Abstract 16615: Reconstitution of CD4 T Cells Causes Cardiac Fibrosis and Myocardial Dysfunction in T Cell Specific S1P Receptor 1 Deficient Diabetic Mice

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Chowdhury S Abdullah ◽  
Zhu-Qiu Jin
Keyword(s):  
T Cells ◽  
Body Weight ◽  
T Cell ◽  
Adoptive Transfer ◽  
Cd4 T Cells ◽  
Cardiac Fibrosis ◽  
Myocardial Dysfunction ◽  
Diabetic Mice ◽  
Knock Out ◽  
S1p Receptor

Involvement of T cells in fibrosis has been reported but modulation of their role in context of diabetic fibrogenesis has yet to be determined. Our previous studies indicated that T cell S1P receptor 1 (S1P1) genetic depletion ensues sustained lymphopenia in circulation and reduced fibrosis in streptozotocin-induced type 1 murine diabetic model. We hypothesized that adoptive transfer of T cells to T-cell S1P1 receptor knock-out (TS1P1KO) mice would abolish cardioprotection and antifibrotic effect as observed earlier. TS1P1KO and littermate wild-type (WT) mice were divided into vehicle and streptozotocin (STZ) (50 mg/kg body weight for five days, i.p.) groups. Naïve CD4 T cells were isolated by positive selection through MS column from CD4 magnetic microbeads-labeled WT mice splenocytes. Isolated CD4 T cells (purity >95%) were adoptively transferred (i.v.) into the mice of above groups at dose of one million cells. Body weight (g) and blood glucose level (mg/dl) were monitored. CD4 and CD8 T cells in blood were counted by flow cytometry. Heart histology was studied in H&E stained sections and pathological grading was given. Masson Trichrome stained heart sections were digitally imaged at 16x magnification and percent of fibrotic area was quantified by using NIH ImageJ. Cardiac contractility was measured in ex-vivo Langendorff’s heart perfusion system at the end of 11 weeks. TS1P1KO mice had ~90% reduced T cells (CD4 cells: 1.15±0.30% vs 25.06±0.64%, CD8 cells: 2.09±0.42% vs 14.72±0.38% in WT, **P<0.01, n=4-5) in blood. KO diabetic mice without adoptive transfer of CD4 T cells exhibited about 70% less fibrotic area (11.86±4.34% vs 46.48±8.06% in WT STZ, *P<0.05, n=7-9) and improved cardiac structure and function. Adoptively CD4 T cells recipient KO diabetic mice presented cardiac structural disorganization (histological score: 9.25±0.95 vs. 1.29±0.52 in KO STZ without transfer, *P<0.05, n=4-7) and increased myocardial fibrosis (37.11±3.22% vs. 11.86±4.34% in KO STZ without transfer, *P<0.05, n=4-7) with reduced cardiac contractile force compared with KO diabetic mice without CD4 T cells transfer. In conclusion, reconstitution of CD4 T cells increases cardiac fibrosis and attenuates cardiac function in lymphopenic T cell S1P1 knock-out diabetic mice.

scholarly journals Generation of a Novel T Cell Specific Interleukin-1 Receptor Type 1 Conditional Knock Out Mouse Reveals Intrinsic Defects in Survival, Expansion and Cytokine Production of CD4 T Cells

PLoS ONE ◽  
2016 ◽  
Vol 11 (8) ◽  
pp. e0161505 ◽  
Author(s):  
Ilgiz A. Mufazalov ◽  
Tommy Regen ◽  
Carsten Schelmbauer ◽  
Janina Kuschmann ◽  
Alisa M. Muratova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cytokine Production ◽  
Interleukin 1 ◽  
Receptor Type ◽  
Intrinsic Defects ◽  
Knock Out ◽  
Knock Out Mouse

scholarly journals Borna disease, a progressive meningoencephalomyelitis as a model for CD4+ T cell-mediated immunopathology in the brain.

1989 ◽  
Vol 170 (3) ◽  
pp. 1045-1050 ◽  
Author(s):  
J A Richt ◽  
L Stitz ◽  
H Wekerle ◽  
R Rott
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Mhc Class Ii ◽  
Adoptive Transfer ◽  
Cd4 T Cells ◽  
Immune Cell ◽  
Cd4 T Cell ◽  
The Brain ◽  
Borna Disease

A homogeneous T cell line NM1 with Borna disease (BD) virus reactivity could be established. The NM1 cells have been characterized as CD4+ T cells. Adoptive transfer revealed that this MHC class II-restricted immune cell is responsible for the immunopathological effect leading to BD, a progressive meningoencephalomyelitis.

