Are we aiming to miss in translational autoimmunity treatments? [version 1; referees: 2 approved, 1 approved with reservations]

Autoimmunity treatments, fruitfully pioneered in mouse models, can be disappointing or result in immunosuppression and opportunistic infections in translational trials. Many possible reasons exist, but one major, overlooked reason may be the treatment timing in relation to circadian oscillations of the immune system. Mice and humans both have immunological circadian clocks and experience the same circulatory oscillations of immune cells with regards to their sleep/wake phases, but have opposite sleep/wake phases with regard to the daylight cycle. Therefore, researchers mainly study mice and potential autoimmunity treatments during the murine sleep/rest phase, which is when pro-inflammatory mediators and more adaptive immune cells are prevalent in the circulation. In translational trials, however, treatment administration happens primarily during a patient’s wake/activity phase, during the daytime, which is when more local and acute immune responses are active in the circulation. Therefore, we believe that the most opportune window for autoimmunity treatment may be missed in translational trials. Shifting the timing, and adjusting dosing to target only immune cells that are active at that time, may result in higher success with minimized immunosuppression or toxicities

Are we aiming to miss in translational autoimmunity treatments? [version 2; referees: 3 approved, 1 approved with reservations]

Autoimmunity treatments, fruitfully pioneered in mouse models, can be disappointing or result in immunosuppression and opportunistic infections in translational trials. Many possible reasons exist, but one major, overlooked reason may be the treatment timing in relation to circadian oscillations of the immune system. Mice and humans both have immunological circadian clocks and experience the same circulatory oscillations of immune cells with regards to their sleep/wake phases, but have opposite sleep/wake phases with regard to the daylight cycle. Therefore, researchers mainly study mice and potential autoimmunity treatments during the murine sleep/rest phase, which is when pro-inflammatory mediators and more adaptive immune cells are prevalent in the circulation. In translational trials, however, treatment administration happens primarily during a patient’s wake/activity phase, during the daytime, which is when more local and acute immune responses are active in the circulation. Therefore, we believe that the most opportune window for autoimmunity treatment may be missed in translational trials. Shifting the timing, and adjusting dosing to target only immune cells that are active at that time, may result in higher success with minimized immunosuppression or toxicities

Galectin-9 Controls CD40 Signaling through a Tim-3 Independent Mechanism and Redirects the Cytokine Profile of Pathogenic T Cells in Autoimmunity

While it has long been understood that CD40 plays a critical role in the etiology of autoimmunity, glycobiology is emerging as an important contributor. CD40 signaling is also gaining further interest in transplantation and cancer therapies. Work on CD40 signaling has focused on signaling outcomes and blocking of its ligand, CD154, while little is known about the actual receptor itself and its control. We demonstrated that CD40 is in fact several receptors occurring as constellations of differentially glycosylated forms of the protein that can sometimes form hybrid receptors with other proteins. An enticing area of autoimmunity is differential glycosylation of immune molecules leading to altered signaling. Galectins interact with carbohydrates on proteins to effect such signaling alterations. Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells. Galectin-9 interacts with CD40 and, at higher concentrations, prevents CD40 induced proliferative responses of CD4loCD40+ effector T cells and induces cell death through a Tim-3 independent mechanism. Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4loCD40+ T cells. Understanding the dynamics of the CD40 receptor(s) and the impact of glycosylation status in immunity will gain insight into how to maintain useful CD40 signals while shutting down detrimental ones

High Distribution of CD40 and TRAF2 in Th40 T Cell Rafts Leads to Preferential Survival of this Auto-Aggressive Population in Autoimmunity

