CD4 Splenocytes Research Articles

IMMU-32. RNA-NANOPARTICLE VACCINES MEDIATE T CELL TRAFFICKING NECESSARY FOR BBB PASSAGE AND ANTI-GLIOMA IMMUNE RESPONSE

Abstract BACKGROUND The blood-brain barrier (BBB) remains a potent obstacle for development of new therapies against glioblastoma (GBM). While activated T cells can cross the BBB, immunotherapy has yet to be fully unlocked for malignant brain tumors due their heterogeneity and immunosuppressive microenvironments. To overcome these challenges, our group has developed a novel treatment platform, which leverages the use of a clinically translatable nanoparticles (NPs) combined with personalized tumor derived mRNA to peripherally activate T cells against a heterogenous source of tumor antigens and reprogram the intratumoral milieu into an immune activated state. OBJECTIVE We sought to assess if RNA-NPs could activate systemic/intratumoral dendritic cells (DCs) and mediate a peripheral T cell response that could penetrate the GBM microenvironment. RESULTS We uncovered that RNA-NPs elicit potent innate immunomodulating effects through release of interferon-α (IFN-α) from plasmacytoid DCs (pDCs). After only a single RNA-NP vaccine, the bulk of systemic and intratumoral DCs in mice display an activated phenotype; DCs, harvested from intracranial tumors, elicit expansion of antigen specific T cell immunity. Tumor-specific RNA-NPs elicited enhanced survival outcomes in immunocompetent animals bearing NF-1/p53 mutant gliomas with increased intratumoral memory CD8+T-cells. Unlike immune checkpoint blockade (anti-PD-L1 mAbs), we found that RNA-NPs increase LFA-1 on peripheral CD8 splenocytes, which is necessary for activated T cell passage across the BBB. RNA-NPs also increased CCR2 on peripheral CD8 cells, which was dependent on IFN-α/β, as the percentage of CCR2+CD8+ splenocytes and anti-tumor activity (mediated by RNA-NPs) was abrogated in animals receiving concomitant type I IFN receptor (IFNAR1) mAbs. CONCLUSION Since LFA-1 is important for T cell trafficking across the BBB and CCR2 may promote chemotaxis to the brain (as GBMs are known to secrete CCL2), RNA-NPs may offer a new treatment modality and immunologic mechanism for unlocking peripheral T cell immunity against malignant gliomas.

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.

Induction of S100A4, S100A6, and galectin-1 during the lineage commitment of CD4+CD8+ thymocyte cell line is suppressed by 2,3,7,8-tetrachlorodibenzo-p-dioxin

To study the mechanisms underlying the linage commitment of CD4+CD8+ thymocytes and the skewed differentiation of CD4+CD8+ into CD4-CD8+ thymocytes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we stimulated with antigen DPK cells, a CD4+CD8+ thymic lymphoma cell line which can differentiate into CD4+CD8- thymocytes and performed a comparative proteomic analysis of DPK cells stimulated with antigen or not. Among the 10 up-regulated or induced proteins upon antigenic stimulation, S100A4, S100A6, and galectin-1 were highly up-regulated. Kinetic studies revealed that expression of S100A4, S100A6, and galectin-1 was dramatically increased as early as 10min after antigen stimulation, similar to that of cKrox and Runx3, transcription factors intimately associated with the lineage commitment. Among four thymocyte subpopulations of the thymus examined, S100A4, S1006, and galectin-1 were most prominently expressed in CD4+CD8+ thymocytes, but not at all in CD4-CD8+ and CD4-CD8- thymocytes. In the spleen, expression of S100A4, S1006, and galectin-1 was greater in CD4 than in CD8 splenocytes. When TCDD was added to antigen-stimulated DPK cells, antigen-induced up-regulation of S100A4, S1006, and galectin-1 were remarkably inhibited, probably partly accounting for the skewed differentiation of CD4+CD8+ into CD4-CD8+ thymocytes induced by TCDD.

