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 MOG_35-55 and NFM_15-35. NFM_15-35 is immunogenic in C57BL/6 mice but fails to induce demyelinating disease by polyclonal T cells despite having the same TCR contact residues as MOG_35-55, a known encephalitogenic antigen. Despite reported cross-reactivity with MOG specific T cells, the polyclonal response to NFM_15-35 did not expand threshold numbers of MOG_38-49 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 MOG_35-55. 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 NFM_18-30 specific T cells in the CNS were predominantly reactive to MOG_38-49. 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 NFM_15-35 core nonamer promoted expansion of high affinity, MOG_38-49 tetramer positive T cells and promoted consistent EAE induction, unlike mice challenged with NFM_15-35. While NFM_15-35 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.

Introduction

Myelin specific T cells exist in all individuals, but it is not clear how these cells initially become triggered to attack self and promote autoimmune disease (1). Multiple factors influence the autoimmune T cell response which can be shaped by positive and negative selection pressures in the thymus and periphery (2, 3), genetic predispositions (4), environmental exposures (5), ability to migrate to the CNS and differentiation into effector and memory subsets (6). Central to these factors is how the TCR sees self-peptides presented on MHC, with MHC being the strongest genetic susceptibility factor currently identified for multiple sclerosis (7-9). One hypothesis for activation of autoreactive T cells is that exposure to structurally related / cross reactive peptides derived from self or foreign origins can break tolerance (10-13). CD4⁺ T cell cross-recognition between MOG_35-55 (myelin oligodendrocyte glycoprotein epitope 35-55) and NFM_15-35 (epitope 15-35 of neurofilament medium protein) in demyelinating autoimmunity is of interest for disease etiology because these two self-epitopes have synergist potential as targets of autoimmune T cell attack due to identical amino acids at proposed TCR contacts (14). Conceptually, T cell recognition and responsiveness to multiple peptides is a critical feature of protective immune surveillance where a limited TCR repertoire is presented with a myriad of peptides displayed on MHC (15-17). In one example, a single T cell clone specific for myelin basic protein (Ac1-11) has the potential to recognize 10⁶ peptides generated from a combinatorial library (18).

Physical interactions between TCR and peptide:MHC (pMHC) provide another level of input for understanding the initiation of TCR signaling. T cell cross-reactivity is unique to the amino acid structure of an individual TCR dictating how it physically recognizes peptides oriented within MHC (19). Alterations in peptides at amino acid residues interfacing with the TCR, known as altered peptide ligands (APL), can change the functional outcome of T cell responses (12, 18, 20-22) by changing affinity of the TCR:pMHC interaction as well as binding kinetics, including on / off rates and bond lifetime (23, 24). Associations between affinity and function of T cells contributing to polyclonal, demyelinating autoimmune disease have identified a breadth of high and low affinity interactions between TCR and pMHC in C57BL/6 and NOD models (25-27). Our goal is to understand how cross-recognition, cross-reactivity and biophysical interactions, such as 2D affinity, define onset of demyelinating autoimmunity in polyclonal models.

The study of polyclonal T cell cross-reactivity to MOG_35-55 and NFM_15-35 is a novel platform to study the etiology of demyelinating autoimmune disease. Currently, polyclonal studies examining T cell specificity to MOG and NFM do not paint a clear model for cross-reactivity and disease because NFM involvement is based on functional responses after MOG_35-55 and not NFM_15-35 priming (28). While this experimental approach stems from earlier reports stating NFM_15-35 does not induce EAE (14), we questioned why this would be the case and chose to comparatively track antigen specific T cells after a MOG_35-55 or NFM_15-35 challenge.

We demonstrate that lack of EAE onset after NFM_15-35 challenge correlated with insufficient expansion of high affinity, MOG_38-49 tetramer positive T cells in spleen and peripheral lymph nodes along with their absence in the CNS. 2D micropipette adhesion frequency assay was used as a more sensitive, monomer based tool to enhance detection of TCR affinity and antigen specificity above tetramer staining in order to tease apart the bispecific CD4⁺ T cell infiltrates in the CNS that are predominantly low affinity and fail to stain with tetramer (25). After MOG_35-55 challenge we found that the CNS T cell infiltrates recognizing NFM_18-30 largely cross-recognized MOG_38-49 and generally lacked the higher affinity cells typically seen with MOG_38-49 specific infiltrates. The functional requirement of MOG and NFM bispecific T cells to EAE onset and severity was tested with MOG_35-55 challenge of NFM^-/- mice. This design revealed MOG and not NFM to be the critical autoantigen for EAE because NFM^-/- mice developed a similar disease course to wild type, NFM sufficient mice. Furthermore, we mapped N-terminal residues of NFM_15-35 that dictated poor encephalitogenicity such that when modified allowed EAE induction as well as detection of MOG_38-49 tetramer positive T cells. The presented data concomitant with the fact that MOG^-/- mice do not develop EAE after MOG_35-55 or NFM_15-35 challenge (29) supported the conclusion that NFM has a minimal role in the onset of EAE and the expansion of encephalitogenic T cells from the polyclonal repertoire where pathogenicity is dictated by expansion of a critical threshold level of higher affinity tetramer positive MOG specific T cells.

Materials and methods

Mice

Female C57BL/6 mice were purchased from Charles River Frederick Facility, formerly National Cancer Institute (NCI). With permission from Dr. Hugh Reid at Monash University, MOG^-/- mice (29) were obtained from Dr. Xue-Feng Bai at The Ohio State University. NFM^-/- mice were obtained from Dr. Julien (30) and backcrossed to NCI C57BL/6N mice due to visual dominance of the 129 background coat color and the presence of an additional nefh knockout. Completion of the backcross was confirmed by DartMouse (Lebanon, NH) and NFM protein deficiency was determined by histology performed at Emory NINDS Neuropathology / Histochemistry Core Facility (Atlanta, GA). IFNγR^-/- (B6.129S7-Ifngr1^tm1Agt/J) mice, Thy1.1 mice (B6.PL-Thy1^a/CyJ), and 2D2 transgenic mice (C57BL/6-Tg(Tcra2D2,Tcrb2D2)1Kuch/J) were initially purchased from Jackson Laboratories. These mice along with SMARTA (Tg(TcrLCMV)Aox) mice (31) were all housed in the Division of Animal Resources at Emory University and handled in accordance with protocols approved by the Institutional Animal Care and Use Committee. Experimental mice were between 6-11 weeks of age.

