Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Jul 15.
Published in final edited form as: J Neuroimmunol. 2019 Mar 21;332:8–15. doi: 10.1016/j.jneuroim.2019.03.013

A CD40 targeting peptide prevents severe symptoms in Experimental Autoimmune Encephalomyelitis

Gisela M Vaitaitis a,b, Martin G Yussman a,b, David H Wagner Jr a,b,*
PMCID: PMC6535109  NIHMSID: NIHMS1525670  PMID: 30925295

Abstract

CD40/CD154-interaction is critical in the development of Experimental Autoimmune Encephalomyelitis (EAE; mouse model of Multiple Sclerosis). Culprit CD4+CD40+ T cells drive a more severe form of EAE than conventional CD4 T cells. Blocking CD40/CD154-interaction with CD154-antibody prevents or ameliorates disease but had thrombotic complications in clinical trials. We targeted CD40 using a CD154-sequence based peptide. Peptides in human therapeutics demonstrate good safety. A small peptide, KGYY6, ameliorates EAE when given as pretreatment or at first symptoms. KGYY6 binds Th40 and memory T cells, affecting expression of CD69 and IL-10 in the CD4 T cell compartment, ultimately hampering disease development.

Keywords: CD40, CD154, EAE, therapeutic peptide

1. Introduction

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Rather than being only a disease of the CNS, MS is primarily an autoimmune disease and it is understood that several autoimmune components drive the disease. This aspect was elucidated primarily from studies in the Experimental Autoimmune Encephalomyelitis (EAE) mouse model and from analysis of lesions in brain and spinal cord of human patients. Sclerotic lesions form in the brain and spinal cord in both MS and EAE and involve infiltrating inflammatory cells, including macrophages (Baeten et al., 2008), mast cells (Hong et al., 2013), and T cells (El-behi et al., 2010, Fletcher et al., 2010, Howard et al., 1999). While CD8+ T cells play a role in EAE (Huseby et al., 2001) and MS (Denic et al., 2013), CD4+ T cells take center stage as drivers of the disease. Th1 cells, which produce IFNγ, TNFα, IL-1β and GM-CSF, are centrally involved in inflammation. IL-6 was originally described as a Th2, non-inflammatory cytokine, but is associated with inflammatory responses in MS (Furuzawa-Carballeda et al., 2007). Th17 cells that produce IL-17A-F, IL-21 and IL-22, likewise are associated with EAE and MS. We recently demonstrated that CD4+CD40+ T cells (Th40), which are capable of producing both Th1 and Th17 cytokines simultaneously (Vaitaitis et al., 2017a), drive a more severe form of EAE than conventional CD4+ T cells (Vaitaitis et al., 2017b).

CD40/CD154 interaction is crucial in EAE (Girvin et al., 2002, Vaitaitis, Yussman, 2017b). The classically described T cell co-stimulus is CD28 on T cells interacting with B7–1 or B7–2 on antigen presenting cells. Immunologic dogma holds that blocking CD28 interactions results in anergy, a total lack of response (Lenschow and Bluestone, 1993, Oliveira-dos-Santos et al., 1999). In EAE, the contrary was shown. Early studies suggested that CD28 co-stimulation was crucial for EAE development (Oliveira-dos-Santos, Ho, 1999) and in a later study, using CD28−/− mice, the initial disease induction challenge resulted in no disease (Girvin, Dal Canto, 2002). However, a second challenge of the CD28−/− mice resulted in a much more severe, fulminant disease (Girvin, Dal Canto, 2002). The treatment that did prevent disease under these conditions was CD40/CD154 block(Girvin, Dal Canto, 2002). The finding suggests CD40 as a central nexus for disease onset and severity, and further suggests that CD40 expression on T cells, as an inflammatory co-stimulus, is the crucial factor for disease development. Preventing the interaction of CD40 and CD154 in mice by using blocking antibodies decreases the severity and delays onset of EAE (Girvin, Dal Canto, 2002, Hong, Kim, 2013) and similar beneficial results from blocking CD40/CD154 interaction occur in other autoimmune disease models (Toubi and Shoenfeld, 2004, Waid et al., 2004).

A problem area however, is that when attempting to block CD40/CD154 interaction with anti-CD154 antibody in human trials, severe clinical problems often arise. In human Systemic Lupus Erythematosus, unfortunate life-threatening thromboembolic events occurred when using anti-CD154 antibodies in clinical trials (Boumpas et al., 2003, Sidiropoulos and Boumpas, 2004, Toubi and Shoenfeld, 2004) and the studies were halted. Antibodies as therapeutics come with inherent problems in that they may be immunogenic in the recipient and they can bind to Fcreceptors on cells such as platelets, neutrophils and macrophages, increasing risk of anaphylaxis. In the case of CD154 antibodies, CD154 interacts with the αIIbβ3 integrin on platelets, which stabilizes thrombi and it is therefore likely that the CD154 antibody blocked that interaction, destabilizing the thrombi. Because of these problems, a better way of blocking CD40/CD154 interactions is necessary. To that end, F(ab) fragments of CD154 antibodies, lacking the Fc-domain, are being pursued (Shock et al., 2015). However, the fragments still are bulky molecules that can be immunogenic. It is also possible that bulky molecules are sterically hindered to contact their targets in a “crowded” situation such as in an immunological synapse. Other strategies include random peptides that bind to CD40 in-vitro (Kitagawa et al., 2005) but those also bind other targets in-vivo, and small organic molecules that also bind CD40 but lose their activity in protein-rich medium (Margolles-Clark et al., 2009b). Additionally, those small organic molecules are highly similar to suramin, which has toxic side effects (Kaur et al., 2002, Margolles-Clark et al., 2009a).

