Fig 1.
Inhibition of T-cell activation by the HTLV FP.
(A) MOG35-55–antigen specific T-cells were activated by irradiated MOG35-55 presenting APCs, in the presence of several viral derived FPs at 10μM. The proliferative responses were assessed by H3-thymidine incorporation assay, and normalized to the proliferation of non-activated T-cells. The HTLV FP inhibits T-cell proliferation with the same potency as the HIV FP. The data is presented as mean inhibition of proliferation. n = 12. (B) MOG35-55–antigen specific T cells were activated by either (i) irradiated MOG35-55 presenting APCs (black), (ii) antibodies against CD3 and CD28 (white), or (iii) PMA and Ionomycin (gray) in the presence of the HTLV and HIV FPs at 10μM. Proliferation was assessed as described above. The HTLV FP inhibits T-cell proliferation induced either through the TCR or downstream from the TCR with equal potencies. The data is presented as mean inhibition of proliferation. n = 12. One-way ANOVA was used for statistical analysis. **P<0.01;***P<0.001.
Table 1.
Designation, sequence, and origin of peptides used in this study.
Fig 2.
Modulation of Th1/Th2 gene expression and cytokine secretion from activated T-cells by the HTLV FP.
MOG35-55–antigen specific T-cells were activated by irradiated MOG35-55 presenting APCs, in the presence of the HTLV FP at 10μM. (A) Total RNA was isolated 24 hours following activation and mRNA expression levels were determined by qRT-PCR. The values of each gene were normalized to Rpl13a as a housekeeping control. The HTLV FP reduces mRNA expression of Th1 specific genes (IFNG, LTA, and STAT4) and elevates mRNA expression of Th2 specific genes (IL4 and IL10). The data is presented as arbitrary units. n≥3. (B) Media was collected 24 hours following activation and secretion of cytokines was measured by ELISA assay. IFN-γ secretion is inhibited, IL4 secretion is elevated and TNFα is not affected by the HTLV FP. n = 12. One-way ANOVA was used for statistical analysis. *P<0.05; **P<0.01;***P<0.001.
Fig 3.
Administration of HTLV FP alleviates MOG35-55-induced EAE.
(A-B) MOG35-55–antigen specific T-cells were activated by irradiated MOG35-55 presenting APCs, in the presence of HTLV FP or HTLV Scr at 10μM. (A) The proliferative responses were assessed by H3-thymidine incorporation assay, and normalized to the proliferation of non-activated T-cells. The data is presented as mean inhibition of proliferation. n = 12. (B) IFN-γ was measured by ELISA assay. n = 12. (C-D) EAE was induced in C57BL/6 female mice that were treated with a single dose of HTLV FP, HTLV Scr or vehicle. Two indexes to measure clinical disease severity are displayed. (C) Direct clinical measurement of EAE phenotype in a 5-point scale with increased disease symptoms correlating with higher score value. The data is presented as mean EAE clinical score. (D) Mice were weighed the day before EAE induction, and the change (as a percentage) in weight was recorded. The data is presented as mean change from the initial weight. One-way ANOVA was used for statistical analysis. a HTLV FP ≠ Vehicle; b HTLV FP ≠ Vehicle & HTLV Scr; c HTLV FP ≠ HTLV Scr. a,b,cP<0.05. (E) Spleenocytes were harvested at 26 DPI and stimulated using MOG35-55. The proliferative responses were assessed by H3-thymidine incorporation assay and normalized to the proliferation of non-activated T-cells. The data is presented as counts per minute (CPM). n = 7. (F-G) Spleenocytes were harvested at 26 DPI and stimulated using MOG35-55. Media was collected 48 hours following activation and secretion of IFN-γ and IL4 was measured by ELISA assay. HTLV FP treatment inhibits IFN-γ and elevates IL4 secretion from MOG35-55-reactive T-cells. n>8. One-way ANOVA was used for statistical analysis. *P<0.05;**P<0.01;***P<0.001; ns, not significant.