Peptides and reagents

Peptides used for T cell priming and EAE induction were generated in our laboratory with the Prelude peptide synthesizer (Protein Technologies, Inc) with the following sequences; MOG_35-55 (MEVGWYRSPFSRVVHLYRNGK), NFM_15-35 (RRVTETRSSFSRVSGSPSSGF), NFM_15-35 T20Y (RRVTEYRSSFSRVSGSPSSGF), NFM_15-35 E19W, T20Y (RRVTWYRSSFSRVSGSPSSGF), NFM_15-35 S23P (RRVTETRSPFSRVSGSPSSGF), NFM_15-35 S28V (RRVTETRSSFSRVVGSPSSGF).

Lymph node priming and T cell proliferation assays

Polyclonal T cell lines were generated by footpad priming mice with emulsions of MOG_35-55 or NFM_15-35 (1 mg/mL) in CFA containing a final concentration of 0.5 mg/mL heat-killed Mycobacterium tuberculosis (H37 RA Mtb) in Incomplete Freund's Adjuvant (BD). Draining lymph nodes were harvested 12-14 days after priming. Single cell suspensions were generated by passing cells through a 100μm cell strainer and directly assessed for antigen specific proliferation by uptake of [³H] tritiated thymidine (VWR International, Inc). 6×10⁵ primed lymph node cells or naïve 2D2 splenocytes were cultured in a 96-well flat bottom plate with indicated peptides and concentrations. After 48 hours, ³H-thymidine (0.4μCi/well) was added to the culture media for 24 hours before the cells were harvested onto a filtermats (PerkinElmer) with the FilterMate 196 harvester (Packard). ³H-thymidine uptake was analyzed with the 1450 Microbeta TriLux microplate liquid scintillation counter (PerkinElmer). Cell culture media contained RPMI 1640 (CellGro) supplemented with 10% heat-inactivated FBS (Gibco-Life Technologies), 4mM L-glutamine (CellGro), 0.01M HEPES (CellGro), 100μg/mL gentamicin (CellGro), 20μM 2-ME (Sigma-Aldrich). Stimulation index was calculated as a ratio of the counts per minute between peptide stimulated versus unstimulated cells.

Induction of EAE

EAE was induced with two subcutaneous injections of emulsion delivering 200μg designated peptide and 0.4mg Mtb in IFA per injection on days 0 and 7. 250ng of pertussis toxin (List Biologicals) was concomitantly administered intraperitoneally to mice on days 0 and 2. Mice were monitored for signs of disease including weight loss and paralytic symptoms. Mice presented with typical ascending paralysis scored accordingly; (0.5) partial tail paralysis, (1.0) complete tail paralysis, (2.0) hindlimb weakness, (2.5) ataxia, (3.0) partial hindlimb paralysis, (3.5) complete hindlimb paralysis, (4.0) inability to right itself, and (5.0) moribund.

Passive EAE was generated by adoptive transfer of 1×10⁷ cells from an NFM T cell line per C57BL/6 mouse, irradiated with 400rads one day before transfer. The T cell line was generated by inducing either wild type C57BL/6 mice (naturally Thy1.2) or Thy1.1 congenic C57BL/6 mice with NFM_15-35 in CFA on days 0 and 7 with pertussis toxin administered on days 0 and 2 as described above. Spleens were harvested 14 days later, mashed into single cell suspensions and cultured as previously described but with the addition of 50 ng/ml of IL-2 and 5ng/mL recombinant mouse IL-12 (Gemini Bio). Splenocytes were cultured for 3 days and with 10×10⁶ blast cells transferred intraperitoneally (i.p.). Mice were then monitored for signs of EAE.

Antibody characterization of T cells enriched by tetramer pulldown

Tetramer enrichment was performed as previously described (32). In brief, spleen and peripheral lymph nodes (inguinal, lumbar, mesenteric, cervical, axillary and brachial) were harvested and processed into a single cell suspension by passing through a 100μm strainer. Cells were washed in cold 1X HBSS (Cellgro) and resuspended with 4μg/mL of tetramer PE conjugated NFM:I-A^b and/or APC conjugated MOG:I-A^b (NIH tetramer core (33, 34)) in Fc block (heat killed mouse and rat serum, Sigma-Aldrich) at a volume 2× the pellet volume. After 1hr at room temperature, cells were wash in cold FACS wash (0.1% BSA, 0.05% NaN3, 1x PBS) and resuspend in 200μL FACS wash plus 50μL of anti-PE and/or anti-APC beads (Miltenyi Biotec) and incubated for 30 min. on ice. Cells were then washed and enriched on a LS column (Miltenyi Biotec). Unbound and column bound cell numbers were determined with AccuCheck microbeads (Invitrogen) alongside with cell surface marker characterization performed with flow cytometry (LSR II, BD) and FlowJo software (Treestar). Antibodies used included; CD3ε-FITC (145-2C11, BD Pharmingen), CD11b- PerCP -Cy5.5 (M1/70, BD Pharmingen), CD11c-PerCP-Cy5.5 (HL3, BD Pharmingen), CD19- PerCP -Cy5.5 (1D3, Tonbo Biosciences), CD4-Brilliant Violet 510 (RM4-5, BioLegend), CD8-Brilliant Violet 785 (53-6.7, BioLegend), CD44-Alexa Fluor 700 (IM7, eBioscience).

T cell isolation from CNS

Mice were euthanized by CO₂ induced asphyxiation and perfused with 1X Dulbecco's PBS (CellGro) through the left ventricle with drainage mediated by laceration of the inferior vena cava. From individual mice, single cell suspensions of the CNS, brain and spinal cord, were generated by passing the tissue through a 100μm strainer. Mononuclear cells were isolated by Percoll density centrifugation (Sigma-Aldrich). CD4⁺ T cells were isolated from pooled CNS mononuclear cells using MACS with L3T4 CD4 positive selecting beads (Miltenyi Biotec). Enriched T cells were assessed for antigen specificity and effective 2D affinities using the 2D micropipette adhesion frequency assay using pMHC coated RBC sensors (25, 27, 35-37).

2D micropipette adhesion frequency assay and analysis

Human RBCs were isolated from healthy volunteers in accordance with the Institutional Review Board at Emory University. RBCs were prepared by first coating the cells with varying concentrations of Biotin-X-NHS (EMD Millipore) followed by 0.5mg/mL streptavidin (ThermoFisher Scientific) and then 1μg of biotinylated monomers MOG_38-49:I-A^b, NFM_18-30:I-A^b, GP_66-77:I-A^b (NIH Tetramer Core Facility). Quantitation of TCR and pMHC densities were determined by flow cytometry (LSR II, BD) after labeling with PE conjugated TCRβ (H57-597, eBioscience) or I-A/I-E (M5/114.15.2, BD) and quantitated with PE-Quantibrite bead standards (BD).

