PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

Different strategies for producing naturally soluble form of common cytokine receptor γ chain.

The common cytokine receptor γ chain (γc) plays an essential role in regulating lymphoid homeostasis. In fact, alteration of this gene causes severe immunodeficiency in humans and animals. Although soluble γc (sγc) was identified in the late 1990s, much remains unknown about its production. This study describes various mechanisms underlying the generation of sγc isoforms in different species. Our data demonstrate that mouse γc and the avian ortholog γc-a did not generate sγc. Moreover, two mouse isoforms, CRA-a and mγc-b, encoded by transcripts lacking a transmembrane region by alternative splicing, did not yield sγc. However, in ducks, sγc was produced from a γc-b transcript lacking a transmembrane region by alternative splicing. In chickens, sγc was produced in normal cells and cell lines by proteolytic shedding of the γc-b isoform containing intron 5, which displayed a relatively high probability of proteolytic cleavage of the ectodomain. This shedding was suppressed by leupeptin, serine and cysteine protease inhibitor. Compared to the chicken ortholog γc-a, expression of γc-b mRNA was differentially regulated according to tissue type, developmental stage, and antigen stimulation. These data demonstrate several mechanisms for producing sγc and suggest a potential role for sγc in avian lymphoid homeostatic responses to environmental antigens.

CD8αα⁺ innate-type lymphocytes in the intestinal epithelium mediate mucosal immunity.

Innate immune responses are critical for mucosal immunity. Here we describe an innate lymphocyte population, iCD8α cells, characterized by expression of CD8α homodimers. iCD8α cells exhibit innate functional characteristics such as the capacity to engulf and kill bacteria. Development of iCD8α cells depends on expression of interleukin-2 receptor γ chain (IL-2Rγc), IL-15, and the major histocompatibility complex (MHC) class Ib protein H2-T3, also known as the thymus leukemia antigen or TL. While lineage tracking experiments indicated that iCD8α cells have a lymphoid origin, their development was independent of the transcriptional suppressor Id2, suggesting that these cells do not belong to the family of innate lymphoid cells. Finally, we identified cells with a similar phenotype in humans, which were profoundly depleted in newborns with necrotizing enterocolitis. These findings suggest a critical role of iCD8α cells in immune responses associated with the intestinal epithelium.

Ex vivo characterization of γδ T-cell repertoire in patients after adoptive transfer of Vγ9Vδ2 T cells expressing the interleukin-2 receptor ß-chain and the common γ-chain.

BACKGROUND AIMS: Adoptive immunotherapy is emerging as a potent anti-tumor treatment modality; Vγ9Vδ2 T cells may represent appropriate agents for such cancer immunotherapy. To improve the currently limited success of Vγ9Vδ2 T-cell-based immunotherapy, we examined the in vivo dynamics of these adoptively-transferred cells and hypothesized that interleukin (IL)-15 is the potential factor for Vγ9δ2 T cell in vivo survival. METHODS: We conducted a clinical trial of adoptive Vγ9Vδ2 T-cell transfer therapy in six colorectal cancer patients who received pulmonary metastasectomy. Patients' peripheral blood mononuclear cells were stimulated with zoledronate (5 µmol/L) and IL-2 (1000 IU/mL) for 14 d. Harvested cells, mostly Vγ9Vδ2 T cells, were given intravenously weekly without additional IL-2 eight times in total. The frequency, phenotype and common γ-chain cytokine receptor expression of Vγ9Vδ2 T cells in peripheral blood was monitored by flow cytometry at each time point during treatment and 4 and 12 weeks after the last administration. RESULTS: Adoptively transferred Vγ9Vδ2 T cells expanded well without exogenous IL-2 administration or lymphodepleting preconditioning. They maintained effector functions in terms of interferon-γ secretion and prompt release of cytotoxic granules in response to PMA/ionomycin or isopentenyl pyrophosphate-positive cells. Because they are IL-2Rα(-)IL-7Rα(-)IL-15Rα(-)IL-2Rß(+)γc(+), it is likely that IL-2 or IL-15 is required for their maintenance. CONCLUSIONS: The persistence of large numbers of functionally active adoptively transferred Vγ9Vδ2 T cells in the absence of exogenous IL-2 implies that an endogenous factor, such as IL-15 transpresentation, is adequate to support these cells in vivo.