Given the aforementioned problems, we developed a more targeted approach to inhibit the CD40/CD154 interaction. Based on the protein domain of CD154 that is essential for interaction with CD40, we designed several peptides that span the amino acids known to be critical (Bajorath et al., 1995a, Bajorath et al., 1995b, Vaitaitis et al., 2014). In the non-obese diabetic (NOD) mouse model of Type 1 Diabetes (T1D), one particular 15-mer peptide was highly efficacious in preventing diabetes onset and even reversed hyperglycemia in 56% of newly hyperglycemic mice (Vaitaitis, Olmstead, 2014). The CD154 sequence spanned by this peptide is 80% identical between the mouse and human sequences (S1Fig), suggesting that it is functionally important. Here we are utilizing a smaller, core region, of the 15-mer, a 6 amino acid sequence (S1Fig; referred to as KGYY6), to prevent or ameliorate EAE symptoms, in the mouse model for MS. The KGYY6 peptide sequence has 83% homology between mouse and human, and the 3 amino acids that are critical for interaction with CD40 are 100% conserved. Peptides are often used in human therapeutics and so far demonstrate a good safety record (Hinke, 2008, Johnson, 2012, Moertl et al., 2011).

2. Materials and Methods

2.1. Mice

C57BL/6 mice were from Taconic (Hudson, NY, USA). B6.CD40KO mice were from The Jackson Laboratory (Bar Harbor, ME, USA). Animals were housed at the University of Colorado Denver Anschutz Medical Campus AALAC approved Vivarium. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at the University of Colorado Denver (Protocol number: B-55817(01)1E). All efforts were made to minimize suffering and euthanasia was performed using carbon-dioxide followed by either cervical dislocation or exsanguination.

2.2. Antibodies and reagents

For flow cytometry: Anti-IFNγ (XMG1.2), anti-IL17A (eBio1787), anti-TNFα (MP6-XT22), anti-IL-10 (JES5–16E3), anti-IL-2 (JES6–5H4), anti-CD3 (145.2C11), anti-CD62L (MEL-14), anti-CD44 (IM7), anti-CD69 (H1.2F3), appropriate isotype control antibodies, and permeabilization buffer for intracellular stains were from eBioscience, Inc. (San Diego, CA, USA). Anti-CD4 (H129.19) was from BioLegend (San Diego, CA, USA). For cell purification: Lympholyte-M and Lympholyte-Mammal were from CedarLane (Burlington, NC, USA). Anti-CD40, 1C10 [22], was produced in-house. For EAE induction: M. Tuberculosis H37 RA was from Becton Dickinson and Company (Franklin Lakes, NJ, USA). Pertussis toxin (PT) was from Sigma-Aldrich® (St. Louis, MO, USA).

2.3. Induction of EAE

The EAE model can be highly variable even when controlling all possible parameters. This is likely due to seasonal variations as well as litter differences. In order to minimize litter-to-litter and seasonal variations, we performed large experiments that had 10 mice per group and randomly mixed the mice once they arrived from the vendor (this is possible with female mice that generally do not show aggression toward new mice). Female 10–12 week old C57BL/6 mice were immunized subcutaneously on the upper back/neck with 100 μl completely emulsified MOG35–55 peptide (50 μg in 50 μl PBS) and complete Freund’s adjuvant (CFA; 75 μg M. Tuberculosis H37 RA in 50 μl incomplete Freund’s adjuvant (mineral oil)), followed by pertussis toxin (PT; 200 ng in 100 μl PBS) injections as described (Miller et al., 2010). Mice were then randomly assigned to a treatment cohort or vehicle control cohort. One cohort of mice received i.v. injections of a 6-mer peptide (AKKGYY; referred to as KGYY6; 25 μg in 100 μl PBS per mouse) on days −3, −1, 6, 11, 13, and 15, with day 0 being the day of EAE induction. Another cohort received peptide on days −3, −1, 13, 20, and 27. Yet another cohort received peptide at the start of visible symptoms and two more times, days 11, 13, and 15, respectively. Vehicle control mice received injections of PBS. All mice were monitored daily for disease and scored: 0 – No abnormalities; 0.5 – Clutching hind limbs; 1 – Limp tail or weak hind limbs and/or wobbly gait; 1.5 – Limp tail and clutching hind limbs; 2 – Limp tail and weak hind limbs and/or wobbly gait. Mouse supports and propels itself using hind limbs; 2.5 – Limp tail and weak hind limbs and/or wobbly gait. Mouse cannot support and propel itself using hind limbs but the paws are moving; 3 – Limp tail and one weak hind limb, while the other is completely paralyzed. Mouse still uses the weak hind limb to propel itself somewhat; 3.5 – Limp tail and one weak hind limb, while the other is completely paralyzed. Mouse does not use the weak limb, which is almost at paralysis; 4 – Limp tail and complete hind limb paralysis. Often the mouse displays spastic hypertonia and involuntarily crosses its hind limbs; 5 – Complete paralysis of hind quarter and weak fore limb(s). The data are reported as the mean daily clinical score for all animals in each group. Mice reaching a level 5 or losing more than 20% of their bodyweight were euthanized.

