Adoptive Cell Transfer

Awareness of the immunological underpinnings of host-parasite interactions may reveal immune signaling pathways that could be used to treat inflammatory disease in humans. Previously we showed that infection with the rat tapeworm, Hymenolepis diminuta , used as a model helminth, or systemic delivery of worm antigen (HdAg) significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice. Extending these analyses, intraperitoneal injection of HdAg dose-dependently suppressed dextran sodium sulfate (DSS)-induced colitis, and this was paralleled by reduced gamma interferon (IFN-γ), interleukin-17 (IL-17), and tumor necrosis factor alpha (TNF-α) production and increased IL-10 production from mitogen-activated splenocytes. Treatment with HdAg resulted in a CCR2-dependent recruitment of CDllb + F4/80 + Ly6C hi Gr-1 lo monocyte-like cells into the peritoneum 24 h later that were predominantly programmed death ligand 1 (PD-L1) positive and CXCR2 negative. In ...

Infection with helminth parasites and treatment with worm extracts can suppress inflammatory disease, including colitis. Postulating that dendritic cells (DCs) participated in the suppression of inflammation and seeking to move beyond the use of helminths per se, we tested the ability of Hymenolepis diminuta antigen-pulsed DCs to suppress colitis as a novel cell-based immunotherapy. Bone-marrow derived DCs pulsed with H. diminuta antigen (HD-DCs), or PBS-, BSA- or LPS-DCs as controls, were transferred into wild-type (WT), interleukin-10 (IL-10) knock-out (KO), and RAG-1 KO mice, and the impact on dinitrobenzene sulphonic acid (DNBS)-induced colitis and splenic cytokine production assessed 72 h later. Mice receiving HD-DCs were significantly protected from DNBS-induced colitis and of the experimental groups only these mice displayed increased Th2 cytokines and IL-10 production. Adoptive transfer of HD-DCs protected neither RAG-1 nor IL-10 KO mice from DNBS-colitis. Furthermore, the t...

Regulatory T cells (Tregs) play a critical role in the maintenance of airway tolerance. We report that inhaled soluble Ag induces adaptive Foxp3+ Tregs, as well as a regulatory population of CD4+ T cells in the lungs and lung-draining lymph nodes that express latency-associated peptide (LAP) on their cell surface but do not express Foxp3. Blocking the cytokine IL-10 or TGF-β prevented the generation of LAP+ Tregs and Foxp3+ Tregs in vivo, and the LAP+ Tregs could also be generated concomitantly with Foxp3+ Tregs in vitro by culturing naive CD4+ T cells with Ag and exogenous TGF-β. The LAP+ Tregs strongly suppressed naive CD4+ T cell proliferation, and transfer of sorted OVA-specific LAP+ Tregs in vivo inhibited allergic eosinophilia and Th2 cytokine expression in the lung, either when present at the time of Th2 sensitization or when injected after Th2 cells were formed. Furthermore, inflammatory innate stimuli from house dust mite extract, nucleotide-binding oligomerization domain c...

Effector cells derived from central memory CD8 + T cells were reported to engraft and survive better than those derived from effector memory populations, suggesting that they are superior for use in adoptive immunotherapy studies. However, previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve, rather than central memory T cells, gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells, but did not acquire the expression of KLRG-1, a marker for terminal differentiation and replicative senescence. Consistent with thi...

CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-γ (IFN-γ)...

Graft-versus-tumor effects can be achieved after allogeneic bone marrow transplantation in patients with malignancies of the kidney or hematopoietic system but are often accompanied by severe graft-versus-host-disease (GVHD). We sought to maximize graft-versus-tumor while minimizing GVHD using tumor-specific allogeneic effector T cells rather than open-repertoire T cells. We transferred allogeneic CD8+ pmel-1 or CD4+ TRP-1 T cells specific for the melanoma-associated antigens, glycoprotein 100 (gp100) and tyrosinase-related protein-1 (TRP-1), respectively, into B16-melanoma–bearing mice. Mice receiving a preparative regimen of nonmyeloablating (5 Gy) total body irradiation experienced the rapid rejection of tumor-specific allogeneic lymphocytes with no impact on tumor growth. However, when mice were given more intense total body irradiation conditioning regimens combined with autologous bone marrow transplantation, adoptively transferred allogeneic tumor-specific T lymphocytes persi...

Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) is an inflammatory demyelination that recapitulates in mouse spinal cord (SC) the human multiple sclerosis disease. We now analyze previously reported cDNA array data from age-matched young female adult control and passively myelin antigen-sensitized EAE mice with regard to organizational principles of the SC transcriptome in autoimmune demyelination. Although AT-EAE had a large impact on immune response genes, broader functional and chromosomal gene cohorts were neither significantly regulated nor showed significant changes in expression coordination. However, overall transcriptional control was increased in AT-EAE and the proportions of transcript abundances were perturbed within each cohort. Striking likenesses and oppositions were identified in the coordination profiles of genes related to myelination, calcium signaling, and inflammatory response in controls that were substantially altered in AT-EAE. We propose that up-or down-regulation of genes linked to those targeted by the disease could potentially compensate for the pathological transcriptomic changes.

