Background/Purpose: Clonal anergy has been proposed as a mechanism to allow for peripheral recognition of self antigens, without the consequence of immunopathology. Model systems have confirmed that CD4 T cells can become unresponsive to self antigen stimulation, but there is currently no evidence for the existence of anergic CD4 T cells in the normal polyclonal repertoire.

Methods: Gene arrays were used to identify mRNAs specifically over-expressed in anergic ovalbumin (OVA)-specific OT-II TCR-transgenic CD4 T cells after adoptive transfer into healthy OVA-transgenic mice. Over-expression of anergy molecules was confirmed by multiparameter flow cytometry in anergic glucose-6-phosphate isomerase (GPI)-specific KRN TCR-transgenic CD4 T cells after adoptive transfer to self antigen-expressing healthy mice. Antigen-experienced polyclonal CD4 T cells expressing these anergy molecules were characterized by flow cytometry, purified, and tested for their capacity to cause immunopathology in lymphopenic hosts. Finally, adoptive transfer recipients of anergic polyclonal CD4 T cells were tested for evidence of autoantibody production and weight loss versus the generation of protective Foxp3⁺ Tregs.

Results: Using microarrays to compare mRNA expression in naive, effector, and anergic OT-II CD4 T cells, we discovered that genes for folate receptor 4 (Folr4; FR4) and ecto-5′-nucleotidase (Nt5e; CD73) were greatly over-expressed following the induction of clonal anergy. Examination of KRN CD4 T cells following their initial adoptive transfer to GPI-expressing hosts confirmed up-regulation of FR4 and CD73 during the induction of anergy, and a reversal of this anergic state following adoptive transfer of FR4^hi CD73^hi anergic KRN T cells to TCRa-deficient lymphopenic hosts. Making use of these FR4 and CD73 anergy markers, 2–3% of the peripheral mature polyclonal CD4 repertoire was discovered to be anergic. Foxp3 was not expressed; however, CD69, PD-1, CTLA4, CD5, and Nur77 up-regulation suggested continuous engagement by high affinity TCRs on these tolerant T cells. Consistent with in vivo anergy induction in response to self antigen recognition, antigen-experienced (CD44^hi) insulin/I-A^g7 tetramer-binding polyclonal CD4 T cells in NOD mice demonstrated high expression of FR4 and CD73. Adoptive transfer of FR4^hi CD73^hi polyclonal CD4 T cells to syngeneic TCRa-deficient lymphopenic hosts led to reconstitution of the Foxp3⁺ Treg compartment. Remarkably, elimination of newly differentiated Tregs in these anergic CD4 T cell recipients led to fatal wasting disease and the production of autoantibodies.

Conclusion: Peripheral self antigen recognition in normal hosts drives autoreactive CD4 T cells into an anergic state that is associated with the up-regulation of FR4 and CD73. Anergic T cells remain in the peripheral repertoire, both as a threat to health upon anergy reversal as well as a reservoir of precursors for protective Foxp3⁺ Tregs. A greater understanding of the role of anergy induction and reversal in the development of rheumatic diseases will offer new insights into strategies designed to restore self tolerance.