CD40-CD154 interactions have proven critical in autoimmunity, with the identification of CD4(lo)CD40(+) T cells (Th40 cells) as harboring an autoaggressive T cell population shedding new insights into those disease processes. Th40 cells are present at contained levels in non-autoimmune individuals but are significantly expanded in autoimmunity. Th40 cells are necessary and sufficient in transferring type 1 diabetes in mouse models. However, little is known about CD40 signaling in T cells and whether there are differences in that signaling and subsequent outcome depending on disease conditions. When CD40 is engaged, CD40 and TNF-receptor associated factors, TRAFs, become associated with lipid raft microdomains. Dysregulation of T cell homeostasis is emerging as a major contributor to autoimmune disease and thwarted apoptosis is key in breaking homeostasis.Cells were sorted into CD4(hi) and CD4(lo) (Th40 cells) then treated and assayed either as whole or fractionated cell lysates. Protein expression was assayed by western blot and Nf-kappaB DNA-binding activity by electrophoretic mobility shifts. We demonstrate here that autoimmune NOD Th40 cells have drastically exaggerated expression of CD40 on a per-cell-basis compared to non-autoimmune BALB/c. Immediately ex-vivo, untreated Th40 cells from NOD mice have high levels of CD40 and TRAF2 associated with the raft microdomain while Th40 cells from NOR and BALB/c mice do not. CD40 engagement of Th40 cells induces Nf-kappaB DNA-binding activity and anti-apoptotic Bcl-X(L) expression in all three mouse strains. However, only in NOD Th40 cells is anti-apoptotic cFLIP(p43) induced which leads to preferential survival and proliferation. Importantly, CD40 engagement rescues NOD Th40 cells from Fas-induced death.CD40 may act as a switch between life and death promoting signals and NOD Th40 cells are poised for survival via this switch. This may explain how they expand in autoimmunity to thwart T cell homeostasis

Galectin-9 interacts with CD40.

<p>CD4^loCD40⁺ T cells were sorted from female NOD, control BALB/c (BALB con.), or agonistic CD40 antibody injected BALB/c (BALB exp.) spleens. Whole cell lysates were prepared. (<b>A</b>) CD40 was immunoprecipitated and resulting proteins were separated by SDS-PAGE. Protein bands were sequenced by LC-LC-MS. (<b>B</b>) Cells were CD40 stimulated (CD40XL) for 0, 30 minutes, 3 hours or overnight (0′, 30′, 3h, o.n.) then lysates were prepared and CD40 immunoprecipitated. Co-imunoprecipitation of galectin-9 was measured in western blots. Data represents three separate experiments on mice ranging from 8 – 10 weeks old. (<b>C</b>) Cells were isotype or CD40 stimulated (CD40XL) overnight then lysates were prepared. Galectin-9 protein conjugated directly to magnetic beads was used in immunoprecipitations. Mock treated beads were used as a control. CD40 was measured in the immunoprecipitated material by western blot. Data represents three separate mice of two different ages (8 and 10 weeks old).</p

Galectin-9 prevents CD40 induced proliferation.

<p>(<b>A</b>) CD4^loCD40⁺ T cells were sorted from 7–13 week old female NOD spleens and were then labeled with CFSE. Cells were either isotype treated (Isotype) or CD40 was stimulated (CD40XL) in the absence/presence of increasing concentrations of galectin-9 (gal-9) for 4 days then CFSE dilution was measured. (<b>B</b>) Cells were sorted and treated as in A and were then stained for necrotic and apoptotic cell death. (<b>C</b>) Cells were sorted and CD40 stimulated as in A for 24 hours then galectin-9 was added at 7.5 µg/ml. Cells were analyzed for proliferation and cell death after a total of 4 days of stimulation. (<b>D and E</b>) Cells were sorted as in A and were pretreated, or not, with a Tim-3 blocking antibody (αTim-3) for 30 miutes then CD40 was stimulated in the absence/presence of galectin-9 for 4 days. Proliferation (D) and cell death (E) was measured, respectively. Percentages in A are means with SEM. Asterisks in B, C, and D denote significant differences determined by two-way Anova; ns – not significant; * – P between 0.01 and 0.05; ** – P <0.01; *** – P <0.001; **** – P<0.0001. Experiments were done at least three separate times.</p

CD40 and CD3 induced cytokine phenotypes differ and galectin-9 alters the production level.

