CTLA-4 controls regulatory T cell peripheral homeostasis and is required for suppression of pancreatic islet autoimmunity

CTLA-4 is a member of the immunoglobulin super-family. It is expressed on the surface of helper T cells and transmits an inhibitory signal to T cells. Earlier studies have linked polymorphisms in CTLA-4 gene with several autoimmune diseases including type1 diabetes, thyroiditis and SLE. Severe disease has been observed in CTLA-4 deficient mouse. Several reasons have been proposed for this, although the exact is not known. These reasons include the ability of CTLA-4 to raise the threshold of T cell activation, to modulate T cell motility, alter T cell thymic selection or control the function of regulatory T cells (Tregs). Much work has been done to identify the role of CTLA-4 in Tregs function.

This paper is from the labs of Dr. Lucy. S. Walker, University of Birmingham Medical School, Birmingham, UK. The authors have tried to analyze the role of CTLA-4 in Tregs and the results are as follows:

• Treg are over-expressed in CTLA4-/- mice: To quantify Tregs in CTLA4 knock out mice (CTLA4-/-), authors used intracellular Foxp3 staining. Foxp3 (Forkhead box P3) is a specific marker for Tregs. The authors observed that Foxp3+ Tregs were over-expressed in CTLA4-/- mice in both peripheral LN and spleen in comparison to controls.
  
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  CTLA4-/-Treg show unaltered thymic selection but increased peripheral proliferation: The increased expression of Tregs in CTLA4-/- mice can possibly indicate thymic selection or enhanced proliferation in the periphery. To answer these questions, the authors stained thymus cells for CD4, CD8 and Foxp3. They found that the proportion of CD4+Foxp3+ cells was not much different in two groups of mice. To determine whether there was an increased proliferation of Tregs in CTLA4-/- mice, spleen sections from CTLA4-/- animals and controls were stained for CD4, Foxp3, and Ki67 (nuclear protein expressed by proliferating cells). The results showed increased proliferation of Foxp3+ cells in CTLA4-/- mice compared to controls.
  
• CTLA4 regulates proliferation of OVA-specific Treg: Interpretation of data obtained from CTLA4-/- animals is complicated by the lethal lymphoproliferative syndrome that they develop. To overcome this, CTLA4-/- mice can be bred onto a TCR transgenic background (DO11.10), directly their specificity to a non self antigen (OVA). These animals can further be maintained in a rag deficient background. Such DO11/CTLA4-/-/rag-/- mice do not develop lymphoproliferative syndrome and their T cells bear a naïve phenotype. But the DO11/rag-/- animals do not develop Treg. The authors therefore crossed these mice with those that expressed OVA under the control of RIP. This strategy allowed the authors to study Treg in mice that lack CTLA4 but have an intact CD28. In DO11+ mice that did not express the RIP-mOVA ag, DO11 T cells did not develop into Tregs. In antigen positive mice, a fraction of DO11+ T cells developed into Tregs. Thus, CTLA4 signaling is not an obligate step in Treg differentiation. The authors next examined whether CTLA4 expression limited Treg proliferation in the periphery. They observed that Treg in peripheral lymphoid organs of DO11xRIP-mOVA/rag-/- mice remained largely undivided even in the absence of CTLA4. In contrast, Tregs isolated from Pancreas exhibited greater proliferation in mice that lacked CTLA4. All these findings support the idea that CTLA4 signaling controls Treg proliferation in response to encounter with self antigens.
  
• Ab-mediated CTLA4 blockade augments Treg proliferation in BALB/c mice: The authors next tested whether Ab-mediated CTLA4 blockade in normal animals enhanced Treg proliferation, as shown in CTLA4 knockout mice. Mice, which were treated with anti-CTLA4 mAb showed increased ki67 within CD4+Foxp3+ cells after 8 days. Three days following blocking, Foxp3+ cells (Tregs) showed significantly increased proliferation than controls (treated with control mAb). The proliferation of Foxp3- T cells was not much different from that seen in control mAB treated mice. By day 8, Foxp3- cells showed an increased proliferation in anti-CTLA4 Ab treated mice, but this increase became significant only at day 14.
  
• Ag-specific Treg deficient in CTLA4 fail to suppress autoimmune diabetes: The results of investigators raised the paradox that a fatal lymphoproliferative syndrome develops in the CTLA4 knockout mice despite an increase in Treg. To resolve this paradox the authors hypothesized two reasons. Firstly, CTLA4 deficiency might render Treg pathogenic and secondly, CTLA4 might be required for Treg suppression. To test the first hypothesis, authors utilized the fact that injection of CTLA4-/- lymphocytes into rag deficient recipient mice can transfer lymphoproliferative syndrome. Thus, they assessed the ability of purified CD4+CD25- or CD4+CD25high cells from CTLA4-/- to transfer this disease. It was observed that introduction of CD25- cells from CTLA4-/- mice induced the lymphoproliferative syndrome while recipients of CD4+CD25+high cells from CTLA4-/- mice remained healthy. To test the second possibility that CTLA4 is necessary for Treg suppression in vivo, the authors took advantage of the fact that Ag-specific Treg lacking CTLA4 can be purified from TCR transgenic mouse. CD4+CD25+ cells were isolated from DO11xRIP-mOVA/rag-/- mice that were either CTLA4 sufficient or deficient. Treg proportion was equivalent between CTLA4 deficient or sufficient groups. To test the ability of these Tregs to control diabetes in an adoptive transfer model, disease was induced by transfer of OVA-specific CD25- cells into mice expressing OVA in the pancreas and then Tregs from both groups were introduced. It was observed that mice that received CTLA4 sufficient Treg were completely protected from diabetes, while mice that received CTLA4 deficient Treg were not protected from diabetes. To further explore the basis for the failure of CTLA4-/- Treg to control diabetes, the investigators analyzed the expression of IL-10 and surface TGF-beta. No difference in IL-10 expression was observed between wild type and CTLA4-/- Tregs. The expression of TGF-beta was higher in CTLA4-/- Tregs. The investigators further investigated the hypothesis that Treg-expressed CTLA4 can deplete antigen presenting cells of co-stimulatory ligands. The authors found that the presence of Treg resulted in marked down-regulation of CD86 expression on APC and this down-regulation could be blocked by using anti-CTLA4 Ab. Expression level of MHC class II on APC was unaltered by Treg. Taken together, these data suggest that Treg-expressed CTLA4 plays important role in the regulation of an anti-islet immune response and this reflects a capacity for Treg to influence APC in a CTLA4 dependent manner.