

BACKGROUND: Melanoma patients vaccinated with tumor-associated antigens frequently develop measurable peptide-specific CD8+ T cell responses; however, such responses often do not confer clinical benefit. Understanding why vaccine-elicited responses are beneficial in some patients but not in others will be important to improve targeted cancer immunotherapies. METHODS AND FINDINGS: We analyzed peptide-specific CD8+ T cell responses in detail, by generating and characterizing over 200 cytotoxic T lymphocyte clones derived from T cell responses to heteroclitic peptide vaccination, and compared these responses to endogenous anti-tumor T cell responses elicited naturally (a heteroclitic peptide is a modification of a native peptide sequence involving substitution of an amino acid at an anchor residue to enhance the immunogenicity of the peptide). We found that vaccine-elicited T cells are diverse in T cell receptor variable chain beta expression and exhibit a different recognition profile for heteroclitic versus native peptide. In particular, vaccine-elicited T cells respond to native peptide with predominantly low recognition efficiency—a measure of the sensitivity of a T cell to different cognate peptide concentrations for stimulation—and, as a result, are inefficient in tumor lysis. In contrast, endogenous tumor-associated-antigen-specific T cells show a predominantly high recognition efficiency for native peptide and efficiently lyse tumor targets. CONCLUSIONS: These results suggest that factors that shape the peptide-specific T cell repertoire after vaccination may be different from those that affect the endogenous response. Furthermore, our findings suggest that current heteroclitic peptide vaccination protocols drive expansion of peptide-specific T cells with a diverse range of recognition efficiencies, a significant proportion of which are unable to respond to melanoma cells. Therefore, it is critical that the recognition efficiency of vaccine-elicited T cells be measured, with the goal of advancing those modalities that elicit T cells with the greatest potential of tumor reactivity

Novel Concept of CD4-Mediated Activation of Regulatory T Cells for the Treatment of Graft-Versus-Host Disease

Allogeneic hematopoietic stem cell transplantation is the only curative treatment option for several hematological malignancies and immune deficiency syndromes. Nevertheless, the development of a graft-versus-host disease (GvHD) after transplantation is a high risk and a severe complication with high morbidity and mortality causing therapeutic challenges. Current pharmacological therapies of GvHD lead to generalized immunosuppression followed by severe adverse side effects including infections and relapse of leukemia. Several novel cell-based immunomodulatory strategies for treatment or prevention of GvHD have been developed. Herein, thymus-derived regulatory T cells (tTreg), essential for the maintenance of peripheral immunologic tolerance, are in the focus of investigation. However, due to the limited number of tTreg in the peripheral blood, a complex, time- and cost-intensive in vitro expansion protocol is necessary for the production of an efficient cellular therapeutic. We demonstrated that activation of tTreg using the CD4-binding human immunodeficiency virus-1 protein gp120 leads to a substantially increased suppressor activity of tTreg without the need for additional expansion. Gp120-activated tTreg prevent GvHD development in a preclinical humanized mouse model. In addition, gp120 is not only effective in prevention but also in therapy of GvHD by suppressing all clinical symptoms and improving survival of treated mice. These data indicate that tTreg activation by gp120 is a feasible and potent strategy for significant functional improvement of tTreg as cellular therapeutic for GvHD treatment without the need of complicated, time-intensive, and expensive in vitro expansion of isolated tTreg

How Soluble GARP Enhances TGFβ Activation.

GARP (glycoprotein A repetitions predominant) is a cell surface receptor on regulatory T-lymphocytes, platelets, hepatic stellate cells and certain cancer cells. Its described function is the binding and accommodation of latent TGFβ (transforming growth factor), before the activation and release of the mature cytokine. For regulatory T cells it was shown that a knockdown of GARP or a treatment with blocking antibodies dramatically decreases their immune suppressive capacity. This confirms a fundamental role of GARP in the basic function of regulatory T cells. Prerequisites postulated for physiological GARP function include membrane anchorage of GARP, disulfide bridges between the propeptide of TGFβ and GARP and connection of this propeptide to αvβ6 or αvβ8 integrins of target cells during mechanical TGFβ release. Other studies indicate the existence of soluble GARP complexes and a functionality of soluble GARP alone. In order to clarify the underlying molecular mechanism, we expressed and purified recombinant TGFβ and a soluble variant of GARP. Surprisingly, soluble GARP and TGFβ formed stable non-covalent complexes in addition to disulfide-coupled complexes, depending on the redox conditions of the microenvironment. We also show that soluble GARP alone and the two variants of complexes mediate different levels of TGFβ activity. TGFβ activation is enhanced by the non-covalent GARP-TGFβ complex already at low (nanomolar) concentrations, at which GARP alone does not show any effect. This supports the idea of soluble GARP acting as immune modulator in vivo

