Insights into the Cytotoxic Potential of Human CD8+ T Cells: Implications for Virologic Control of HIV

CD8+ T cells are often referred to as cytotoxic T lymphocytes because of their ability to induce the apoptosis of cells infected with an intracellular pathogen, thereby limiting the spread of an infection to previously uninfected cells. CD8+ T cells produce many proteins - including perforin, various granzymes, and granulysin - that are responsible for inducing target cell cytolysis. These cytolytic proteins are found pre-packaged into secretory granules within many resting CD8+ T cells, but their de novo synthesis can also occur after activation. In the setting of HIV infection, a rare group of HIV-positive patients, termed elite controllers (EC), naturally control HIV viremia to virtually undetectable levels without the intervention of drug therapy. Some evidence has implicated HIV-specific CD8+ T cells in achieving or maintaining this virologic control; however, the mechanism(s) to explain these findings remains largely undefined. In this work, we report that HIV-specific CD8+ T cells from EC demonstrated a superior ability to express perforin and granzyme B after activation compared to patients with progressive disease. Therefore, HIV-specific CD8+ T cells from EC possessed a greater cytotoxic potential by expressing higher levels of two principal cytolytic mediators, which may at least partially explain the ability of EC to suppress the replication of HIV in vivo. One critical upstream regulator of effector functionality and differentiation is the T-box transcription factor T-bet. We demonstrated a clear link between levels of T-bet and the presence of perforin and granzyme B within human CD8+ T cells. Notably, HIV-specific CD8+ T cells from EC expressed higher amounts of T-bet than progressors, suggesting that therapeutic modulation of T-bet may restore the cytolytic potential that is deficient among patients with uncontrolled viremia. Collectively, our results imply that the underlying defect(s) in effector function by HIV-specific CD8+ T cells from progressors lie not in the cytolytic proteins themselves, but rather in the elements controlling their expression, including T-bet

Human GUCY2C-Targeted Chimeric Antigen Receptor (CAR)-Expressing T Cells Eliminate Colorectal Cancer Metastases.

One major hurdle to the success of adoptive T-cell therapy is the identification of antigens that permit effective targeting of tumors in the absence of toxicities to essential organs. Previous work has demonstrated that T cells engineered to express chimeric antigen receptors (CAR-T cells) targeting the murine homolog of the colorectal cancer antigen GUCY2C treat established colorectal cancer metastases, without toxicity to the normal GUCY2C-expressing intestinal epithelium, reflecting structural compartmentalization of endogenous GUCY2C to apical membranes comprising the intestinal lumen. Here, we examined the utility of a human-specific, GUCY2C-directed single-chain variable fragment as the basis for a CAR construct targeting human GUCY2C-expressing metastases. Human GUCY2C-targeted murine CAR-T cells promoted antigen-dependent T-cell activation quantified by activation marker upregulation, cytokine production, and killing of GUCY2C-expressing, but not GUCY2C-deficient, cancer cells in vitro. GUCY2C CAR-T cells provided long-term protection against lung metastases of murine colorectal cancer cells engineered to express human GUCY2C in a syngeneic mouse model. GUCY2C murine CAR-T cells recognized and killed human colorectal cancer cells endogenously expressing GUCY2C, providing durable survival in a human xenograft model in immunodeficient mice. Thus, we have identified a human GUCY2C-specific CAR-T cell therapy approach that may be developed for the treatment of GUCY2C-expressing metastatic colorectal cancer

Perforin Expression Directly Ex Vivo by HIV-Specific CD8+ T-Cells Is a Correlate of HIV Elite Control

Many immune correlates of CD8+ T-cell-mediated control of HIV replication, including polyfunctionality, proliferative ability, and inhibitory receptor expression, have been discovered. However, no functional correlates using ex vivo cells have been identified with the known ability to cause the direct elimination of HIV-infected cells. We have recently discovered the ability of human CD8+ T-cells to rapidly upregulate perforin—an essential molecule for cell-mediated cytotoxicity—following antigen-specific stimulation. Here, we examined perforin expression capability in a large cross-sectional cohort of chronically HIV-infected individuals with varying levels of viral load: elite controllers (n = 35), viremic controllers (n = 29), chronic progressors (n = 27), and viremic nonprogressors (n = 6). Using polychromatic flow cytometry and standard intracellular cytokine staining assays, we measured perforin upregulation, cytokine production, and degranulation following stimulation with overlapping peptide pools encompassing all proteins of HIV. We observed that HIV-specific CD8+ T-cells from elite controllers consistently display an enhanced ability to express perforin directly ex vivo compared to all other groups. This ability is not restricted to protective HLA-B haplotypes, does not require proliferation or the addition of exogenous factors, is not restored by HAART, and primarily originates from effector CD8+ T-cells with otherwise limited functional capability. Notably, we found an inverse relationship between HIV-specific perforin expression and viral load. Thus, the capability of HIV-specific CD8+ T-cells to rapidly express perforin defines a novel correlate of control in HIV infection

Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells

Citation: Hand, E. S., Haller, S. L., Peng, C., Rothenburg, S., & Hersperger, A. R. (2015). Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells. Plos One, 10(3), 15. doi:10.1371/journal.pone.0119189As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV

Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV

Late replication cycle events and cell surface virion assembly of ECTV are diminished in RK13 cells.

