GA is used as an immunomodulatory treatment in patients with MS. It is thought to deviate GA-specific CD4+
T cell responses toward the Th2 phenotype, thereby causing bystander suppression of the pathogenic myelin-specific Th1 responses (21
). However, direct immunophenotypic analysis of GA-induced responses has not been carried out thus far. In the present study, we characterized the immunophenotype of GA-induced T cell responses.
Using CFSE-based flow cytometric proliferation assays, we detected T cell responses to GA in both healthy individuals and MS patients, as described in previous studies (23
). However, we observed significant differences in the phenotypic subsets of T cells responding to this drug. Whereas the CD4+
GA-specific T cell responses were comparable in all groups, the CD8+
responses were significantly lower in untreated MS patients (Figure ). To our knowledge, this is the first direct immunophenotypic evidence of a CD8+
proliferative response to GA.
Following treatment with GA, the GA-induced CD4+ and CD8+ T cell responses displayed differential regulation. The overall pattern of CD4+ responses appeared to be a transient upregulation around the 3-month time point followed by a gradual decline (Figure ; Table ). The CD8+ T cell responses, however, showed distinct upregulation after treatment and were significantly higher compared with baseline levels, more comparable to those seen in healthy individuals (Figures and ). Since CD4+ responses to GA show temporal downregulation, in contrast to upregulation of CD8+ responses, it is possible that the immunomodulatory effect of this drug may be mediated, at least in part, by the GA-induced CD8+ T cells.
Prior studies have used traditional 3
H-thymidine–based in vitro proliferation assays that measure antigen-induced incorporation of 3
H-thymidine in proliferating cell cultures. Using these assay procedures, we found a transient increase in GA-specific bulk proliferation followed by a decline during the treatment course (Figure ), similar to previously reported observations (24
). Thus, the temporal dynamics of 3
H-thymidine–based proliferation appears to predominantly mirror the CD4+
GA-specific T cell responses. Whereas dividing CD8+
T cells would undoubtedly incorporate 3
H-thymidine during DNA synthesis, it is important to note that the enhanced CD8+
T cell responses are not reflected in bulk proliferation. The CD8+
T cell population forms a minor component of the overall proliferating fraction due to the high CD4/CD8 ratio. This is especially true in patients with MS, who may show a skewed CD4 preponderance (36
). As an example, in Figure a, at the 3-month time point for patient no. MS1, we observed a prominent GA-specific CD8+
T cell response with a proliferating fraction of 21.32%. However, this represents only 3.08% of the total live cells in culture, whereas the concurrent CD4+
response with a proliferating fraction of 42.43% represents 26.21% of the total live cells. Thus, the CD4+
T cell response contributes a far greater proportion of the bulk proliferation. To detect shifts in the CD8+
responses, it is essential to specifically analyze that population of T cells. Additionally, the CFSE-based proliferation assay measures cumulative proliferation over the entire culture period, whereas the 3
H-thymidine–based assay measures incorporation only over the last 12–18 hours of culture. These factors explain why a previous report using comparative bulk proliferation assays before and after depletion of CD8+
T cells from untreated MS patients did not detect significant differences, leading to the interpretation that there are no CD8+
responses to GA (23
). However, it is still possible that such a difference may be detected in fractionated cells from healthy individuals or treated MS patients who have a robust CD8+
T cell response. This emphasizes the need to specifically evaluate this subset of cells by using direct phenotypic assays or pre-sorted populations of cells.
Several investigators have reported that MS patients have defective suppressor function in their CD8+
T cell compartment (36
). A regulatory role for CD8+
T cells has been demonstrated in EAE (39
). Upregulation of such suppressive ability has also been seen following combined therapy with etretinate and IFN-β1b (43
). From these studies, it is tempting to speculate that the diminished CD8+
T cell response to GA in untreated MS patients may be reflective of the global defect of regulatory cells in this subset. As a corollary, treatment with GA may function, in part, through the induction and restoration of this regulatory population of cells.
To investigate the functional profile of GA-induced T cell responses, we performed flow cytometric staining on GA-responsive cells as well as molecular evaluation of flow-sorted populations of GA-reactive CD4+
T cells (Figures –). There were changes in the cytokine profiles of post-treatment GA-specific cells. IL-4 was detected predominantly in post-treatment CD4+
T cells. This finding corroborates previous reports that have shown a shift toward the Th0/Th2 phenotype of long-term T cell lines derived from treated patients and in ELISPOT assays performed on bulk PBMC specimens (24
T cells were positive for IFN-γ protein and message (Figures –). One previous report, using ELISPOT assays performed on one GA-treated patient, suggested that GA-specific IFN-γ production may be prominent in the CD8+
). Using short-term cytokine flow cytometry assays (6–8 hours of culture), we have also observed such GA-specific IFN-γ production in CD8+
T cells at the protein level in some patients (unpublished observations). Thus, GA treatment appears to induce an IFN-γ–secreting CD8+
T cell response. Inter-estingly, the suppressive activity of CD8+
T cells in mitogen-driven in vitro proliferation assays is thought to be mediated by IFN-γ secretion (37
We also detected the presence of TGF-β message in GA-specific CD4+
T cells (Figure ). This is a significant finding, as TGF-β has been shown to be an important mediator of bystander suppressive effects of Th2/Th3–type responses in EAE (44
). However, TNF-α was also detected in these cells, which may be pro- or antiinflammatory in different systems. Thus, the overall cytokine profile of GA-induced CD8+
T cells appears to have a component that may mediate regulatory inhibition of disease-mediating responses.
Of note, we did not observe statistically significant changes in the MBP-specific proliferative responses in these patients during the treatment course, although individual patients showed mild variation. This finding is consistent with previous reports (24
). Thus, at least in the PBMC compartment, there does not appear to be an obvious suppression of the MBP-specific response. It is possible that GA-specific T cells exert their regulatory effect locally at the site of tissue damage or through cross-reactive competition in these microenvironments. Further investigation is required to determine the exact mechanisms and the extent to which GA-specific CD4+
T cells may play a role in the modulatory effect of this drug.
To summarize, our observations provide the first direct immunophenotypic evidence, to our knowledge, of the presence of GA-induced CD8+ T cell responses. These responses are deficient in untreated MS patients and are upregulated in patients on GA therapy. The overall cytokine profiles of GA-induced responses are suggestive of, but do not provide conclusive evidence for, a regulatory functional profile. These findings support the notion that GA-induced CD8+ T cells may play an important regulatory role in the immunomodulatory effects of this drug and warrant further characterization. Finally, the upregulation of CD8+ T cell responses may serve as a useful marker in the monitoring of GA therapy.