2.4. Flow cytometry

Peripheral blood mononuclear cells (PBMC), draining lymph nodes (dLN; cervical), and spleens were processed over Lympholyte (Cedarlane, Hornby, Ontario, Canada) according to manufacturer’s protocol to generate purified lymphocytes. Cells were stained for extracellular proteins, washed and fixed in paraformaldehyde, then permeabilized and stained for intracellular proteins. Gates were set from appropriate isotype controls. Samples were run on a MACSQuant flow cytometer (Miltenyi Biotec Inc., Auburn, CA, USA). Data was analyzed with FlowJo software (FlowJo LLC, Ashland, OR, USA).

3. Results

3.1. KGYY6 ameliorates the disease course of EAE.

KGYY6 comprises the core region of the CD154 protein and contains 3 of the amino acids required for interaction with CD40 (Bajorath, Chalupny, 1995a, Bajorath, Marken, 1995b). In previous work we tested a 15-mer version of the peptide, KGYY15, which prevented diabetes onset in NOD mice, and reversed hyperglycemia in 56% of newly onset diabetic mice (Vaitaitis, Olmstead, 2014). The 15-mer has 80% homology between mouse and human (S1Fig). The KGYY6 peptide has 83% homology between mouse and human and the amino acids known to be critical for interaction with CD40 are 100% conserved (S1Fig). We chose to test the 6-mer in a model of multiple sclerosis (EAE).

One group of mice received KGYY6 i.v. injections 3 and 1 day(s) prior to EAE induction while another group received vehicle PBS. EAE was then induced and the mice started to show symptoms on day 10, which is normal for our laboratory (Fig. 1). On days 13, 20 and 27, the KGYY6 group received a boost of peptide while the other group again received vehicle PBS. The disease course was significantly less severe in the KGYY6 treated group compared to the control group (Fig. 1; p<0.0001; two-way ANOV6A). Already on day 13 there was a statistically significant difference that lasted throughout the experiment (Fig. 1; asterisks denote p-value; * - <0.05, **- <0.01, *** - <0.001, **** - <0.0001; two-way ANOVA with Sidak’s multiple comparisons test).

Figure 1. KGYY6 ameliorates EAE.

Figure 1.

Open in a new tab

C57BL/6 mice were either pretreated, or not, with KGYY6 peptide then EAE was induced. KGYY6 pretreated mice received boosters of KGYY6 on days 13, 20, and 27 (blue arrows). Mice were disease scored daily. Statistical significance was calculated by Two-Way ANOVA with Sidak’s multiple comparison test (asterisks denote p-value; * - <0.05, **- <0.01, *** - <0.001, **** - <0.0001).

3.2. Starting treatment at first sign of symptoms ameliorates the EAE disease course

In humans, it is not possible to know who is going to develop MS and, therefore, we addressed whether starting treatments at early signs of EAE would ameliorate symptoms in the test model. In our laboratory, disease symptoms typically start on day 10 or 11 post disease induction challenge. We treated a group of EAE induced mice starting on day 11 and then on days 13 and 15. We pretreated another group at days −3 and −1, and then further treated on days 6, 11, 13, and 15. Yet another group served as control for disease induction and received no treatment. The control group demonstrated a disease course identical to the previous experiment while the pretreated mice, again, had a significantly less severe disease course (Fig. 2; p < 0.0001; Two-way ANOVA with Tukey’s multiple comparison test). The treat-at-symptom group also demonstrated a less severe disease course compared to control but was more severe than the pretreated group (Fig. 2; p<0.0001 for both comparisons; Two-way ANOVA with Tukey’s multiple comparison test).

Figure 2. Starting treatment at first symptoms ameliorates EAE.

Figure 2.

Open in a new tab

C57BL/6 mice were either pretreated at days −3 and −1, or not, with KGYY6 peptide then EAE was induced. KGYY6 pretreated mice received boosters of KGYY6 on days 6, 11, 13, and 15 (blue arrows). One group, that was not pretreated, received KGYY6 peptide at first symptom on day 11 as well as boosters on days 13 and 15. Mice were disease scored daily. Statistical significance was calculated by Two-Way ANOVAwith Tukey multiple comparison test. Control vs. Treat at symptom, Control vs. Pretreat, and Treat at symptom vs. Pretreat were all significantly different; p<0.0001.

3.3. Earlier boosts with KGYY6 have a better impact on the EAE disease course

The disease course in the pretreated group in figure 1 appeared to be more severe than the disease course of the pretreated group in figure 2. The difference between the two was the timing of the peptide boosts that were administered after the initial pretreatment at days −3 and −1. When comparing the disease courses of the groups with the different boost schedules, clearly there was a significant difference in disease severity, with overall less severe disease if the boosts started early, at 6 days, rather than at 13 days (Fig. 3; < 0.0001; Two-way ANOVA with Tukey’s multiple comparison test). In fact, administering the boosts later, at days 13, 20, and 27, caused a disease course similar to that of the treat-at-symptom group in figure 2.