<p><b>(A)</b> Cells were grown on sterile glass coverslips, infected (MOI = 5) with ECTV, fixed 24 hours later, and then stained for the presence of cell surface B5 protein. Representative images are shown only for infected conditions because staining of uninfected control cells yielded no detectable signal. All images are at 400x total magnification. <b>(B)</b> Cells, which had been infected (MOI = 5) for 24 hours with ECTV, were stained for surface B5 and intracellular E3 following the protocol outlined in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119189#sec002" target="_blank">Methods</a> section. All data shown are representative of three independent experiments.</p

ECTV does not form plaques on RK13 cells.

<p>Monolayers of BSC1 and RK13 cells were initially infected with a low amount of ECTV-GFP (MOI = 0.001). On day 5 post-infection, the monolayers were examined for the formation of plaques. As expected, ECTV formed plaques on BSC1 cells (a representative example is shown on the bottom row). However, no plaque formation was observed on RK13 cells (a representative example is shown on the top row). All images are at 100x total magnification.</p

Growth of VACVΔE3LΔK3L is restored in RK13 cells expressing VACV E3 and K3 proteins.

<p><b>(A)</b> VACVΔE3LΔK3L was constructed using homologous recombination techniques and engineered to express GFP in place of the E3L gene. No plaque formation was visualized in wild-type RK13 cells whereas replication capacity was restored in RK13 cells stably expressing E3 and K3 proteins derived from VACV. Representative images are shown. <b>(B)</b> The total yield of VACV wild-type, VACVΔE3L, VACVΔK3L, and VACVΔE3LΔK3L was determined using wild-type RK13 (red bars) and RK13+E3L+K3L (gray bars) cells. Cells were initially infected with a low amount of virus (MOI = 0.01). Virus was harvested 30 hours post-infection and the total yield was determined using standard plaque assays with RK13+E3L+K3L cells. The data is the average of two independent experiments with error bars representing the standard deviation. P-values were determined in Microsoft Excel using the Student’s t-test.</p

Virus factory formation during ECTV infection is more robust in BSC1 compared to RK13 cells.

<p><b>(A)</b> Cells were grown on sterile glass coverslips, infected (MOI = 5) with ECTV, and fixed 24 hours later. Cell nuclei (N) and virus factories (F) were visualized following DAPI staining. Representative images are shown for both uninfected and infected cells. All images are at 1,000x total magnification. <b>(B)</b> One hundred randomly viewed cells within each infection condition were scored as either being positive or negative for the presence of a virus factory as compared to the uninfected control. The bars depict the average values and the error bars represent the standard deviations of three independent trials. Statistical analysis [performed using GraphPad Prism software (version 5.0a)] was carried out using a one-way ANOVA (nonparametric; Kruskal-Wallis) followed by a Dunns test for multiple comparisons. * denotes a p value <0.05.</p

ECTV- and VACV-induced phosphorylation of eIF2α in RK13 cells.

<p><b>(A)</b> Wild-type RK13 or RK13+E3L+K3L cells were mock infected or infected with the indicated VACV strains at an MOI of 5. Cell lysates were collected 6 hours post infection and subjected to SDS-PAGE and immunoblot analysis. Membranes were first probed with antibodies against eIF2α phosphorylated at Ser51 (eIF2α-P; top row), stripped, and then re-probed with anti-eIF2α antibodies (total eIF2α; bottom row). Band intensities were measured using the Kodak Image Station 4000MM and the ratios of phosphorylated eIF2α to total eIF2α were calculated as a percentage and listed below the panels. <b>(B)</b> The average ratios of two independent western blots are indicated in the bar graph with error bars indicating the standard deviation. <b>(C and D)</b> Wild-type RK13 <b>(C)</b> or RK13+E3L+K3L cells <b>(D)</b> were mock infected or infected with the indicated viruses at an MOI of 5 for 12 hours at which time cells were collected, permeabilized, and stained for phosphorylated eIF2α. The histograms depict relative fluorescence intensity among total cells and are representative of three independent trials.</p