MECHANISTIC AND FUNCTIONAL CHARACTERIZATION OF B AND T LYMPHOCYTE ATTENUATOR (BTLA) IN CD8 TUMOR INFILTRATING LYMPHOCYTES

This dissertation project focused on understanding the functional role of BTLA on CD8+ Tumor Infiltrating Lymphocytes (TIL) from metastatic melanoma patients. Clinical trials of adoptive T-cell therapy (ACT) using autologous ex vivo expanded TIL have demonstrated the great potential of this immunotherapy with an overall clinical response rate 40-50% for stage IV metastatic melanoma patients. We have investigated a number of biomarkers in both the infused TIL and the tumor microenvironment for their association with clinical response. Surprisingly, a subset of CD8+ TIL expressing the co-inhibitory molecule BTLA (B-and-T lymphocyte attenuator) was highly associated with clinical response, while expression of other co-inhibitory molecules such as PD-1, TIM-3, and Lag3 did not associate with response. BTLA is expressed by T cells, B cells, and NK cells and serves as a T cell differentiation maker whereby high expression of BTLA associates with less differentiated T cell phenotype. While the suppressive function of the ITIM and ITSM motifs of BTLA are well described, the Grb2 motif’s function remains understudied. In this study, we sought to determine the functional characteristics of the CD8+BTLA+TIL subset and define the contribution of the Grb2 motif of BTLA in T cell co-stimulation. We have uncovered a survival advantages of the BTLA+ subset that allows for serial killing of target tumor cells, which may explain our previous correlation between this subset and response to TIL ACT. BTLA-HVEM interaction during T cell activation led to the specific activation of SRC kinase. In addition, our results unveiled a role for the BTLA-associated Grb2-binding motif in T cell proliferation and IL-2 production following TCR engagement that was independent of the inhibitory function of ITIM/ITSM motifs. Overall, our study first unveil the dual role of BTLA as both a co-stimulatory and co-inhibitory molecule. The integration of the positive and negative signals transduced by BTLA promotes IL-2 secretion while reducing certain effector function of T cell. Altogether, the combination of both BTLA signaling and inherent attributes of less differentiated T cells could promote T cell survival, persistence, and anti-tumor function

FUNCTIONAL CHARACTERIZATION OF THE VIRAL FLICE INHIBITORY PROTEIN OF RHESUS MONKEY RHADINOVIRUS

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus closely related to Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV is associated with several malignant diseases, including Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Here we found that RRV viral FLICE inhibitory protein (vFLIP) inhibits apoptosis. In HeLa cells with vFLIP expression, cleavage of poly [ADP-ribose] polymerase 1 (PARP-1) and activities of caspase 3, 7, and 9 were much lower than controls. RRV vFLIP was able to enhance cell survival under starved condition or apoptosis induction. After apoptosis induction, autophagosome formation was enhanced in cells with vFLIP expression and when autophagy was inhibited, these cells underwent apoptosis. Moreover, RRV latent infection of BJAB B-lymphoblastoid cells protects the cells against apoptosis. Knockdown of vFLIP expression in the RRV-infected BJAB cells with siRNA abolished the protection against apoptosis. These findings indicate that RRV vFLIP protects cells against apoptosis by enhancing autophagosome formation

Multifaceted role of BTLA in the control of CD8+ T cell fate after antigen encounter

Purpose: Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40%–50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte associated) expression on transferred CD8+ TILs was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8+BTLA+TIL subset and define the contribution of the Grb2 motif of BTLA in T-cell costimulation. Experimental Design: We determined the functional role and downstream signal of BTLA in both human CD8+ TILs and mouse CD8+ T cells. Functional assays were used including single-cell analysis, reverse-phase protein array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as patient-derived xenograft (PDX) model using immunodeficient NOD-scid IL2Rgammanull (NSG) tumor-bearing mice treated with autologous TILs. Results: CD8+BTLA? TILs could not control tumor growth in vivo as well as their BTLA+ counterpart and antigen-specific CD8+BTLA? T cells had impaired recall response to a vaccine. However, CD8+BTLA+ TILs displayed improved survival following the killing of a tumor target and heightened “serial killing” capacity. Using mutants of BTLA signaling motifs, we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation. Conclusions: Our data portrays BTLA as a molecule with the singular ability to provide both costimulatory and coinhibitory signals to activated CD8+ T cells, resulting in extended survival, improved tumor control, and the development of a functional recall response. Clin Cancer Res; 23(20); 6151–64. ©2017 AACR

Viral FLICE Inhibitory Protein of Rhesus Monkey Rhadinovirus Inhibits Apoptosis by Enhancing Autophagosome Formation

