Paclitaxel may inhibit autoimmune diseases by sparing or increasing the number of CD4(+) CD25(+) regulatory T cells

Paclitaxel, a representative of taxanes, exhibits cytotoxic effects against a broad range of tumors. Strikingly, an emerging body of data suggests that paclitaxel also exerts effects on immune system by stimulating anti-tumor and anti-autoimmunity effects, supporting the idea that paclitaxel suppresses tumor through several mechanisms and not solely through inhibiting cell division. Based on the accumulating data, we hypothesized that paclitaxel may inhibit autoimmune diseases by sparing or actively increasing the number of CD4(+) CD25(+) Treg cells. The hypothesis, if proved to be correct, will significantly improve our understanding of the tumor immunity, autoimmunity and its related pathological effects. It will influence our choice on immunosuppressive drugs for cancer patients with autoimmune diseases. It will also impact the immunotherapy for tumors.     

The ACE inhibitors enalapril and captopril modulate cytokine responses in Balb/c and C57Bl/6 normal mice and increase CD4CD103CD25 splenic T cell numbers

Increasing evidence implies beneficial effects of angiotensin-converting enzyme (ACE) inhibitors beyond those of their original indications to control hypertension. One of the most attractive non-hemodynamic properties of ACE inhibitors is their ability to regulate cytokine production. The mechanism(s) underlying the role of ACE inhibitors on cytokine synthesis are not well understood but they have traditionally been attributed to the inhibition of angiotensin (Ang) II formation. In fact, it has been extensively demonstrated that ACE inhibitors decrease Ang II-induced production of proinflammatory cytokines and chemokines. However, it is not well described if inhibition of endogenous Ang II generation by ACE inhibitors modulates systemic cytokine production in mice. To verify that, in this work, we investigated the effects of treatment with the ACE inhibitors enalapril and captopril on cytokine synthesis in C57Bl/6 and Balb/c mice. Our results show that enalapril up regulates IL-10 produced by splenocytes from Balb/c and C57Bl/6 mice and captopril increased it only in Balb/c mice. Furthermore, CD4⁺CD103⁺ presented increased IL-10 production after enalapril treatment. Enalapril as well as captopril short-term treatment enhanced IL-2 synthesis in Balb/c mice. Besides, enhanced IL-2 and IL-10 levels correlates with increased CD4⁺CD103⁺CD25^negative T cells numbers in spleens from enalapril-treated mice.     

CD4 T cell dependent tumor immunity stimulated by dendritic cell based vaccine

CD8 CTLs have been accountable for the major effector cells responsible for the rejection of tumor cells. And CD40 signaling and IL-12 have been shown to be the essential pathways involved in the activation process. Immunizing mice with dendritic cells transduced with an adenovirus expressing the human melanoma antigen gp100, an immunization strategy of xenoimmunization, stimulated potent tumor protection dependent on effective CD4 T cells in the absence of CD8 T cells. Further studies revealed that neither CD40 signaling nor IL-12 was indispensable for the activation of dendritic and CD4 T cells in this model. Stimulation of effective antitumor immunity targeting the self-antigen did not elicit autoimmunity. The implications of this study were discussed.     

Induction of FoxP3+CD4+CD25+ regulatory T cells by a bone marrow population distinct from plasmacytoid-DC

Facilitating cells (FC) are bone marrow-derived cells that facilitate allogeneic hematopoietic stem cell (SC) engraftment and induce transplantation tolerance without causing graft vs. host disease. Although there is evidence for FC directing the development of FoxP3⁺CD4⁺CD25⁺ regulatory T cells, the specific FC subsets that control regulatory T cell development have not been defined. The current study investigates the role of FC-CD3ε⁺ and FC-CD3ε⁻ subpopulations in the development of FoxP3⁺CD4⁺CD25⁺ regulatory T cells. Here, we demonstrate that the induction of FoxP3⁺CD4⁺CD25⁺ regulatory T cells in coculture is mediated by not only the FC-CD3ε⁻ subset but also the FC-CD3ε⁺ subset, which is distinct from plasmacytoid precursor dendritic cells (p-preDC). The identification of cell populations distinct from p-preDC that efficiently induce the generation of FoxP3⁺CD4⁺CD25⁺ regulatory T cells may prove useful for future therapeutic applications for the induction of tolerance following allogeneic SC transplantation.     

CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R gamma-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-gamma in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-gamma production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25- lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-gamma. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.     

CD4+ T cells in aged or thymectomized recipients of allogeneic stem cell transplantations

Background

CD4+CD25highFOXP3+ regulatory T (Treg) cells, which include thymus-derived and peripherally induced cells, play a central role in immune regulation, and are therefore crucial to prevent graft-versus-host disease (GVHD). The increasing use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for elderly patients with thymus regression, and our case of allo-HSCT shortly after total thymectomy, raised questions about the activity of thymus-derived Treg cells and peripherally induced Treg cells, which are otherwise indistinguishable.

Results

We found that despite pre-transplant thymectomy or older age, both naïve and effector Treg cells, as well as naïve and effector conventional T cells, proliferated in allo-HSCT recipients. Higher proportions of total Treg cells 1 month post allo-HSCT, and naïve Treg cells 1 year post allo-HSCT, appeared in patients achieving complete chimera without developing significant chronic GVHD, including our thymectomized patient, compared with patients who developed chronic GVHD.

Conclusions

Treg cells that modulate human allogeneic immunity may arise peripherally as well as in the thymus of allo-HSCT recipients.     

Heterologous CD8 T cell immune response to HSV induced by toll like receptor ligands

A memory response is established following primary antigen exposure that stays more or less constant. It appears to adopt a set-point in magnitude but upon re-exposure the response is quicker and better and there is an upward shift in memory frequency that varies with individuals based on the exposure pattern to other microbes or its components. Our investigations were designed to test such differences of non-specific stimulation by PAMPs in lowering the threshold of activation. Neonatal mice were pre-exposed to TLR-ligands intermittently and later analyzed for its resilience to challenge with virus during adult-life. Secondly, adult mice with pre-existing memory to virus were exposed to various TLR-ligands and analyzed for their quality of memory response. The TLR-ligands exposed animals were better responders to a new agent exposure compared to the animals kept in sterile surroundings. Moreover, immune memory recall and the viral specific CD8⁺ T cells response with TLR-ligands were comparable to the recall response with the cognate antigen. The results provide insights into the role of hyper-sanitized environment versus PAMPs mediated signaling in adaptive immunity and long-term immune memory.     

CD137 signaling interferes with activation and function of CD4+CD25+ regulatory T cells in induced tolerance to experimental autoimmune thyroiditis

Experimental autoimmune thyroiditis (EAT), a model for Hashimoto's thyroiditis, is a T cell-mediated disease inducible with mouse thyroglobulin (mTg). Pretreatment with mTg, however, can induce CD4+ T cell-mediated tolerance to EAT. We demonstrate that CD4+CD25+ regulatory cells are critical for the tolerance induction, as in vivo depletion of CD25+ cells abrogated established tolerance, and CD4+CD25+ cells from tolerized mice suppressed mTg-responsive cells in vitro. Importantly, administration of an agonistic CD137 monoclonal antibody (mAb) inhibited tolerance development, and the mediation of established tolerance. CD137 mAb also inhibited the suppression of mTg-responsive cells by CD4+CD25+ cells in vitro. Signaling through CD137 likely resulted in enhancement of the responding inflammatory T cells, as anti-CD137 did not enable CD4+CD25+ T cells to proliferate in response to mTg in vitro.     

Induction of a T helper cell response against the tumor-associated antigen HER2 using monocyte-derived dendritic cells

Tumor-reactive CD4⁺ T helper (Th) cells play a criticalrole in antitumor immunity, due to their ability to induceCD8⁺ T cell-mediated cytotoxic activity and humoralresponse. This study focuses on the in vitro generationand expansion of Th cells specific for the tumor-associatedantigen `human epidermal growth factor receptor-2' (HER2). Aprotocol for efficient HER2 presentation was developed usingautologous monocyte-derived dendritic cells (DC) as antigenpresenting cells (APC) and purified HER2 protein as antigensource. Our data suggest that DC pulsed with recombinantprotein of the extracellular domain (ECD) of HER2 (ECD/HER2)induce an ECD/HER2-specific Th cell response. This finding mayfacilitate the development of immunotherapy regimens withoutrequiring defined immunogenic epitopes of the antigen.     

