Accumulating evidence from both animal and human studies point to an important role of regulatory T cells in the aetiology and pathogenesis of SLE.48
We have previously shown that nasal tolerance to histone peptide H471 suppresses lupus via
and that nasal anti-CD3 suppresses lupus via
In this study, we show oral anti-CD3 induced CD4+CD25-LAP+ regulatory T cells that suppressed effector T-cell function in a TGF-β-dependent, cell-contact independent fashion. Oral anti-CD3 in mice prone to developing lupus significantly reduced disease pathology and extended survival of mice beyond their life expectancy. Furthermore, suppression of disease was associated with down regulation of inflammatory IL-17+CD4+ICOS+CXCR5+ follicular helper T cells, plasma cells and memory B cells. We have previously shown that oral anti-CD3 suppresses murine models of EAE23
We now demonstrate that oral anti-CD3 can suppress lupus, a prototypic antibody-mediated disease in mice. We studied the therapeutic effects of oral anti-CD3 in a peptide-accelerated lupus model in SNF1 mice. Lupus prone SNF1 mice uniformly produce pathogenic anti-DNA autoantibodies under the influence of certain Th cells and develop severe lupus glomerulonephritis.50-52
Th clones derived from nephritic SNF1 mice rapidly induce immune deposit glomerulonephritis when transferred in vivo
into young preautoimmune SNF1 mice.53
Approximately 50% of such pathogenic T-cell clones are highly responsive to nucleosomal antigens54
and one of the determinants is in histone protein H4 sequence 71-94.31
Systemic immunisation of histone peptide H471 in SNF1 mice leads to early development of autoantibodies and glomerulonephritis.30
Oral anti-CD3 prior to systemic immunisation of H471 in SNF1 mice suppressed the development of severe glomerulonephritis and proteinuria. This was associated with a downregulation of IgG anti-dsDNA and anti-H471 autoantibodies.
We previously showed oral anti-CD3 led to the induction of CD4+CD25-LAP+ regulatory T cells.23,24
The anti-CD3 antibody was rapidly taken up in the intestinal tract and appeared in the villous epithelium within 30 min and increased at 1 h and 3 h after feeding.23
After oral anti-CD3, LAP+ regulatory T cells are first induced in the mesenteric lymph nodes after which they migrate to sites of inflammation. Thus, we observed increased numbers of LAP+ T cells in the popliteal lymph nodes in EAE animals immunised with proteolipid protein in CFA.23
In addition, in our oral anti-CD3 study in animals with streptozocin-induced diabetes, we found an increase of LAP+ T cells in the pancreatic lymph nodes.24
Thus, it appears that one of the properties of LAP+ regulatory T cells is their ability to migrate to sites of inflammation where they exert their regulatory effects. Furthermore, we eluted anti-CD3 antibody from the upper intestine of anti-CD3-fed animals and found that the eluted anti-CD3 antibody was biologically active in that it was able to stimulate naive T-cell proliferation to an equivalent of ~0.01 μg/mL of anti-CD3 antibody in vitro.24
The CD4+CD25-LAP+ regulatory T cell express surface LAP which identifies a class of regulatory T cells that function in a TGF-β-dependent fashion.22,25-28
LAP is the aminoterminal domain of the TGF-β precursor peptide and remains non-covalently associated with the TGF-β peptide after cleavage, forming the latent TGF-β complex. We studied purified (above 95% purity) CD4+CD25-LAP+ T cells from oral anti-CD3-treated SNF1 mice by real time RTPCR (Reverse Transcription Polymerase Chain Reaction) and found the relative expression of TGF-β was five times higher than CD4+CD25-LAP-T cell (data not shown). This finding was also demonstrated in our previous study on oral anti-CD3 in EAE mice.23
CD4+LAP+ T cells appear to be distinct from naturally occurring CD25+
regulatory T cells, though it has been reported that CD4+CD25+ T cells may express TGF-β on their surface and mediate their suppressive function by presenting TGF-β to a receptor on target cells via cell-to-cell contact.22,26,28,29
In this study, we also find that oral anti-CD3 in lupus prone SNF1 mice led to the upregulation of CD4+CD25-LAP+ regulatory T cells that were still detectable 90 days after treatment. This suggests that, like the naturally occurring CD4+CD25+ regulatory T cells, the inducible CD4+CD25-LAP+ regulatory T cells are able to persist in the periphery. We did not observe changes in frequency of CD4+CD25+ regulatory T cells following oral anti-CD3. In addition, the inducible LAP+ regulatory T cells suppress effector T-cell function in a TGF-β-dependent fashion. However, in contrast to the former, the latter do not express the molecular marker foxp3, and suppression does not require direct contact between the regulator and responder. These findings suggest that there are fundamental differences between naturally occurring and inducible regulatory T cells. Generation of inducible regulatory T cells can be beneficiary to SLE in that they can restore the deficit in immune regulation due to a lack of naturally occurring CD4+CD25+ regulatory T cells.16,17
The production of nephritogenic antinuclear autoantibodies in SLE is driven by cognate interactions between select populations of autoimmune Th and B cells.51,55-57
A distinct subset of T cells, follicular helper T cells, selects mutated, high affinity B cells within germinal centers,7
and they are needed to initiate plasma cell generation58,59
and antibody production.60,61
Numerous recent studies have shown that IL-17 producing T-helper cells constitute a distinct CD4+ T-helper population that differs in function and phenotype from classical T-helper type-1 and type-2 populations.62
IL-17 is linked to the induction of autoreactive humoral immune response because a deficiency in or blockade of IL-17 is associated with a decline in the autoantibody response.63-65
Here, we find that follicular helper T cells in SNF1 mice express the inflammatory cytokine IL-17. This result correlates with findings by Hsu et al
which show autoimmune BXD2 mice express heightened levels of IL-17 and that IL-17 is required for the spontaneous development of germinal centres and production of pathogenic autoantibodies in these mice. Injection of antagonistic antibody to IL-17 in mice disrupted T and B cell interaction and the formation of germinal centres and autoantibodies.66
Oral anti-CD3 suppressed the formation of CD4+ICOS +CXCR5+ follicular helper T cells. This correlates with a downregulation of CD73+ memory B cells and autoantibodies following oral anti-CD3. Oral anti-CD3 also led to a downregulation of IL-17 expression by the follicular helper T cells. Oral anti-CD3 in mice with spontaneous lupus also resulted in reduced plasma cells in spleens and IL-17+CD4+ T-cell infiltration in kidneys. Thus, it appears that oral anti-CD3 suppresses lupus in mice by disrupting cognate interaction of follicular helper T cells and B cells thus reducing autoantibody production and antibody-antigen immune complex mediated glomerulonephritis.
We performed our experiments with a F(ab’)2 antibody to eliminate any potential side effects related to the Fc portion of the molecule that might occur after multiple administrations of the antibody orally. We observed no mitogenic effect of oral hamster CD3-specific F(ab’)2 antibody in mice and no evidence of cytokine release syndrome (wasted appearance, ruffled fur) even after 30 oral administrations. Further more, we did not observe an anti-globulin response against anti-CD3 in mice orally treated with anti-CD3 (data not shown). Oral administration of CD3-specific antibody is applicable for chronic therapy and would not be expected to have side effects including cytokine release syndromes and anti-globulin responses.
In summary, we have shown that oral anti-CD3 antibody induces a CD4+CD25-LAP+ regulatory T cell that suppressed T-helper cell function leading to downregulation of autoantibody production and glomerulonephritis in murine lupus and it is applicable for treatment of human SLE.