Novel CD28 antagonist mPEG PV1-Fab’ mitigates experimental autoimmune uveitis by suppressing CD4+ T lymphocyte activation and IFN-γ production

Autoimmune Uveitis is an important chronic inflammatory disease and a leading cause of impaired vision and blindness. This ocular autoimmune disorder is mainly mediated by T CD4+ lymphocytes poising a TH1 phenotype. Costimulatory molecules are known to play an important role on T cell activation and therefore represent interesting therapeutical targets for autoimmune disorders. CD28 is the prototypical costimulatory molecule for T lymphocytes, and plays a crucial role in the initiation, and maintenance of immune responses. However, previous attempts to use this molecule in clinical practice achieved no success. Thus, we evaluated the efficacy of mPEG PV1-Fab’ (PV1), a novel selective CD28 antagonist monovalent Fab fragment in the treatment of Experimental Autoimmune Uveitis (EAU). Here, we showed that PV1 treatment decreases both average disease score and incidence of EAU. A decrease in the activation profile of both T CD4+ and T CD8+ eye-infiltrating lymphocytes was evidenced. In the periphery, T CD4+ cells from PV1-treated mice also showed a decrease in their activation status, with reduced expression of CD69, CD25, and PD-1 molecules. This suppression was not dependent on Treg cells, as both their frequency and absolute number were lower in PV1-treated mice. In addition, frequency of CD4+IFN-γ+ T cells was significantly lower in PV1-treated group, but not of IL-17-producing T cells. Moreover, after specific restimulation, PV1 blockade selectively blocked IFN-γ production by CD4+ lymphocytes Taken together, our data suggest that mPEG PV1-Fab’ acts mainly on IFN-γ-producing CD4+ T cells and emphasize that this specific CD28 blockade strategy is a potential specific and alternative tool for the treatment of autoimmune disorders in the eye.


Introduction
Autoimmune uveitis is an important inflammatory disease of the eye and it is responsible for approximately 10% of visual deficit and legal blindness cases in the USA [1]. Uveitis is characterized by an inflammation of the uvea-a layer comprising the tissues between the sclera and a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 working as a classical antagonist. This antibody has been used in different models, with promising results. Perrin and colleagues [20] showed that mAb PV1 targets encephalomyelitogenic T cell clones and mitigates Experimental Autoimmune Encephalomyelitis. In a heart transplant model the use of PV1 IgG3 was shown to improve graft survival through modulation of the cytokine milieu [19]. Based on these results, Poirier and colleagues developed FR104, a novel humanized CD28 antagonist composed of a monovalent Fab fragment conjugated with polyethylene glycol (PEG), aiming for tolerance restoration in autoimmune conditions and in transplantation [21]. Similar to mAb PV1, FR104 failed to induce T cell responses and showed efficacy in suppressing effector T cells in a humanized Graft versus Host Disease model [27]. However, further knowledge of the mechanism of action of FR104 is still needed. In this study, using mPEG PV1-Fab' (PV1), a FR104 murine analogue comprising a monovalent fragment of the mAb PV1 antibody conjugated with PEG molecules, we investigated its effect on the treatment of EAU. Here we show that PV1 decreases both disease average score and incidence, whereas decreasing overall T cell activation in the uveitic eyes and the periphery. The observed immunosuppression is not due to generation of T reg cells or induction of anergy but is directed against IFN-γ production by T H 1 cells.

Material and methods
Mice B10.RIII mice were obtained from Jackson Laboratories and were maintained under specific pathogen-free conditions at Hospital Israelita Albert Einstein animal facility (CETEC), an institution certified by the Association for Assessment and Accreditation of Laboratory Animal Care. The Animal Care Committee of the Institute of Biomedical Sciences at the University of São Paulo and the Animal Care Committee of the Hospital Israelita Albert Einstein approved all the procedures utilized in this study; all procedures are in accordance to international rules of animal care as defined by the International Animal Welfare Recommendations [28].

Histological analysis and disease scoring
On day 21, eyes were collected and prepared for histological analysis as described elsewhere [30]. Disease severity affecting both untreated and PV1-treated mice was evaluated in a double-blinded fashion by examining four sections from each eye, cut at different levels. Disease was scored according to a scale from 0 (no disease) to 4 (maximum disease), in half-point increments, according to a semi-quantitative system described previously [13]. The minimal EAU score considered was characterized by inflammatory cell infiltration of the ciliary body, choroids, or retina (score = 0.5). Progressively higher scores were assigned according to the numbers and severity of findings such as vasculitis, granuloma, retinal folding, and detachment and damage to the photoreceptor layer. The individual average score of both eyes was then calculated for the final individual scores.

