Homeostasis in the ileum, which is commonly disrupted in patients with Crohn's disease, involves ongoing immune responses. To study how homeostatic processes of the ileum impact CD4T cell responses, we used TCR transgenic tools to breed mice that spontaneously produced CD4T cells reactive to an antigen expressed in the ileum. At an early age, the ilea of these mice exhibit crypt hyperplasia and accumulate increased numbers of T17 cells bearing non-transgenic clonotypes. Half of these mice subsequently developed colitis linked to broad mucosal infiltration by T17 and T1 cells expressing non-transgenic clonotypes, chronic wasting disease and loss of ileal crypt hyperplasia. By contrast, adult mice with normal growth continued to exhibit T17-associated ileal crypt hyperplasia and additionally accumulated ileal-reactive Treg cells. Both IL-17A and IFNγ were protective, as their deficiency precluded ileal-reactive Treg accumulation and exacerbated colitic disease. IL-23R blockade prevented progression to colitis, whereas nTreg cell transfers prevented colitic disease, ileal crypt hyperplasia and ileal-reactive Treg accumulation. Thus, our studies identify an IL-17A and IFNγ-dependent homeostatic process that mobilizes ileal-reactive Treg cells and is disrupted by IL-23.

A unimolecular oligonucleotide switch, termed here an AlloSwitch, binds the mature HIV-1 nucleocapsid protein, NCp7. This switch can be used as an indicator for the presence of free NCp7 and NC domains in precursor and fusion proteins. It is thermodynamically stable in two conformations, H and O. A FRET pair is covalently attached to the strands to report on the molecular state of the switch. The results show that NC has an affinity for O 170 times higher than its affinity for H and that in the absence of NC the equilibrium ratio K1 = [O]/[H] = 0.10 +/- 0.03 for the switch sequence reported here. The change between the two states happens on a rapid kinetic time scale. A framework is introduced to aid in the design of AlloSwitches aimed at other targets. A high-affinity probe segment must be available to bind the target in the O-form, while a cover segment hides the probe in H. A key is adjusting the cover sequence to favor the H-form by a factor of 10-1000. This affords a robust response to small changes in target concentration, while saturation produces more than 90% of the maximal change in fluorescence. When a competitor displaces the switch from the NC-O complex, the released switch reverts to the H-form. This is the basis for a mix-and-read strategy for high-throughput screening of anti-nucleocapsid drug candidates that is much simpler to execute than traditional assays that require immobilization and washing steps.

Homeostasis in the ileum involves ongoing immune responses. To study how homeostatic processes of the ileum impact CD4+T cell responses, we used TCR transgenic tools to breed “Bigenic” mice that spontaneously produced CD4+T cells reactive to a model antigen expressed in the ileum. Bigenic mice were grossly similar to control littermates at weaning. By DOL 50, the Bigenic cohort showed reduced weight, increased circulating CD4+TEM frequencies and TH17-associated ileal hyperplasia. An extended natural history study to DOL 150 revealed that only half of Bigenic mice exhibit chronic wasting disease marked by mucosal infiltration by TH17 and TH1 cells expressing non-transgenic clonotypes, colitis and loss of ileal crypt hyperplasia. By contrast, adult Bigenic mice with normal growth maintained the TH17-associated ileal crypt hyperplasia phenotype and additionally accumulated ileal-reactive Treg cells. Adoptive transfer of nTreg cells prevented both ileal crypt hyperplasia and ileal-reactive Treg cell accumulation. Both IL-17A and IFNγ were required for ileal-reactive Treg accumulation and regulation of ileal-reactive CD4+Tconv cells. Ifng−/− Bigenic mice also exhibited an IL-23R-dependent, acutely progressive wasting disease that was >95% penetrant by DOL 50. Thus, our studies identify an IL-17A and IFNγ-dependent homeostatic inflammatory process that mobilizes ileal-reactive Treg cells and is disrupted by IL-23.