Enhanced Tumor Eradication by Combining CTLA-4 or PD-1 Blockade With CpG Therapy

Tumor immunotherapy aims to break effector T-cell anergy and to block suppressive cell types and ligands allowing effector cells to exert tumor eradication. Previous reports demonstrate that cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibodies promote T-cell activation and render T effector cells resistant to T regulatory cells (Tregs) whereas programmed death receptor-1 (PD-1)/PD-L1 blockade results in loss of peripheral tolerance. Herein, we explored single or combined antibody blockade of CTLA-4 and PD-1 alone or combined with the toll-like receptor agonists CpG or bacillus Calmette-Guérin for treatment of murine experimental bladder cancer. In therapeutic studies, tumors were rejected by anti-CTLA-4 (aCTLA-4) while anti-PD-1 (aPD-1) suppressed tumor growth. The combination had no additive effect compared with aCTLA-4 alone. However, elevated levels of circulating CD107a expressing CD8 T cells were found in the aCTLA-4 plus aPD-1 group. In addition, levels of antinuclear antibodies correlated inversely with tumor size. Next, we combined CpG or bacillus Calmette-Guérin with aCTLA-4, aPD-1, or aPD-L1 and found that CpG in combination with aCTLA-4 or aPD-1 increased the survival of mice, with aPD-1 plus CpG being superior to either agent alone. CpG plus aCTLA-4 or aPD-1 increased the numbers of circulating tumor-specific CD107a expressing CD8 T cells as well as activated (CD25FoxP3-) CD4 splenocytes. Further, we investigated the numbers of Tregs in the tumor area of treated animals and detected decreased levels after aCTLA-4 or aPD-1 plus CpG therapy. Thus, the combination of CpG with CTLA-4 or PD-1 blockade improved long-term survival and led to increased levels of tumor-reactive T cells and reduced numbers of Tregs at the tumor site.

Leptin Receptor–Related Immune Response in Colorectal Tumors: The Role of Colonocytes and Interleukin-8

We have shown that ObRb, the leptin receptor, is overexpressed in colorectal cancer cells, and that this may influence the patients' outcome. We investigated colonocytes as leptin targets and characterized their pivotal role in antitumor immune response. Cytokine and chemokine mRNAs in HT29 cells were measured by targeted arrays. In vitro, normal colonocytes and human colon cancer cells (HT29, Caco-2, SW480, and HCT116) were used to investigate ObRb transduction system and cytokine releases. Animal colonocytes and CD8 splenocytes and human HT29, HCT116, and CD8(+) cells from blood donors were used to investigate the lymphocyte response to the colonocytes when stimulated by leptin. Leptin-induced cytokine releases in the normal colonic mucosa and tumor growth and cytokine releases within tumors in vivo were measured in male rats and nude mice, respectively. Statistical analysis was done by Fisher's exact and Mann-Whitney U tests. Various cytokines and their receptors were produced in normal and tumoral colonocytes in response to leptin by increasing nuclear factor-kappaB activation. Interleukin-8 (IL-8) was the main cytokine produced in vitro. The levels of IL-8 and its receptor, CXCR1, were higher in tumors than in homologous normal mucosa. Systemic leptin enhanced the proinflammatory cytokines in normal colonocytes and in HT29 xenografted tumor colonocytes. Colonocyte-derived products after leptin treatment stimulated perforin and granzyme B expressions in normal CD8(+) T cells in vitro. Leptin triggers an inflammatory response in tumor tissue by directly stimulating colonocytes, which can recruit T cytotoxic cells in the tumor microenvironment.

Induction of Indefinite Survival of Fully Mismatched Cardiac Allografts and Generation of Regulatory Cells by Sarpogrelate Hydrochloride

At initiation of the immunologic response, platelets rapidly release chemical mediators such as serotonin (5-hydroxytryptamine, [5-HT]) and cytokines. Sarpogrelate hydrochloride (SH), a selective 5-HT2-receptor antagonist, is used to treat patients with peripheral arterial disease. We investigated the effect of SH on the alloimmune response in a murine cardiac transplantation model. CBA mice underwent transplantation of a C57BL/10 heart and received a short course of SH treatment. Survival of the allograft was recorded. An adoptive transfer study was performed to determine whether regulatory cells were generated. Immunohistochemistry studies of intercellular adhesion molecule 1 (ICAM-1), histological, cell-proliferation, and cytokine assessments were performed. Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 8 days). In mice given 10 mg/kg of SH, all allografts survived indefinitely (MST, >100 days); these mice also had significantly prolonged survival of donor-specific skin grafts but acute rejection of third-party skin grafts. Secondary CBA recipients given not only whole but also CD4 splenocytes from primary SH-treated CBA recipients with C57BL/10 cardiac allograft had indefinite survival of C57BL/10 hearts (MST, >100 days). SH inhibited upregulation of ICAM-1 on endothelial cells in the allografts. Graft acceptance and hyporesponsiveness were confirmed by the histological and cell-proliferation studies, respectively. Production of interleukin-4 and interleukin-10 from splenocytes of SH-treated transplant recipients increased compared to that from splenocytes of untreated recipients. SH induced indefinite survival of fully allogeneic cardiac allografts, generated CD4 regulatory cells, inhibited ICAM-1 expression in the allografts, and upregulated IL-4 and IL-10 production.