Figure 3. Earlier boosts with KGYY6 have a better impact on the EAE disease course.

Figure 3.

Open in a new tab

C57BL/6 mice were either pretreated, or not, with KGYY6 peptide then EAE was induced. KGYY6 pretreated mice received boosters of KGYY6 either on days 6, 11, 13, and 15 (green arrows) or on days 13, 20, and 27 (purple arrows). Mice were disease scored daily. Statistical significance was calculated by Two-Way ANOVAwith Tukey multiple comparison test. Control vs. Treat at symptom, Control vs. Pretreat, and Treat at symptom vs. Pretreat were all significantly different; p<0.0001.

3.4. T cells from KGYY6 treated mice express more CD69

CD69 expression traditionally is described as an early or very early activation marker on T cells and is induced by TCR recognition of antigen and subsequent CD3 stimulation (Cibrian and Sanchez-Madrid, 2017). However, studies that are more recent have focused on the function of the molecule. CD69 is a membrane-bound, type II, C-lectin receptor that interacts with galectin-1 as a trans-ligand, and interacts with sphingosine-1-phosphate receptor (S1P1R) as a cis-ligand (Cibrian and Sanchez-Madrid, 2017). S1P1 promotes egress of T cells from lymph nodes and from tissues (Mackay et al., 2015) and CD69 interaction with S1P1R prolongs tissue retention. This is particularly relevant given that a S1P1R super-agonist, Gilenya, is one of the disease modulating therapies that is FDA approved for MS (Mao-Draayer et al., 2017). Gilenya’s mechanism of action is to bind S1P1R and to down regulate its surface expression, which then retards T cells in lymph nodes. Based on the above, CD69 expression on cells from lymph nodes indicates cells poised for nodal retention. We determined the number of CD69 expressing cells in draining lymph nodes (dLN) and spleens harvested from the EAE mice at the end of the experiment, after 33 days. The number of Th40 cells in dLN of treated mice expressing CD69 was increased when compared to vehicle treated controls, but the difference did not yet achieve significance (Fig. 4A; top left panel). However, one sample in the vehicle treated controls was an outlier and, if it was not taken into consideration, there was a significant difference (p=0.0387, t-test). A significantly greater number of Th40 cells from spleens of KGYY6 treated mice expressed CD69 compared to untreated mice (Fig. 4A; top right panel; p = 0.0139; t-test). Overall, fewer of the conventional CD4 T cells from dLN and spleen expressed CD69 compared to Th40 cells (Fig. 4A), but the number of conventional CD4 T cells expressing CD69 was increased by KGYY6 treatment compared to the same cells from controls (Fig. 4A; bottom panels; p = 0.0021 (dLN) and p = 0.0450 (spleen); t-test). We considered CD69 expression on a per cell basis and found that in Th40 cells there was an increase in the amount of CD69 expressed per cell (Fig. 4B). As in figure 4A, the increase did not reach significance in the dLN, however, when the same outlier was taken out of consideration, there was a significant difference (p=0.0400; t-test). Th40 cells from spleens of treated mice expressed significantly more CD69 per cell than the same cells from vehicle controls (Fig. 4B; right panel; p=0.0452; t-test). While the percentage of conventional CD4 T cells that expressed CD69 was increased by KGYY6 treatment, that treatment did not change the amount of CD69 per cell in this cell type (Fig. 4B; bottom panels).

Figure 4. T cells from KGYY6 treated mice express more CD69.

Figure 4.

Open in a new tab

Lymphocytes from dLN or spleen were stained and gated on Th40 or conventional CD4 T cells then (A, B) CD69 and (C) CD62L expression was analyzed. Significance was calculated by t-test and p-values are indicated in the figure and the text.

L-selectins are expressed on T cells and, depending upon activation status, expression rapidly cycles between high and low levels, thus allowing cells to crawl to areas of inflammation, and to extravasate into the tissue (Butcher and Picker, 1996) (Chao et al., 1997). CD62L is an L-selectin that has generally been associated with naïve T cells, given its importance in lymph node retention (Kansas, 1992, Tedder et al., 1993). Rather than considering CD62L as a biomarker for naïve T cells, we considered that it may be involved in lymph node retention of potentially pathogenic effector cells. We examined the number of CD62L expressing Th40 and conventional CD4 T cells from both spleens and dLN of KGYY6 treated mice and compared to vehicle treated mice. There was no difference in number of CD62L expressing cells between treated and untreated (Fig. 4C).

3.5. KGYY6 treatment alters IL-10 production by conventional CD4 T cells

We examined intracellular cytokine expression profiles in Th40 and conventional CD4 T cells immediately ex-vivo at the end of the experiment, after 33 days. In conventional CD4 T cells from dLN, but not spleen, of KGYY6 treated animals there was an increase in intracellular IL-10 compared to the same cells from untreated animals (Fig. 5; p=0.0145; t-test). In Th40 cells, there was no differences in intracellular IL-10 expression between KGYY6 treated and untreated mice in dLN or spleen (Fig. 5). Other cytokines (TNFα, IFNγ, IL-2, and IL-17A) were unchanged in response to KGYY6 treatment in both cell types (Fig. S2).