Therapeutic efficacy of antitumor dendritic cell vaccinations correlates with persistent Th1 responses,high intratumor CD8+ T cell recruitment and low relative regulatory T cell infiltration

Despite the increasing number of immunotherapeutic strategies for the treatment of cancer, most approaches have failed to correlate the induction of an anti-tumor immune response with therapeutic efficacy. We therefore took advantage of a successful vaccination strategy-combining dendritic cells and irradiated GM-CSF secreting tumor cells-to compare the immune response induced against 9L gliosarcoma tumors in cured rats versus those with progressively growing tumors. At the systemic level, the tumor specific cytotoxic responses were quite heterogeneous in uncured vaccinated rats, and were surprisingly often high in animals with rapidly-growing tumors. IFN-gamma secretion by activated splenic T cells was more discriminative as the CD4+ T cell-mediated production was weak in uncured rats whereas high in cured ones. At the tumor level, regressing tumors were strongly infiltrated by CD8+ T cells, which demonstrated lytic capacities as high as their splenic counterparts. In contrast, progressing tumors were weakly infiltrated by T cells showing impaired cytotoxic activities. Proportionately to the T cell infiltrate, the expression of Foxp3 was increased in progressive tumors suggesting inhibition by regulatory T cells. In conclusion, the main difference between cured and uncured vaccinated animals does not depend directly upon the induction of systemic cytotoxic responses. Rather the persistence of higher CD4+ Th1 responses, a high intratumoral recruitment of functional CD8+ T cells, and a low proportion of regulatory T cells correlate with tumor rejection.     

Lifelong dynamics of human CD4CD25 regulatory T cells: Insights from in vivo data and mathematical modeling

Despite their limited proliferation capacity, regulatory T cells (T_regs) constitute a population maintained over the entire lifetime of a human organism. The means by which T_regs sustain a stable pool in vivo are controversial. Using a mathematical model, we address this issue by evaluating several biological scenarios of the origins and the proliferation capacity of two subsets of T_regs: precursor CD4⁺CD25⁺CD45RO⁻ and mature CD4⁺CD25⁺CD45RO⁺ cells. The lifelong dynamics of T_regs are described by a set of ordinary differential equations, driven by a stochastic process representing the major immune reactions involving these cells. The model dynamics are validated using data from human donors of different ages. Analysis of the data led to the identification of two properties of the dynamics: (1) the equilibrium in the CD4⁺CD25⁺FoxP3⁺T_regs population is maintained over both precursor and mature T_regs pools together, and (2) the ratio between precursor and mature T_regs is inverted in the early years of adulthood. Then, using the model, we identified three biologically relevant scenarios that have the above properties: (1) the unique source of mature T_regs is the antigen-driven differentiation of precursors that acquire the mature profile in the periphery and the proliferation of T_regs is essential for the development and the maintenance of the pool; there exist other sources of mature T_regs, such as (2) a homeostatic density-dependent regulation or (3) thymus- or effector-derived T_regs, and in both cases, antigen-induced proliferation is not necessary for the development of a stable pool of T_regs. This is the first time that a mathematical model built to describe the in vivo dynamics of regulatory T cells is validated using human data. The application of this model provides an invaluable tool in estimating the amount of regulatory T cells as a function of time in the blood of patients that received a solid organ transplant or are suffering from an autoimmune disease.     

Delivery of mengovirus-derived RNA replicons into tumoural liver enhances the anti-tumour efficacy of a peripheral peptide-based vaccine

Hepatocellular carcinoma is a deadly cancer with growing incidence for which immunotherapy is one of the most promising therapeutic approach. Peptide-based vaccines designed to induce strong, sustained CD8+ T cell responses are effective in animal models and cancer patients. We demonstrated the efficacy of curative peptide-based immunisation against a unique epitope of SV40 tumour antigen, through the induction of a strong CD8+ T cell-specific response, in our liver tumour model. However, as in human clinical trials, most tumour antigen epitopes did not induce a therapeutic effect, despite inducing strong CD8+ T cell responses. We therefore modified the tumour environment to enhance peptide-based vaccine efficacy by delivering mengovirus (MV)-derived RNA autoreplicating sequences (MV-RNA replicons) into the liver. The injection of replication-competent RNA replicons into the liver converted partial tumour regression into tumour eradication, whereas non-replicating RNA had no such effect. Replicating RNA replicon injection induced local recruitment of innate immunity effectors (NK and NKT) to the tumour and did not affect specific CD8+ T cell populations or other myelolymphoid subsets. The local delivery of such RNA replicons into tumour stroma is therefore a promising strategy complementary to the use of peripheral peptide-based vaccines for treating liver tumours.     