Adoptive cell transfers (ACTs) can interact specifically with inflamed tissues, but lack a mechanism for transport through viscous biological barriers such as mucus when administered locally. Further, maintaining cell function is challenging due to the loss of cellular phenotypes in diseased microenvironments. In this work, the use of magnetically controlled helical microrobots is examined to transport macrophages through physiologically representative mucus and maintain functional phenotypes through drug elution for improved cell delivery. Two-photon lithography and sputter coating are used to fabricate helical particles that are embedded with a small molecule drug, dexamethasone, and coated with nickel and titanium. The attachment of macrophages to the helices and the analysis of how the cells influence the trajectories of cell-helix complexes when exposed to a rotating magnetic field are investigated. Additionally, the transport of complexes is compared in aqueous solutions and artificial mucus, noting a nonzero cell-helix complex velocity in the most viscous mucus for potential therapeutic implications. It is found that attached macrophages display no loss of viability after actuation and exhibit directed polarization toward a prescribed anti-inflammatory phenotype due to the sustained release of dexamethasone from the microrobots. This system provides a proof-of-concept for using magnetically controlled ACTs to enhance the treatment of inflammatory diseases.

Influenza infection results in considerable pulmonary pathology, a significant component of which is mediated by CD8(+) T cell effector functions. To isolate the specific contribution of CD8(+) T cells to lung immunopathology, we utilized a non-viral murine model in which alveolar epithelial cells express an influenza antigen and injury is initiated by adoptively transferring influenza-specific CD8(+) T cells. Here, we report that IFN-γ production by adoptively transferred influenza-specific CD8(+) T cells is a significant contributor to acute lung injury following influenza antigen recognition, in isolation from its impact on viral clearance. CD8(+) T cell production of IFN-γ enhanced lung epithelial cell expression of chemokines, and the subsequent recruitment of inflammatory cells into the airways. Surprisingly, Stat1-deficiency in the adoptive-transfer recipients exacerbated the lung injury that was mediated by the transferred influenza-specific CD8(+) T cells but was still depe...

Pediatric patients with recurrent brain tumors have a poor prognosis and limited therapeutic options. We investigated the use of high-dose chemotherapy with adoptive immunotherapy for recurrent brain tumors. Three pediatric patients with recurrent brain tumors received high-dose chemotherapy. This was followed by adoptive transfer of ex-vivo expanded T-cells. The T-cells were generated from peripheral blood after immunization with autologous cancer cells. The objectives of this study included (1) establishing the safety and feasibility of this potential treatment, (2) measuring changes in immune response after high-dose chemotherapy and adoptive immunotherapy, and (3) determining whether adoptive immunotherapy would be able to translate into a clinical response. Immune function was tested in all patients at the time of enrollment into the study. Humoral responses to recall antigens delayed-type hypersensitivity (DTH) were intact in all patients. After immunizing patients with autolo...

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of alphabeta T cells. Nevertheless, antibody treatment did not prevent the development of lethal encephalitis. On the contrary, the adoptive transfer of primed CD4(+) T cells was necessary to prevent lethal encephalitis in mice lacking alphabeta T cell receptor.

The pathways underlying dendritic cell (DC) activation in allergic asthma are incompletely understood. Here we demonstrate that adoptive transfer of ovalbumin-pulsed wild-type (wt) but not of C5a receptor-deficient (C5aR À / À ) bone marrow (BM)-derived DCs (BMDCs) induced mixed T helper type 2 (Th2)/Th17 maladaptive immunity, associated with severe airway hyperresponsiveness, mucus production, and mixed eosinophilic/neutrophilic inflammation. Mechanistically, antigen uptake, processing, and CD11b expression were reduced in C5aR À / À BMDCs. Further, interleukin (IL)-1b, -6, and -23 production were impaired resulting in reduced Th17 cell differentiation, associated with accelerated activated T-cell death in vitro and in vivo. Surprisingly, we found an increased frequency of CD11b hi CD11c int Gr1 þ F4/80 þ cells, expressing arginase and nitric oxide synthase in C5aR À / À BM preparations. Intratracheal administration of ovalbumin-pulsed wt DCs and sorted CD11b hi CD11c int Gr1 þ F4/80 þ C5aR À / À cells reduced Th2 immune responses in vivo. Together, we uncover novel roles for C5aR in Th17 differentiation, T-cell survival, and differentiation of a DC-suppressor population controlling Th2 immunity in experimental allergic asthma.

CD4 + helper T (Th) cells play pivotal roles in induction of CD8 + CTL immunity. However, the mechanism of CD4 + T cell help delivery to CD8 + T cells in vivo is still elusive. In this study, we used ovalbumin (OVA)-pulsed dendritic cells (DC OVA ) to activate OT-II mouse CD4 + T cells, and then studied the help effect of these CD4 + T cells on CD8 + cytotoxic T lymphocyte (CTL) responses. We also examined CTL mediated islet  cell destruction which led to diabetes in wild-type C57BL/6 mice and transgenic rat insulin promoter (RIP)-mOVA mice expressing  cell antigen OVA with self OVA-specific tolerance, respectively. In adoptive transfer experiments, we demonstrated that help, in the form of peptide/major histocompatibility complex (pMHC) I acquired from DC OVA by DC OVA activation, was required for induction of OVA-specific CTL responses in C57BL/6 mice. However, in combination with TCR transgenic OT-I mouse CD8 + T cells, the tolerogenic dosage of CD4 + Th cells with acquired pMHC I, but not CD4 + (K b-/-) Th cells without acquired pMHC I were able to cause diabetes in 8/10 (80%) RIP-mOVA mice. This study thus expands the current knowledge in T cell-mediated autoimmunity and provides insight into the nature of CD4 + T cell-mediated help in CD8 + CTL induction.

