NFM Cross-Reactivity to MOG Does Not Expand a Critical Threshold Level of High-Affinity T Cells Necessary for Onset of Demyelinating Disease

Abstract

Of interest to the etiology of demyelinating autoimmune disease is the potential to aberrantly activate CD4+ T cells due to cross-recognition of multiple self-epitopes such as has been suggested for MOG35-55 and NFM15-35. NFM15-35 is immunogenic in C57BL/6 mice but fails to induce demyelinating disease by polyclonal T cells despite having the same TCR contact residues as MOG35-55, a known encephalitogenic antigen. Despite reported cross-reactivity with MOG specific T cells, the polyclonal response to NFM15-35 did not expand threshold numbers of MOG38-49 tetramer positive T cells. Furthermore, NFM lacked functional synergy with MOG to promote EAE because NFM-/- mice developed an identical disease course to wild type mice after challenge with MOG35-55. Single cells analysis of encephalitogenic T cells using the pMHC monomer based 2D micropipette adhesion frequency assay confirmed that NFM was not a critical antigen driving demyelinating disease because NFM18-30 specific T cells in the CNS were predominantly reactive to MOG38-49. The absence of NFM contribution to disease allowed mapping of the amino acids required for encephalitogenicity and expansion of high affinity, MOG specific T cells that defined the polyclonal response. Alterations of N-terminal residues outside of the NFM15-35 core nonamer promoted expansion of high affinity, MOG38-49 tetramer positive T cells and promoted consistent EAE induction, unlike mice challenged with NFM15-35. While NFM15-35 is immunogenic and cross-reactive with MOG at the polyclonal level, it fails to expand a threshold level of encephalitogenic, high affinity MOG specific T cells.

A GMCSF-Neuroantigen Tolerogenic Vaccine Elicits Systemic Lymphocytosis of CD4+ CD25high FOXP3+ Regulatory T Cells in Myelin-Specific TCR Transgenic Mice Contingent Upon Low-Efficiency T Cell Antigen Receptor Recognition

“…To test whether low-efficiency TCR ligands are optimal for induction of Tregs, we devised an alternative experimental system based on the observation that the 2D2 TCR recognizes two distinct NAg, including MOG35-55 as a low affinity antigen and NFM13-37 as a high affinity antigen (60, 61). We derived an expression system for GMCSF-NFM, which exhibited GM-CSF activity equivalent to that of GMCSF-MOG, GMCSF-OVA and GM-CSF in bone marrow proliferation assays (Figure 7A).…”

“…The low affinity nature of myelin peptides may reflect generalized mechanisms of self-tolerance, whereby high-affinity self-reactive clones are deleted during maturation and only low-affinity self-reactive clones persist in the periphery. Although high-affinity NFM13-37 peptide may represent an exception in regard to the specific 2D2 clonotype, the overall NFM-reactive repertoire may primarily comprise low-affinity clones because NFM13-37 lacks encephalitogenic activity in C57BL/6 mice (60).…”

Relationship of 2D Affinity to T Cell Functional Outcomes

“…When binding occurs between the two opposing cells an adhesion frequency is calculated [68]. T cells are additionally probed with RBCs coated with antigen-irrelevant pMHC monomers (Figure 2b) and those devoid of pMHC (Figure 2c) to determine any rates of non-specific binding [63,[69][70][71]. In the case of CD4+ T cells this control is often Class II invariant chain peptide (CLIP) as it is provided by the NIH tetramer core facility as the control for MHC class II tetramers due to the property that it binds to most MHC class II alleles.…”

“…Data from the 2D-micropipette assay in a variety of systems have revealed that lower affinity cells not only expand, but can also dominate polyclonal T cell expansion in the majority of systems tested [63,69,70,81,82,[84][85][86][87]. Thus, it is pertinent to consider the functional ramifications for a T cell that becomes activated via a lower affinity TCR.…”