Abstract P225: Rupp Rat Model Cd4+ T Cells Activate Nk Cells And Cause Mitochondrial Oxidative Stress And Hypertension In Normal Pregnant Rats

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Evangeline M Deer ◽  
Kristin Reeve ◽  
Lorena M Amaral ◽  
Venkata Ramana Vaka ◽  
Michael Franks ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Rat Model ◽  
Adoptive Transfer ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Pregnant Rats ◽  
Atp Production

Preeclampsia (PE) is new onset hypertension during pregnancy and is associated with elevated inflammatory response such as CD4+ T cells, NK cells, and cytokines. We have previously shown women with PE exhibit increases in circulating and placental CD4+T cells and placental mitochondrial (mt) dysfunction/ROS compared to normal pregnant (NP) women. The Reduced Uterine Perfusion Pressure (RUPP) rat model produces many characteristics of PE such as hypertension, increases in CD4+ cells, increases in renal and placental NK cells, and mt dysfunction/ROS. We have previously demonstrated that RUPP CD4+T cells cause hypertension in NP rats, however the role of RUPP CD4+ T cells in stimulating NK cells to cause mt dysfunction/ROS are not elucidated. Therefore, we examined the effect of adoptive transfer of RUPP CD4+ T cells to activate NK cells in NP rats. Splenic CD4+ T cells were isolated from RUPP rats, cultured, and injected into NP rats on GD 13. On GD19, MAP values and blood/tissue samples were collected from both RUPP CD4+ T cell recipients and NP controls. Mitochondrial respiration and mtROS were measured in isolated mitochondria using the Oxygraph 2K and fluorescent microplate reader, respectively. A student’s t-test was used for statistical analysis. On GD19, MAP increased to 110±2 mmHg (n=13) in RUPP CD4+ T cell recipients compared to control NP rats 102±2 mmHg (n=7, p<0.05). Circulating cytolytic NK cells increased to 3±0.6% in RUPP CD4+ T cell recipients (n=8) compared to NP controls 0.3±0.2% (n=7, p<0.05). Placental state 3 (209.3±31.3 vs 422.7 ±83.3 pmol/sec/mg, p<0.05) and maximal (152.1±46.2 vs 229.7±58.9 pmol/sec/mg) and renal state 3 (133.4 ±21.4 vs 289.8±43.4 pmol/sec/mg, p<0.05) and maximal (61.8±18 vs 242.4±27.7 pmol/sec/mg, p<0.05) respiration rates, indicative of ATP production and electron transport chain efficacy respectively, were reduced with RUPP CD4+ T cells (n=6; n=9) compared to NP (n=5; n=5). Collectively, the data indicate that the adoptive transfer of RUPP CD4+ T cells stimulates cytolytic NK cells and placental and renal mitochondrial dysfunction/ROS during pregnancy as important mechanisms of hypertension in the pathophysiology of preeclampsia. Keywords: Preeclamspia, Hypertension, Oxidative stress

Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection

Blood ◽  
2005 ◽  
Vol 106 (9) ◽  
pp. 2995-3003 ◽  
Author(s):  
Maria Moeller ◽  
Nicole M. Haynes ◽  
Michael H. Kershaw ◽  
Jacob T. Jackson ◽  
Michele W. L. Teng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cd4 T Cells ◽  
Therapeutic Potential ◽  
Lung Metastases ◽  
Tumor Rejection ◽  
Specific Gene ◽  
Transfer Strategies

AbstractBecause CD4+ T cells play a key role in aiding cellular immune responses, we wanted to assess whether increasing numbers of gene-engineered antigen-restricted CD4+ T cells could enhance an antitumor response mediated by similarly gene-engineered CD8+ T cells. In this study, we have used retroviral transduction to generate erbB2-reactive mouse T-cell populations composed of various proportions of CD4+ and CD8+ cells and then determined the antitumor reactivity of these mixtures. Gene-modified CD4+ and CD8+ T cells were shown to specifically secrete Tc1 (T cytotoxic-1) or Tc2 cytokines, proliferate, and lyse erbB2+ tumor targets following antigen ligation in vitro. In adoptive transfer experiments using severe combined immunodeficient (scid) mice, we demonstrated that injection of equivalent numbers of antigenspecific engineered CD8+ and CD4+ T cells led to significant improvement in survival of mice bearing established lung metastases compared with transfer of unfractionated (largely CD8+) engineered T cells. Transferred CD4+ T cells had to be antigen-specific (not just activated) and secrete interferon γ (IFN-γ) to potentiate the antitumor effect. Importantly, antitumor responses in these mice correlated with localization and persistence of geneengineered T cells at the tumor site. Strikingly, mice that survived primary tumor challenge could reject a subsequent rechallenge. Overall, this study has highlighted the therapeutic potential of using combined transfer of antigen-specific gene-modified CD8+ and CD4+ T cells to significantly enhance T-cell adoptive transfer strategies for cancer therapy.