Recent clinical studies demonstrate the high potency of regulatory T cells (Tregs) to control graft-versus-host disease in hematopoietic stem cell transplantation (SCT). However, the adoptive transfer of Tregs is limited by their low frequency in unstimulated donors and considerable concerns that G-CSF induced SC mobilization might have negative effects on the stability and function of Tregs. The isolation of Tregs from the G-CSF mobilized SC grafts would extend this novel strategy for tolerance induction to the unrelated setting and simplify global clinical application. We characterized CD4 + CD25 high CD127 2 Tregs from SC donors before and after G-CSF mobilization for their phenotype, function, and stability. After G-CSF application the Treg cell yield increased significantly. Donor Tregs retained their cytokine profile, phenotypic characteristics and in vitro expansion capacity after SC mobilization. Most importantly, in vivo G-CSF stimulated Tregs remained highly suppressive on the proliferation of effector T cells, also after in vitro expansion, and displayed a stable phenotype in epigenetic studies. The surface expression of CXCR3 is transiently reduced. However, donor-derived Tregs maintain their migratory properties after G-CSF stimulation. Therefore, the adoptive transfer of Tregs from G-CSF mobilized SC donors seems to be a feasible and safe strategy for clinical application in allogeneic SCT.

Pancreatic cancer is the fourth leading cause of cancer mortality in both men and women. Approximately 32,000 Americans each year will develop and also die from this disease . Despite aggressive surgical and medical management, the mean life expectancy is approximately 15-18 months for patients with local and regional disease, and 3-6 months for patients with metastatic disease 1-2. Even in case of radical surgery it is associated with a poor prognosis and a 5-year survival rate of less than 4%. Early detection methods are under development but do not yet exist in practice for pancreatic cancer. Therefore, most patients present with advanced disease that cannot be cured by surgery (pancreaticoduodenectomy). Clinically, pancreatic cancer is characterized by rapid tumor progression, early metastatization and unresponsiveness to most conventional treatment modalities. In a recent analysis using a database from 1973 to 2003 based on modeled period analysis, 5-year survival of pancreatic cancer patients was 7.1% and 10-year survival was below 5%3. The survival rate is apparently related to the disease stage with a low rate at 1.6-3.3% among patients with distant metastases. Curative resection remains the most important factor determining outcome for resectable tumors. However, the resection rate for pancreatic carcinoma is only 10% and the overall five-year survival rate after resection is still only 10 to 20%. Early diagnosis and effective treatment to control the advanced stages of disease may prolong the survival rate of pancreatic cancer. Otherwise pancreatic cancer remains a disease with high mortality despite numerous efforts that have been made to improve its survival rates. In developing cancer immunotherapy, the following aims must be considered: (1) detection of immune response to autologous tumor cells, (2) identification of tumor antigens and analysis of the immune responses in patients, (3) analysis of tumor escape mechanisms and development of methods to overcome them, and (4) development of a more efficient immune intervention system by way of animal model experiments and clinical trials. Identification of tumor antigens in the first objective is important because it subsequently allows their use not only as targets for immunotherapy in a more immunogenic form but also enables quantitative and qualitative monitoring of immune responses to tumor cells during immunotherapy. In many animal tumors and in human melanoma, T cells play an www.intechopen.com Pancreatic Cancer -Clinical Management 138 important role in in vivo tumor rejection. Because of their expression of MHC class I, CD8+ T cells are integral in the eradication of most solid tumors. However, CD4+ T cells are also important in the induction and maintenance of final effectors, such as CD8+ T cells and macrophages, as well as for the accumulation of CD8+ T cells in tumor tissues. Thus, we are applying various methods to identify human tumor antigens recognized by T cells. Immunotherapy has an advantage over radiation therapy and chemotherapy because it can act specifically against the tumor without damaging normal tissue. Immunotherapeutic approaches to PC have included the use of monoclonal antibodies 47, cytokines 8, vaccine 9 and lymphokine activated killer (LAK) cells (10). The extraordinary features of the immune system make it possible to discern self from nonself. However, most human cancers, and pancreatic cancer in particular, are known to be poorly immunogenic, as crucial somatic genetic mutations can generate pancreatic cancer proteins that are essentially altered self proteins. Furthermore, promising immunotherapeutic approaches that have been used for relatively immunogenic cancers such as melanoma have met with variable success 6 . These observations have revealed that for tumours to form and progress, they must develop local and/or systemic mechanisms that subsequently allow them to escape the normal surveillance mechanisms of the intact immune system. Immune-based therapies must therefore incorporate at least one agent against a pancreatic cancer target as well as one or more agents that will modify both local and systemic mechanisms of pancreatic-cancer-induced IMMUNE TOLERANCE. It is now clear that both local characteristics of the tumour microenvironment as well as systemic factors are important for the immune evasion of tumours. For example, T-cell recognition of pancreatic tumours might be inhibited or suppressed due to the downregulation of human leukocyte antigen (HLA) CLASS I tumour-antigen complexes on tumour cells by a range of intracellular mechanisms 4, 7 -upregulation of immuneinhibition molecules 11,

Professor Anahid Jewett, Co-chair Professor Ichiro Nishimura, Co-chair NK cells are known to limit growth and expansion of cancer stem cells and oral tumors by providing key cytokines such as IFN-γ and TNF-α, which drive differentiation of stem-like/poorly differentiated cancer stem cells. The detailed understanding of how and why their function is affected by the cancer microenvironment is very important for outlining the most efficient NK cell-based immunotherapies for cancer patients. In this regard, we first characterized NK cells from the cancer patients for their IFN-γ secretion, cytotoxic function against the cancer stem cells, and their surface receptors. In previous studies, we demonstrated that IFN-γ plays a dominant role in up-regulation of B7H1, CD54 and MHC1 surface receptor expression and inhibition of tumor growth by differentiating the cancer stem cells, and via inflammatory cytokine release when they are in contact with cancer stem cells. We also showed that induction of NK cell-mediated I owe my deepest gratitude to my advisors, Dr. Anahid Jewett and Dr. Ichiro Nishimura, for their excellent guidance, mentoring, support and encouragement from day I joined the lab. I was fortunate enough to work and develop under tutelage of two successful and well respected researchers in their own field. Dr. Jewett is patient, caring, and provides an excellent atmosphere for professional development in her lab. Her intellectual knowledge, thinking, enthusiasm and devotion to work have been and will always be a source of inspiration to me in both my professional and personal life. On other hand, Dr. Ichiro Nishimura nurtured me to be an independent thinker and provides different prospective to my project. His dedication to detail and passion for science always motivates me to give my best. A special thanks to my committee members Dr. Nicholas Cacalano, Dr. Rueben Kim, and Dr. Gwack Yousang for their expertise and precious time. They were always there for their generous help and great advice. Special thanks to my parents, parent-in-laws, brothers, brother-in-law and sister-in-laws. Last, but definitely not least, my lab members and colleagues, especially Jessica Cook and Brett Ploussard, for being the are best students.