Efficient xenoengraftment in severe immunodeficient NOD/Shi-scid IL2rγnull mice is attributed to a lack of CD11c+B220+CD122+ cells.

Xenograft animal models using immunodeficient mice have been widely applied in medical research on various human diseases. NOD/Shi-scid-IL2rγ(null) (NOG) mice are known to show an extremely high engraftment rate of xenotransplants compared with conventional immunodeficient mice. This high engraftment rate of xenotransplants in NOG mice was substantially suppressed by the transfer of spleen cells from NOD-scid mice that were devoid of NK cells. These results indicate that cell types other than splenic NK cells present in NOD-scid mice but not in NOG mice may be involved in this suppression. To identify the cell types responsible for this effect, we transferred subpopulations of spleen cells from NOD-scid mice into NOG mice and assessed the levels of human cell engraftment after human PBMC (hPBMC) transplantation. These experiments revealed that CD11c(+)B220(+) plasmacytoid dendritic cells (pDCs) from NOD-scid mice markedly inhibited engraftment of human cells. The CD11c(+)B220(+)CD122(+) cells further fractionated from the pDCs based on the expression of CD122, which is an NK cell marker strongly inhibited during hPBMC engraftment in NOG mice. Moreover, the CD122(+) cells in the pDC fraction were morphologically distinguishable from conventional CD122(+) NK cells and showed a higher rejection efficiency. The current results suggest that CD11c(+)B220(+)CD122(+) cells play an important role in xenograft rejection, and their absence in NOG mice may be critical in supporting the successful engraftment of xenotransplants.

Rapid down-regulation of γc on T cells in early SIV infection correlates with impairment of T-cell function.

The common γ(c) subunit molecule is shared among all γ(c) cytokines and clearly involved in T-cell function, but its role in HIV infection and immunity is not well understood. Here, we examined expression and function of γ(c) on T cells during SIV infection in Rhesus macaques. Surface γ(c) distribution was differentially expressed on CD4(+) and CD8(+) T cells, and CD4(+) naive/memory cell populations in various lymphoid tissues of normal macaques. However, surface γ(c) expression was rapidly and significantly down-regulated on T cells in acute infection with pathogenic SIV, compared to infection with a less virulent SHIV or controls and did not recover on CD8(+) T cells in the chronic stage. Moreover, the peripheral and CD4(+)T cell loss was inversely correlated with γ(c)(+) CD8(+) T cells in individual tissues. γ(c)(+) T cells were mainly functional as evidenced by higher cytokine secretion and proliferative capacity. Further in vitro experiments found that surface γ(c) expression could be down-regulated following high level of IL-7 treatment by both internalization and shedding. Down-regulation of γ(c) during early HIV/SIV infection may inhibit T-cell function, particularly of CD8(+) T cells, and, may be linked with immune failure and loss of viral containment.

Molecular identification of duck and quail common cytokine receptor γ chain genes.

Common cytokine receptor γ chain (γ(c)) family cytokines play crucial roles in the regulation of innate and adaptive immune responses. Unlike mammals, chickens possess two different γ(c) transcripts. To determine if this difference is present in other avian species, γ(c) cDNA and genomic clones from ducks and quails were investigated. Two different γ(c) transcripts were identified in both species and designated as duck γ(c)-a (duγ(c)-a), duγ(c)-b, quail γ(c)-a (quγ(c)-a), and quγ(c)-b. Comparisons between the duck and quail γ(c) cDNA and genomic sequences indicated that the two transcripts were produced by alternative splicing. Unexpectedly, the duγ(c)-b contained the fifth intron, a frame-switching 88-bp insertion, resulting in a receptor molecule lacking a transmembrane region. These findings indicate a possibility that avian species, unlike mammals, express two different γ(c) transcripts due to alternative splicing. This study is the first demonstration of an alternatively spliced γ(c) isoform that lacks a transmembrane domain.

Implications for gene therapy-limiting expression of IL-2R gamma c delineate differences in signaling thresholds required for lymphocyte development and maintenance.