Figure 5. KGYY6 treatment alters IL-10 production by conventional CD4 T cells.

Figure 5.

Open in a new tab

Lymphocytes from dLN or spleen were stained and gated on Th40 or conventional CD4 T cells then intracellular IL-10 levels were analyzed. Significance was calculated by t-test and p-values are indicated in the figure and the text.

3.6. KGYY6 treatment does not affect memory cell levels

In previous work, we showed that memory cell levels increase in response to induction of EAE. Therefore, we determined whether treatment with KGYY6 had an effect on memory cell levels. When examining the expression of CD44+CD62L− (effector memory) and CD44+CD62L+ (central memory) in both spleens and dLN there was no difference in the levels of those memory phenotypes between treated and untreated Th40 or conventional CD4 T cells (Fig. S3A and B).

3.7. KGYY6 peptide binds to EAE induced Th40 cells

To confirm that KGYY6 binds to the intended target cells we harvested peripheral blood mononuclear cells (PBMC), dLN lymphocytes, and splenic lymphocytes from mice at 6 and 13 days out from receiving the full EAE regimen and stained them with a FITC-conjugated KGYY6. Clearly, the peptide stained the CD4+CD40+ cells but not the CD40− cells in PBMC, dLN, and spleen samples (Fig. 6A, left panels). When gating on memory cells (CD62L versus CD44), the peptide stained primarily effector (CD44+CD62L−) and central (CD44+CD62L+) memory cells in PBMC and spleen samples but had a more diffuse pattern in dLN samples (Fig. 6A, right panels).

Figure 6. KGYY6 binds Th40 and memory cells.

Figure 6.

Open in a new tab

C57BL/6 mice were EAE induced then PBMC, dLN, and splenic lymphocytes were harvested on day 6 or 13 and stained. (A) Dot plots on the left are CD4 vs. CD40 with heat-mapping for KGYY6. Dot plots on the right are CD62L vs. CD44 with heat-mapping for KGYY6. The more yellow and orange, the more staining for KGYY6. The results shown are from the 6 days post EAE induction. (B) Histogram depicting KGYY6 staining of B6.CD40KO, control non-induced C57BL/6, and EAE-induced C57BL/6 mice 6 and 13 days post induction. Data represents 2 mice at 6 days and 3 mice at 13 days, all with similar results.

To confirm that the KGYY6 peptide binds specifically to CD40, we stained PBMC from C57BL/6 mice that have the CD154-interacting domain of CD40 knocked out (B6.CD40KO). Clearly, the peptide did not stain the B6.CD40KO cells while it readily stained both the non-induced and EAE-induced C57BL/6 mice (Fig. 6B), demonstrating the specificity of the peptide for CD40.

4. Discussion

Early and ongoing treatment with disease-modifying therapy is currently the best care in relapsing remitting MS. Such treatments can reduce the number of relapses, delay progression of disability, and limit new lesions as seen on MRI. When severe relapses occur, corticosteroids are used to manage and reduce the inflammation in the CNS. Many of the current treatments for MS often have the side effect of broadly suppressing the immune system. While that may decrease disease symptoms, it also leads to an increased susceptibility to opportunistic infections. Therefore, the search for treatments that have a more targeted effect on the actual culprit immune cells is ongoing.

We recently demonstrated that Th40 cells drive a more severe form of EAE than conventional CD4 T cells do (Vaitaitis, Yussman, 2017b). Th40 cells express the alternative costimulatory molecule CD40 (Baker et al., 2008, Munroe and Bishop, 2007), which is also expressed by other immune cells and is known to be a major target in autoimmune diseases (Peters et al., 2009). Interestingly, there appears to be several different forms of CD40 receptor, either because of glycosylation differences in the protein itself (Vaitaitis and Wagner, 2010) or because of the interaction of CD40 with other molecules (Vaitaitis and Wagner, 2010, 2012, 2013) or both. Certainly, a less glycosylated form of CD40 appears to be associated with more activated cells (Vaitaitis and Wagner, 2010). Therefore, it would be ideal to target such activated immune cells specifically. It is known that EAE symptoms can be reduced by anti-CD154 antibody injections, which block CD40 signaling (Girvin, Dal Canto, 2002), but anti-CD154 antibody treatments had serious and life-threatening side effects in clinical trials (Boumpas, Furie, 2003, Sidiropoulos and Boumpas, 2004, Toubi and Shoenfeld, 2004). Therefore, we set out to find a different, yet specific way of targeting CD40-signals. We previously demonstrated that a CD40-targeting peptide binds to CD40 and prevents and even reverses T1D in the NOD mouse model (Vaitaitis, Olmstead, 2014). This peptide avoids many of the problems that are inherent to antibodies, e.g. immunogenicity and anaphylaxis causality, and patients generally tolerate peptide treatments well.