Differential partitioning and trafficking of GM gangliosides and cholesterol-rich lipid rafts in thymic and splenic CD4 T cells

The GM gangliosides and cholesterol components of plasma membrane lipid rafts play an important role in the recruitment and signaling of protein receptors in eukaryotic cells. Herein, we have analyzed at the single-cell level the partitioning and intracellular trafficking of GM gangliosides and cholesterol in quiescent (CD4 +CD69 −) and CD3-activated (CD4 +CD69 +) thymic and splenic T cells. First, regardless the gender and the quiescent or activated status of T cells, the GM and cholesterol content in cytosol and plasma membrane as well as the expression levels of GM synthase, Sphingomyelin phosphodiestarase 2 and HMG Co-A reductase genes involved in GM and cholesterol synthesis were constantly lower in CD4 thymocytes than in CD4 splenocytes. Second, we detected variations in the balance between GM and cholesterol in plasma membrane depending on aging, and found that deprivation of cellular cholesterol does not necessarily affect the GM content in both quiescent CD4 thymocytes and splenocytes. Third, CD3 stimulation up-regulated the GM and little if any the cholesterol content in both thymic and splenic CD4 T cells, suggesting a cross talk between the CD3 signaling and GM but not cholesterol biosynthesis pathway. Fourth, partitioning and trafficking of GM to the plasma membrane depended on the transport of ceramide precursors from endoplasmic reticulum to Golgi network, as well as on the synthesis, glycosylation and vesicular assembly in trans-Golgi, and less on the cytoskeleton architecture in both quiescent and activated CD4 thymic and splenic T cells. Together, these findings suggest that the differential partitioning and intracellular trafficking of GM and cholesterol in thymic and splenic CD4 T cells may account for the stage of functional maturation.

687. Tracking Transgene Product-Specific T Cells In Vivo

Immune responses to the transgene product are a major hurdle for successful treatment of genetic disease by gene transfer. Characterization of T helper cell activation has been largely relied on in vitro assays due to the inability to track antigen-specific T cells in vivo. We sought to develop a system to visualize in vivo activation and trafficking of T cells after gene transfer. Previously, we documented that intramuscular administration of an AAV vector expressing chicken ovalbumin (ova) resulted in a cytotoxic T lymphocyte response that destroyed ova-transduced muscle fibers within 3-4 weeks (Mol. Ther. 9:S71). Ova was transiently detectable in the systemic circulation during the first 2 weeks until the ensuing inflammatory reaction had destroyed transduced myocytes. Theoretically, immune responses to the systemically delivered antigen or the antigen introduced into skeletal muscle tissue could have triggered T cell activation, thereby limiting duration of transgene expression. Ova-specific CD4 T cells extracted from BALB/c mice transgenic for DO11.10 T cell receptor (TCR, specific for amino acids 323-339), can be identified by flow cytometry (using anti-murine CD4 and clonotypic KJ1-26 monoclonal antibody specific to the DO11.10 TCR). Twenty-four hrs after adoptive transfer of CFSE-labeled CD4 splenocytes from DO11.10 transgenic to wild-type BALB/c mice, we injected AAV2-ova vector into the muscles of one hind limb, while the contra-lateral leg received AAV2-null vector (no transgene). At day 5 after gene transfer, CFSE cells were detected by flow cytometry in the draining lymph nodes (LN) of the AAV2-null injected leg and in the spleen at a frequency of 0.2-0.4% of total cells. As expected, around 70% of CFSE cells were also TCR. However, there was no evidence for cell division at any of the vector doses tested (1|[times]|1012, 3|[times]|1012, and 1|[times]|1013 vg/leg). In contrast, in the LN draining the AAV2-ova transduced leg, vector dose-dependent in vivo proliferation of CFSE+-TCR+- cells was evident (14-68% had been proliferating, showing 1-5 cell divisions), while CFSE+-TCR- cells did not proliferate. These data illustrate local antigen-specific T cell activation in the draining LN of the ova-expressing muscle. By day 10, an inflammatory infiltrate had developed in the AAV2-ova, but not the AAV2-null transduced muscle. Inflammation was more severe at higher vector doses and contained CD8 and IFN-g expressing cells. Immuofluorescence staining showed that the infiltrate contained a large number of CD4+TCT//Q cells, indicating that ova-specific CD4 T cells activated in the draining LN had migrated into the muscle, likely providing T help for cytotoxic T lymphocytes. Isolation of dendritic cells (DCs) followed by co-culture with BDZ.OVA.26.2 hybridoma cell line showed that the CD8a subset of DCs had activated ova-specific CD4 T cells in draining LNs. We now have an experimental system that allows us to pinpoint transgene product-specific T cell activation and migration in vivo. The implications are that systemic delivery of a protein by muscle-derived expression is limited by a local immune response in the transduced muscle tissue.