Adhesion frequencies between T cells and pMHC RBC sensors were determined if binding, visualized by RBC membrane distension, was observed after individual T cells were brought into contact 25-50 times with movement controlled by a piezo actuator (38). Background or non-specific adhesion frequencies are considered to be below 0.1 in this antigen specific system (25). Adhesion frequencies along with molecule surface densities were used to calculate effective 2D affinities (A_cK_a) using A_cK_a= - ln[1-P_a(∞)}/m_rm_l with m_r and m_l representing TCR and pMHC surface densities, respectively.

Testing individual T cells among multiple pMHC is done by sequential binding. First, RBC sensors with different pMHC are concentrated at opposing locations within the microscope chamber containing purified T cells. Next, one T cell is aspirated into the micropipette attached to the piezo actuator and the MOG_38-49:I-A^b RBC sensor is aspirated into the second micropipette. After 25-50 touches the MOG RBC sensor is expelled and then the NFM_18-30:I-A^b RBC sensor can be aspirated and tested against the same aspirated T cell. The next individual T cell will be tested in reverse order from the previous assessment, i.e. the NFM sensor first followed by the MOG sensor in order to assess if the sequential order influences subsequent antigen binding.

Statistics

Statistical analyses were performed with Prism version 6 (GraphPad Software). Disease onset was assessed by unpaired, nonparametric t tests with Mann-Whitney post hoc tests. Two-tailed unpaired parametric t tests with F test variances were used for analyses between 2 groups while comparison of multiple means were performed with one-way ANOVA. Paired t tests were run where single cell detection of MOG versus NFM specificity was directly compared.

Results

NFM^-/- mice reveal MOG_35-55 is the primary encephalitogenic antigen

The experiments highlighted here are aimed at clarifying the encephalitogenic potential of NFM based on the work initially describing T cell cross-reactivity between MOG_35-55 and NFM_15-35 in polyclonal MOG and NFM cell lines (14). We also found both MOG_35-55 and NFM_15-35 to be immunogens based on proliferation of polyclonal lymph node cells directly ex vivo using a range of peptide doses to determine sensitivity of the T cell response, Fig. 1A. Restimulation of lymph node cells with the cognate priming antigen revealed that higher peptide concentrations (10μM to 100μM) were needed to see proliferation above background. Interestingly, cross-reactive proliferation to the non-cognate antigen was poorly detected, if at all, despite 6 of the 9 putative core epitopes being identical between NFM_20-28 and MOG_40-48. This indicated that T cell expansion via the cross-reactive, non-cognate epitope was not significant on the polyclonal level, questioning the relevance of cross-reactive expansion to disease. C57BL/6 mice actively challenged with NFM_15-35 using a conventional induction regimen lacked the weight loss and encephalitogenic potential exhibited by MOG_35-55, Fig. 1B (14, 28).

Figure 1. NFM is immunogenic but not encephalitogenic.

A) Lymph nodes cells were harvested 12-14 days after priming C57BL/6 mice with MOG_35-55 or NFM_15-35 in CFA. Cells were directly assessed for antigen specific proliferation and ³H-thymidine incorporation. Counts per minute were assessed and reported as stimulation index in order to average multiple experiments. Data is averaged from at least 3 experiments and 5 or more replicates per condition. B) EAE was induced in C57BL/6 mice using MOG_35-55 (n=7) or NFM_15-35 (n=15) with mice being monitored for paralytic severity and weight loss. Data is representative of 2 experiments.

To more clearly tease apart the contribution of NFM_15-35 to the polyclonal response, we used mice deficient in NFM (NFM^-/-) (30). Agouti mice double deficient in NFM and NFH proteins were generously obtained from Dr. Julien and subsequently backcrossed in our laboratory with our progeny confirmed as NFM^-/-, NFH^+/+ and 99% C57BL/6N by speed congenics (DartMouse, Fig. 2A), PCR (Fig. 2B) and histology (Fig. 2C). NFM deficient mice were used to eliminate potential thymic and peripheral selection pressures due to the expression of NFM by medullary and cortical thymic epithelial cells (39, 40). NFM_15-35 primed lymph node cells from wild type or NFM deficient mice gave a similar ex vivo proliferative response between the mice, Fig. 2D. This showed that NFM_15-35 was equally immunogenic regardless of NFM expression. The course of MOG induced EAE was then characterized in the NFM deficient mice in order to eliminate potential T cell cross-reactivity with NFM. We hypothesized that if T cell cross-reactivity to NFM contributed to EAE onset or severity then a diminished disease course would be seen in the absence of NFM. NFM^-/- mice however developed EAE similarly to wild type NFM sufficient mice with no difference in day of symptom onset and overall disease course, Fig. 2E. Furthermore, NFM_15-35 challenge was unable to induce EAE in MOG^-/- mice clearly indicating that NFM alone is not sufficient to cause EAE, Fig. 2F.

Figure 2. Characterization of NFM deficient mice suggests MOG is the critical autoantigen for induced EAE.

A) Mice were backcrossed to C57BL/6 and confirmed by DartMouse to be 99% C57BL/6N (n=3) with analyses of 1 mouse shown. SNPs from C57BL/6N are shown in green and variances are highlighted in yellow (129X 1/Sv) or pink (heterozygous for C57BL/6N and 129). B) WT control mice or progeny of the NFM^-/- backcross to C57BL/6 were individually PCR tested for the presence (Neo Primers) or absence (WT Primers) of the neomycin cassette disrupting exon 1 of Nefm, shown are 2 representative gels. C) Representative light microscopy images (10× or 20× magnification) of the pyramidal cell layer in the hippocampus of WT C57BL/6 mice versus NFM^-/- mice. NFM expression was evaluated by immunostaining with NFM specific antibody NN18 (Millipore MAB5254) and compared to control slides where the primary antibody was withheld. NFM (dark brown) was visualized in cell bodies and fiber tracts of WT mice (n=1) but not NFM^-/- mice (n=2). Sections were counterstained with hematoxylin (blue/purple) to highlight nuclei. D) Lymph nodes cells were harvested 12-14 days post NFM_15-35 / CFA priming of C57BL/6 or NFM^-/- mice. Counts per minute were assessed 24 hours after the addition of ³H-thymidine. Data is the average of 2 replicates per condition. E) MOG_35-55 EAE was induced in WT and NFM^-/- mice with disease course and weight loss were subsequently monitored. The data here represents 1 of 3 experiments with a total of n=10 WT mice and n=28 NFM^-/- mice. There was no significant difference in day of symptom onset p=0.093 using an unpaired, nonparametric T test with Mann-Whitney post hoc test. F) MOG^-/- mice were challenged with a NFM_15-35 EAE induction in order to assess ability of T cells to recognize NFM alone and cause EAE, n=19. WT mice were challenged with MOG_35-55 as a positive control n=5. Data shown is representative of 2 experiments.