X-linked SCID patients are deficient in functional IL-2Rgamma(c) leading to the loss of IL-2/IL-4/IL-7/IL-9/IL-15/IL-21 signaling and a lack of NK and mature T cells. Patients treated with IL-2Rgamma(c) gene therapy have T cells develop; however, their NK cell numbers remain low, suggesting antiviral responses may be compromised. Similarly, IL-2Rgamma(c)(-/-) mice reconstituted with IL-2Rgamma(c) developed few NK cells, and reconstituted T cells exhibited defective proliferative responses suggesting incomplete recovery of IL-2Rgamma(c) signaling. Given the shift toward self-inactivating long terminal repeats with weaker promoters to control the risk of leukemia, we assessed NK and T cell numbers and function in IL-2Rgamma(c)(-/-) mice reconstituted with limiting amounts of IL-2Rgamma(c). Reconstitution resulted in lower IL-2/-15-mediated STAT5 phosphorylation and proliferation in NK and T cells. However, TCR costimulation restored cytokine-driven T cell proliferation to wild-type levels. Vector modifications that improved IL-2Rgamma(c) levels increased cytokine-induced STAT5 phosphorylation in both populations and increased NK cell proliferation demonstrating that IL-2Rgamma(c) levels are limiting. In addition, although the half-lives of both NK and T cells expressing intermediate levels of IL-2Rgamma(c) are reduced compared with wild-type cells, the reduction in NK cell half-live is much more severe than in T cells. Collectively, these data indicate different IL-2Rgamma(c) signaling thresholds for lymphocyte development and proliferation making functional monitoring imperative during gene therapy. Further, our findings suggest that IL-2Rgamma(c) reconstituted T cells may persist more efficiently than NK cells due to compensation for suboptimal IL-2Rgamma(c) signaling by the TCR.

The cellular amount of the common gamma-chain influences spontaneous or induced cell proliferation.

Mutations of the IL2RG encoding the common gamma-chain (gamma(c)) lead to the X-linked SCID disease. Gene correction through ex vivo retroviral transduction restored the immunological impairment in the most of treated patients, although lymphoproliferative events occurred in five of them. Even though in two cases it was clearly documented an insertional mutagenesis in LMO2, it is conceivable that gamma(c) could have a role per se in malignant lymphoproliferation. The gamma(c) is a shared cytokine receptor subunit, involved also in growth hormone (GH) receptor signaling. Through short interfering RNA or using X-linked SCID B lymphoblastoid cell lines lacking gamma(c), we demonstrate that self-sufficient growth was strongly dependent on gamma(c) expression. Furthermore, a correlation between gamma(c) amount and the extent of constitutive activation of JAK3 was found. The reduction of gamma(c) protein expression also reduced GH-induced proliferation and STAT5 nuclear translocation in B lymphoblastoid cell lines. Hence, our data demonstrate that gamma(c) plays a remarkable role in either spontaneous or GH-induced cell cycle progression depending on the amount of protein expression, suggesting a potential role as enhancing cofactor in lymphoproliferation.

Pharmacological immunomodulation of surgical trauma.

Surgery and accidental trauma induce changes in the immune response, showing a predominant pattern of activation through the Th2 cell pathway to the detriment of Th1 cell pathway activation. Anapsos is a hydrosoluble extract obtained from Polypodium leucotomos. Anapsos has shown immunomodulating effects in vitro. On a rat experimental model (tibia and fibula fracture), cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, and IL-12) (enzyme-linked immunosorbent assay, ELISA) and cell percentages of CD4, CD8 CD25, CD122, and CD132 (monoclonal antibodies, MoAb) were determined in peripheral blood 7 days before surgery (PRE), 1 day after surgery (1PO), and 7 days after surgery (7PO). On postoperative day 1, rats undergoing fracture show an increase of CD8 percent expression and IL-6 and IL-10 levels, in contrast to rats undergoing fracture plus anapsos treatment. On postoperative day 7, rats undergoing fracture show an increase of IL-6 levels, whereas rats undergoing fracture plus anapsos do not. The IL-12 level decreases on postoperative day 7 in the group with fracture but not in the fracture plus anapsos group. Thus, we conclude that anapsos is able to modulate the immune response after trauma, inhibiting Th2 pathway activation with no effect on Th1 pathway activation. In trauma, Anapsos could prevent the shifting Th1/Th2 balance.

Cell-specific interleukin-15 and interleukin-15 receptor subunit expression and regulation in pneumococcal pneumonia--comparison to chlamydial lung infection.