Isolation of eye-infiltrating cells
Uveitic eyes were collected from at least three mice per group, either on day 14 or 21 after immunization. Both eyes were washed with phosphate buffer-saline (PBS) plus 2% fetal bovine serum (FBS) and were carefully mashed between two sterile nylon membranes with 2mL of PBS plus 2% FBS in a Petri dish; cells were then centrifuged. The supernatant was discarded and the cell pellet resuspended in 1mL of Lysing Buffer (BD Biosciences, San Diego, USA) for 3 minutes at 37˚C. Subsequently, 10mL of PBS plus 2% FBS was added and cells were again centrifuged. The supernatant was discarded and the cell pellet was resuspended in 1mL of PBS plus 2% FBS for posterior cell counting and immunophenotyping as described below.
For intracellular detection of IFN-γ and IL-17, dLN cells were cultured overnight with 100ng/mL of phorbol myristate acetate (PMA) and 500ng/mL of ionomycin (Sigma-Aldrich, St. Louis, USA) in the presence of Golgi Plug (brefeldin A) at the recommended concentrations (BD Biosciences, San Diego, USA). Non-stimulated cells were used as controls. PV1-treated and untreated mice cells were then harvested and labeled for anti-CD3, anti-CD4 and anti-CD8 (BioLegend Inc., San Diego, USA). Cells were then fixed and permeabilized using Cytofix/Cytoperm reagents from BD Biosciences (San Diego, USA). For intracellular staining of cytokines the following antibodies were used: anti-IFN-γ, anti-IL-17 and anti-IL-2 (BD Biosciences, San Diego, USA). Cells were acquired using a LSR Fortessa (BD Biosciences, San Diego, USA) and analyzed with FlowJo (TreeStar Inc., Ashland, USA) and Pestle (version 1.7)/ SPICE (version 5.3; M. Roederer, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health) as described elsewhere [31].

Statistical analysis
Experiments were repeated at least three times. Experimental groups were typically composed of five mice. Figures show data combined from three independent experiments, unless stated otherwise. For statistical analysis of EAU scores, each mouse (average of both eyes) was considered as one statistical event. Groups were analyzed using two-tailed Mann-Whitney tests and statistical significance was set for p<0.05. All the analyses were performed using GraphPad Prism 4.0 software (GraphPad Software Inc., La Jolla, USA).

PV1 treatment decreases EAU scores
To evaluate the effect of PV1 on the progression of EAU, mice were immunized with 161-180 IRBP peptide and, on day 9 post-immunization, were either treated with PV1 or left untreated. At this time point, disease is in the end of its afferent phase and inflammatory T lymphocytes are already found in the eyes. Mice were treated with PV1 every four days and were sacrificed on day 21 to score EAU disease by histological analysis. PV1 treatment significantly (p<0.05, two-tailed Mann-Whitney test) decreased both average EAU score (0.95±1.07) and incidence (69%) when compared with the untreated group (1.65±0.75 and 100%, respectively) ( Fig 1A). Accordingly, the lower score was characterized by a less pronounced inflammatory response with lower incidence of vasculitis, granuloma formation, and retinal folding when compared with untreated animals (Fig 1B). No apparent adverse effects, such as weight loss, lethargy or death were observed in PV1-treated and controls mice.

Diminished EAU severity is accompanied by a decrease in the activation profile of eye-infiltrating T lymphocytes
Since PV1 decreased EAU, the effects of PV1 on T cells infiltrating the eyes of B10.RIII mice were investigated. On day 14, when the disease is already established and the inflammatory infiltrate is at its peak, eyes were collected for immunophenotyping of infiltrating cells. Both untreated and PV1-treated mice showed similar number of eye-infiltrating cells (Fig 2A), and of the CD4 + and CD8+ T cell populations (Fig 2C and 2D) as well. However, it was observed a lower frequency of CD4 + T cells in the PV1-treated group ( Fig 2B). Of note, no differences between PV1-treated mice and controls were found for B lymphocyte and NK cell frequencies (S1A Fig).
As PV1 treatment did not seem to interfere with T cell migration to the eyes (Fig 2A), we next sought to investigate the activation profile (assessed by the expression of CD44 and CD62L) of eye-infiltrating T lymphocytes. PV1-treated mice showed a lower T effector/T naïve ratio for both CD4 + (Fig 2E and 2F) and CD8 + (Fig 2E and 2G) cells when compared to the controls. Additionally, CD25 and PD-1 in CD4 + and CD8 + cells, both markers of T cell activation, were decreased in PV1-treated animals (Fig 2H and 2I).
Taken together, the results indicate that the CD28 blockade achieved with PV1 dampens the activation of eye-infiltrating T cells rather than interfering with their homing to the eye.