Tetramer positive, MOG specific T cells expand poorly after NFM_15-35 priming

The absence of NFM induced EAE was similar to our previously published reports with the MOG 45D variant peptide, which like NFM shares all primary TCR contact residues with wild type MOG but differs at MHC anchor residues (33, 41). Interestingly, MOG 45D could induce EAE and expand MOG_38-49 specific, tetramer positive T cell when IFNγ signaling was deficient in mice (33). We therefore tested whether NFM_15-35 challenge of IFNγR^-/- would induce EAE and found NFM_15-35 was still not encephalitogenic, Fig. 3A. Since the MOG 45D data suggested a threshold level of MOG tetramer positive T cells was needed for onset of polyclonal EAE, we monitored antigen specific T cell expansion in C57BL/6 mice after MOG_35-55 or NFM_15-35 to assess why NFM_15-35 challenge does not induce EAE.

Figure 3. MOG specific T cell expansion is poor after NFM challenge.

A) EAE was induced in IFNγ receptor knockout mice after MOG_35-55 (n=6) or NFM_15-35 (n=14) challenge. Disease course is representative of 2 experiments. B) WT mice were induced with an EAE challenge of MOG_35-55 or NFM_15-35 in CFA plus pertussis toxin. MOG_38-49 or NFM_18-30 tetramer pulldowns were performed on days 14 (n=6-7 mice / group) or 22 (n=8-15 mice / group) post challenge. MOG_38-49 detection of CD4⁺ CD44^hi T cells differed significantly between MOG_35-55 and NFM_15-35 induction 14 or 22 days post injection. CD4⁺ T cells were identified with an initial lymphocyte gate (FSC-A / SSC-A) followed by a singlet gate (SSC-W / SSC-H), CD3⁺ CD11b^- CD11c^- CD19^- gate and CD4⁺ versus CD8⁺ gate. C) Day 22 T cell activation status was evaluated by observing the number of activated CD44^hi versus unactivated CD44^low MOG specific T cell numbers. NFM_15-35 induction did not significantly induce the numbers of CD44^hi MOG specific T cells (p<0.0001) expanded by MOG_35-55 induction. D) WT or NFM^-/- mice were challenged with NFM_15-35 in CFA, as in (A). 22 days later, no significant differences were found in expansion of MOG_38-49 T cells between WT and deficient mice (n=10 / strain, p=0.41). E) No significant differences in NFM_18-30 detection were seen between MOG_35-55 versus NFM_15-35 induction groups at d14 (p=0.09) or d22 (p=0.25). Representative flow plots from the bound fraction of the tetramer enrichment at d14 post challenge are shown (F). G) MOG_38-49 tetramer was used to identify antigen specific T cells in the CNS of 5-6 mice / group d22 after induction. There was no significant difference seen in the CD3⁺ CD4⁺ CD44⁺ population p=0.06 despite significant differences in enumerating MOG and NFM tetramer positive cells. All statistics were done using 2-tailed unpaired, parametric t tests assuming both populations had equal standard deviations.

Cell numbers of tetramer positive T cells were examined at days 14 and 22 post challenge with MOG_35-55 and NFM_15-35, days typical of onset and peak paralytic symptoms in our laboratory (33, 41-43) using the tetramer pulldown technique to enhance detection and quantitation of T cells within the polyclonal repertoire (32), Fig. 3. At day 14 after MOG_35-55 challenge, the MOG specific T cell population expanded to 16,000 cells ± 2,200 (mean ± SEM) with only a subtle decline in numbers to 9,200 cells ± 1,800 by day 22 (p=0.02, Fig. 3B). NFM_15-35 challenge maximally expanded cross-reactive MOG_38-49 specific T cells to a number 5-times lower than MOG_35-55 challenge at day 14, respectively 3,200 cells ± 770 which further diminished 34% by day 22 to 1,100 cells ± 410, p=0.018. Breakdown of the day 22 MOG_38-49 tetramer positive populations into CD4⁺ CD44^hi versus CD4⁺ CD44^low indicated how well T cells were being activated by the antigen priming regimen, Fig. 3C. MOG_35-55 challenge significantly expanded a greater number of CD44^hi (9,200 cells ± 1,800) than CD44^low cells (1,360 cells ± 290), while NFM priming did not with 1,100 cells ± 410 versus 4,300 cells ± 2,000 respectively.

We considered that endogenous NFM expression could potentially regulate the expansion of MOG_38-49 tetramer positive T cells after NFM_15-35 challenge. We used the tetramer pulldown method to enumerate any changes in T cell expansion between NFM deficient and wild type mice 22 days post challenge. We found a similar expansion (p=0.41) of MOG_38-49 specific T cells after NFM_15-35 challenge between wild type C57BL/6 mice (1,500 cells ± 580) and NFM-/- mice (890 cells ± 460), Fig. 3D suggesting that endogenous expression of NFM was not negatively regulating expansion of MOG_38-49 tetramer positive cells in peripheral spleen and lymph nodes at this time point.

The identification of polyclonal NFM_18-30 specific T cells in the spleen and lymph nodes was difficult without enriching for the cells by tetramer pulldown. Low level detection was possible with this method, most notably at day 14 post challenge, Fig. 3E & F. There was no significant difference in the detection of NFM_18-30 tetramer positive cells between MOG_35-55 versus NFM_15-35 challenge at days 14 (respectively 6,100 cells ± 1,000 and 3,500 cells ± 980, p=0.09) and 22 (p=0.25 at 1,400 cells ± 490 and 760 cells ± 210 respectively). Yet NFM_18-30 specific T cells numbers were significantly diminished by day 22 when compared to the numbers seen at day 14 following MOG_35-55 challenge (p=0.0004) and NFM_15-35 challenge (p=0.008).

The notable difference in peripheral detection of MOG_38-49 T cells identified 22 days after NFM_15-35 challenge, Fig. 3B, spurred the question whether the missing peripheral MOG specific T cells had migrated to the CNS by this time point, Fig. 3G. CD4⁺ CD44^hi MOG_38-49 specific T cells were found in the CNS after MOG_35-55 (760 cells ± 240) but not NFM_15-35 challenge (2.7 cells ± 1.7) despite a similar number of CD3⁺ CD4⁺ CD44^hi T cell infiltrates (25,000 cells ± 7,200 and 9,800 cells ± 3,000 respectively). There was limited detection of tetramer positive NFM_18-30 specific T cells after MOG_35-55 challenge (32.8 cells ± 7.0) with no detection of this population in the CNS following NFM_15-35 challenge (2.2 cells ± 1.5). The antigen specificity of the remaining CD3⁺ infiltrates could not be determined by MOG_38-49 and NFM_18-30 tetramer.

