Targeting of TLRs inhibits CD4+ regulatory T cell function and activates lymphocytes in human peripheral blood mononuclear cells.

Accumulating evidence suggests elements within tumors induce exhaustion of effector T cells and infiltration of immunosuppressive regulatory T cells (Tregs), thus preventing the development of durable antitumor immunity. Therefore, the discovery of agents that simultaneously block Treg suppressive function and reinvigorate effector function of lymphocytes is key to the development of effective cancer immunotherapy. Previous studies have shown that TLR ligands (TLRLs) could modulate the function of these T cell targets; however, those studies relied on cell-free or accessory cell-based assay systems that do not accurately reflect in vivo responses. In contrast, we used a human PBMC-based proliferation assay system to simultaneously monitor the effect of TLRLs on T cells (CD4(+), CD8(+), Tregs), B cells, and NK cells, which gave different and even conflicting results. We found that the TLR7/8L:CL097 could simultaneously activate CD8(+) T cells, B cells, and NK cells plus block Treg suppression of T cells and B cells. The TLRLs TLR1/2L:Pam3CSK4, TLR5L:flagellin, TLR4L:LPS, and TLR8/7L:CL075 also blocked Treg suppression of CD4(+) or CD8(+) T cell proliferation, but not B cell proliferation. Besides CL097, TLR2L:PGN, CL075, and TLR9L:CpG-A, CpG-B, and CpG-C) were strong activators of NK cells. Importantly, we found that Pam3CSK4 could: 1) activate CD4(+) T cell proliferation, 2) inhibit the expansion of IL-10(+) naturally occurring FOXP3(+) Tregs and induction of IL-10(+) CD4(+) Tregs (IL-10-producing type 1 Treg), and 3) block naturally occurring FOXP3(+) Tregs suppressive function. Our results suggest these agents could serve as adjuvants to enhance the efficacy of current immunotherapeutic strategies in cancer patients.

Selective targeting of Toll-like receptors and OX40 inhibit regulatory T-cell function in follicular lymphoma.

Immunotherapeutic strategies are promising approaches for the treatment of follicular lymphoma (FL). However, their efficacy may be limited by immunosuppressive elements in the immune system and tumor microenvironment. Therefore, strategies to reverse the effects of the immunosuppressive elements are needed. We observed that regulatory T cells (Tregs) were increased in the peripheral blood at diagnosis and persisted in high numbers after induction of clinical remission with a cyclophosphamide and doxorubicin-containing chemotherapy regimen in FL patients. High levels of peripheral blood Tregs prior to therapy were associated with decreased progression-free survival in FL patients treated with either chemotherapy or combination immunotherapy that targeted CD20 and PD-1 with monoclonal antibodies rituximab and pidilizumab, respectively. Intratumoral and peripheral blood Tregs potently suppressed autologous antitumor effector T cells in FL. However, the effects of FL Tregs could be reversed by triggering Toll-like receptors (TLR) with TLR ligands Pam3 CSK4 (TLR 1/2), flagellin (TLR 5), and CpG-B (TLR 9), and/or OX40. The TLR ligands synergized with each other as well as OX40 signaling to inhibit Tregs. Furthermore, they restored the function of FL tumor-specific effector T cells. Our results suggest that a state of tolerance exists in FL patients at diagnosis and after induction of clinical remission, and agents that activate TLRs 1/2, 5, and 9, and OX40 may serve as adjuvants to enhance the efficacy of antitumor immunotherapeutic strategies and preventive vaccines against infectious diseases in these patients.

IL-25 augments type 2 immune responses by enhancing the expansion and functions of TSLP-DC-activated Th2 memory cells.

Interleukin (IL) 25 (IL-17E), a distinct member of the IL-17 cytokine family, plays important roles in evoking T helper type 2 (Th2) cell-mediated inflammation that features the infiltrations of eosinophils and Th2 memory cells. However, the cellular sources, target cells, and underlying mechanisms remain elusive in humans. We demonstrate that human Th2 memory cells expressing distinctive levels of IL-25 receptor (R) are one of the responding cell types. IL-25 promotes cell expansion and Th2 cytokine production when Th2 central memory cells are stimulated with thymic stromal lymphopoietin (TSLP)-activated dendritic cells (DCs), homeostatic cytokines, or T cell receptor for antigen triggering. The enhanced functions of Th2 memory cells induced by IL-25 are associated with sustained expression of GATA-3, c-MAF, and JunB in an IL-4-independent manner. Although keratinocytes, mast cells, eosinophils, and basophils express IL-25 transcripts, activated eosinophils and basophils from normal and atopic subjects were found to secrete bioactive IL-25 protein, which augments the functions of Th2 memory cells. Elevated expression of IL-25 and IL-25R transcripts was observed in asthmatic lung tissues and atopic dermatitis skin lesions, linking their possible roles with exacerbated allergic disorders. Our results provide a plausible explanation that IL-25 produced by innate effector eosinophils and basophils may augment the allergic inflammation by enhancing the maintenance and functions of adaptive Th2 memory cells.

