Transfer Of Antigen-specific CD8 Research Articles

Novel engineered B lymphocytes targeting islet-specific T cells inhibit the development of type 1 diabetes in non-obese diabetic Scid mice.

In this study, we report a novel therapeutic approach using B lymphocytes to attract islet-specific T cells in the non-obese diabetic (NOD) mouse model and prevent the development of autoimmune diabetes. Rather than using the antibody receptor of B cells, this approach utilizes their properties as antigen-presenting cells to T cells. Purified splenic B cells were treated with lipopolysaccharide, which increases regulatory B (Breg) cell function, then electroporated with mRNA encoding either chimeric MHC-I or MHC-II molecules covalently linked to antigenic peptides. Immunoregulatory functions of these engineered B cells (e-B cells) were tested by in vitro assays and in vivo co-transfer experiments with beta-cell-antigen-specific CD8+ or CD4+ T cells in NOD.Scid mice, respectively. The e-B cells expressing chimeric MHC-I-peptide inhibited antigen-specific CD8+ T-cell cytotoxicity in vitro. The e-B cells expressing chimeric MHC-II-peptide induced antigen-specific CD4+ T cells to express the regulatory markers, PD-1, ICOS, CTLA-4, Lag3, and Nrp1. Furthermore, e-B cells encoding the chimeric MHC-I and MHC-II peptide constructs protected NOD.Scid mice from autoimmune diabetes induced by transfer of antigen-specific CD8+ and CD4+ T cells. MHC-peptide chimeric e-B cells interacted with pathogenic T cells, and protected the host from autoimmune diabetes, in a mouse model. Thus, we have successfully expressed MHC-peptide constructs in B cells that selectively targeted antigen-specific cells, raising the possibility that this strategy could be used to endow different protective cell types to specifically regulate/remove pathogenic cells.

Oral immunization induces a novel CXCR6+ β7+ intraepithelial lymphocyte subset predominating in the small intestine.

Intestinal T cells form a central part of the front-line defence against foreign organisms and need to be situated in the mucosa where infection occurs. It is well accepted that immunization by a mucosal route favours localization of antigen-specific effector T cells in the mucosal epithelium, while systemic immunization does not. The aim of the study is to determine how homing receptors are specifically involved in retaining effector T cells in the small intestine after oral immunization. We here demonstrate that the chemokine receptor CXCR6, integrins β7 and CD29 contribute differentially to the epithelial retention phenotype of CD8+ T cells in the small intestine of mice. CD8+ intraepithelial lymphocytes (IELs) of unvaccinated mice are predominantly β7 single positives, and subcutaneous immunization-induced antigen-specific CD8+ effector IELs are mainly composed of CXCR6+ , CD29+ and CXCR6+ CD29+ cells. Strikingly, the majority of oral immunization-induced antigen-specific CD8+ effector IELs exhibit a distinct, tissue-specific CXCR6+ β7+ double-positive phenotype, cytotoxic potential and enhanced intraepithelial localization. Transfer of antigen-specific CD8+ T cells preactivated with certain immuno-stimuli (such as monophosphoryl lipid A) results in increased accumulation of donor IELs with the CXCR6+ β7+ phenotype. As β7 exclusively paired with αE on IELs, our results strongly suggest that CXCR6 may cooperate with the heterodimer αEβ7 to preferentially retain intestinally induced effector IELs in the epithelium. The identification of this novel IEL phenotype has significant implications for the development of vaccines and therapeutic strategies to enhance gut immunity.

NKG2D stimulation of CD8+ T cells during priming promotes their capacity to produce cytokines in response to viral infection in mice.

Natural killer group 2 member D (NKG2D) is an activating receptor that is expressed on most cytotoxic cells of the immune system, including NK cells, γδ, and CD8+ T cells. It is still a matter of debate whether and how NKG2D mediates priming of CD8+ T cells in vivo, due to a lack of studies where NKG2D is eliminated exclusively in these cells. Here, we studied the impact of NKG2D on effector CD8+ T-cell formation. NKG2D deficiency that is restricted to murine CD8+ T cells did not impair antigen-specific T-cell expansion following mouse CMV and lymphocytic choriomeningitis virus infection, but reduced their capacity to produce cytokines. Upon infection, conventional dendritic cells induce NKG2D ligands, which drive cytokine production on CD8+ T cells via the Dap10 signaling pathway. T-cell development, homing, and proliferation were not affected by NKG2D deficiency and cytotoxicity was only impaired when strong T-cell receptor (TCR) stimuli were used. Transfer of antigen-specific CD8+ T cells demonstrated that NKG2D deficiency attenuated their capacity to reduce viral loads. The inability of NKG2D-deficient cells to produce cytokines could be overcome with injection of IL-15 superagonist during priming. In summary, our data show that NKG2D has a nonredundant role in priming of CD8+ T cells to produce antiviral cytokines.