Based on data obtained from oral, pancreatic and lung cancers, glioblastoma, and melanoma, we have established that Natural Killer (NK) cells target cancer stem-like cells (CSCs). CSCs displaying low MHC class I, CD54, and PD-L1 are killed by cytotoxic NK cells, and are differentiated by split anergized NK cells through both membrane bound and secreted forms of TNF-α and IFN-γ. NK cells select and differentiate both healthy and transformed stem-like cells, resulting in target cell maturation and shaping of their microenvironment. In our recent studies, we have observed that oral, pancreatic and melanoma CSCs were capable of forming large tumors in humanized bone marrow, liver, thymus (hu-BLT) mice with fully reconstituted human immune system. In addition, major human immune subsets including NK cells, T cells, B cells and monocytes were present in the spleen, bone marrow, blood, and tumor microenvironment. Similar to our previously published in vitro data, CSCs differentiated with split anergized NK cells prior to implantation in mice formed smaller tumors. Intravenous injection of functionally potent osteoclast-expanded NK cells inhibited tumor growth through differentiation of CSCs in humanized mice. In this review, we present current approaches, advances and existing limitations in studying interactions of the immune system with tumor, in particular NK cells with CSCs in, in vivo preclinical hu-BLT mouse model. In addition, we discuss the use of osteoclast-expanded NK cells in targeting cancer stem-like tumors in humanized mice -a strategy that provides a muchneeded platform to develop effective cancer immunotherapies.

Development of mucosal immunity and tolerance requires coordinated expression of a number of genes within the mucosaassociated lymphoid tissue (MALT). To study the roles of these genes in the MALT, we have established a MALT-specific gene transfer model using replication-defective adenovirus as vector. In this model, the target gene of interest is directly delivered into the Peyer's patch by intra-Peyer's patch injection of the recombinant virus. Using this gene transfer model, we investigated the roles of B7-1 and IL-12 in the development of mucosal tolerance. We found that intra-Peyer's patch injection of OVA induced Ag-specific T cell hyporesponsiveness, as manifested by decreased T cell proliferation and IL-2/IFN-␥ production upon subsequent immune challenge. Intra-Peyer's patch B7-1 gene transfer at the time of OVA administration partially reversed the inhibition of T cell proliferation and IL-2 secretion, but had no effect on IFN-␥ production. By contrast, intra-Peyer's patch IL-12 gene transfer completely restored T cell proliferation and IFN-␥ secretion and partially reversed IL-2 inhibition. Using an adoptive TCR transgenic model, we further demonstrated that B7 and IL-12 played distinct roles during the inductive phase of mucosal tolerance. B7 selectively increased T cell proliferation and IL-2 secretion without affecting IFN-␥ production, whereas IL-12 increased both IL-2 and IFN-␥ production. These results indicate that B7 alone may not be sufficient to abrogate mucosal tolerance, and that cytokines such as IL-12 may also be required. Based on these findings, we propose a new model to explain the paradoxical roles of B7 in mucosal immunity and tolerance.

Development of mucosal immunity and tolerance requires coordinated expression of a number of genes within the mucosa-associated lymphoid tissue (MALT). To study the roles of these genes in the MALT, we have established a MALT-specific gene transfer model using replication-defective adenovirus as vector. In this model, the target gene of interest is directly delivered into the Peyer’s patch by intra-Peyer’s patch injection of the recombinant virus. Using this gene transfer model, we investigated the roles of B7-1 and IL-12 in the development of mucosal tolerance. We found that intra-Peyer’s patch injection of OVA induced Ag-specific T cell hyporesponsiveness, as manifested by decreased T cell proliferation and IL-2/IFN-γ production upon subsequent immune challenge. Intra-Peyer’s patch B7-1 gene transfer at the time of OVA administration partially reversed the inhibition of T cell proliferation and IL-2 secretion, but had no effect on IFN-γ production. By contrast, intra-Peyer’s patch...

The adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has produced tumor responses even in patients with refractory diseases. However, the paucity of antigens that are tumor selective has resulted, on occasion, in "on-target, off-tumor" toxicities. To address this issue, we developed an approach to render T cells responsive to an expression pattern present exclusively at the tumor by using a trio of novel chimeric receptors. Using pancreatic cancer as a model, we demonstrate how T cells engineered with receptors that recognize PSCA, TGFβ, and IL4, and whose endodomains recapitulate physiologic T cell signaling by providing signals for activation, co-stimulation and cytokine support, produce potent anti-tumor effects selectively at the tumor site. In addition, this strategy has the benefit of rendering our cells resistant to otherwise immunosuppressive cytokines (TGFβ and IL4) and can be readily extended to other inhibitory molecules present at the tumor...