CD4<Superscript>+</Superscript> T-cell recognition of human 5T4 oncofoetal antigen: implications for initial depletion of CD25<Superscript>+</Superscript> T cells

Background The human 5T4 (h5T4) oncofoetal antigen is expressed by a wide variety of human carcinomas including colorectal, ovarian, gastric and renal, but rarely on normal tissues. Its restricted expression on tumour tissues as well as its association with tumour progression and bad prognosis has driven the development of a MVA-based vaccine (TroVax) which has been tested in several early phase clinical trials and these studies have led to the start of a phase III trial in renal cell carcinoma patients. We have recently shown that CD8⁺ T cells recognizing h5T4 can be generated in the absence of CD4⁺ T cells from peripheral blood lymphocytes of human healthy individuals. Results We report the existence and expansion of human CD4⁺ T cells against h5T4 by stimulation with autologous monocyte-derived dendritic cells infected with a replication defective adenovirus encoding the h5T4 cDNA (Ad-h5T4). The h5T4-specific T-cell responses in normal individuals are enhanced by initial depletion of CD25⁺ cells (putative T regulatory cells) prior to the in vitro stimulation. We have identified a novel h5T4-derived 15-mer peptide recognized by CD4⁺ T cells in HLA-DR4 positive healthy individuals. Interestingly, CD4⁺ T cells spontaneously recognizing a different 5T4 epitope restricted by HLA-DR were identified in tumour-infiltrating lymphocytes isolated from a regressing renal cell carcinoma lung metastasis. Conclusion Our data show that CD4⁺ T cells recognizing h5T4 can be expanded and detected in healthy individuals and a renal cell carcinoma patient. Such h5T4-specific CD4⁺ T cells boosted or induced by vaccination could act to modulate both cell or antibody mediated anti-tumour responses. This work was supported by Cancer Research UK.     

Anti-tumor activity and trafficking of self, tumor-specific T cells against tumors located in the brain

It is commonly believed that T cells have difficulty reaching tumors located in the brain due to the presumed "immune privilege" of the central nervous system (CNS). Therefore, we studied the biodistribution and anti-tumor activity of adoptively transferred T cells specific for an endogenous tumor-associated antigen (TAA), gp100, expressed by tumors implanted in the brain. Mice with pre-established intracranial (i.c.) tumors underwent total body irradiation (TBI) to induce transient lymphopenia, followed by the adoptive transfer of gp100(25-33)-specific CD8+ T cells (Pmel-1). Pmel-1 cells were transduced to express the bioluminescent imaging (BLI) gene luciferase. Following adoptive transfer, recipient mice were vaccinated with hgp100(25-33) peptide-pulsed dendritic cells (hgp100(25-33)/DC) and systemic interleukin 2 (IL-2). This treatment regimen resulted in significant reduction in tumor size and extended survival. Imaging of T cell trafficking demonstrated early accumulation of transduced T cells in lymph nodes draining the hgp100(25-33)/DC vaccination sites, the spleen and the cervical lymph nodes draining the CNS tumor. Subsequently, transduced T cells accumulated in the bone marrow and brain tumor. BLI could also detect significant differences in the expansion of gp100-specific CD8+ T cells in the treatment group compared with mice that did not receive either DC vaccination or IL-2. These differences in BLI correlated with the differences seen both in survival and tumor infiltrating lymphocytes (TIL). These studies demonstrate that peripheral tolerance to endogenous TAA can be overcome to treat tumors in the brain and suggest a novel trafficking paradigm for the homing of tumor-specific T cells that target CNS tumors.     

