Silencing T-bet Defines a Critical Role in the Differentiation of Autoreactive T Lymphocytes

AbstractAs a means of developing therapies that target the pathogenic T cells in multiple sclerosis (MS) without compromising the immune system or eliciting systemic side effects, we investigated the use of T-bet-specific antisense oligonucleotides and small interfering RNAs (siRNA) to silence T-bet expression in autoreactive encephalitogenic T cells and evaluated the biological consequences of this suppression in experimental autoimmune encephalomyelitis, a model for MS. The T-bet-specific AS oligonucleotide and siRNA suppressed T-bet expression, IFNγ production, and STAT1 levels during antigen-specific T cell differentiation. In vitro suppression of T-bet during differentiation of myelin-specific T cells and in vivo administration of a T-bet-specific antisense oligonucleotide or siRNA inhibited disease. T-bet was shown to bind the IFNγ and STAT1 promoters, but did not regulate the IL-12/STAT4 pathway. Since T-bet regulates IFNγ production in CD4+ T cells, but to a lesser extent in most other IFNγ-producing cells, T-bet may be a target for therapeutics for Th1-mediated diseases

Demarcated thresholds of tumor-specific CD8 T cells elicited by MCMV-based vaccine vectors provide robust correlates of protection.

The capacity of cytomegalovirus (CMV) to elicit long-lasting strong T cell responses, and the ability to engineer the genome of this DNA virus positions CMV-based vaccine vectors highly suitable as a cancer vaccine platform. Defined immune thresholds for tumor protection and the factors affecting such thresholds have not well been investigated in cancer immunotherapy. We here determined using CMV as a vaccine platform whether critical thresholds of vaccine-specific T cell responses can be established that relate to tumor protection, and which factors control such thresholds. We generated CMV-based vaccine vectors expressing the E7 epitope and tested these in preclinical models of HPV16-induced cancer. Vaccination was applied via different doses and routes (intraperitoneal (IP), subcutaneous (SC) and intranasal (IN)). The magnitude, kinetics and phenotype of the circulating tumor-specific CD8 Immunization with CMV-based vaccines via the IP or SC route eliciting vaccine-induced CD8 This study highlight the effectiveness of CMV-based vaccine vectors, and shows that demarcated thresholds of vaccine-specific T cells could be defined that correlate to tumor protection. Together, these results may hold importance for cancer vaccine development to achieve high efficacy in vaccine recipients

Glatiramer acetate (Copaxone) therapy induces CD8(+) T cell responses in patients with multiple sclerosis

Glatiramer acetate (GA; Copaxone) is a random copolymer of glutamic acid, lysine, alanine, and tyrosine that is used therapeutically in patients with multiple sclerosis (MS). To investigate the mechanism of the drug’s immunomodulatory effect, we used immunophenotypic approaches to characterize the precise nature of GA-induced T cell responses. We demonstrate here that healthy individuals and untreated MS patients exhibit prominent T cell proliferative responses to GA. However, these responses are different in distinct subsets of T cells. Whereas GA-induced CD4(+) T cell responses are comparable in healthy individuals and MS patients, CD8(+) T cell responses are significantly lower in untreated MS patients. Treatment with GA results in upregulation of these CD8(+) responses with restoration to levels observed in healthy individuals. Both CD4(+) and CD8(+) GA-specific responses are HLA-restricted. GA therapy also induces a change in the cytokine profile of GA-specific CD4(+) and CD8(+) T cells. This study provides the first direct immunophenotypic evidence, to our knowledge, of GA-specific CD8(+) T cell responses and their upregulation during the course of therapy, which may suggest a role for these responses in the immunomodulatory effects of the drug

Peptide Processing Is Critical for T-Cell Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine Vectors.

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy

C-terminal localization of the HGIRNASFI peptide allows its presentation on the surface of infected cells.