MP72-05 TOLL-LIKE RECEPTOR ACTIVATION IS ASSOCIATED WITH BLADDER INFLAMMATION, PELVIC PAIN AND VOIDING DYSFUNCTION IN A TRANSGENIC INTERSTITIAL CYSTITIS/BLADDER PAIN SYNDROME-LIKE ANIMAL MODEL: A MAPP RESEARCH NETWORK ANIMAL MODEL STUDY

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Interstitial Cystitis1 Apr 2016MP72-05 TOLL-LIKE RECEPTOR ACTIVATION IS ASSOCIATED WITH BLADDER INFLAMMATION, PELVIC PAIN AND VOIDING DYSFUNCTION IN A TRANSGENIC INTERSTITIAL CYSTITIS/BLADDER PAIN SYNDROME-LIKE ANIMAL MODEL: A MAPP RESEARCH NETWORK ANIMAL MODEL STUDY Yaoqin Wang, Suming Xu, Michael O'Donnell, Susan Lutgendorf, Pengchao Li, Lang Feng, Catherine Bradley, Bradley Erickson, Vincent Magnotta, Karl Kreder, and Yi Luo Yaoqin WangYaoqin Wang More articles by this author , Suming XuSuming Xu More articles by this author , Michael O'DonnellMichael O'Donnell More articles by this author , Susan LutgendorfSusan Lutgendorf More articles by this author , Pengchao LiPengchao Li More articles by this author , Lang FengLang Feng More articles by this author , Catherine BradleyCatherine Bradley More articles by this author , Bradley EricksonBradley Erickson More articles by this author , Vincent MagnottaVincent Magnotta More articles by this author , Karl KrederKarl Kreder More articles by this author , and Yi LuoYi Luo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1618AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Toll-like receptor (TLR) activation has been demonstrated in several chronic pain conditions but has not been well explored in IC/BPS. We previously showed that peripheral blood mononuclear cells from IC/BPS patients produced elevated proinflammatory cytokines in response to TLR2/4 stimulations, indicating the presence of altered peripheral TLR activation in IC/BPS. Here we used our validated transgenic autoimmune cystitis model (URO-OVA) that reproduces many clinical features of IC/BPS to further investigate whether central and peripheral TLR activations are associated with bladder inflammation, pelvic pain and voiding dysfunction seen in human IC/BPS. METHODS URO-OVA mice (n=8) were induced for cystitis by adoptive transfer of antigen specific CD8+ T cells. At day 7 after cystitis induction mice were analyzed for pelvic pain (von Frey filament stimulation), voiding dysfunction (micturition cages) and bladder inflammation (H&E staining). Splenocytes were prepared and stimulated with escalating doses of Pam3CSK4 (a TLR2 agonist) or lipopolysaccharide (LPS, a TLR4 agonist) ranging 10-5-102 µg/ml for 24 hrs, followed by ELISA analysis of proinflammatory cytokines in the conditioned culture supernatants. Lumbar spinal cords were processed for total RNA extraction and RT-PCR analysis of mRNAs for proinflammatory cytokines, TLR and glial activation markers. URO-OVA mice (n=8) not induced for cystitis were analyzed in parallel. RESULTS After cystitis induction URO-OVA mice developed profound bladder histopathology, increased pelvic responses to von Frey filament stimulation (p<0.05 for all applied forces ranging 0.04-4 g), increased urinary frequency (control 4.86±0.634 vs. cystitis 9.0±0.447 voids, p<0.001), and decreased average volume voided per micturition (control 0.26±0.036 vs. cystitis 0.14±0.016 g, p=0.022). These changes were associated with increased splenocyte production of IL-1ß, IL-6 and TNF-a in response to TLR2/4 stimulations. Elevated expressions of IL-6, TNF-a, TLR4 and CD11b mRNAs in the lumbar spinal cords were observed in cystitis-induced mice, which was also associated with phenotypic and functional changes. CONCLUSIONS Central and peripheral TLR activations are associated with bladder inflammation, pelvic pain and voiding dysfunction in URO-OVA mice, providing a potential mechanistic insight for human IC/BPS. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e953-e954 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yaoqin Wang More articles by this author Suming Xu More articles by this author Michael O'Donnell More articles by this author Susan Lutgendorf More articles by this author Pengchao Li More articles by this author Lang Feng More articles by this author Catherine Bradley More articles by this author Bradley Erickson More articles by this author Vincent Magnotta More articles by this author Karl Kreder More articles by this author Yi Luo More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Antigen export during liver infection of the malaria parasite augments protective immunity.