Adult T-cells impair neonatal cardiac regeneration

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T Dolejsi ◽  
T Schuetz ◽  
M Delgobo ◽  
L Tortola ◽  
A Bauer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Functional Impairment ◽  
Adoptive Transfer ◽  
Age Groups ◽  
Cardiac Regeneration ◽  
Public Institution ◽  
Funding Source ◽  
Γδt Cells ◽  
Knock Out

Abstract Background Cardiac remodeling and subsequent heart failure remain critical issues after myocardial infarction (MI). Complete cardiac regeneration was shown recently in a neonatal mouse model of MI. This cardiac regenerative potential is limited to the first few postnatal days and its decline parallels with the maturation of the adaptive immune system. Purpose Herein, we hypothesized that the T-cell maturation status critically impacts the myocardial healing outcomes in neonates and contributes to the shift from regenerative to scarring phenotype observed shortly after birth. Methods The post-MI immune responses were characterized in postnatal day one (P1, regenerative) compared to seven-day old (P7, scarring) mice subjected to permanent left anterior descending artery (LAD) ligation. The myocardial leukocyte infiltrate was phenotyped by flow cytometry at 36 hours and five days after LAD ligation. Next, we studied neonatal post-MI repair in lymphocyte-deficient Rag2 knock-out (KO) mice subjected to LAD ligation. Moreover, we adoptively transferred syngeneic splenic Thy 1.1+ T-cells obtained from adult donors into P1 versus P7 recipients and then assessed their impact on post-MI healing. Results LAD ligation induced a robust early inflammatory response (36h post-MI) in both age groups. The in situ inflammation was, nevertheless, rapidly resolved in P1-, but not in P7-infarcted animals. The distinct age groups showed a similar profile of cardiac myeloid cell infiltration but showed remarkable differences in the lymphoid compartment. P1-infarcted mice showed an early recruitment of γδT-cells, whereas P7-infarcted mice exhibited a prominent infiltration of αβT-cells. Of note, neonatal cardiac regeneration was not altered in neonatal lymphocyte-deficient (Rag2 KO) animals. However, the adoptive transfer of adult T-cells had several consequences in neonatal and one week old mice subjected to ischemic injury. P1-infarcted mice transferred with adult T-cells showed an adult-like healing phenotype, marked by an irreversible cardiac functional impairment (assessed by echocardiography) and increased fibrosis. This is in sharp contrast to the regenerative phenotype typically observed in untreated age-matched controls. Furthermore, P7-infarcted mice transferred with adult T-cell showed significantly decreased survival rate after LAD ligation. Conclusion Neonatal hearts demonstrate rapid clearance of the ischemia-induced leukocyte infiltration, further reflecting the known fact of fast cardiac regeneration in newborn rodents. Of note, the adoptive transfer of adult T-cells into neonate recipients partially blocked cardiac regeneration and promoted an irreversible functional impairment. These data indicate that the cardiac repair process, and its related “regeneration vs. scarring” dichotomy, is critically impacted by the T-cell development status. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Innsbruck Medical University, Medizinischer Forschungsfonds Tirol

scholarly journals Expression of the Butyrate/Niacin Receptor, GPR109a on T Cells Plays an Important Role in a Mouse Model of Graft Versus Host Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 61-61 ◽  
Author(s):  
Melissa D Docampo ◽  
Christoph K. Stein-Thoeringer ◽  
Amina Lazrak ◽  
Marina D Burgos da Silva ◽  
Justin Cross ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Effector Memory ◽  
Knock Out ◽  
Graft Versus Host