<p>(<b>A</b>) Cell surface expression of MHC class I molecule (D^b). LSECs (C57BL/6) were infected with the indicated viruses at MOI of 5 with centrifugal enhancement as described. D^b expression was measured by flow cytometry at 16h p.i‥ Fluorescence histograms for a representative experiment are shown. (<b>B</b>) LSECs were infected with the indicated viruses or incubated with a corresponding dose of UV inactivated virus (UV dose– 150J) at an MOI of 0.2 with centrifugal enhancement and co-cultured with HGIRNASFI-specific CTLs at an E:T ratio 3:1. Co-culture was performed for 15h, upon which the T cells were collected and stained for intracellular IFNγ. Where indicated, virus was inactivated by UV light. Two independent experiments were performed, with 4 or 5 wells per experimental condition. Representative dot plots are shown. (<b>C</b>) Relative intensity of signals measured by targeted nanoLC-MS³ from D^b-immunoprecipitates of cells infected for 24 hours with indicated viruses at an MOI of 2 with centrifugal enhancement. Two high abundant endogenous control peptides (KALINADEL and AALENTHLL) and a low abundant (FGPVNHEEL) endogenous control peptide were present in all IP samples, indicating valid sample processing in all cases. The MCMV target peptide HGIRNASFI was detected only in the cells infected with the MCMV^M45Cterm recombinant. (<b>D</b>) Grouped means +/- SEM of blood CD8 T-cells stained by HGIRNASFI-D^b tetramers in bone-marrow chimeric mice, where C57BL/6 mice received TAP^-/- or C57BL/6 bone marrow at 3 months before infection with 10⁶ PFU/mouse of MCMV^WT (top panel) or MCMV^M45Cterm (bottom panel). The experiment was performed three times at 5 mice per group and pooled results are shown. Significance on day 7 p.i. was assessed by a Mann—Whitney <i>U</i> test. ****<i>p</i> < 0.0001, ns—not significant.</p

Constitutive proteasomal processing is critical for the induction of inflationary CD8 T-cell responses.

<p>(<b>A</b>) IC-21 cells were infected with indicated viruses at an MOI of 0.2 with centrifugal enhancement. Splenocytes obtained from gBT-I.1 mice were used as effector cells at an E:T ratio of 3:1. Splenocytes were not restimulated upon isolation from the mice and used untouched for the assay. Co-culture was performed overnight (15h). Columns represent the mean percentage of IFNγ⁺ or TNFα⁺ cells from triplicate experiments, and error bars show the SEM. (<b>B</b>) 129/Sv mice were infected intraperitoneally (i.p.) with 2x10⁵ PFU of indicated MCMV recombinants and 10⁶ PFU of VACV^SL. Blood of mice was analysed for the fraction of CD8 T cells responding to <i>in vitro</i> re-stimulation with the SSIEFARL peptide for 6 hours, followed by intracellular staining for IFNγ. Grouped means +/- SEM of cells responding to the SSIEFARL peptide at 7, 14, 28, 60, 90, 120, 180 dpi are shown. The experiment was performed two times independently, at 5 mice per group in each experiment, and grouped averages from two experiments are shown. Difference in responses between groups infected with either MCMV^M45SL or MCMV^M45ASL was identified. Significance was assessed by Kruskal-Wallis test followed by Dunns post-analysis for MCMV^M45SL and MCMV^M45ASL infected mice (*<i>p</i><0.05, **<i>p</i><0.01). (<b>C</b>) Treatment with proteasomal inhibitors (but not protease inhibitors) impairs target cell recognition by HGIRNASFI-specific CTL. Target cells (LSECs) were pretreated for 5h with indicated inhibitors, washed twice with PBS and infected with MCMV^WT or MCMV^M45Cterm at an MOI 0.2 with centrifugal enhancement. Co-culture with HGIRNASFI-specific CTLs was performed at an E:T ratio 3:1 for 15h, upon which the T cells were collected and stained for intracellular IFNγ. The y-axis shows percentages of CTL responding by IFNγ to co-culture with target cells (mean +/- SEM from three experiments is shown). Labels below the x-axis show the deployed inhibitor and its concentration in μM; LC—lactacystin, MG—MG132, LP—leupeptin; Pos–positive control, target cells infected with indicated viruses without pretreatment with inhibitors; DMSO—infection in presence of the diluent for inhibitors Neg–negative control, untreated cells. (<b>D</b>) LMP7^-/- and C57BL/6 adult mice were infected i.v. with 10⁵ PFU of MCMV^WT (top panel) or MCMV^M45Cterm (bottom panel). The percentage of blood CD8 T cells stained by HGIRNASFI-D^b tetramers was measured at the indicated time points. The data show the mean values +/- SD from of pooled results from 2 independent experiments (in total 8 mice per each group) Significance on indicated time points was assessed by a Mann—Whitney <i>U</i> test. *<i>p</i> < 0.05. ***<i>p</i> < 0.0001, ns—not significant.</p