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) can induce objective clinical responses in patients with malignant diseases. The option of providing a proliferative and survival advantage to adoptively transferred CTLs remains a challenge to improve their efficacy. Host lymphodepletion and administration of recombinant interleukin-2 (IL-2) are currently used to improve CTL survival and expansion after adoptive transfer, but these approaches are frequently associated with significant side effects and may increase proliferation of T regulatory cells. IL-7 is a crucial homeostatic cytokine that has been safely administered as a recombinant protein. However, while IL-7 induces robust expansion of naive and memory T lymphocytes, the lack of expression of the IL-7 receptor α chain (IL-7Rα) by CTLs precludes their response to this cytokine. We found that CTLs can be genetically modified to re-express IL-7Rα, and that this manipulation restores the response of these cells to IL-7 without apparent modification of their antigen specificity or dependency, and without changing their response to other common γ (γc) chain cytokines. This approach may allow selective expansion of CTLs without the unwanted effects associated with IL-2.

SummaryThe adoptive transfer of T cells specific for native tumor antigens (TAs) is an increasingly popular cancer treatment option because of the ability of these cells to discriminate between normal and tumor tissues and the corresponding lack of short or long‐term toxicities. Infusions of antigen‐specific CD4+ and CD8+ T cells targeting viral antigens derived from Epstein–Barr virus (EBV) induce sustained complete tumor remissions in patients with highly immunogenic tumors such as post‐transplant lymphoproliferative disease, although resistance occurred when the infused T‐cell population had restricted antigen specificity. T cells specific for EBV antigens have also produced complete remissions of EBV‐positive nasopharyngeal carcinomas and lymphomas developing in immunocompetent individuals, even though in these patients tumor survival is dependent on their ability to evade T‐cell immunity. Adapting this strategy to non‐viral tumors is more challenging, as the target antigens exp...

Objective: Conventional chemotherapy and radiotherapy produce marginal survival benefits in pancreatic cancer, underscoring the need for novel therapies. The aim of this study is to develop an adoptive T cell transfer approach to target tumours expressing prostate stem cell antigen (PSCA), a tumourassociated antigen that is frequently expressed by pancreatic cancer cells. Methods: Expression of PSCA on cell lines and primary tumour samples was confirmed by immunohistochemistry. Healthy donor-and patient-derived T cells were isolated, activated in vitro using CD3/ CD28, and transduced with a retroviral vector encoding a chimeric antigen receptor (CAR) targeting PSCA. The ability of these cells to kill tumour cells was analysed by chromium-51 (Cr 51 ) release. Results: Prostate stem cell antigen was expressed on >70% of the primary tumour samples screened. Activated, CAR-modified T cells could be readily generated in clinically relevant numbers and were specifically able to kill PSCA-expressing pancreatic cancer cell lines with no non-specific killing of PSCA-negative target cells, thus indicating the potential efficacy and safety of this approach. Conclusions: Prostate stem cell antigen is frequently expressed on pancreatic cancer cells and can be targeted for immune-mediated destruction using CAR-modified, adoptively transferred T cells. The safety and efficacy of this approach indicate that it deserves further study and may represent a promising novel treatment for patients with pancreatic cancer.

There has been interest in generating T cells expressing chimeric artificial receptors (CARs) targeting CD19/CD20 antigens to treat B-cell lymphomas. If successful, however, this approach would likely impair humoral immunity because T cells may persist long-term. Most low-grade lymphoma and chronic lymphocytic leukemia (B-CLL) cells express monoclonal immunoglobulins carrying either κ or λ light chains. We, therefore, explored whether T lymphocytes could be genetically modified to target the tumor-associated light chain, sparing B lymphocytes expressing the reciprocal light chain, and consequently reduce impairment of humoral immunity. We found that T lymphocytes expressing the anti-κ light chain CAR showed cytotoxic activity against Igκ+ tumor cell lines and B-CLL cells both in vitro and in vivo. We also found that the incorporation of the CD28 endodomain within the CAR enhanced the in vitro and in vivo expansion of transgenic T cells after tumor-associated antigen stimulation. Fre...

Hodgkin lymphoma (NHL). However, long-term outcomes of auto HCT for HL and NHL have not been well described. We conducted a retrospective cohort study to evaluate the probability and risk factors for long-term survival and late relapse in this population. Recipients of auto HCT for HL (N 5 407) and NHL (N 5 960) between 1990 and 1998 reported to the CIBMTR and in continuous complete remission (CR) for at least 2 years post-HCT were included in this study . The median followup was 104 (range, 25-203) months for HL and 107 (range, months for NHL. NHL subtypes were follicular (30%), diffuse large cell (45%), mantle cell (6%) and other (7%). The median age of HL patients was 31 years compared to 47 years for NHL; more HL patients were \20 years (15% vs 5%) while NHL patients were .50 years (6% vs 40%). Patients were predominantly White (87%), male (60%), with chemosensitive disease (82%),and KPS score $90 (67%). A greater proportion of HL patients had history of B symptoms (52% vs 34%) and were $12 months from diagnosis (90% vs 59%) while use of total body irradiation (TBI) was more prevalent for NHL (94% vs 64%). The probability of overall survival (OS) and cumulative incidence of relapse and non-relapse mortality (NRM) at 10-years after HCT was 76% (95% CI, 73-79%), 17% (13-22) and 13% (9-17%) for HL. The 10-year rates of OS, relapse and NRM for NHL were 77% (72-82%), 26% (23-30%) and 11% (9-13%). On multivariate analysis, OS (Table ) was significantly associated with histology, age at HCT and time from diagnosis to HCT. Factors predictive for relapse included histology (compared to HL, relative risk (RR) 1.5 (1.0-2.2) for follicular, 1.4 (0.9-1.9) for diffuse large cell, 1.4 (0.8-2.8) for lymphoblastic/Burkitts, 4.6 (2.8-7.6) for mantle cell and 1.5 (0.9-2.4) for other NHL), male gender (RR 1.3 (1.0-1.6)), use of TBI (RR 1.6 (1.2-2.1)), use of growth factors (RR 1.5 (1.1-2.0)) and time since diagnosis of $12 months (RR 1.9 (1.5-2.6)) while KPS score of $90 at HCT decreased relapse risk (RR 0.8 (0.6-1.0)). Recipients of auto HCT for HL and NHL who remain in remission for at least 2 years have a very favorable subsequent long-term survival. However, they remain at risk for late relapse and NRM. Mantle cell histology, older age at HCT and increasing time between diagnosis and HCT are predictive of adverse long-term survival.