Fig 4.
Primary human T-cell activation is inhibited upon HTLV FP treatment.
Human peripheral T lymphocytes were isolated from whole blood and activated using CD3 and CD28 antibodies, in the presence of HTLV FP or HTLV Scr at 10μM. Media was collected 48 hours following activation and secretion of IL2, IFN-γ and IL10 was measured by ELISA assay. HTLV FP treatment inhibits IL2 and IFN-γ and elevates IL10 secretion from primary human peripheral T-cells. n = 12. One-way ANOVA was used for statistical analysis. *P<0.05; ***P<0.001; ns, not significant.
Fig 5.
Reduced T-bet expression and elevated Gata3 expression induced by the HTLV FP.
(A-B) MOG35-55–antigen specific T-cells were activated by irradiated MOG35-55 presenting APCs, in the presence of the HTLV FP at 10μM. Total RNA was isolated 24 hours following activation and mRNA expression levels were determined by qRT-PCR. The values of each gene were normalized to Rpl13a as a housekeeping control. The HTLV FP reduces TBX21 (T-bet) mRNA expression and elevates GATA3 mRNA expression. The data is presented as arbitrary units. n≥3. (C-H) MOG35-55–antigen specific T-cells were activated by irradiated MOG35-55 presenting APCs, in the presence of either HTLV or HIV FP at 10μM. Samples were fixed in 4% PFA and stained with anti T-bet-APC and anti Gata3-FITC antibodies (C-D) 24, (E-F) 48 and (G-H) 72 hours following activation. Each time point is represented as cell count vs. APC or FITC fluorescence histogram. Analysis was performed using LSR-II flow cytometer (BD) and FlowJo cell analysis software (FlowJo, LLC). The HTLV FP reduces T-bet expression and elevates Gata3 expression. n = 3. One-way ANOVA was used for statistical analysis. ns, not significant; *P<0.05;** P<0.01.
Fig 6.
Detection of the immune modulatory region within the HTLV FP.
(A) Sequences of shorter peptides derived from the full length HTLV FP. (B) MOG35-55–antigen specific T-cells were activated by APCs in the presence of HTLV FP derived peptides at 10μM and proliferative responses were assessed as described above. The HTLV FP and HTLV FP5-13 inhibit T-cell proliferation with higher potency than HTLV FP9-22 and HTLV FP14-22. The data is presented as mean inhibition of proliferation. n = 12. (C) MOG35-55–antigen specific T cells were activated by either (i) irradiated MOG35-55 presenting APCs (black), (ii) antibodies against CD3 and CD28 (white), or (iii) PMA and Ionomycin (gray) in the presence of HTLV FP5-13 and HIV FP5-13 at 10μM. Proliferative responses were assessed as described above. The HTLV FP inhibits T-cell proliferation induced either through the TCR or downstream from the TCR with equal potencies. The data is presented as mean inhibition of proliferation. n = 12. (D) Secondary structures of HTLV FP derived peptides as revealed by CD spectroscopy. CD spectra were measured at 25μM in 5mM HEPES buffer containing 1% lyso-phosphatidylcholine (LPC). The HTLV FP and HTLV FP5-13 exhibit a typical α-helical curve while the HTLV FP9-22 and HTLV FP14-22 exhibit a typical random coil. (E) MOG35-55–antigen specific T-cells were activated by APCs in the presence of HTLV FP5-13 D/L enantiomers at 10μM and proliferative responses were assessed as described above. Both HTLV FP5-13 enantiomers inhibit T-cells proliferation with equal potencies. The data is presented as mean inhibition of proliferation. n = 12. One-way ANOVA was used for statistical analysis. *P<0.05;**P<0.01;***P<0.001.
Table 2.
CD spectra analysis of the HTLV FP derived peptides by the CDNN secondary structure analysis program.