<p>CD4^loCD40⁺ T cells were sorted from 7–20 weeks old female NOD spleens. Cells were either isotype treated, CD40- or CD3-stimulated in the absence/presence of indicated concentrations of galectin-9 (gal-9; µg/ml) for 3 days then cytokines were measured. Bar graphs depict means with SEM. Asterisks denote significant differences determined by one-way Anova; * – P between 0.01 and 0.05; ** – P <0.01; *** – P <0.001. Measurements were done on four individual mice of different ages.</p

In-vivo CD40 signals cause non-autoimmune CD4^loCD40⁺ T cells to appear more like autoimmune CD4^loCD40⁺ T cells.

<p>(<b>A</b>) 10–12 week old, female BALB/c mice were injected i.p. with 1C10+FGK45 agonistic CD40 antibodies (CD40-inj.) or with isotype control (control) antibody. Three days post-injection, splenic CD4^loCD40⁺ and CD4^hi T cells were purified and stained with L-PHA. Graph depicts the difference in L-PHA stain compared to control. Data are represented as means with SEM. Images are H&E stained sections of pancreata. (<b>B</b>) BALB/c mice were treated and CD4^loCD40⁺ T cells purified as in A. CD4^loCD40⁺ T cells from 10–12 weeks old female NOD spleens were purified for comparison. Cells were CFSE labeled then CD40-stimulated for 4 days. CFSE dilution was measured. Dotted line – isotype treated; solid line – CD40-stimunlated. (<b>C</b>) 10 week old female BALB/c mice were injected and CD4^loCD40⁺ T cells purified as in A, then cells were labeled with CFSE and isotype treated (Isotype) or CD40 stimulated (CD40XL) in the absence/presence of galectin-9. CFSE dilution was measured. Percentages in B and C are means +/− SEM. Experiments were performed at least three separate times.</p

Galectin-9 causes autoimmune CD4^loCD40⁺ T cells to appear more like CD4^hi T cells and increases CD3 induced proliferation.

<p>CD4^loCD40⁺ and CD4^hi T cells were sorted from 7–13 weeks old female NOD spleens. (<b>A</b>) CD4^loCD40⁺ T cells were isotype treated (Isotype) or CD40-stimulated for 2 days in the absence/presence of indicated concentrations of galectin-9 then photographed under a microscope. (<b>B</b>) NOD CD4^loCD40⁺ T cells were treated for 2 hours with 2.5 µg/ml galectin-9 then stained for CD3, CD4, TCR, CD5 and CD8. Tinted – untreated; black line – 2.5 µg/ml galectin-9. Gates were set based on appropriate isotype controls. Below each histogram is a corresponding bar graph depicting the cumulative data. (<b>C</b>) NOD CD4^hi T cells were either stained immediately for CD28 or CD40 (left two histograms; grey tinted – stain-isotype; black line – CD28 or CD40 as indicated in figure; percentages are means with SEM) or isotype treated or stimulated with CD3+CD28+CD40 overnight then stained for CD4 (bar graph and right histogram; grey tinted – stain-isotype; black line – isotype treated; dotted line – CD3+CD28+CD40 stimulated). (<b>D</b>) NOD CD4^loCD40⁺ T cells were CFSE labeled then isotype treated (Isotype) or CD3-stimulated (CD3XL) in the absence/presence of indicated concentrations of galectin-9 (gal-9) for 8 days. Proliferation was measured by CFSE dilution. All bar graphs in this figure depict means with SEM. Asterisks denote significant differences determined by one- or two-way Anova as appropriate; ns – not significant; * – P between 0.01 and 0.05; ** – P <0.01; *** – P <0.001; **** – P<0.0001. Experiments were done at least three separate times.</p