Abstract INTRODUCTION: The intestinal microbiota is essential for the fermentation of fibers into the short-chain fatty acids (SCFA) butyrate, acetate and propionate. SCFA can bind to G-protein-coupled receptors GPR41, GPR43 and GPR109a to activate downstream anti-inflammatory signaling pathways. In colitis or graft versus host disease (GVHD), GPR43 signaling has been reported as an important regulator of intestinal homeostasis by increasing the pool of regulatory T cells. In contrast to GPR43, which binds preferentially propionate and acetate, GPR109a is the major receptor for butyrate. We and others have demonstrated that butyrate can ameliorate gastrointestinal injury during GVHD through enterocyte protection. Therefore, we hypothesized that GPR109a plays an important role in the pathophysiology of intestinal GVHD, focusing specifically on alloreactive T cells. METHODS AND RESULTS: Using mouse models of GVHD, we examined the role of the butyrate/niacin receptor, GPR109a in allogeneic hematopoietic cell transplantation (allo-HCT). First, we studied whether a genetic knock-out (KO) of GPR109a in transplant recipient mice affected GVHD, but GPR109a-KO recipient mice did not exhibit increased mortality from GVHD compared to wild type (WT) mice. We next investigated the role of GPR109a in the donor compartment by transplanting either BM or T cells from WT or GPR109a-KO mice into major MHC mismatched BALB/c host mice. Mice transplanted with B6 BM, with T cells from a GPR109a-KO mouse into BALB/c hosts displayed a lower incidence of lethal GVHD (Fig. 1A). To determine whether the attenuation of GVHD is intrinsic to GPR109a-KO T cells, we established BM chimeras and performed a secondary transplant by transplanting B6 BM + (B6 à Ly5.1) or (GPR109a à Ly5.1) T cells into BALB/c hosts. We observed the same improvement in survival in mice that received GPR109a-KO T cells. This indicates an intrinsic role for GPR109a specifically in the donor hematopoietic compartment. Having identified a T-cell specific requirement for GPR109a we next examined expression of GPR109a on WT T cells in vitro at baseline and following stimulation with CD3/28 and found GPR109a significantly upregulated on both CD4+ and CD8+ T cells after 72 hours of stimulation (Fig 1B). At steady state in vivo, we observed the same numbers and percentages of splenic effector memory, central memory, and naïve CD4+ T cells as well as regulatory T cells in WT B6 mice and GPR109a-KO mice, suggesting normal T cell development in the knockout mice. In an in vitro mixed lymphocyte reaction (MLR), GPR109a-KO CD4+ T cells become activated, proliferate, polarize and secrete cytokine (specifically IFNg) to the same level as WT CD4+ T cells, suggesting normal functional capacity. However, after allo-HCT in mice we observed significantly fewer CD4+ and CD8+ T cells, and specifically fewer effector memory CD4+ T cells (Fig. C), in the small and large intestines of mice that received GPR109a-KO T cells at day 7 post transplant. In contrast, we found significantly more regulatory T cells in the intestines (Fig. 1D) and the spleen of GPR1091-KO T cell recipients, while numbers and percentages of polarized Th1 and Th17 T cells were similar between the two groups. We further 16S rRNA sequenced the gut microbiota of mice at day 7 after transplant and observed an increased relative abundance of bacteria from the genus Clostridium (Fig. 1D) along with an increased concentration of cecal butyrate as measured by GC-MS (Fig. 1E). In a preliminary graft versus tumor (GVT) experiment, we found that mice that received A20 tumor cells and GPR109a-KO T cells exhibited increased survival compared to mice that received A20 tumor cells and WT T cells. These preliminary findings suggest that GPR109a-KO T cells maintain their graft versus tumor response while causing less GVHD, and exclude a defective functional capacity. CONCLUSIONS: We report a novel role of the butyrate/niacin receptor GPR109a on donor T cells in allo-HCT as a genetic knock-out on T cells attenuates lethal GVHD. As these T cells are tested as functionally intact, we propose that the reduction in overall T cells of KO T cell recipients may underlie the attenuation in GVHD. Furthermore, such a reduction in allograft-induced gut injury is accompanied by maintenance of the gut commensal Clostridium and butyrate production, which is known to protect the intestinal epithelium and increases the regulatory T cell pool. Disclosures No relevant conflicts of interest to declare.

scholarly journals Helper T Cell Response to Factor VIII In Vivo Requires Several Anatomically Distinct Types of Antigen Presenting Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 440-440
Author(s):  
Radoslaw Kaczmarek ◽  
Annie R Pineros ◽  
Matthew Carl Arvin ◽  
Thais Bertolini ◽  
Rodney M. Camire ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Adoptive Transfer ◽  
Cd4 T Cells ◽  
Hemophilia A ◽  
T Cell Proliferation