Reduced L-selectin (CD62LLow) expression identifies tumor-specific type 1 T cells from lymph nodes draining an autologous tumor cell vaccine

Reduced expression of CD62L can identify tumor-specific T cells in lymph nodes draining murine tumors. Here, we examined whether this strategy could isolate tumor-specific T cells from vaccinated patients. Tumor vaccine-draining lymph node (TVDLN) T cells of seven patients were separated into populations with reduced (CD62LLow) or high levels of CD62L (CD62LHigh). Effector T cells generated from CD62LLow cells maintained or enriched the autologous tumor-specific type 1 cytokine response compared to unseparated TVDLN T cells in four of four patients showing tumor-specific cytokine secretion. Interestingly, effector T cells generated from CD62LLow or CD62LHigh TVDLN were polarized towards a dominant type 1 or type 2 cytokine profile, respectively. For CD62LLow T cells the type 1 cytokine profile appeared determined prior to culture. Since a tumor-specific type 1 cytokine profile appears critical for mediating anti-tumor activity in vivo, this approach might be used to isolate T cells for adoptive immunotherapy.     

Innate memory phenotype CD4+ T cells play a role in early protection against infection by Listeria monocytogenes in a CD30L-dependent manner

CD30 ligand (CD30L, CD153) is a type II membrane-associated glycoprotein belonging to the tumor necrosis factor family. It is shown here that CD30L knock out (KO) mice are highly susceptible to primary infection with Listeria monocytogenes as assessed by the survival rate. There were significantly more bacteria on day 3 after infection in the peritoneal cavity, spleen and liver of CD30LKO mice than in wild type (WT) mice. The innate function of memory phenotype (MP) CD44+ CD4+ T cells for interferon-gamma production was significantly lower in CD30LKO mice than in WT mice in response to interleukin (IL)-12 and IL-15 in vitro. Depletion of CD4+ T cells by in vivo administration of anti-CD4 mAb at an early stage after infection hampered protection against Listeria. Furthermore, in vivo administration of agonistic anti-CD30 mAb restored protection against Listeria in CD30LKO mice, whereas treatment with soluble mCD30-Ig hampered protection in WT mice. Taken together, it appears that CD30L/CD30 signaling plays an important role in innate MPCD4+ T cell-mediated protection against infection with L. monocytogenes.     

Modified tumour antigen-encoding mRNA facilitates the analysis of naturally occurring and vaccine-induced CD4 and CD8 T cells in cancer patients

The development of effective anti-cancer vaccines requires precise assessment of vaccine-induced immunity. This is often hampered by low ex vivo frequencies of antigen-specific T cells and limited defined epitopes. This study investigates the applicability of modified, in vitro-transcribed mRNA encoding a therapeutically relevant tumour antigen to analyse T cell responses in cancer patients. In this study transfection of antigen presenting cells, by mRNA encoding the tumour antigen NY-ESO-1, was optimised and applied to address spontaneous and vaccine-induced T cell responses in cancer patients. Memory CD8+ T cells from lung cancer patients having detectable humoral immune responses directed towards NY-ESO-1 could be efficiently detected in peripheral blood. Specific T cells utilised a range of different T cell receptors, indicating a polyclonal response. Specific killing of a panel of NY-ESO-1 expressing tumour cell lines indicates recognition restricted to several HLA allelic variants, including a novel HLA-B49 epitope. Using a modified mRNA construct targeting the translated antigen to the secretory pathway, detection of NY-ESO-1-specific CD4+ T cells in patients could be enhanced, which allowed the in-depth characterisation of established T cell clones. Moreover, broad CD8+ and CD4+ T cell responses covering multiple epitopes were detected following mRNA stimulation of patients treated with a recombinant vaccinia/fowlpox NY-ESO-1 vaccine. This approach allows for a precise monitoring of responses to tumour antigens in a setting that addresses the breadth and magnitude of antigen-specific T cell responses, and that is not limited to a particular combination of known epitopes and HLA-restrictions.     

Functional adaptation to oxidative stress by memory T cells: an analysis of the role in the cardiovascular disease process

T cells participate in combating infection and critically determine the outcomes in any given disease process. Impaired immune response occurs in a number disease processes such as in cancer and atherosclerosis although the underlying mechanisms are still not fully understood. This article gives an up-to-date review of T cells development and functional adaptation to pathophysiological stimuli and participation in the cardiovascular disease process. In addition, we have discussed the signaling pathways controlled by the microenvironment that determine T cells function and resultant type of immune response. We have also discussed in detail how oxidative stress is a key component of the micro environmental interaction.