GARP Regulates the Immune Capacity of a Human Autologous Platelet Concentrate

Autologous platelet concentrates, like liquid platelet rich fibrin (iPRF), optimize wound healing; however, the underlying immunological mechanisms are poorly understood. Platelets, the main cellular component of iPRF, highly express the protein, Glycoprotein A repetitions predominant (GARP), on their surfaces. GARP plays a crucial role in maintaining peripheral tolerance, but its influence on the immune capacity of iPRF remains unclear. This study analyzed the interaction of iPRF with immune cells implicated in the wound healing process (human monocyte derived macrophages and CD4+ T cells) and evaluated the distinct influence of GARP on these mechanisms in vitro. GARP was determined to be expressed on the surface of platelets and to exist as a soluble factor in iPRF. Platelets derived from iPRF and iPRF itself induced a regulatory phenotype in CD4+ T cells, shown by increased expression of Foxp3 and GARP as well as decreased production of IL-2 and IFN-γ. Application of an anti-GARP antibody reversed these effects. Additionally, iPRF polarized macrophages to a “M0/M2-like” phenotype in a GARP independent manner. Altogether, this study demonstrated for the first time that the immune capacity of iPRF is mediated in part by GARP and its ability to induce regulatory CD4+ T cells

Combined treatment of hidradenitis suppurativa with intense pulsed light (IPL) and radiofrequency (RF)

Background: Hidradenitis suppurativa is a chronic inflammatory disease with high burden. Treatment options are often unsatisfactory. We assessed the effect of a combination therapy of intense pulsed light (IPL) and radiofrequency (RF). Methods: The explorative study included 47 patients and was performed as a prospective, monocentric, randomized, three-arm parallel-group design trial with a prior 12?weeks observation period. Treatment arms were IPL and RF monotherapies or IPL?+?RF combination therapy. After 12?weeks, all patients received IPL?+?RF for additional 12?weeks (cross-over). Primary endpoint was the change in active lesion numbers, secondary endpoint the change in Dermatology Quality of Life Index (DLQI). Results: After 12?weeks, active lesion counts of the IPL?+?RF group decreased more than in the IPL group (p?=?.044); the decrease in DLQI was significantly higher in the IPL?+?RF and RF groups compared to IPL. Prolonged 24-week treatment with IPL?+?RF obtained better results as 12?weeks. Overall, disease burden after 24?weeks of treatment compared to disease fluctuation during the observation period was significantly lower (change in active lesions ?3.6, p?=?.001; in DLQI ?5.2, p?=?.003). Conclusions: IPL?+?RF treatment appears to represent a promising therapeutic option that leads to reduction of disease activity without severe side effects

Platelet-Derived GARP Induces Peripheral Regulatory T Cells—Potential Impact on T Cell Suppression in Patients with Melanoma-Associated Thrombocytosis

Platelets have been recently described as an important component of the innate and adaptive immunity through their interaction with immune cells. However, information on the platelet–T cell interaction in immune-mediated diseases remains limited. Glycoprotein A repetitions predominant (GARP) expressed on platelets and on activated regulatory T cells (Treg) is involved in the regulation of peripheral immune responses by modulating the bioavailability of transforming growth factor β (TGF-β). Soluble GARP (sGARP) exhibits strong regulatory and anti-inflammatory capacities both in vitro and in vivo, leading to the induction of peripheral Treg. Herein, we investigated the effect of platelet-derived GARP on the differentiation, phenotype, and function of T effector cells. CD4+CD25− T cells cocultured with platelets upregulated FoxP3, the master transcription factor for Treg, were anergic, and were strongly suppressive. These effects were reversed by using a blocking anti-GARP antibody, indicating a dependency on GARP. Importantly, melanoma patients in different stages of disease showed a significant upregulation of GARP on the platelet surface, correlating to a reduced responsiveness to immunotherapy. In conclusion, our data indicate that platelets induce peripheral Treg via GARP. These findings might contribute to diseases such as cancer-associated thrombocytosis, wherein poor prognosis and metastasis are associated with high counts of circulating platelets