ABSTRACTProtective immunity against preerythrocytic malaria parasite infection is difficult to achieve. Intracellular Plasmodium parasites likely minimize antigen presentation by surface-expressed major histocompatibility complex class I (MHC-I) molecules on infected cells, yet they actively remodel their host cells by export of parasite factors. Whether exported liver-stage proteins constitute better candidates for MHC-I antigen presentation to CD8+ T lymphocytes remains unknown. Here, we systematically characterized the contribution of protein export to the magnitude of antigen-specific T-cell responses against Plasmodium berghei liver-stage parasites in C57BL/6 mice. We generated transgenic sporozoites that secrete a truncated ovalbumin (OVA) surrogate antigen only in the presence of an amino-terminal protein export element. Immunization with live attenuated transgenic sporozoites revealed that antigen export was not critical for CD8+ T-cell priming but enhanced CD8+ T-cell proliferation in the liver. Upon transfer of antigen-specific CD8+ T cells, liver-stage parasites secreting the target protein were eliminated more efficiently. We conclude that Plasmodium parasites strictly control protein export during liver infection to minimize immune recognition. Strategies that enhance the discharge of parasite proteins into infected hepatocytes could improve the efficacy of candidate preerythrocytic malaria vaccines.

Enhancing the Efficacy of T Cell Receptor (TCR) Gene Therapy by Co-Transfer of TCR and Additional CD3 Molecules Into CD4+ T Cells

AbstractAbstract 2044 Introduction:The specificity of T cells can be redirected using retroviral T cell receptor (TCR) gene transfer. This has the potential to generate tumour specific T cells that can be adoptively transferred to target defined tumour antigens. The majority of TCR gene therapy studies have focused on the transfer of TCR genes into CD8 T cells. However the transfer of antigen specific CD8 T cells in the absence of antigen specific CD4 T cells leads to impaired anti-tumour responses and impaired memory development in vivo. Class I restricted TCR can be used to transduce CD4 T cells for use in adoptive transfer. The majority of class I restricted TCRs are CD8 dependent and thus require co-transduction of CD8 to be fully functional in CD4 T cells. CD4 T cells transduced with the class I restricted F5-TCR (specific for influenza peptide NP presented by H2-Dbclass I molecules) produce IL-2 and proliferate in vitro in response to class II negative tumour cells expressing NP peptide but these cells were not able to generate an IFN-γ response. In vivo, F5-TCR CD4 T cells could provide help for F5-TCR CD8 T cell mediated tumour eradication. These F5-TCR CD4 T cells persisted in vivo for up to 90 days post tumour regression and were able to re-expand following tumour challenge. In order to improve the function of class I restricted TCR expressing CD4 T cells, we co-transduced a vector containing all 4 chains of the CD3 complex. High surface expression of TCR has been shown to correlate with increased responsiveness to specific antigen. When additional TCR is introduced into a T cell, the introduced T cell must compete with the endogenous TCR for binding to CD3. The amount of CD3 within the cell will thus be rate limiting for the level of surface expression of the introduced TCR. Method:The retroviral vectors pMP71-F5α-2A-F5β (F5-TCR) and pMP71-CD3-ζ-2A-ε-2A-δ-2A-γ-IRES-GFP (CD3) were used for retroviral transduction. CD4 splenocytes obtained from C57BL/6 mice were activated with CD3/CD28 magnetic beads for 24 hours prior to transduction with either F5-TCR alone or F5-TCR and CD3. 5 days post transduction, transduced T cells were stimulated with C57BL/6 splenocytes loaded with NP (relevant) peptide or WT1 (irrelevant) peptide and cytokine production was measured by ELISA and intracellular cytokine staining and proliferation by [3H] thymidine incorporation. For in vivo tumour challenge, C57BL/6 recipient mice were irradiated with 5.5Gy and injected subcutaneously with 1 × 106 EL4-NP-luciferase cells (a lymphoma cell line stably transfected with NP peptide and luciferase) on day 0. On day 1, mice received 1 × 106 F5-TCR CD3 CD4 T cells or 1 × 106 F5-TCR CD4 T cells or 1 × 106 Mock Transduced T cells. Tumour area was measured by calipers and by bioluminescence imaging. For T cell trafficking experiments, the experimental set up was as above but transgenic CD4 luciferase T cells were used for adoptive transfer and EL4-NP luciferase negative cells were used for tumour challenge. Results:CD4 T cells transduced with F5-TCR and CD3 had a 5-fold higher expression of F5-TCR compared to cells transduced with F5-TCR alone. In vitro, F5-TCR CD3 CD4 T cells showed increased proliferation and increased production of IL-2 and IFN-γ in response to specific antigen compared to F5-TCR CD4 T cells. F5-TCR CD3 CD4 T cells responded to at a 2-fold lower concentration of specific peptide than F5-TCR CD4 T cells. Following adoptive transfer in murine models, F5-TCR CD3 CD4 T cells eradicated NP expressing EL4 tumours but transfer of equivalent doses of F5-TCR CD4 T cells did not lead to tumour regression. Using bioluminescence imaging, F5-TCR CD3 CD4 T cells trafficked to tumour site faster and accumulated in greater numbers than F5-TCR CD4 T cells. Following tumour challenge, there were higher numbers of F5-TCR CD3 CD4 T cells persisting in bone marrow, lymph node and peripheral blood than in mice that received F5-TCR CD4 T cells. Conclusion:Increased surface expression of class I restricted TCR in CD4 T cells leads to increased sensitivity to peptide in vitro and higher levels of proliferation and cytokine production in response to specific peptide. This translates in vivo to enhanced persistence of F5-TCR CD3 CD4 T cells and more efficient trafficking to tumour site and superior tumour protection. Therefore, the co-transduction of additional CD3 can improve the function of class I restricted TCR in CD4 T cells. Disclosures:Stauss:Cell Medica: Scientific Advisor Other.