Abstract Inhibitor formation is the most serious complication of factor (F)VIII replacement therapy for hemophilia A. It has long been clear that FVIII inhibitors arise in a CD4 + T cell-dependent manner, but early events in the immune response leading to MHC-II presentation and CD4 + T cell activation remain obscure. Several types of antigen presenting cells (APCs) have been implicated in the uptake of FVIII, but their relevance in T cell activation has been unclear. This study aimed to pinpoint the roles of APCs in priming FVIII-specific CD4 + T cells in vivo. Several transgenic strains of mice on the C57BL/6J background were employed to perform in vivo antigen presentation assays involving one intravenous (IV) injection of 5 µg FVIII-OVA fusion protein (FOVA), which contained an MHC-II I-A b peptide epitope of chicken ovalbumin in place of the B domain of FVIII, and an adoptive transfer (via IV injection on the following day) of 5E6 transgenic OT-II CD4 + T cells, which express chicken ovalbumin-specific T cell receptor (TCR). Before adoptive transfers, OT-II T cells were stained with CellTrace Violet (CTV). Spleens were collected for flow cytometry analyses four days after the adoptive transfer. FOVA shows identical specific activity to that of B domain deleted FVIII. CD11c-DTR/GFP mice (which express diphtheria toxin receptor and green fluorescent protein under the CD11c promoter) (n=3) received two intraperitoneal (IP) injections of 100 ng diphtheria toxin (DT) or PBS only (n=3) one day before and on the day of FVIII-OVA injection to deplete CD11c high cells (i.e. dendritic cells, DCs, marginal zone, sinusoidal and metallophilic macrophages, MFs). DT treatment completely abrogated T cell proliferation in the animals (p=0.03), while on average 11.1% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. XCR1-DTRvenus mice (which express DT receptor and the Venus variant of yellow fluorescent protein under the XCR1 promoter) were similarly treated with DT or PBS (n=4/group) one day before FOVA injection and one day after adoptive transfer to deplete type I conventional DCs (which make up ~80% of XCR1 + cells). XCR1 + cell depletion reduced T cell proliferation by ~50% (p=0.02). CD4 +CTV + cells from the DT-treated and control mice included 19.4% and 38.6% progenies, respectively. Hemophilia A (F8e16-/-) mice received 100 µg of marginal zone (MZ) B cell-depleting antibodies anti-CD11a and anti-CD49d (n=4) or isotype control antibodies (n=3) four and two days before FOVA injection. MZ B cell depletion completely abrogated T cell proliferation (p=0.02), while on average 31% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. In another experiment, hemophilia A mice were injected with GdCl 3 (n=4), which inactivates MFs, or PBS (n=3) one day before and on the day of FOVA administration. MF inactivation completely abrogated T cell proliferation in all but 1 animal (p=0.03), while on average 15.9% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. To visualize APC-T cell interactions in vivo, we performed multiphoton (MP) intravital microscopy (IVM) of inguinal lymph nodes (LNs) in CD11c-DTR/GFP mice. The animals received adoptive transfers of 1E7 CTV-stained OT-II CD4 + T cells IV ~24 hours and 5 µg FOVA 20, 5 or 1 hour before IVM delivered intradermally (ID) to target the skin-draining LNs. Control animals received adoptive cell transfer only. For IVM, the LNs were surgically exposed in live, anesthetized animals. Five hours after FOVA injection, multiple CTV + OT-II T cells formed clusters around GFP + cells throughout the T cell zone with several motile T cells surveying the B cell follicle. Twenty hours after FOVA injection, CTV + T cells densely populated and demarcated the T-B border in the LNs. CTV + T cells were nearly absent from the inguinal LNs in the control animals. We propose that activation of CD4 + T cells in response to FVIII requires a complex interaction of multiple types of APCs, which occupy distinct compartments in the secondary lymphoid organs that FVIII antigen traverses en route to cognate CD4 + T cells. In the spleen, where response to FVIII primarily takes place, antigens larger than 60 kDa (such as FVIII at 280 kDa) do not freely flow into the white pulp and instead need to be ferried by APCs. While DCs ultimately activate CD4 + T helper cells, MFs shuttle the antigen to DCs and may also provide innate activation immune signals. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2044-2044
Author(s):  
Emma K Nicholson ◽  
Maryam Ahmadi ◽  
Angelika Holler ◽  
Rebecca Pike ◽  
Ben Carpenter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Cd4 T Cells ◽  
Specific Antigen ◽  
Surface Expression ◽  
Class I ◽  
Tumour Regression

Abstract Abstract 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.

Sign in / Sign up

Export Citation Format

Share Document