N-terminal alterations in the NFM peptide restore encephalitogenicity and highlight residues critical for expansion of cross-reactive, tetramer positive, MOG specific T cells

The poor expansion and sustainability of cross-reactive, MOG_38-49 specific T cells after NFM_15-35 challenge allowed us to probe which amino acids within the NFM_20-28 core nonamer would expand encephalitogenic T cells that cross recognized MOG_38-49. Published data indicated NFM poorly binds I-A^b, therefore we first modified NFM_20-28 at proposed MHC anchor residues (positions P1, P4, and P9 within the NFM_20-28 core) and tested their ability to promote EAE. The P6 anchor residue was not modified because serine is found at this position in both MOG_35-55 and NFM_15-35 peptides. The NFM specific amino acids were swapped with the respective residues found within MOG_35-55, Table I. P1 (40Y) of MOG_35-55 is a critical MHC anchor residue shown by I-A^b peptide-competition assays such that, functionally, an alanine substitution at this position results in a loss of MOG specific T cell responsiveness (42, 44-46). We therefore made NFM variant T20Y at P1, challenged mice and found that encephalitogenic potential increased 33% incidence in comparison to the complete absence of disease compared to wild type NFM_15-35 challenge (Fig. 4A and Table II) confirming a previously reported (40). We generated additional NFM_15-35 MHC variant peptides at P4 and P9 to have the MHC anchor residues of the MOG_40-48 nonamer, respectively S23P and S28V, but these peptides did not promote EAE Fig. 4A. The suboptimal induction of disease with the P1 change alone suggested that P1 along with residues outside of the core nonamer may be important for inducing 100% incidence of EAE.

Table I. Amino acid sequences of MOG_35-55, NFM_15-35 and NFM_15-35 variant peptides. The nine core amino acids (P1-P9) are denoted along with P-1. Substitutions in NFM_15-35 were made at P-1, P1, P4, and P9.

					-1	1	2	3	4	5	6	7	8	9
NFM 15-35	R	R	V	T	E	T	R	S	S	F	S	R	V	S	G	S	P	S	S	G	F
MOG 35-55	M	E	V	G	W	Y	R	S	P	F	S	R	V	V	H	L	Y	R	N	G	K
NFM T20Y	R	R	V	T	E	Y	R	S	S	F	S	R	V	S	G	S	P	S	S	G	F
NFM E19W, T20Y	R	R	V	T	W	Y	R	S	S	F	S	R	V	S	G	S	P	S	S	G	F
NFM S23P	R	R	V	T	E	T	R	S	P	F	S	R	V	S	G	S	P	S	S	G	F
NFM S28V	R	R	V	T	E	T	R	S	S	F	S	R	V	V	G	S	P	S	S	G	F

Figure 4. NFM variants at amino residues P1 and P-1 restored MOG_38-40 tetramer detection and encephalitogenic incidence to 100%.

A) Synthesized variant peptides of NFM_15-35 were used to induce EAE in C57BL/6 mice and disease course was monitored (panels A-B and Table II). Panel A is the graphical version of Exp. 1 and panel B is the graphical version of Exp. 5 detailed in Table II. The experiments represented in A-B were performed 2 times. C) 2D2 T cell dose response curves for NFM_15-35, MOG_35-55 and NFM variants. This experiment was performed 1 time in duplicate. D) MOG specific T cell numbers from the periphery at d22 post challenge were enumerated with MOG_38-49 and NFM_18-30 tetramers. Statistics between indicated peptide challenges were performed with 2-tailed unpaired, parametric t tests with the following designations ** p<0.0001 with F test variance p<0.0001, * p<0.0001 with F test variance of p=0.01 and T20Y versus NFM_15-35 was not significant with p=0.245 and F test variance p=0.857. MOG_35-55 versus ‘NFM E19W, T20Y’ has p=0.455 and exhibited no significant differences in means or F test variances. Expansion of NFM specific T cell numbers in the periphery were also assessed and no overall differences were seen by ordinary one-way ANOVA p=0.63.

Table II. Summary table of EAE experiments with NFM variant peptides compared to MOG_35-55.

Synthesized variant peptides of NFM_15-35 were used to induce EAE in C57BL/6 mice. Disease was monitored in experimental mice (N) and percent incidence of disease along with average day of symptom onset and maximal disease score +/- standard deviations are reported. Disease incidences after challenge with MOG_35-55 versus T20Y were summarized in the combined (Exp. 1 & 2) section.

Exp. #	Peptide	N	% Incidence	Avg. Day Onset	Avg. Max Score
1	MOG 35-55	2	50	13	3
	NFM T20Y	2	50	15	2.5
	NFM S23P	2	0	-	-
	NFM S28V	2	0	-	-
2	MOG 35-55	2	100	14.5 +/- 0.7	3 +/- 0.7
	NFM T20Y	4	25	32	2.5
	NFM S23P	2	0	-	-
	NFM S28V	2	0	-	-
Combined Exp. 1 & 2	MOG 35-55	4	75
	NFM T20Y	6	33.3
3	MOG 35-55	3	100	10 +/- 3.5	3.7 +/- 0.29
	NFM E19W, T20Y	5	100	10.6 +/- 1.5	2.5 +/- 1.2
4	MOG 35-55	5	100	8.2 +/- 1.1	3.9 +/- 0.22
	NFM E19W, T20Y	8	100	10.3 +/- 2.0	3.3 +/- 0.46

Published data shows that I-A^b can tolerate changes in the peptide MHC anchor positions through predicted hydrogen bonding with peptide N-terminal residues at P-1 and -2 (45) and that alanine substitutions at P-1 reduce proliferation of MOG specific T cell lines (44). We subsequently engineered NFM_15-35 with the amino acids found at MOG_35-55 P-1 concomitantly with the T20Y mutation at P1generating the double mutant NFM E19W, T20Y (Table I). Introduction of aromatic side chains at P-1 and P1 restored encephalitogenicity to 100% incidence compared to NFM_15-35 (Fig. 4B & Table II). The 2D2 TCR transgenic model used to initially define MOG and NFM cross-reactivity showed the dose response curve for NFM E19W, T20Y to be most similar to MOG_35-55, exhibiting maximal proliferation at 10μM peptide. The NFM T20Y proliferative response was most similar to NFM_15-35 and marked by maximal proliferation at 1μM peptide, Fig. 4C. This reiterates the importance of W at P-1 for the characteristic MOG responsiveness.