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) has shown promising anti-tumor activity in early phase clinical studies, especially for hematological malignancies. However, most preclinical models do not reliably mimic human disease. We reasoned that developing an adoptive T-cell therapy approach for spontaneous osteosarcoma (OS) occurring in dogs would more closely reproduce the condition in human cancer. To generate CAR-expressing canine T cells we developed expansion and transduction protocols that allow for the generation of sufficient numbers of CAR-expressing canine T cells for future clinical studies in dogs within 2 weeks of ex vivo culture. To evaluate the functionality of CAR-expressing canine T cells we targeted HER2-positive OS. We demonstrate that canine OS is positive for HER2, and that canine T cells expressing a HER2-specific CAR with human-derived transmembrane and CD28.ζ signaling domains recognize and kill HER2-positive canine OS cell lines in an antigen-dependent manner. To reduce the potential immunogenicity of the CAR we evaluated a CAR with caninederived transmembrane and signaling domains, and found no functional difference between human and canine CARs. Hence, we have successfully developed a strategy to generate CAR-

An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease. We used ovalbumin (OVA)-based mouse models of asthma and primary CD4(+) T cells. C57BL/6J WT or PARP-1(-/-) mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge. Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiv...

T-bet-expressing Th17 (T-bet+RORγt+) cells are associated with the induction of pathology during experimental autoimmune encephalomyelitis (EAE) and the encephalitic nature of these Th17 cells can be explained by their ability to produce GM-CSF. However, the upstream regulatory mechanisms that control Csf2 (gene encoding GM-CSF) expression are still unclear. In this study, we found that Th17 cells dynamically expressed GATA3, the master transcription factor for Th2 cell differentiation, during their differentiation both in vitro and in vivo. Early deletion of Gata3 in three complimentary conditional knockout models by Cre-ERT2, hCd2Cre and Tbx21Cre, respectively, limited the pathogenicity of Th17 cells during EAE, which was correlated with a defect in generating pathogenic T-bet-expressing Th17 cells. These results indicate that early GATA3-dependent gene regulation is critically required to generate a de novo encephalitogenic Th17 response. Furthermore, a late deletion of Gata3 via...

We herein report retargeting of T-helper (Th) cells against the universal cancer antigen telomerase for use in adoptive cell therapy. The redirected Th cells may counter tumor tolerance, transform the inflammatory milieu, and induce epitope spreading and cancer senescence. We have previously conducted a series of trials evaluating vaccination with telomerase peptides. From long-term survivors, we isolated >100 CD4 C Th-cell clones recognizing telomerase epitopes. The clones were characterized with regard to HLA restriction, functional avidity, fine specificity, proliferative capacity, cytokine profile, and recognition of naturally processed epitopes. DP4 is the most prevalent HLA molecule worldwide. Two DP4-restricted Tcell clones with different functional avidity, C13 and D71, were selected for molecular T-cell receptor (TCR) cloning. Both clones showed a high proliferative capacity, recognition of naturally processed telomerase epitopes, and a polyfunctional and Th1-weighted cytokine profile. TCR C13 and D71 were cloned into the retroviral vector MP71 together with the compact and GMP-applicable marker/suicide gene RQR8. Both TCRs were expressed well in recipient T cells after PBMC transduction. The transduced T cells co-expressed RQR8 and acquired the desired telomerase specificity, with a polyfunctional response including production of TNFa, IFNg, and CD107a. Interestingly, the DP4-restricted TCRs were expressed and functional both in CD4 C and CD8 C T cells. The findings demonstrate that the cloned TCRs confer recipient T cells with the desired hTERT-specificity and functionality. We hypothesize that adoptive therapy with Th cells may offer a powerful novel approach for overcoming tumor tolerance and synergize with other forms of immunotherapy.

Adoptive cell transfers (ACTs) constitute an emerging platform for improving the systemic delivery of nano-and microparticle systems. Macrophages (M𝝋) are an attractive cell type for particle-carrying ACTs because their attachment, phagocytosis, and chemotaxis can improve pharmacokinetics and reduce off-target effects. However, little is known about how macrophage transport and function change when carrying particles of different shapes, or whether these changes can be leveraged for improving ACTs. This work investigates macrophage interactions with biodegradable spherical and discoidal particles to promote or suppress phagocytosis, respectively. Adoptively transferred macrophages with internalized spheres (M𝝋-S) or surface-bound discs (M𝝋-D) possess enhanced targeted delivery to solid tumors compared to conventional free microparticle administration by 5.2-fold and exhibit distinct phenotypic profiles within the cold B16-F10 tumor microenvironment. Moreover, phenotypic changes are evaluated upon particle association by profiling the transcriptional, chromatin accessibility, and protein state. M𝝋-S complexes adopt epigenetic changes and key biomarkers associated with a proinflammatory phenotype. In M𝝋-D complexes, a diverse chromatin and protein landscape with simultaneous upregulation of both pro-and anti-inflammatory biomarkers is observed, suggesting functional flexibility is observed. These findings suggest particle shape can be used as a design parameter to influence the function of adoptive macrophage transfers without compromising their delivery performance.