Here we used a 6-mer peptide, which is predicted to be stable for at least 4.4 hours (calculated by the ExPasy ProtParam tool) and spans the core region of the domain in the CD154 protein that interacts with CD40, to affect CD40 signals during the EAE disease process. Clearly, administration of the peptide ameliorated the symptoms whether pretreated then boosted or just treated at the first sign of symptoms. Pretreatment followed by boosts worked better than just treating at signs of symptoms. Obviously, by treating at the first signs of symptoms, we are trying to approximate human disease where it is desirable to treat only when disease symptoms onset rather than continually administering therapy. The limitation with the EAE studies is that onset can be gaged only by visual clues such as a limp tail, wobbly gait, paralysis etc. In human disease, an MS patient who is experiencing a relapse may very well be aware of telltale signs much earlier than the appearance of actual disability. Peptide therapy could therefore commence at much earlier stages, possibly having a greater impact. In the EAE experiments here, there was certainly a significantly better outcome if the peptide boosts were administered earlier, before any visible signs of disease. It would also be possible to keep those with diagnosed MS who are in remission in treatment with periodic peptide doses and then increase the dosing frequency if the patient reports early signs of a relapse.

Interestingly, treating EAE mice with KGYY6 induced a significant increase in CD69 expression in dLN and spleen CD4 T cells (Th40 and conventional CD4 T cells), which recently was shown to prolong tissue retention of T cells (Cibrian and Sanchez-Madrid, 2017, Mackay, Braun, 2015). Also recently, circadian oscillations of lymphocytes trafficking to/from the vascular space and tissues/lymph nodes were demonstrated to have a significant effect on EAE disease severity, where EAE induction during the daytime caused more severe symptoms than if done 1 hour after the start of the dark cycle (Druzd et al., 2017). We have shown previously that CD40-signaling influences trafficking of leukocytes (Vaitaitis, Waid, 2017a). While not tested here, we speculate that the KGYY6 treatment may affect the timely trafficking of culprit cells to the CNS for optimal disease induction. Previous data led us to the same speculation about timely trafficking (Vaitaitis, Yussman, 2017b). It will be highly important to understand whether alterations in trafficking is a mechanism in the KGYY6 treatment and whether time of day of administration impacts the efficacy.

KGYY6 treatment induced an increase in IL-10 expression in conventional CD4 T cells from dLN samples. While IL-10 is considered anti-inflammatory, it is not clear from the current data whether the cells expressing IL-10 are exerting such an effect. Contrary to what we previously demonstrated when a similar peptide was used to prevent T1D (Vaitaitis, Olmstead, 2014), KGYY6 did not cause a prevention of expansion in the Th40 cell population. NOD mice spontaneously develop T1D while EAE has to be induced in mice. It is unclear if the rather robust immune stimulation employed to induce EAE is simply to strong and overcomes an “expansion-prevention” aspect of the peptide treatment. In any case, KGYY6 appears to directly target the culprit cells as we could demonstrate binding specifically to Th40 cells from EAE induced mice, the cells that we previously showed were causing a more severe disease in this model (Vaitaitis, Yussman, 2017b). However, other cells express CD40 and inflammatory conditions can induce CD40 expression on previously surface-CD40-negative cells (Vaitaitis, Yussman, 2017b). Therefore, it is likely that B cells, macrophages, and other immune cells would also bind the peptide at certain points throughout the disease course. In future studies, it will be important to assess the effect of KGYY6 on those cells as well as target cells in the CNS, such as microglia, during EAE.

Whether the target cells are only Th40 cells or also other CD40 expressing cells the peptide does not eliminate or depress all T cells or even all Th40 cells. Therefore, it is likely that the KGYY6 peptide would not be generally immunosuppressive. However, it will be important to confirm that that is the case. If so, KGYY6 therapy would be advantageous compared to many current MS drug therapies that often immune suppress the patients.

Future large studies will be necessary to elucidate the exact mechanism(s) of action of the KGYY6 peptide. Currently it is in toxicity and kinetics studies in mice with the ultimate aim of filing an Investigational New Drug application with the Food and Drug Administration such that clinical testing can commence.

Supplementary Material

1
2
3

Highlights.

  • A CD154-derived, CD40-targeting, peptide (KGYY6) ameliorates EAE.

  • KGYY6 alters the phenotype of CD4 T cells but does not deplete those cells.

  • Being a peptide, KGYY6 avoids problems common to antibodies.

  • KGYY6 directly targets CD40, not CD154, avoiding possible thrombotic problems.

  • KGYY6 does not include the αIIbβ3 interacting domain of CD154.

Acknowledgments

FlowJo software was licensed through the University of Colorado Cancer Center Support Grant (P30CA046934) and the Skin Diseases Research Cores Grant (P30AR057212).

Funding

This work was funded by grants from the National Institute of Allergy and Infectious Diseases (https://www.niaid.nih.gov; grant number R41 AI113977) and the National Multiple Sclerosis Society (http://www.nationalmssociety.org/; grant number RG 5070-A-1) awarded to DHW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Competing interests

DMW is the co-founder and co-owner of Op-T LLC. MGY is the chief medical officer of Op-T LLC. GMV performs consultant work for Op-T LLC.