Immune signature as predictive marker for response to checkpoint inhibitor immunotherapy and overall survival in melanoma

Abstract Background Malignant melanoma is an immunogenic skin cancer with an increasing global incidence. Advanced stages of melanoma have poor prognoses. Currently, there are no reliable parameters to predict a patient's response to immune checkpoint inhibitor (ICI) therapy. Methods This study highlights the relevance of a distinct immune signature in the blood for response to ICI therapy and overall survival (OS). Therefore, the immune cell composition in the peripheral blood of 45 melanoma patients prior to ICI therapy was analyzed by flow cytometry and complete blood count. Results Responders to ICI therapy displayed an abundance of proliferating CD4+ T cells, an increased lymphocyte‐to‐monocyte ratio, a low platelet‐to‐lymphocyte ratio, low levels of CTLA‐4+ Treg, and (arginase 1+) polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSC). Nevertheless, non‐responders with similar immune cell compositions also benefited from therapy displaying increased long‐term OS. Conclusions Our study demonstrated that the observed immune signature in the peripheral blood of melanoma patients prior to treatment could identify responders as well as non‐responders that benefit from ICI immunotherapies

Oxidative Stress Differentially Influences the Survival and Metabolism of Cells in the Melanoma Microenvironment

The cellular composition of the tumor microenvironment, including tumor, immune, stromal, and endothelial cells, significantly influences responses to cancer therapies. In this study, we analyzed the impact of oxidative stress, induced by cold atmospheric plasma (CAP), on tumor cells, T cells, and macrophages, which comprise part of the melanoma microenvironment. To accomplish this, cells were grown in different in vitro cell culture models and were treated with varying amounts of CAP. Subsequent alterations in viability, proliferation, and phenotype were analyzed via flow cytometry and metabolic alterations by Seahorse Cell Mito Stress Tests. It was found that cells generally exhibited reduced viability and proliferation, stemming from CAP induced G2/M cell cycle arrest and subsequent apoptosis, as well as increased mitochondrial stress following CAP treatment. Overall, sensitivity to CAP treatment was found to be cell type dependent with T cells being the most affected. Interestingly, CAP influenced the polarization of M0 macrophages to a “M0/M2-like” phenotype, and M1 macrophages were found to display a heightened sensitivity to CAP induced mitochondrial stress. CAP also inhibited the growth and killed melanoma cells in 2D and 3D in vitro cell culture models in a dose-dependent manner. Improving our understanding of oxidative stress, mechanisms to manipulate it, and its implications for the tumor microenvironment may help in the discovery of new therapeutic targets

Kinome Profiling of Regulatory T Cells: A Closer Look into a Complex Intracellular Network.

Regulatory T cells (Treg) are essential for T cell homeostasis and maintenance of peripheral tolerance. They prevent activation of auto-reactive T effector cells (Teff) in the context of autoimmunity and allergy. Otherwise, Treg also inhibit effective immune responses against tumors. Besides a number of Treg-associated molecules such as Foxp3, CTLA-4 or GARP, known to play critical roles in Treg differentiation, activation and function, the involvement of additional regulatory elements is suggested. Herein, kinase activities seem to play an important role in Treg fine tuning. Nevertheless, our knowledge regarding the complex intracellular signaling pathways controlling phenotype and function of Treg is still limited and based on single kinase cascades so far. To gain a more comprehensive insight into the pathways determining Treg function we performed kinome profiling using a phosphorylation-based kinome array in human Treg at different activation stages compared to Teff. Here we have determined intriguing quantitative differences in both populations. Resting and activated Treg showed an altered pattern of CD28-dependent kinases as well as of those involved in cell cycle progression. Additionally, significant up-regulation of distinct kinases such as EGFR or CK2 in activated Treg but not in Teff not only resemble data we obtained in previous studies in the murine system but also suggest that those specific molecular activation patterns can be used for definition of the activation and functional state of human Treg. Taken together, detailed investigation of kinome profiles opens the possibility to identify novel molecular mechanisms for a better understanding of Treg biology but also for development of effective immunotherapies against unwanted T cell responses in allergy, autoimmunity and cancer