Among pediatric cancers, malignancies of the brain carry significant morbidity and mortality, harboring dismal prognosis and poor outcomes. Our group has previously shown that adoptive cellular therapy (ACT) provides therapeutic benefit against central nervous system (CNS) malignancies, including medulloblastoma and high-grade glioma. In this study, we characterized cell cycling differences of immune sub-populations in peripheral lymphoid and tumor tissue compartments in vivo. ACT was conducted in orthotopic KR158 luciferase tumor-bearing mice, a syngeneic murine glioma model. Five days following intracranial implantation of tumor cells, mice were treated with ACT which included the following elements: total body irradiation, hematopoietic stem cell transplantation, one dose of tumor-activated splenocytes and three weekly doses of dendritic cells that had been electroporated with tumor-derived ribonucleic acid. One week after the completion of ACT, bromodeoxyuridine (BRDU) was admin...

Esophageal cancer is an exceedingly aggressive and malignant cancer that imposes a substantial burden on patients and their families. It is usually treated with surgery, chemotherapy, radiotherapy, and molecular-targeted therapy. Immunotherapy is a novel treatment modality for esophageal cancer wherein genetically engineered adoptive cell therapy is utilized, which modifies immune cells to attack cancer cells. Using chimeric antigen receptor (CAR) or T cell receptor (TCR) modified T cells yielded demonstrably encouraging efficacy in patients. CAR-T cell therapy has shown robust clinical results for malignant hematological diseases, particularly in B cell-derived malignancies. Natural killer (NK) cells could serve as another reliable and safe CAR engineering platform, and CAR-NK cell therapy could be a more generalized approach for cancer immunotherapy because NK cells are histocompatibility-independent. TCR-T cells can detect a broad range of targeted antigens within subcellular com...

Purpose: Most patients diagnosed with ovarian cancer will ultimately die from their disease. For this reason, novel approaches to the treatment of this malignancy are needed. Adoptive transfer of a patient's own T cells, genetically modified ex vivo through the introduction of a gene encoding a chimeric antigen receptor (CAR) targeted to a tumor-associated antigen, is a novel approach to the treatment of ovarian cancer. Experimental Design: We have generated several CARs targeted to the retained extracellular domain of MUC16, termed MUC-CD, an antigen expressed on most ovarian carcinomas. We investigate the in vitro biology of human T cells retrovirally transduced to express these CARs by coculture assays on artificial antigen-presenting cells as well as by cytotoxicity and cytokine release assays using the human MUC-CD+ ovarian tumor cell lines and primary patient tumor cells. Further, we assess the in vivo antitumor efficacy of MUC-CD–targeted T cells in SCID-Beige mice bearin...

We have previously reported that oral administration of allogeneic rat spleen cells before kidney allotransplantation significantly prolongs graft survival. This prolongation was alloantigen specific and was associated with a decrease in graft-infiltrating cells (GIC) and an increase in transcription of IL-4 mRNA in the GIC. In this study increased splenic mixed lymphocyte responses from animals orally exposed to alloantigen before kidney transplantation suggested that the kidney allograft prolongation was not due to a masking of allorecognition, but to an immunomodulation of the immune response. We have assessed GIC T cell subsets on day 5 post-transplant and found decreased numbers of CD4+ T cells in fed animals compared with controls, but there was no change in CD8+ T cell numbers. The CD8+ GIC from fed animals transcribed substantial levels of perforin, granzyme, and Fas ligand mRNA, indicating the presence of active CTL. Direct CTL assays showed that the GIC from fed recipients...

Objective Tracking the autoreactive T-cell migration in the pancreatic region after labeling with fluorinated nanoparticles (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate]-perfluoro-15-crown-5-ether nanoparticles, PDP-PFCE NPs) in a diabetic murine model using 19 F MRI. Materials and methods Synthesis of novel PDP-PFCE fluorine tracer was performed for in vitro labeling of T cells. Labeling conditions were optimized using different PDP-PFCE NPs concentrations. For in vivo 19 F MRI, mice were longitudinally followed after adoptive transfer of activated, autoreactive, labeled T cells in NOD.SCID mice. Results Established MR protocols were used for challenging T cell labeling to track inflammation in a model of diabetes after successful labeling of CD4+ and CD8+ T cells with PDP-PFCE NPs. However, T cells were difficult to be detected in vivo after their engraftment in animals. Discussion We showed successful in vitro labeling of T cells using novel fluorinated liposomal nanoparticles. However, insufficient and slow accumulation of labeled T cells and subsequent T cell proliferation in the pancreatic region remains as limitations of in vivo cell imaging by 19 F MRI.

ObjectiveDendritic cells (DCs) modulated with lipopolysaccharide (LPS) are able to reduce inflammation when therapeutically administered into mice with collagen‐induced arthritis (CIA). The aim of this study was to uncover the mechanisms that define the tolerogenic effect of short‐term LPS‐modulated DCs on CIA.MethodsBone marrow–derived DCs were stimulated for 4 hours with LPS and characterized for their expression of maturation markers and their cytokine secretion profiles. Stimulated cells were treated with SB203580 or SB431542 to inhibit the p38 or transforming growth factor β (TGFβ) receptor pathway, respectively, or were left unmodified and, on day 35 after CIA induction, were used to inoculate mice. Disease severity was evaluated clinically. CD4+ T cell populations were counted in the spleen and lymph nodes from inoculated or untreated mice with CIA. CD4+ splenic T cells were transferred from mice with CIA treated with LPS‐stimulated DCs or from untreated mice with CIA into ot...