We appreciated that the N terminal substitutions were influencing polyclonal encephalitogenicity as well as MOG versus NFM responsiveness in 2D2 T cells and wanted to further clarify how these substitutions affected expansion of MOG_38-49 and NFM_18-30 tetramer positive cells within the polyclonal EAE response, Fig. 4D. High affinity, MOG_38-49 tetramer positive cells expanded similarly (p=0.45) between NFM E19W, T20Y (7,300 cells ± 1,400) and wild type MOG_35-55 induced EAE (9,200 cells ± 1,800), Fig. 4D left panel. Expansion of MOG_38-49 specific T cells was significantly reduced after challenge with NFM variant T20Y (2,000 cells ± 540) or wild type NFM_15-35 (1,100 cells ± 410) when compared to the numbers expanded by MOG_35-55 (p=0.006 and p<0.0001 respectively) or NFM E19W, T20Y (p=0.003 and p<0.0001 respectively). There was no significant difference between expansion of MOG_38-49 specific T cells by T20Y and NFM_15-35, p=0.244, which could relate to inconsistent EAE onset with this antigen. Detection of NFM_18-30 specific T cells by tetramer was not enhanced by any amino acid substitution examined with no significant difference among the groups p=0.68, Fig. 4D right panel. Overall, the encephalitogenicity of NFM variant peptides was dependent on expansion of higher affinity, MOG_38-49 tetramer positive T cells and not NFM_18-30 specific T cells.

Polyclonal NFM specific T cells are predominantly cross-reactive to MOG during the course of MOG_35-55 induced EAE as assessed by the 2D micropipette adhesion frequency assay

We previously reported that MOG tetramer enriches for high affinity T cells, so reduced detection of higher affinity, NFM_18-30 specific T cells by tetramer suggested that these T cells display TCR with generally low affinity or avidity interactions with pMHC (25). The 2D micropipette adhesion frequency assay provides a more sensitive technology to sample antigen specificity and effective 2D affinity interactions among individual T cells within the polyclonal response without using avidity based tetramers. We previously reported that tetramer poorly detected MOG_38-49 : I-A^b or MOG_42-55 : I-A^g7 specific T cells when compared to the 2D micropipette adhesion frequency assay (25, 26). Similarly, tetramer also underestimated the percent detection of NFM_18-30 : I-A^b specific T cells ∼100 times less when compared to detection by the 2D micropipette adhesion frequency assay, respectively 0.5% versus 55% Fig. 5A. NFM_18-30:I-A^b monomeric detection of T cells was specific as demonstrated by lack of binding to CD4⁺ SMARTA T cells, specific for LCMV glycoprotein epitope 61-80, Fig. 5B.

Figure 5. CNS T cell infiltrates that recognize NFM_18-30 are largely specific for MOG_38-49.

A) Splenocytes from a mouse primed with NFM_15-35 were cultured for 1 week and tested for NFM_18-30 specificity by tetramer (A) or the 2D micropipette adhesion frequency assay with n=28 cells. B) NFM specificity of the 2D micropipette adhesion frequency assay was shown via comparison to LCMV specific SMARTA T cells cultured on GP_66-80 (n=16 cells). Densities of the NFM_18-30 or GP_66-77 I-A^b monomers coated on the RBC sensor are reported on the x-axis and each symbol represents one T cell. C) EAE was induced with MOG_35-55 and CD4⁺ T cells were isolated from the CNS at indicated time points where ‘onset’ designates days 12-16 (n=56 cells), ‘peak’ days 20-23 (n=70 cells), and ‘chronic’ days 28-32 (n=53 cells) post induction. Each dot represents one T cell. Log transformed affinities were analyzed by two-tailed, unpaired parametric t-tests with assumption of equal standard deviations were used to compare MOG_38-49 versus NFM_18-30 specific T cells at each time point; onset (p=0.51), peak (p=0.058) and chronic (p=0.55). Asterisks mark significant differences in population breadth between MOG_38-49 and NFM_18-30 specific T cells at onset (p=0.004) and chronic (p=0.029) disease using unpaired, parametric t tests with F tests to compare variances. D-F) Breakdown of the data collected in (C). D) Bar graphs indicate the average percent of detection among the individual T cells analyzed per 2D micropipette experiment; average experiments include 7 for onset, 4 for peak and 5 for chronic time points. No significant difference was seen between MOG and NFM specific detection using a two-tailed paired t tests p>0.1864. E) Each dotted line links one individual T cell between its affinities for MOG_38-49 versus NFM_18-30. Zero binding of a T cell to a given antigen could not be graphed on a log-scale and were given an arbitrary value of 1×10^-7 and are graphically visualized below the solid y-intercept line. F) Breakdown of individual T cell specificity for one or both antigens using the data in panel D. Double binders is a category of cells that recognize both MOG_38-49 and NFM_18-30.

CD4⁺ T cells isolated from the CNS were monitored by the 2D micropipette adhesion frequency assay for MOG_38-49 and NFM_18-30 specificity and affinity throughout the course of MOG_35-55 induced EAE in C57BL/6 mice, particularly at onset of paralytic symptoms (days 12-16 post MOG_35-55 induction), peak (days 20-23 post induction) and more chronic time points (days 28-32 post induction), Fig. 5C. NFM_18-30: I-A^b and MOG_38-49: I-A^b RBC sensors were placed at opposing ends of the media chamber, to prevent mixing, and individual T cells were tested in random sequence with the sensors. This means one cell was tested for MOG_38-49 specificity first followed by NFM_18-30 assessment while the second T cell was tested in the opposite order to rule out potential biasing of TCR:pMHC recognition based on memory of a previous pMHC (47). The order of TCR contact with MOG or NFM pMHC did not alter the overall adhesion frequency (Supplementary Figure 1). Geometric mean affinities of MOG_38-49 specific T cells at onset, peak and chronic time points are reported as 1.1×10^-5 μm⁴, 6.2×10^-6 μm⁴ and 1.1×10^-5 μm⁴ respectively. NFM_18-30 specific T cells exhibited means of 1.4×10^-5 μm⁴ at onset, 1.1×10^-5 μm⁴ at peak and 9.5×10^-6 μm⁴ at chronic time points. Unpaired parametric t-test comparisons of the mean MOG and NFM affinities per time point indicated no significant differences between the groups assuming equal standard deviations (p≥0.058).

Although the means were similar, the breadth of affinities seen within the MOG_38-49 specific population differed from NFM_18-30 specific cells, Fig. 5C. Significant differences in affinity ranges between these two populations were seen at onset (F(52,30)=2.7, p=0.004) and chronic (F(48,36)=2.0, p=0.029) time points as measured by the F-test to compare variances. The greatest breadth in maximal / minimal 2D affinities were seen at onset with breadths of 1,927 for MOG_38-49 : I-A^b and 158 for NFM_18-30 : I-A^b, a finding we also reported during EAE onset in NOD mice (26). NFM_18-30 specific T cells with high 2D affinities were absent from detection at peak and chronic time points using 1.1 × 10^-4 μm⁴ as a cutoff for high affinity previously published for MOG_38-49 : I-A^b T cells (25). At onset, the few high affinity NFM_18-30 specific T cells found remained close to this cutoff with a geometric mean of 1.3×10^-4 μm⁴, an affinity below the MOG_38-49 specific population at 6.1×10^-4 μm⁴. Overall, there is a deficit in the number of higher affinity NFM specific T cells detected by both the 2D micropipette adhesion frequency assay and tetramer, Fig. 3.