HDAC11 plays an essential role in regulating OX40 ligand expression in Hodgkin lymphoma.

In Hodgkin lymphoma (HL), the malignant cells are surrounded by a large number of reactive infiltrating inflammatory cells, including OX40-expressing T cells and interleukin 10 (IL-10)-producing regulatory T (T-reg) cells. These T-reg cells can suppress the immune response and thus contribute to the maintenance of immune tolerance and to insufficient antitumor response. The engagement of OX40L with the OX40 receptor is essential for the generation of antigen-specific memory T cells and for the induction of host antitumor immunity. In the present study, we investigated whether histone deacetylase inhibitors (HDACis) may induce a favorable antitumor immune response by regulating the expression of OX40L in HL. We found that HDACis up-regulated OX40L surface expression in HL cell lines in a dose-dependent manner. Small interfering RNAs (siRNAs) that selectively inhibited HDAC11 expression, significantly up-regulated OX40L and induced apoptosis in HL cell lines, and silencing HDAC11 transcripts increased the production of tumor necrosis-α (TNF-α) and IL-17 in the supernatants of HL cells. Furthermore, HDACI-induced OX40L inhibited the generation of IL-10-producing type 1 T-reg cells. These results demonstrate for the first time that HDAC11 plays an essential role in regulating OX40L expression. Pharmacologic inhibition of HDAC11 may produce a favorable antitumor immune response in patients with HL.

Thymic stromal lymphopoietin-activated plasmacytoid dendritic cells induce the generation of FOXP3+ regulatory T cells in human thymus.

Human thymus contains major dendritic cell (DC) subsets, myeloid DCs (mDCs), and plasmacytoid DCs (pDCs). We previously showed that mDCs, educated by thymic stromal lymphopoietin (TSLP) produced by the epithelial cells of the Hassall's corpuscles, induced differentiation of CD4(+)CD25(-) thymocytes into Forkhead Box P3(+) (FOXP3(+)) regulatory T cells (T(R)) within the medulla of human thymus. In this study, we show that pDCs expressed the TSLP receptor and IL-7 receptor alpha complexes upon activation and became responsive to TSLP. TSLP-activated human pDCs secrete macrophage-derived chemokine CCL-22 and thymus- and activation-regulated chemokine CCL-17 but not Th1- or Th2-polarizing cytokines. TSLP-activated pDCs induced the generation of FOXP3(+) T(R) from CD4(+)CD8(-)CD25(-) thymocytes, which could be strongly inhibited by Th1-polarizing cytokine IL-12 or Th2-polarizing cytokine IL-4. Interestingly, the FOXP3(+) T(R) induced by the TSLP-pDCs expressed more IL-10 but less TGF-beta than that induced by the TSLP-mDCs. These data suggest that TSLP expressed by thymic epithelial cells can activate mDCs and pDCs to positively select the FOXP3(+) T(R) with different cytokine production potential in human thymus. The inability of TSLP to induce DC maturation without producing Th1- or Th2-polarizing cytokines may provide a thymic niche for T(R) development.

Identification of IL-17-producing FOXP3+ regulatory T cells in humans.

IL-17-producing CD4(+) T helper (Th17) cells have recently been defined as a unique subset of proinflammatory helper cells whose development depends on signaling initiated by IL-6 and TGF-beta, autocrine activity of IL-21, activation of STAT3, and induction of the orphan nuclear receptor RORgammat. The maintenance, expansion, and further differentiation of the committed Th17 cells depend on IL-1beta and IL-23. IL-17 was originally found produced by circulating human CD45RO(+) memory T cells. A recent study found that human Th17 memory cells selectively express high levels of CCR6. In this study, we report that human peripheral blood and lymphoid tissue contain a significant number of CD4(+)FOXP3(+) T cells that express CCR6 and have the capacity to produce IL-17 upon activation. These cells coexpress FOXP3 and RORgammat transcription factors. The CD4(+)FOXP3(+)CCR6(+) IL-17-producing cells strongly inhibit the proliferation of CD4(+) responder T cells. CD4(+)CD25(high)-derived T-cell clones express FOXP3, RORgammat, and IL-17 and maintain their suppressive function via a cell-cell contact mechanism. We further show that human CD4(+)FOXP3(+)CCR6(-) regulatory T (Treg) cells differentiate into IL-17 producer cells upon T-cell receptor stimulation in the presence of IL-1beta, IL-2, IL-21, IL-23, and human serum. This, together with the finding that human thymus does not contain IL-17-producing Treg cells, suggests that the IL-17(+)FOXP3(+) Treg cells are generated in the periphery. IL-17-producing Treg cells may play critical roles in antimicrobial defense, while controlling autoimmunity and inflammation.

Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes.

The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated tumors, making it an important target for immunotherapy. However, evidence for major histocompatibility complex (MHC) class I-restricted EBNA1 peptides endogenously presented by EBV-transformed B and tumor cells remains elusive. Here we describe for the first time the identification of an endogenously processed human histocompatibility leukocyte antigen (HLA)-B8-restricted EBNA1 peptide that is recognized by CD8+ T cells. T cell recognition could be inhibited by the treatment of target cells with proteasome inhibitors that block the MHC class I antigen processing pathway, but not by an inhibitor (chloroquine) of MHC class II antigen processing. We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes. Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes. These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

CD4+ T-cell response to mitochondrial cytochrome B in human melanoma.

Mitochondrial DNA (mtDNA) is highly susceptible to mutations due to the low level of DNA repair and the presence of a high level of reactive oxygen species in the organelle. Although mtDNA mutations have been implicated in degenerating diseases, aging, and cancer, very little is known about the role of T cells in immunosurveillance for mtDNA aberrations. Here, we describe T-cell recognition of a peptide translated from an alternative open reading frame of the mitochondrial cytochrome b (cyt b) gene in melanoma cells established from a patient. To understand how the cyt b gene is transcribed and translated in tumor cells, we found that cyt b-specific CD4(+) T cells only recognized protein fractions derived from cytoplasm and not from mitochondria. However, T-cell recognition of tumor cells could be inhibited by treatment of tumor cells with rhodamine 6G inhibitor, which depletes mitochondria. These findings suggest that cyt b mRNA is leaked out of the mitochondria and then translated in the cytoplasm for presentation to CD4(+) T cells. The cyt b cDNAs from this patient contain highly heteroplasmic transition mutations compared with control cell lines, suggesting a compromise of mitochondrial integrity that may have contributed to melanoma induction or progression. These findings provide the first example of a mitochondrial immune target for CD4(+) T cells and therefore have implications for the immunosurveillance of mitochondrial aberrations in cancer patients.

Critical role of EBNA1-specific CD4+ T cells in the control of mouse Burkitt lymphoma in vivo.

CD4+ T cells play important roles in orchestrating host immune responses against cancer and infectious diseases. Although EBV-encoded nuclear antigen 1-specific (EBNA1-specific) CD4+ T cells have been implicated in controlling the growth of EBV-associated tumors such as Burkitt lymphoma (BL) in vitro, direct evidence for their in vivo function remains elusive due to the lack of an appropriate experimental BL model. Here, we describe the development of a mouse EBNA1-expressing BL tumor model and the identification of 2 novel MHC H-2I-A(b)-restricted T cell epitopes derived from EBNA1. Using our murine BL tumor model and the relevant peptides, we show that vaccination of mice with EBNA1 peptide-loaded DCs can elicit CD4+ T cell responses. These EBNA1-specific CD4+ T cells recognized peptide-pulsed targets as well as EBNA1-expressing tumor cells and were necessary and sufficient for suppressing tumor growth in vivo. By contrast, EBNA1 peptide-reactive CD8+ T cells failed to recognize tumor cells and did not contribute to protective immunity. These studies represent what we believe to be the first demonstration that EBNA1-specific CD4+ T cells can suppress tumor growth in vivo, which suggests that CD4+ T cells play an important role in generating protective immunity against EBV-associated cancer.

Functional characterization of EBV-encoded nuclear antigen 1-specific CD4+ helper and regulatory T cells elicited by in vitro peptide stimulation.