Correlation of effector function with phenotype and cell division after in vitro differentiation of naive MART-1-specific CD8+ T cells

Adoptive transfer of antigen-specific CD8+ T cells may represent an effective strategy for immunotherapy of tumors such as melanoma, but is limited by the number and functionality of in vitro expanded T cells. Here, we document that although ELAGIGILTV-specific CD8+ T cells from different donors initially possessed a naive phenotype, after antigen-induced in vitro expansion two distinct phenotypes correlating with cell proliferation rate emerged in the different donors. Those cultures achieving fewer cumulative population doublings (CPDs) were cytotoxic and displayed a CD45RA+CCR7- phenotype. In contrast, cultures reaching higher CPDs were non-cytotoxic T cells with a CD45RA-CCR7- phenotype. Thus, the generation of larger numbers of ELAGIGILTV-specific CD8+ T cells correlates negatively with the acquisition of a CD45RA+CCR7- phenotype and cytotoxic capacity. A better understanding of the differentiation pathways of cytotoxic T cells to obtain optimally efficient cells for adoptive transfer will allow the development of new immunotherapy protocols.

Persistent Antigen Presentation after Acute Vesicular Stomatitis Virus Infection

Long-term antigen expression is believed to play an important role in modulation of T-cell responses to chronic virus infections. However, recent studies suggest that immune responses may occur late after apparently acute infections. We have now analyzed the CD8 T-cell response to vesicular stomatitis virus (VSV), which is thought to cause to an infection characterized by rapid virus clearance by innate and adaptive immune system components. Unexpectedly, virus-encoded antigen was detectable more than 6 weeks after intranasal VSV infection in both draining and nondraining lymph nodes by adoptively transferred CD8 T cells. Infection with Listeria monocytogenes expressing the same antigen did not result in prolonged antigen presentation. Weeks after VSV infection, discrete T-cell clustering with dendritic cells within the lymph node was observed after transfer of antigen-specific CD8 T cells. Moreover, memory CD8 T cells as defined by phenotype and function were generated from naïve CD8 T cells entering the response late after infection. These findings suggested that protracted antigen presentation after an apparently acute virus infection may contribute to an ongoing antiviral immune response.

Transgenic Mouse Model of Intestine-Specific Mucosal Injury and Repair

Most studies of injury and repair to mucosal tissue have used nonspecific mediators to induce injury. Damage to the mucosal epithelium resulting from chemical or radiation treatment associated with cancer therapy may fall into this category of injury. When such treatments are applied, it is generally not possible to predict or control the extent of possible injury. This fact makes analysis of inductive and reparative processes difficult. In addition, the role of the immune system in the etiology and subsequent healing of mucosal tissue following cancer therapy with or without bone marrow transplantation remains unclear. To study tissue- and antigen-specific immune damage of intestinal mucosal tissue, we generated transgenic mice that express a nominal antigen exclusively in intestinal epithelial cells. The transfer of antigen-specific CD8 T cells with concomitant virus infection resulted in the destruction of intestinal epithelial cells and disease. The destructive phase in some cases was followed by complete recovery and tolerance induction. This model will provide a system that can be regulated for analysis of the mediators of mucosa-specific tissue damage and repair.