Interleukin (IL)-15 has critical impact on the homeostasis and activation of natural killer cells, natural killer T cells, gammadeltaT cells, and CD8(+)T cells, and contributes to antimicrobial defenses particularly at mucosal sites. The respiratory tract comprises a large mucosal surface and harbors significant amounts of lymphocytes, however the expression pattern of IL-15 in the lung and its role in local immune responses are largely unknown. We therefore analyzed the differential expression of IL-15 and the IL-15 receptor (IL-15R) complex in the lungs of mice and demonstrated substantial constitutive expression in bronchial and alveolar epithelial cells, alveolar macrophages, and vascular smooth muscle cells, implicating contribution to pulmonary immune cell homeostasis already under normal conditions. The induction of pneumococcal pneumonia but not the infection with Chlamydophila pneumoniae evoked a significant up-regulation of IL-15 on alveolar macrophages and bronchial epithelial cells, with the latter presenting de-novo expression of IL-15 on their basolateral surface and additional up-regulation of IL-15Ralpha. Moreover, transcriptome analysis as well as semi-quantitative PCR indicated at least partial transcriptional regulation in mice lungs. In conclusion IL-15 is suggested being of functional importance in the pulmonary immune response against pneumococcal pneumonia.

Limiting {gamma}c expression differentially affects signaling via the interleukin (IL)-7 and IL-15 receptors.

X-linked severe combined immunodeficiency (SCID-X1) results from mutations in the IL2RG gene, which encodes the common gamma chain (gammac) of the receptors for interleukin (IL)-2, 4, 7, 9, 15, and 21. Affected infants typically lack T and natural killer (NK) cells as a consequence of loss of signaling via the IL-7 receptor (IL-7R) and the IL-15R, respectively. In some infants, however, autologous NK cells are observed despite failure of T-cell ontogeny. The mechanisms by which mutations in gammac differentially impact T- and NK-cell ontogeny remain incompletely understood. We used SCID-X1 patient-derived EBV-transformed B cells to test the hypothesis that the IL-15R-mediated signaling is preferentially retained as gammac expression becomes limiting. Signal transduction via the IL-15R was readily detected in control EBV-transformed B cells, and via the IL-7R when modified to express IL-7Ralpha. Under the same experimental conditions, patient-derived EBV-transformed B cells expressing trace amounts of gammac proved incapable of signal transduction via the IL-7R while retaining the capacity for signal transduction via the IL-15R. An equivalent result was obtained in ED-7R cells modified to express varying levels of gammac. Collectively, these results confirm that signal transduction via the IL-15R, and hence NK ontogeny, is preferentially retained relative to the IL-7R as gammac expression becomes limiting.

Curcumin prevents tumor-induced T cell apoptosis through Stat-5a-mediated Bcl-2 induction.

Patients with advanced cancer exhibit multifaceted defects in their immune capacity, which are likely to contribute to an increased susceptibility to infections and disease progression. We demonstrated earlier that curcumin inhibits tumor growth and prevents immune cell death in tumor-bearing hosts. Here we report that tumor-induced immunodepletion involves apoptosis of thymic CD4+/CD8+ single/double positive cells as well as loss of circulating CD4+/CD8+ T cells. Administration of curcumin to tumor-bearing animals resulted in restoration of progenitor, effecter, and circulating T cells. In fact, tumor burden decreased the expression level of the pro-proliferative protein Bcl-2 while increasing the pro-apoptotic protein Bax in T cells. Curcumin down-regulated the Bax level while augmenting Bcl-2 expression in these cells, thereby protecting the immunocytes from tumor-induced apoptosis. A search for the upstream mechanism revealed down-regulation of the common cytokine receptor gamma chain (gammac) expression in T cells by tumor-secreted prostaglandin E2. As a result, Jak-3 and Stat-5a phosphorylation and to a lesser extent Stat-5b phosphorylation were also decreased in T cells. These entire phenomena could be reverted back by curcumin, indicating that this phytochemical restored the cytokine-dependent Jak-3/Stat-5a signaling pathway in T cells of tumor bearers. Overexpressed Stat-5a/constitutively active Stat-5a1*6 but not Stat-5b could efficiently elevate Bcl-2 levels and protect T cells from tumor-induced death, whereas C-terminal truncated Stat-5a713 overexpression failed to do so, indicating the importance of Stat-5a signaling in T cell survival. Thus, these results raise the possibility of inclusion of curcumin in successful therapeutic regimens against cancer.