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus closely related to human herpesvirus 8 (HHV8). RRV encodes viral FLICE inhibitory protein (vFLIP), which has death effector domains. Little is known about RRV vFLIP. This study intended to examine its function in apoptosis. Here we found that RRV vFLIP inhibits apoptosis induced by tumor necrosis factor-α (TNF-α) and cycloheximide. In HeLa cells with vFLIP expression, the cleavage of poly [ADP-ribose] polymerase 1 (PARP-1) and activities of caspase 3, 7, and 9 were much lower than those in controls. Cell viability of HeLa cells with vFLIP expression was significantly higher than control cells after apoptosis induction. However, RRV vFLIP appears unable to induce NF-κB signaling when tested in NF-κB reporter assay. RRV vFLIP was able to enhance cell survival under starved conditions or apoptosis induction. At early time points after apoptosis induction, autophagosome formation was enhanced and LC3-II level was elevated in cells with vFLIP and, when autophagy was blocked with chemical inhibitors, these cells underwent apoptosis. Moreover, RRV latent infection of BJAB B-lymphoblastoid cells protects the cells against apoptosis by enhancing autophagy to maintain cell survival. Knockdown of vFLIP expression in the RRV-infected BJAB cells with siRNA abolished the protection against apoptosis. These results indicate that vFLIP protects cells against apoptosis by enhancing autophagosome formation to extend cell survival. The finding of vFLIP’s inhibition of apoptosis via the autophagy pathway provides insights of vFLIP in RRV pathogenesis

Tracing the Genetic Evolution of Canine Parvovirus Type 2 (CPV-2) in Thailand

Canine parvovirus type 2 (CPV-2) is responsible for hemorrhagic gastroenteritis in dogs worldwide. High genomic substitution rates in CPV-2 contribute to the progressive emergence of novel variants with increased ability to evade the host immune response. Three studies have analyzed the genomic mutations of CPV-2 variants in Thailand. These investigations were independently conducted at different timepoints. Thus, a retrospective integrated analysis of CPV-2 genomic mutations has not been fully performed. Our study aimed at evaluating the evolutionary changes in CPV-2 in Thailand from 2003 to 2019. Two hundred and sixty-eight Thai CPV-2 nucleotide sequences were used for multiple amino acid sequence alignment and phylogenetic analyses. From 2003 to 2010, CPV-2a and -2b were the only variants detected. CPV-2c, emerged in 2014, replacing CPV-2a and -2b, and has become a major variant in 2019. Phylogenetic analysis revealed that the proposed mutation pattern of VP2 amino acid residues could help distinguish Thai CPV-2 variants. This comprehensive examination provides insight into the genomic evolution of CPV-2 in Thailand since its first reporting in 2003, which may facilitate the surveillance of the potential genetic alteration of emergent CPV-2 variants

RRV vFLIP enhances autophagosome formation after apoptosis induction.

<p>A. RRV vFLIP expression leads to more punctated autophagosomes in HeLa cells after apoptosis induction. HeLa cells were co-transfected with mCherry-LC3 and VenusN1-vFLIP or empty vector (EV), and induced with TNF-α and cycloheximide for 3 h the next day. The cells were observed under confocal fluorescence microscopy. Nuclear DNA was counterstained with DAPI. Mock: no apoptosis induction. TNF: 3 h after addition of TNF-α and cycloheximide. B. Elevation of LC3-II level in cells with vFLIP expression after apoptosis induction. HeLa-vFLIP stable and HeLa cells with EV were transfected with CFP-LC3, and induced with TNF-α and cycloheximide to undergo apoptosis the next day. The cells were harvested 0, 4, and 6 h after the induction for Western blotting of LC3. Tubulin was blotted for normalization. The relative ratios of LC3-II (the lower band of LC3 image) in comparison with HeLa-EV cells 0 h after apoptosis induction are shown below the image. C. RRV vFLIP is unable to inhibit apoptosis when cells are treated with 3-MA. Addition of 3-MA to HeLa-vFLIP stable cells was done 1 h before addition of TNF-α and cycloheximide. The cells were harvested 10 h after TNF-α addition for Western blotting of PARP-1 cleavage. D. Treatment with NH4Cl reduces vFLIP’s capability to inhibit apoptosis. NH4Cl was added to HeLa-vFLIP or HeLa-EV cells at 4, 6, and 8 h after apoptosis induction. The cells without apoptosis induction were included as control.</p

Primers used for ORF71 cloning and qPCR.

a<p>F, forward; R, reverse.</p>b<p>Restriction sites of <i>Bam</i>HI, <i>Xho</i>I and <i>Eco</i>RI included in the primers are italicized.</p

RRV vFLIP inhibits apoptosis in HeLa cells.