The total percentage of cells that recognize MOG_38-49 versus NFM_18-30 on average (SD) is not significantly different at onset, peak or chronic time points p ≥ 0.1864, Fig. 5D. This suggested that there was significant T cell cross-recognition between MOG and NFM. Analysis of individual T cells from the CNS indicated that each cell has a unique, intrinsic capacity to cross-recognize MOG_38-49 and NFM_18-30. Importantly, individual T cells tested against multiple pMHC revealed that TCR affinity for one antigen did not dictate strength of recognition to the second antigen, meaning high affinity for MOG does not indicate high affinity for NFM or vice versa, Fig. 5E. Breakdown of single cell cross-recognition, Fig. 5F, indicated that among all time points it was rare to find T cells in the CNS that recognized NFM_18-30 only and not also MOG_38-49, occurring at 5.7% to 11.3% of the population. The majority of T cells in the CNS at peak (58.6%) or chronic (56.6%) disease actually recognized both MOG_38-49 and NFM_18-30, denoted as double binders. Therefore, the NFM_18-30 specific T cells at peak and chronic time points are predominantly MOG_38-49 specific at 91.1% and 83.3% respectively. One should note that the NFM only T cells could be functionally MOG reactive but below the limit of detection by the 2D micropipette adhesion frequency assay, a phenotype exhibited by 2D2 T cells (34). At onset (Fig. 5E) it was rare to find T cells displaying a 2D2-like phenotype of measurable affinity for NFM and not MOG, only 4 of the 56 cells tested at onset. Overall, 12 of the 56 T cells displayed heteroclitic affinities for NFM over MOG compared to 26 of the 56 cells displaying affinities dominant for MOG over NFM.

The dominant cross-reactivity of NFM specific cells with MOG led us to track MOG specificity in a NFM T cell line previously reported to promote EAE (14). Krishnamoorthy, et al. showed a NFM line could proliferate to both MOG and NFM and we confirm this cross-recognition with the 2D micropipette adhesion frequency assay, Fig. 6A. A Thy1.1⁺ NFM_15-35 T cell line was adoptively transferred into irradiated C56BL/6 Thy1.2⁺ recipients with no additional administration of CFA, peptide antigen or pertussis toxin. After EAE onset (Fig. 6B), we tested antigen specificity of the CNS infiltrates and found MOG_38-49 but not NFM_18-30 tetramer positive cells present in the CNS, Fig. 6C. The CNS cell population was a mix of Thy1.1 positive and negative cells with the Thy1.1 NFM cell line being the source of the CD44^hi MOG tetramer positive T cells, Fig. 6D. NFM_18-30 specific T cell contributions to EAE are strongly associated with MOG_38-49 specific cross-recognition and not stand alone NFM reactivity. These data in total point to MOG_35-55 as the dominant autoantigen for polyclonal T cell mediated EAE in C57BL/6 mice even when heteroclitic responsiveness to NFM is present.

Figure 6. Encephalitogenicity of a NFM T cell line is concomitant with infiltration of tetramer positive MOG specific T cell in the CNS.

A) Lymph nodes from NFM_15-35 primed C57BL/6 wild type mice were harvested and cultured for 1 week on NFM_15-35. 2D micropipette adhesion frequency assay was used to assess MOG versus NFM specificity. This figure represents one of 2 experiments, displaying data from 36 cells. B) Irradiated (400 rads) C57BL/6 mice received 10×10⁶ cells from a NFM T cell line (i.p.). 25 days post transfer, the CNS of mice with scores from 1.0 to 3.5 were harvested and antigen specificity was quantitated with MOG_38-49 or NFM_18-30 tetramer (C). D) Representative flow plots from adoptive transfer of a Thy1.1⁺ NFM T cell line in to a Thy1.2 recipient. C & D are data representative from 1 of 2 experiments. All statistics were done using 2-tailed unpaired, parametric t tests assuming both populations had equal standard deviations.

Discussion

Given the identical TCR contact residues of NFM_15-35 and MOG_35-55, it was surprising that NFM does not induce EAE (Fig. 1). Antigen specific T cell recognition is critical for generating a robust immune response where increased cell numbers influence the onset and magnitude of the response (48, 49). The idea that autoreactive T cell expansion is influenced by TCR cross-recognition and reactivity to peptides with alterations at TCR or MHC residues introduced through amino acid substitutions has been well documented, particularly in multiple sclerosis and EAE research (5, 15, 19, 42, 50-53). In one example, expansion of tetramer positive MOG specific T cells in the polyclonal repertoire has been reported in mice challenged with foreign derived peptides that are structurally similar to MOG_40-48 (19). These mice developed EAE with varying degrees of severity and the weaker disease courses were associated with slightly diminished expansion of higher affinity, MOG tetramer positive T cells (19). In the case of NFM_15-35 challenge there were reduced numbers of CD44^hi MOG_38-49 tetramer positive T cells in the spleen and peripheral lymph nodes which were not sustained long term when compared to MOG_35-55 challenge (Fig. 3). Expansion of a reduced number of MOG specific cells ultimately failed to promote T cell enrichment in the CNS and EAE after NFM_15-35 challenge (Figures 1, 3 & 4).

Important differences between the core nonamers of NFM_15-35 and MOG_35-55 that influence T cell reactivity are the amino acid residues proposed to interface with I-A^b molecule. In fact, replacement of the P1 MHC anchor residue partially restored the disease potential of the NFM peptide. We previously reported that a MHC variant peptide of MOG_35-55 at peptide position P6, MOG 45D, also resulted in poor encephalitogenic potential despite sharing 5 TCR contact residues with MOG_35-55 (33). Of interest was the finding that IFNγ deficient signaling rendered mice permissive to 45D mediated EAE and that disease was concomitant with enhanced detection of MOG_38-49 specific tetramer positive T cells. These data suggested that a threshold of high affinity, MOG_38-49 tetramer positive T cells was needed for polyclonal T cells to establish EAE in the IFNγ deficient mice. Since IFNγ deficiency did not restore encephalitogenic potential of NFM_15-35 as it did for 45D (Fig. 3), we addressed the role of additional amino acid differences between NFM_15-35 and MOG_35-55 that hindered T cell encephalitogenicity.