Recombinant lemA without adjuvant induces extensive expansion of H2-M3-restricted CD8 effectors, which can suppress primary listeriosis in mice.

Mice infected with Listeria monocytogenes (LM) produce large numbers of H2-M3-restricted CD8 T cells directed against the formylated peptides, f-MIGWII and f-MIVIL. To examine responsiveness to these epitopes in the absence of infection, we inoculated mice with recombinant lemA (r-lemA) containing f-MIGWII or r-vemA (a variant of r-lemA containing f-MIVIL in place of f-MIGWII) without adjuvant. To monitor responses, we measured peptide-specific cytoplasmic IFN-gamma production ex vivo by freshly harvested splenocytes at varying times post-inoculation. B6 mice inoculated with r-lemA produced substantial numbers of epitope-specific CD8 cells with peak levels on day 7 when there were 1.1 x 10(6) f-MIGWII-specific CD8 cells in the spleen (8.2% of total CD8 splenocytes). The r-vemA-treated animals accumulated 0.25 x 10(6) cells (1.8% of total CD8 cells) at this time point. Comparable responses were observed after rechallenge of immunized animals. Other elements in the lemA moiety distinct from the immunogenic peptide were required since mice did not respond to equimolar amounts of synthetic f-MIGWII or f-MIVIL alone. In comparative studies, B6 and C3H/HeJ mice responded to r-lemA much more vigorously than BALB/c animals. When r-lemA- or r-vemA-treated B6 animals were challenged i.v. with LM 7 days later, they suppressed splenic accumulation of bacteria much more effectively than controls. On the other hand, antigen-treated animals were not protected against infection 1 month later. Thus, responsive strains of mice respond vigorously to H2-M3-restricted epitopes, even in the absence of bacterial infection or adjuvant. The resulting effectors acutely enhance antimicrobial resistance but do not confer long-term memory protection.

Intestinal intraepithelial lymphocytes exert potent protective cytotoxic activity during an acute virus infection.

After systemic infection of mice with 104 PFU of lymphocytic choriomeningitis virus (LCMV), infected cells are detected simultaneously in various organs, including spleen and intestinal mucosa. Most notably, virus-infected cells are also present among CD11c+ dendritic cells in the subepithelial area of the small intestinal mucosa. Some of these virus-infected cells are in close spatial association with intestinal intraepithelial lymphocytes (IEL). Therefore, we compared virus-specific cytotoxic activity of CD8 splenocytes with that of IEL subsets. While ex vivo isolated TCRalphabeta+CD8alphaalpha+ IEL exert only minimal virus-specific cytotoxicity, maximum specific killing mediated by TCRalphabeta+CD8alphabeta+ IEL on day 8 postinfection exceeds maximum cytotoxic activity observed with CD8 splenocytes when assessed in vitro. Maximum cytotoxic activity of IEL is preceded by peak perforin and granzyme B mRNA expression in IEL around day 6 postinfection, suggesting a recent activation in situ. The antivirus cytotoxicity of in vivo primed IEL is further demonstrated by the protection from virus production in the spleen of mice infected with LCMV 10 h before adoptive cell transfer. These data indicate a potent priming of LCMV-specific IEL in situ after systemic LCMV infection and suggest that cytotoxic IEL markedly contribute to the elimination of virus-infected cells in the intestinal mucosa.

Effects of Ganoderma lucidum and krestin on subset T-cell in spleen of gamma-irradiated mice.

Effects of Ganoderma lucidum (Gl) and Krestin (PSK) extracts on spleen, thymus and splenocytes in gamma-irradiated mice were investigated in this study. ICR strain male mice were divided into five groups. Group A was the normal control. Group B, the experimental control, was treated with Gl. Group C, the radiation treatment control, was treated with whole body exposure to 4 Gy gamma-irradiation (RT). Group D was treated with RT and Gl. Group E was treated with RT and PSK. The dosage of Gl was 400 mg/day/kg body weight and PSK was 500 mg/day/kg body weight. Our results indicated that the relative thymus weight in groups D and E were higher than group C on day 28 after gamma-irradiation. Group D was the highest in all the experimental groups. CD4 and CD8 splenocytes in group D were higher than group C on days 7 and 28. Gl was better than PSK in repairing the damage of subset T-cells in the spleen of gamma-irradiated mice.