<p>A. Reduction of PARP-1 cleavage in HeLa cells detected by Western blotting. HeLa and HeLa-vFLIP stable cells were treated with tumor necrosis factor-α (TNF-α) and cycloheximide to induce apoptosis, and harvested at 0, 6, 9, and 12 h after the treatment. PARP-1 cleavage was detected as a marker of apoptosis. Tubulin was detected on the same membrane for loading normalization. B. Reduction of activities of caspase 3/7, 8, and 9. HeLa-vFLIP stable cells and HeLa cells were treated with TNF-α and cycloheximide for 6 h. Relative percentages of caspase activities in comparison with normal HeLa cells are shown. Significant differences between HeLa-vFLIP and HeLa cells are denoted by “**", which indicates <i>P</i><0.01. C. Up-regulation of transcript of MnSOD in HeLa-vFLIP stable cells after apoptosis induction detected by real-time PCR. Relative fold in comparison with control HeLa cells under the same treatment is shown. D. RRV vFLIP is unable to activate the NF-κB luciferase reporter. HeLa cells were transfected with NF-κB reporter plasmid pGL4.32[LUC2P/NF-κB-RE/HYGRO], VenusN1-vFLIP (vF1), VenusC1-vFLIP (vF2), empty vector VenusN1 (EV1), or VenusC1 (EV2). TNF-α was used to activate NF-κB as a positive control. Prokaryotic expression vector pGEX-3X was included as a control. Luciferase signals were measured 4 h after TNF-α addition. Relative folds in comparison with EV1 control are shown. Significant differences between cells with vF1 in the presence or absence of TNF-α induction are denoted by “**". E. RRV vFLIP is unable to induce nuclear translocation of NF-κB subunit p65. HeLa cells (top panel image) and HeLa-vFLIP stable cells (lower panel image) were treated with TNF-α or combination of TNF-α and cycloheximide for 4 h and harvested for fractionation of cytoplasmic and nuclear portions, followed by Western blotting with p65 antibody. Tubulin and PARP-1 were detected on the same membrane to confirm the separation of cytoplasmic and nuclear fractions. F. Extension of cell viability after apoptosis induction. HeLa, HeLa-empty vector (EV) and HeLa-vFLIP cells were tested by CellTiter-Glo Cell Viability Assay at 0 and 38 h after apoptosis induction. Relative folds in comparison with normal HeLa cells at 0 h are shown. Significant differences between HeLa-vFLIP and HeLa-EV cells are denoted by “**".</p

RRV latent infection of BJAB cells protects the cells from apoptosis.

<p>A. RRV latent infection of BJAB cells protects the cells against apoptosis and inhibition of autophagy abolishes the protective effect. BJAB cells latently infected with RRV (BJAB-RRV) were either untreated, treated with 3-MA for 3 h prior apoptosis induction by TNF-α and cycloheximide, or treated with ammonium chloride at the same time of the apoptosis induction. The cells were harvested 2 h post-apoptosis induction for Western blotting of PARP-1 cleavage. Similar treatment of uninfected BJAB cells was included as a control. The ratio of top PARP-1 band to low band is shown under the image. B. Detection of vFLIP in RRV-infected BJAB cells by Western blotting with rabbit anti-vFLIP antibody. C. Cell viability assay of BJAB and BJAB-RRV cells 3 h after apoptosis induction. Relative folds in comparison with uninfected BJAB cells at 0 h are shown. Significant differences between BJAB and BJAB-RRV cells after apoptosis induction are denoted by “**", which indicates <i>P</i><0.01. D. Suppression vFLIP expression in RRV-infected BJAB cells by siRNA. BJAB cells latently infected with RRV were transfected with a siRNA against vFLIP. An irrelevant siRNA was included as a control. Real-time RT-PCR was conducted to assess vFLIP transcript level. Relative percentages in comparison with mock-treated control are shown. Significant differences between siRNA-treated and mock-treated BJAB-RRV cells are denoted by “**". E. Suppression of RRV vFLIP gene expression in BJAB-RRV cells leads to loss of the capability against apoptosis. BJAB cells latently infected with RRV were transfected with siRNA against vFLIP 15 h before apoptosis induction. Treatment of uninfected BJAB cells was included as a control. The cells were harvested 2 h after treatment with TNF-α and cycloheximide for Western blotting of PARP-1 cleavage. The ratios of top PARP-1 band to low band are shown under the image.</p