Fig 1.
Methodology for the generation of murine GA-responsive Th2-polarized T cells.
In vivo immunization of naive mice with Copaxone was followed by 13 rounds of ex vivo restimulation of the CD4+ T-cell population over 6 months for development of the Th2-455 line. APCs, antigen-presenting cells; GA, glatiramer acetate; IL-2, interleukin 2; Th, T-helper.
Fig 2.
Characterization of the murine Th2-polarized T cells.
Cytokine profile and dose response. A: Cytokine profile of the conditioned medium from the Th2-455 cell line after 24 hours of treatment with Copaxone at a single concentration of 20 μg/mL, demonstrating Th2 polarization. B: Copaxone dose response at 24 hours of a single cytokine, IL-4. IFN-γ; interferon gamma; KC, keratinocyte chemoattractant; IL, interleukin; TGF-β1, transforming growth factor-beta 1; Th, T-helper.
Table 1.
Number of different probes between Copaxone, Glatopa, nonequivalent glatiramoid molecule ACN, and media (univariate analysis, P < 0.05*).
Table 2.
Number of significantly perturbed probes (q-value < 0.05 and fold change > 1.3) when comparing sample groups.
An initial set of 4176 probes was detected by comparing Copaxone and media groups. This set yielded significant differences between Glatopa and media and between nonequivalent glatiramoid ACN and glatiramer acetate (Copaxone or Glatopa), but no significant difference between Copaxone and Glatopa.
Table 3.
P-values for multivariate statistic t inferred by permutation control (105 random assignments of samples between compared groups) based on the 4176 Copaxone-responsive probes.
In agreement with results presented in Table 2, there were significant differences when comparing Copaxone with media, Glatopa with media, nonequivalent glatiramoid ACN with media, and ACN with glatiramer acetate (Copaxone or Glatopa), but no significant difference for Copaxone vs Glatopa.
Fig 3.
MDS plots based on the 4176 Copaxone-responsive probes.
As expected, clear separation is obtained between Copaxone and media groups. There is also clear separation between media and copolymer groups (ACN, Copaxone, or Glatopa), some separation between GA (Copaxone or Glatopa) and ACN and no visible separation between Copaxone and Glatopa. ACN, acetonitrile nonconforming copolymer; GA, glatiramer acetate; MDS, multidimensional scaling.
Fig 4.
Statistical comparisons between Copaxone and Glatopa based on all probes.
A: distribution of t-test P-values across all array spots. The proportion of small P-values (P < 0.05) is small and the largest obtained q-value is greater than 0.99. B: multivariate statistic t (MVT) results. The observed value of MVT for Copaxone vs Glatopa is not significantly large as compared to values expected when randomly mixing samples between these two groups (P = 0.38).
Fig 5.
Visual comparisons between Copaxone, Glatopa and ACN treatment groups based on all probes.
A: The MDS plot shows separation between glatiramer acetate (GA; Copaxone or Glatopa) and media, separation between ACN and media, and no separation between Copaxone and Glatopa. B: Hierarchical clustering yields separation between GA (Copaxone or Glatopa) or ACN and media and no separation between Copaxone and Glatopa. C: Box plot of principal component analysis (PCA) component 1. Includes samples from media control, Copaxone, Glatopa, and ACN.
Fig 6.
T-helper cell pathway diagram.
Transcripts measured in the current study are shown in the diagram as nodes and are colored based on the differences observed when cells are stimulated with Copaxone in comparison with cell culture media alone (P < 1e-3 [Student’s t-test]; red for increase, green for decrease). Molecules with P > 1e-3 are shown in gray. Genes refer to the human ortholog. Human HLA-DMA, HLA-DMB, and HLA-DQB1 represent murine H2-Dma, H2-Dmb2, and H2-Ab1, respectively. Blue arrows show the flow of activation by major Th1- and Th2-influencing molecules in the pathway. For example, APCs produce IL-4, which binds to and activates the IL-4 receptor, leading to the phosphorylation and activation of the transcription factor STAT6. Red and green arrows show the expected transcriptional outcomes of Th1 and Th2 polarization; red and green arrows indicate that activation will cause the transcript to increase and decrease, respectively. Expected transcriptional outcomes are based on reports in the literature on Th1/Th2 T-cell polarization and on studies conducted with GA [3,4,8,10,17,29–34]. Gray lines indicate members of a group. APCs, antigen-presenting cells; CXCR1, chemokine (C-X-C motif) receptor 1; CXCR3, chemokine (C-X-C motif) receptor 3; CXCR5, chemokine (C-X-C motif) receptor 5; CXCL10, chemokine (C-X-C motif) ligand 10; GA, glatiramer acetate; HLADMA, major histocompatibility complex class II, DM alpha; HLADMB, major histocompatibility complex class II, DM beta; HLADQB1, major histocompatibility complex class II, DQ beta 1; ICOSLG, inducible T-cell costimulatory ligand; IL-4, interleukin-4; MHC class II, major histocompatibility complex class II; LTB, lymphotoxin beta (tumor necrosis factor superfamily, member 3); MYD, myeloid differentiation primary response protein; S100A10, S100 calcium-binding protein A10; STAT, signal transducer and activator of transcription; TCR, T-cell receptor; Th, T-helper.
Table 4.
Gene expression levels in Th2-polarized cells exposed to Copaxone and Glatopa vs media alone.
Table 5.
Gene expression levels in Th2-polarized cells exposed to Glatopa vs Copaxone.
Fig 7.
Box plots for gene expression changes for key Th2 cytokines IL-4 and IL-3 and additional genes related to immune cell function.
No statistically significant differences between Glatopa and Copaxone were observed for any of these genes.