References

  1. Baeten K, Hendriks JJ, Hellings N, Theunissen E, Vanderlocht J, Ryck LD, et al. Visualisation of the kinetics of macrophage infiltration during experimental autoimmune encephalomyelitis by magnetic resonance imaging. Journal of neuroimmunology. 2008;195:1–6. [DOI] [PubMed] [Google Scholar]
  2. Bajorath J, Chalupny NJ, Marken JS, Siadak AW, Skonier J, Gordon M, et al. Identification of residues on CD40 and its ligand which are critical for the receptor-ligand interaction. Biochemistry. 1995a;34:1833–44. [DOI] [PubMed] [Google Scholar]
  3. Bajorath J, Marken JS, Chalupny NJ, Spoon TL, Siadak AW, Gordon M, et al. Analysis of gp39/CD40 interactions using molecular models and site-directed mutagenesis. Biochemistry. 1995b;34:9884–92. [DOI] [PubMed] [Google Scholar]
  4. Baker RL, Wagner DH Jr., Haskins K. CD40 on NOD CD4 T cells contributes to their activation and pathogenicity. Journal of autoimmunity. 2008;31:385–92. [DOI] [PubMed] [Google Scholar]
  5. Boumpas DT, Furie R, Manzi S, Illei GG, Wallace DJ, Balow JE, et al. A short course of BG9588 (anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis Rheum. 2003;48:719–27. [DOI] [PubMed] [Google Scholar]
  6. Butcher EC, Picker LJ. Lymphocyte homing and homeostasis. Science (New York, NY. 1996;272:60–6. [DOI] [PubMed] [Google Scholar]
  7. Chao CC, Jensen R, Dailey MO. Mechanisms of L-selectin regulation by activated T cells. J Immunol. 1997;159:1686–94. [PubMed] [Google Scholar]
  8. Cibrian D, Sanchez-Madrid F. CD69: from activation marker to metabolic gatekeeper. European journal of immunology. 2017;47:946–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Denic A, Wootla B, Rodriguez M. CD8(+) T cells in multiple sclerosis. Expert Opin Ther Targets. 2013;17:1053–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Druzd D, Matveeva O, Ince L, Harrison U, He W, Schmal C, et al. Lymphocyte Circadian Clocks Control Lymph Node Trafficking and Adaptive Immune Responses. Immunity. 2017;46:120–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. El-behi M, Rostami A, Ciric B. Current views on the roles of Th1 and Th17 cells in experimental autoimmune encephalomyelitis. J Neuroimmune Pharmacol. 2010;5:189–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fletcher JM, Lalor SJ, Sweeney CM, Tubridy N, Mills KH. T cells in multiple sclerosis and experimental autoimmune encephalomyelitis. Clinical and experimental immunology. 2010;162:1–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Furuzawa-Carballeda J, Vargas-Rojas MI, Cabral AR. Autoimmune inflammation from the Th17 perspective. Autoimmun Rev. 2007;6:169–75. [DOI] [PubMed] [Google Scholar]
  14. Girvin AM, Dal Canto MC, Miller SD. CD40/CD40L interaction is essential for the induction of EAE in the absence of CD28-mediated co-stimulation. Journal of autoimmunity. 2002;18:83–94. [DOI] [PubMed] [Google Scholar]
  15. Hinke SA. Diamyd, an alum-formulated recombinant human GAD65 for the prevention of autoimmune diabetes. Curr Opin Mol Ther. 2008;10:516–25. [PubMed] [Google Scholar]
  16. Hong GU, Kim NG, Jeoung D, Ro JY. Anti-CD40 Ab- or 8-oxo-dG-enhanced Treg cells reduce development of experimental autoimmune encephalomyelitis via down-regulating migration and activation of mast cells. Journal of neuroimmunology. 2013;260:60–73. [DOI] [PubMed] [Google Scholar]
  17. Howard LM, Miga AJ, Vanderlugt CL, Dal Canto MC, Laman JD, Noelle RJ, et al. Mechanisms of immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple sclerosis. The Journal of clinical investigation. 1999;103:281–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huseby ES, Liggitt D, Brabb T, Schnabel B, Ohlen C, Goverman J. A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis. The Journal of experimental medicine. 2001;194:669–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Johnson KP. Glatiramer acetate for treatment of relapsing-remitting multiple sclerosis. Expert review of neurotherapeutics. 2012;12:371–84. [DOI] [PubMed] [Google Scholar]
  20. Kansas GS. Structure and function of L-selectin. APMIS. 1992;100:287–93. [DOI] [PubMed] [Google Scholar]
  21. Kaur M, Reed E, Sartor O, Dahut W, Figg WD. Suramin’s development: what did we learn? Inv estigational new drugs. 2002;20:209–19. [DOI] [PubMed] [Google Scholar]
  22. Kitagawa M, Goto D, Mamura M, Matsumoto I, Ito S, Tsutsumi A, et al. Identification of three novel peptides that inhibit CD40-CD154 interaction. Modern rheumatology / the Japan Rheumatism Association. 2005;15:423–6. [DOI] [PubMed] [Google Scholar]
  23. Lenschow DJ, Bluestone JA. T cell co-stimulation and in vivo tolerance. Current Opinion in Immunology. 1993;5:747–52. [DOI] [PubMed] [Google Scholar]