We found that amino acid substitutions within NFM_15-35 enabled us to map the residues critical for encephalitogenicity and expansion of MOG_38-49 tetramer positive T cells in a polyclonal setting (Fig. 4). For MOG_35-55, evidence suggested that amino acid residues P-1 and P-2 outside of the core nonamer contribute to MHC anchoring and to TCR binding in individual clones (44, 45, 54, 55) with implications for T cell recognition of NFM_15-35 (28). We focused on modulating NFM at P1 (T20Y) to mirror MOG at that position because Petersen et al. reported that MOG Y40 was important for MHC binding and T cell responsiveness (44); S23P and S28V MHC anchor substitutions were tested in comparison. S23P and S28V did not promote EAE in C57BL/6 mice and T20Y exhibited a low incidence of disease (Table II), a phenotype published during this time (40). We further showed that dual alterations at NFM_15-35 P-1 (E19W) and P1 (T20Y) enhanced EAE incidence to 100%, Table II. It was interesting that EAE incidence of NFM E19W, T20Y was concomitant with expansion of polyclonal MOG_38-49 tetramer positive T cells and a MOG-like proliferation profile by 2D2 monoclonal T cells (Fig. 4). Expansion of high numbers of polyclonal MOG_38-49 tetramer positive T cells was clearly dependent on the Y20 and W19 amino acids at P1 and P-1 respectively. Mapping of the amino acids required for expansion of tetramer positive T cells suggested that a threshold number of MOG_38-49 specific T cells was required for consistent onset of disease, namely an average of 7,324 to 9,244 cells seen with NFM E19W, T20Y and MOG_35-55.

NFM deficient mice were used to indicate whether antigen specific selection pressures influenced encephalitogenicity. Although it was possible that T cells educated in these mice would be hyperresponsive to NFM based on reports showing enhanced antigen specific T cell responses in MBP^-/- and MOG^-/- mice (29, 56), we saw no significant difference in NFM specific T cell proliferation between wild type and NFM deficient mice (Fig. 2). This could likely relate to the findings that peptides with low affinity for MHC do not mediate thymocyte negative selection, which first came to prominence with acetylated MBP (57). Our group also found this true for a model antigen system (58). It is not totally unexpected that deletion failed to alter the NFM specific T cell response considering the MHC alterations inherent in NFM _15-35 elicit a weaker association for I-A^b than MOG_35-55 (28), which is consistent with the high concentration of NFM required to tolerize EAE compared to MOG (59). In support of this, our studies revealed that cross-reactivity was not altered between wild type and NFM^-/- mice because MOG_35-55 induced EAE similarly in both mouse strains (Fig. 2). Furthermore, a report published during this time showed that ex vivo T cell cross-reactivity between MOG and NFM could be enhanced in MOG^-/- but not NFM^-/- mice (40). These data combined with the finding that NFM_15-35 challenge of MOG^-/- mice still did not induce EAE through cross-reactivity (Fig. 2) supported the finding that NFM is not a critical T cell antigen in polyclonal EAE.

The equivalent disease severity exhibited by our NFM^-/- mice after MOG_35-55 challenge differed somewhat from another report showing reduced EAE severity in the absence of NFM (59). Our mice (30) backcrossed to C57BL/6N had fulminant EAE and it is not known whether environmental or genetic factors caused this point of phenotypic divergence. However, our data still supports the observations of Ramadan et al. that NFM expression was not required for T cell mediated EAE (59). This is consistent with the reports that proliferation and cytokine production by MOG specific T cells were markedly diminished by restimulation with NFM compared to cognate MOG (28, 40, 59). We support that CNS T cell infiltrates are functionally responsive to NFM during EAE (14, 28), because pMHC monomers detected T cell cross-recognition of MOG_38-49 and NFM_18-30 by the majority of infiltrates at peak and chronic time points (Fig. 5).

Dissecting the role of individual cross-reactive clones in EAE lends to observations of heteroclitic responses where there is enhanced recognition and responsiveness to a second epitope above the initial priming antigen (Fig. 5). Heteroclitic behavior is best documented with clonal TCR in response to peptides with amino acid substitutions at TCR or MHC contacts but how one clone reflects on the bulk polyclonal response is less clear and dependent on previous antigenic exposures (12, 57, 60-62). Furthermore the priming antigen whether NFM_15-35 or MOG_35-55 could lead to asymmetry or skewedness of the immune response based on divergent expansion of unique T cell clones. Certainly there are reported differences in percentage detection of individual TCRα and TCRβ usage after priming with these respective peptides (14), yet overlap exists with divergence being dictated by a few amino acids at P1 and P-1 at the N-terminus of the core nonamer, Fig. 4. The sequence similarities between MOG_35-55 and NFM_15-35 and the current data in the field support that T cells are largely bispecific between these two peptides. We would argue that our analyses question the functional significance of NFM_15-35 on a polyclonal level because NFM monospecific T cells are rare in the population, Fig.5. Even in the presence of heteroclitic recognition of NFM, we found MOG to be the dominant autoantigen for disease onset in the absence of stand-alone encephalitogenicity by NFM_15-35 (Fig. 2).

The expansion of high affinity, MOG tetramer positive T cells are a relatively small population within the polyclonal repertoire (25-27, 63) and expansion or engraftment numbers has been associated with spontaneous EAE in retrogenic models (49). While NFM can expand a limited number of MOG tetramer positive T cells, these cells are not able to migrate to the CNS and cause EAE. We would argue that NFM is not providing a strong enough stimulus to evoke de novo EAE based on the weak interaction with I-A^b, which can be overcome with amino acid substitutions (Fig. 4). Culturing NFM primed splenocytes with NFM was a means to enhance T cell recognition and activation on NFM to enhance expansion encephalitogenic NFM or MOG specific T cell clones (14, 64, 65). As previously reported, NFM expanded T cells were able to passively transfer EAE, but we found this was associated with transfer of cross-reactive MOG_38-49 tetramer positive T cells capable of enrichment in the CNS (Fig. 6). This again showcased NFM without stand-alone encephalitogenic potential and supported MOG as the critical determinant in the bispecific model required for onset of demyelinating autoimmune disease. Overall, our experiments here indicate that expansion of a threshold number of high affinity, MOG tetramer positive T cells within the polyclonal response is a readout of encephalitogenic potential.

Abbreviations

MOG

myelin oligodendrocyte glycoprotein

NFM

neurofilament medium protein

pMHC

peptide: MHC

2D

2-dimensional

CNS

central nervous system

EAE

experimental autoimmune encephalomyelitis

-/-

deficient synonymous with knockout

WT

wild type C56BL/6 mice