CD4(+) helper and regulatory T (Treg) cells play important but opposing roles in regulating host immune responses against cancer and other diseases. However, very little is known about the antigen specificity of CD4(+) Treg cells. Here we describe the generation of a panel of EBV-encoded nuclear antigen 1 (EBNA1)-specific CD4(+) T-cell lines and clones that recognize naturally processed EBNA1-P(607-619) and -P(561-573) peptides in the context of HLA-DQ2 and HLA-DR11, -DR12, and -DR13 molecules, respectively. Phenotypic and functional analyses of these CD4(+) T cells revealed that they represent EBNA1-specific CD4(+) T helper as well as Treg cells. CD4(+) Treg cells do not secrete interleukin (IL)-10 and transforming growth factor beta cytokines but express CD25, the glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), and Forkhead Box P3 (Foxp3), and are capable of suppressing the proliferative responses of naive CD4(+) and CD8(+) T cells to stimulation with mitogenic anti-CD3 antibody. The suppressive activity of these CD4(+) Treg cells is mediated via cell-cell contact or in part by a cytokine-dependent manner. Importantly, these Treg cells suppress IL-2 secretion by CD4(+) effector T cells specific for either EBNA1 or a melanoma antigen, suggesting that these CD4(+) Treg cells induce immune suppression. These observations suggest that the success of peptide-based vaccines against EBV-associated cancer and other diseases may likely depend upon our ability to identify antigens/peptides that preferentially activate helper T cells and/or to design strategies to regulate the balance between CD4(+) helper and Treg cells.

Identification of HLA-DP3-restricted peptides from EBNA1 recognized by CD4(+) T cells.

The EBV-encoded nuclear antigen 1 (EBNA1) is required for the maintenance and replication of the viral episome in EBV-transformed human B-lymphoblastoid cell lines. It is expressed in all EBV-associated tumors, making it a potentially important target for immunotherapy. However, this promise has not been realized, because an endogenously processed MHC class I-restricted T-cell epitope remains to be identified, and relatively little is known about MHC class II-restricted helper epitopes in the molecule. In this report, we identify a T-cell peptide derived from EBNA1 that is recognized by CD4(+) T cells. More importantly, EBNA1-specific, HLA-DP3-restricted CD4(+) T cells are capable of recognizing MHC class II-matched Burkitt's lymphoma cells, autologous peripheral blood mononuclear cells loaded with the purified EBNA1 protein, as well as target cells transfected with Ii-EBNA1 cDNA. These new findings demonstrate that EBNA1 is processed endogenously and presented to T cells by MHC class II molecules, and, hence, may be useful to incorporate into cancer vaccines to enhance antitumor immunity against EBV-associated tumors.

A novel subset of CD4(+) T(H)2 memory/effector cells that produce inflammatory IL-17 cytokine and promote the exacerbation of chronic allergic asthma.

The inflammatory cytokine interleukin (IL)-17 is involved in the pathogenesis of allergic diseases. However, the identity and functions of IL-17-producing T cells during the pathogenesis of allergic diseases remain unclear. Here, we report a novel subset of T(H)2 memory/effector cells that coexpress the transcription factors GATA3 and RORγt and coproduce T(H)17 and T(H)2 cytokines. Classical T(H)2 memory/effector cells had the potential to produce IL-17 after stimulation with proinflammatory cytokines IL-1ß, IL-6, and IL-21. The number of IL-17-T(H)2 cells was significantly increased in blood of patients with atopic asthma. In a mouse model of allergic lung diseases, IL-17-producing CD4(+) T(H)2 cells were induced in the inflamed lung and persisted as the dominant IL-17-producing T cell population during the chronic stage of asthma. Treating cultured bronchial epithelial cells with IL-17 plus T(H)2 cytokines induced strong up-regulation of chemokine eotaxin-3, Il8, Mip1b, and Groa gene expression. Compared with classical T(H)17 and T(H)2 cells, antigen-specific IL-17-producing T(H)2 cells induced a profound influx of heterogeneous inflammatory leukocytes and exacerbated asthma. Our findings highlight the plasticity of T(H)2 memory cells and suggest that IL-17-producing T(H)2 cells may represent the key pathogenic T(H)2 cells promoting the exacerbation of allergic asthma.

Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function.

CD4+ regulatory T (Treg) cells have a profound ability to suppress host immune responses, yet little is understood about how these cells are regulated. We describe a mechanism linking Toll-like receptor (TLR) 8 signaling to the control of Treg cell function, in which synthetic and natural ligands for human TLR8 can reverse Treg cell function. This effect was independent of dendritic cells but required functional TLR8-MyD88-IRAK4 signaling in Treg cells. Adoptive transfer of TLR8 ligand-stimulated Treg cells into tumor-bearing mice enhanced anti-tumor immunity. These results suggest that TLR8 signaling could play a critical role in controlling immune responses to cancer and other diseases.