Immune protection by C-terminal epitope localization in a CMV vaccine vector.

<p>(<b>A-C</b>) Mice were prime/boosted at 4 weeks intervals with recombinant MCMVs or control virus and challenged with 2.5x10⁴ TC-1 cells/mouse at least 10 weeks after priming. Tumor size measured by caliper (mean +/- SEM is shown). <b>(A)</b> Immunization was performed with 10⁶ PFU of MCMV^E6+E7 (n = 9) and tumor growth compared to unvaccinated (naïve) controls (n = 10) (<b>B</b>) Mice were immunized with 10⁵ PFU of MCMV^ie2E7, and compared to MCMV^ie2SL or PBS control (n = 10 in each group) (<b>C</b>) Mice were prime/boosted with 10⁵ PFU of MCMV^ieE6-7Full (n = 9) and compared to unvaccinated controls (n = 12) (<b>D</b>) Representative flow cytometry plots of dextramer-stained blood lymphocytes from mice infected with 10⁵ PFU of MCMV^E6+E7, MCMV^ie2E7, MCMV^ie2E6-7Full or MCMV^ie2SL and analyzed by D(b) E7_49-57 dextramer staining for the presence of E7-specific CD8 T cells at 21 weeks post-priming. (<b>E</b>) Group values from dextramer staining as in panel D are shown (each symbol is a mouse; horizontal line shows the median). Significance was assessed by Kruskal—Wallis test followed by Dunn’s post hoc analysis for indicated columns. **<i>p</i> < 0.01, ns—not significant.</p

Peptide C-terminal localization results in better protection and induction of effector memory CD8 T-cell response.

<p><b>(A</b>) 129Sv female mice were i.p. infected with 2x10⁵ PFU MCMV^WT, MCMV^ie2SL or MCMV^M45SL (n = 10 in each group) and 8 months later challenged with 10⁶ PFU of VACV^SL. Seven days post challenge, ovaries were titrated for infectious vaccinia by plaque assay. Histograms show group means, error bars are standard deviations. Significance was assessed by Kruskal—Wallis test followed by Dunn’s post hoc analysis for indicated columns. **<i>p</i> < 0.01, ns—not significant. <b>(B-C)</b> 129/Sv mice were infected intraperitoneally (i.p.) with 2x10⁵ PFU of MCMV^ie2SL, MCMV^M45SL or 10⁶ PFU of VACV^SL. Blood leukocytes were stimulated with the SSIEFARL peptide at 7, 14, 28, 60, 90, 120, 180 dpi. Cells were surface-stained for CD3, CD4, CD8, CD11a, CD44, KLRG1, CD127 and intracellularly for IFNγ expression and analyzed by flow cytometry. (<b>B</b>) Representative dot plots of KLRG1 and CD127 expression in IFNγ producing cells upon 6h SSIEFARL in vitro re-stimulation on days 7 and 180 p.i‥ (<b>C</b>) Left graph—epitope specific cells with the CM phenotype (CD127⁺KLRG1^-). Right graph—epitope specific cells with the EM phenotype (CD127^-KLRG1⁺). The experiment was performed three times independently, at 5 mice per group in each experiment, and grouped averages +/- SEM from all three experiments are shown. Significance on day 180 p.i. was assessed by Kruskal—Wallis test followed by Dunn’s post hoc analysis. *<i>p</i><0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, ****<i>p</i> < 0.0001, ns—not significant.</p