  24. Mackay LK, Braun A, Macleod BL, Collins N, Tebartz C, Bedoui S, et al. Cutting edge: CD69 interference with sphingosine-1-phosphate receptor function regulates peripheral T cell retention. J Immunol. 2015;194:2059–63. [DOI] [PubMed] [Google Scholar]
  25. Mao-Draayer Y, Sarazin J, Fox D, Schiopu E. The sphingosine-1-phosphate receptor: A novel therapeutic target for multiple sclerosis and other autoimmune diseases. Clinical immunology (Orlando, Fla. 2017;175:10–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Margolles-Clark E, Jacques-Silva MC, Ganesan L, Umland O, Kenyon NS, Ricordi C, et al. Suramin inhibits the CD40-CD154 costimulatory interaction: a possible mechanism for immunosuppressive effects. Biochemical pharmacology. 2009a;77:1236–45. [DOI] [PubMed] [Google Scholar]
  27. Margolles-Clark E, Umland O, Kenyon NS, Ricordi C, Buchwald P. Small-molecule costimulatory blockade: organic dye inhibitors of the CD40-CD154 interaction. Journal of molecular medicine (Berlin, Germany). 2009b;87:1133–43. [DOI] [PubMed] [Google Scholar]
  28. Miller SD, Karpus WJ, Davidson TS. Experimental autoimmune encephalomyelitis in the mouse. Curr Protoc Immunol. 2010;Chapter 15:Unit 15 1. [DOI] [PubMed] [Google Scholar]
  29. Moertl MG, Friedrich S, Kraschl J, Wadsack C, Lang U, Schlembach D. Haemodynamic effects of carbetocin and oxytocin given as intravenous bolus on women undergoing caesarean delivery: a randomised trial. BJOG : an international journal of obstetrics and gynaecology. 2011;118:1349–56. [DOI] [PubMed] [Google Scholar]
  30. Munroe ME, Bishop GA. A costimulatory function for T cell CD40. J Immunol. 2007;178:671–82. [DOI] [PubMed] [Google Scholar]
  31. Oliveira-dos-Santos AJ, Ho A, Tada Y, Lafaille JJ, Tonegawa S, Mak TW, et al. CD28 costimulation is crucial for the development of spontaneous autoimmune encephalomyelitis. J Immunol. 1999;162:4490−5. [PubMed] [Google Scholar]
  32. Peters AL, Stunz LL, Bishop GA. CD40 and autoimmunity: the dark side of a great activator. Seminars in immunology. 2009;21:293–300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shock A, Burkly L, Wakefield I, Peters C, Garber E, Ferrant J, et al. CDP7657, an anti-CD40L antibody lacking an Fc domain, inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study. Arthritis Res Ther. 2015;17:234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sidiropoulos PI, Boumpas DT. Lessons learned from anti-CD40L treatment in systemic lupus erythematosus patients. Lupus. 2004;13:391–7. [DOI] [PubMed] [Google Scholar]
  35. Tedder TF, Luscinskas W, Kansas GS. Regulation of leukocyte migration by L-selectin: mechanisms, domains and ligands. Behring Inst Mitt. 1993:165–77. [PubMed] [Google Scholar]
  36. Toubi E, Shoenfeld Y. The role of CD40-CD154 interactions in autoimmunity and the benefit of disrupting this pathway. Autoimmunity. 2004;37:457–64. [DOI] [PubMed] [Google Scholar]
  37. Vaitaitis GM, Olmstead MH, Waid DM, Carter JR, Wagner DH Jr. A CD40-targeted peptide controls and reverses type 1 diabetes in NOD mice. Diabetologia. 2014;57:2366–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vaitaitis GM, Wagner DH Jr. CD40 glycoforms and TNF-receptors 1 and 2 in the formation of CD40 receptor(s) in autoimmunity. Mol Immunol. 2010;47:2303–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vaitaitis GM, Wagner DH Jr. Galectin-9 Controls CD40 Signaling through a Tim-3 Independent Mechanism and Redirects the Cytokine Profile of Pathogenic T Cells in Autoimmunity. PLoS One. 2012;7:e38708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vaitaitis GM, Wagner DH Jr. CD40 interacts directly with RAG1 and RAG2 in autoaggressive T cells and Fas prevents CD40-induced RAG expression. Cellular & molecular immunology. 2013;10:483–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vaitaitis GM, Waid DM, Yussman MG, Wagner DH Jr. CD40-mediated signalling influences trafficking, T-cell receptor expression, and T-cell pathogenesis, in the NOD model of type 1 diabetes. Immunology. 2017a;152:243–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vaitaitis GM, Yussman MG, Waid DM, Wagner DH Jr. Th40 cells (CD4+CD40+ Tcells) drive a more severe form of Experimental Autoimmune Encephalomyelitis than conventional CD4 T cells. PLoS One. 2017b;12:e0172037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Waid DM, Vaitaitis GM, Wagner DH Jr. Peripheral CD4loCD40+ auto-aggressive T cell expansion during insulin-dependent diabetes mellitus. European journal of immunology. 2004;34:1488–97. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1525670-supplement-1.pptx (34.4KB, pptx)
2
NIHMS1525670-supplement-2.pptx (364KB, pptx)
3
NIHMS1525670-supplement-3.pptx (213.3KB, pptx)

RESOURCES