The airway epithelial cell (AEC) response to allergens helps initiate and propagate allergic inflammation in asthma. CARMA3 is a scaffold protein that mediates G protein-coupled receptor-induced NF-kB activation in airway epithelium. In this study, we demonstrate that mice with CARMA3-deficient AECs have reduced airway inflammation, as well as reduced type 2 cytokine levels in response to Alternaria alternata. These mice also have reduced production of IL-33 and IL-25, and reduced numbers of innate lymphoid cells in the lung. We also show that CARMA3-deficient human AECs have decreased production of proasthmatic mediators in response to A. alternata. Finally, we show that CARMA3 interacts with inositol 1,4,5-trisphosphate receptors in AECs, and that inhibition of CARMA3 signaling reduces A. alternata-induced intracellular calcium release. In conclusion, we show that CARMA3 signaling in AECs helps mediate A. alternata-induced allergic airway inflammation, and that CARMA3 is an important signaling molecule for type 2 immune responses in the lung.

Background and Aims: Non-alcoholic steatohepatitis (NASH) is a multisystem condition, involving the liver, adipose tissue, and immune system. Regulatory T (Treg) cells are a subset of T cells that exert an immune-controlling effect. Previously, a reduction of Treg cells in the visceral adipose tissue (VAT) was shown to be associated with a more severe degree of liver disease. We aimed to correct this immune disruption through adoptive cell transfer (ACT) of Treg cells. Methods: Male 8-week-old C57BL/6J mice were fed a high-fat high-fructose diet (HFHFD) for 20 weeks. Treg cells were isolated from the spleens of healthy 8 to 10-week-old C57BL/6J mice and were adoptively transferred to HFHFD-fed mice. PBS-injected mice served as controls. Plasma ALT and lipid levels were determined. Liver and adipose tissue were assessed histologically. Cytotoxic T (Tc), Treg, T helper (Th) 1 and Th17 cells were characterized in VAT, liver, subcutaneous adipose tissue (SAT), blood, and spleen via flow cytometry. Gene expression analysis was performed in SAT and VAT of mice fed either the HFHFD or a control diet for 10-32 weeks. Results: ACT increased Treg cells in SAT, but not in any of the other tissues. Moreover, the ACT induced a decrease in Th1 cells in SAT, liver, blood, and spleen. Higher plasma ALT levels and a higher degree of steatosis were observed in ACT mice, whereas the other HFHFD-induced metabolic and histologic disruptions were unaffected. Expression analysis of genes related to Treg-cell proliferation revealed a HFHFD-induced decrease in all investigated genes in the SAT, while in the VAT the expression of these genes was largely unaffected, except for a decrease in Pparg. : ACT of Treg cells in HFHFD-fed mice exacerbated hepatic steatosis, which was possibly related to the increase of Treg cells in the SAT and/or the general decrease in Th1 cells. Moreover, the HFHFD-induced decrease in Pparg expression appeared critical in the decrease of Treg cells at the level of the VAT and the inability to replenish the amount Van Herck et al. Treg Cells in NASH of Treg cells by the ACT, while the mechanism of Treg cell accumulation at the level of the SAT remained unclear.

The steady flow of lactic acid (LA) from tumor cells to the extracellular space via the monocarboxylate transporter symport system suppresses antitumor T cell immunity. However, LA is a natural energy metabolite that can be oxidized in the mitochondria and could potentially stimulate T cells. Here we show that the lactate-lowering mood stabilizer lithium carbonate (LC) can inhibit LA-mediated CD8+ T cell immunosuppression. Cytoplasmic LA increased the pumping of protons into lysosomes. LC interfered with vacuolar ATPase to block lysosomal acidification and rescue lysosomal diacylglycerol–PKCθ signaling to facilitate monocarboxylate transporter 1 localization to mitochondrial membranes, thus transporting LA into the mitochondria as an energy source for CD8+ T cells. These findings indicate that targeting LA metabolism using LC could support cancer immunotherapy.

This study evaluates Arabic grammar (qawāʿid) instruction using visual wall charts in class XD at MA NU 03 Sunan Katong, Kaliwungu, Kendal, during the 2023/2024 academic year, assessing alignment with the SECTIONS framework. Employing a qualitative descriptive approach, the study involved the Arabic teacher, principal, and 33 students, with data collected via observations, interviews, documentation, and assessment records, analyzed using triangulation. Arabic knowledge, and enhanced engagement among 60.6% visual learners. Per the SECTIONS framework, the wall chart aligns with seven criteria (Students, Ease of Use, Cost, Teaching and Media Selection, Interaction, Organizational Issues, Security and Privacy). However, it lacks Networking alignment due to its non-digital nature. Wall charts are effective for qawāʿid in resource-limited settings, though digital integration could enhance learning resources. Future research should explore multimodal approaches to accommodate diverse learning styles.

This study evaluates Arabic grammar (qawāʿid) instruction using visual wall charts in class XD at MA NU 03 Sunan Katong, Kaliwungu, Kendal, during the 2023/2024 academic year, assessing alignment with the SECTIONS framework. Employing a qualitative descriptive approach, the study involved the Arabic teacher, principal, and 33 students, with data collected via observations, interviews, documentation, and assessment records, analyzed using triangulation. Arabic knowledge, and enhanced engagement among 60.6% visual learners. Per the SECTIONS framework, the wall chart aligns with seven criteria (Students, Ease of Use, Cost, Teaching and Media Selection, Interaction, Organizational Issues, Security and Privacy). However, it lacks Networking alignment due to its non-digital nature. Wall charts are effective for qawāʿid in resource-limited settings, though digital integration could enhance learning resources. Future research should explore multimodal approaches to accommodate diverse learning styles.