CD28 blockade with PV1 decreases overall T cell activation in periphery
To evaluate if our findings were confined to the inflammatory environment of uveitic eyes, dLN and spleen were collected on day 14 for analysis of T lymphocyte activation profile. Again, total cell count was similar in both dLN and spleen in both groups (S2A Fig). Moreover, similar frequencies of CD4 + , CD8 + , CD19 + and NK cells were found in spleen and dLN from both untreated and PV1-treated group (S2B Fig). However, similar to findings in the eye, CD4 + T cells from PV1-treated mice exhibited lower frequencies of effector cells evaluated here as CD44 + CD62Lcells and higher frequencies of naïve cells (CD44 -CD62L + cells), in both dLN and spleen (Fig 3A and 3B). Furthermore, other activation markers, expressed at different time points of a T cell response were also altered in PV1-treated mice. The frequency of CD4 + CD69 + (Fig 3C and 3D), CD4 + CD25 + (Fig 3E and 3F), CD4 + PD-1 + (Fig 3G and 3H), and CD4 + Tim-3 + cells (Fig 3I and 3J) in PV1-treated animals was lower than in their untreated counterparts, in both spleen and dLN. Likewise, CD69, CD25 and PD-1 mean fluorescence intensity (MFI) were lower in CD4 + cells from spleen and dLN from PV1-treated mice, when compared to their untreated counterparts (S3 Fig  Therefore, we conclude that PV1 decreases overall T cell activation in the periphery with decreased expression of different costimulatory and activation molecules.

PV1 treatment decreases the T reg population in peripheral lymphoid organs
Regulatory T cells are responsible for controlling T cell activation and effector responses and have been shown to reduce the severity of several autoimmune disorders [32]. In addition to well-known mechanisms, T reg cell generation can also occur due to incomplete activation of T lymphocytes [33,34]. Consequently, it was conceivable that CD28 blockade by PV1 might increase T reg frequency and mitigate progression of autoimmunity in our uveitis model. Therefore, we evaluated Treg lymphocytes population in dLN and spleen ( Fig 4A). Surprisingly, PV1 treatment led to a decrease in both frequency and absolute numbers of T reg in both dLN (Fig 4B  and 4C) and spleen (Fig 4D and 4E). Taken together, these results showed that PV1 exert a blocking effect on T reg generation and that the decrease in disease severity seemingly occurs due to a direct effect upon the effector T lymphocytes. PV1 treatment decreases T H 1 cell population, but has no effect on T H 17 cells EAU pathogenesis is greatly dependent on T H 1 lymphocytes [8] but T H 17 cells also were shown to participate in the progression of disease [11]. As PV1 effects did not seem to be mediated by T reg cells in EAU, we next explored the effects of PV1 treatment on IFN-γ, IL-17, and IL-2 production by CD4 + T lymphocytes (Fig 5A). In PV1-treated mice CD4 + IFN-γ + T cells (Fig 5B, 5C and 5D) were significantly reduced both in number and frequency in dLN. In contrast, there were no changes in the frequencies of CD4 + IL-17 + (Fig 5B and 5C), CD4 + IL-2 + T cells (Fig 5B and 5C), or in IFN-γ-, IL-2-, and IL-17-producing T CD8+ cells (S5A, S5B and S5C Fig). However, a decrease in the numbers of CD4 + IL-17 + was also observed (Fig 5E).
These results suggest that PV1 acts mainly on the IFN-γ production by CD4+ T cells.

CD28 blockade with PV1 prevents T H 1 cell expansion after antigen reencounter
Finally, T cells primed for ocular antigens in dLN migrate to the eyes, where they exert their effector functions [8] leading to disease. The decrease in T H 1 lymphocytes population observed in PV1-treated mice could be due to a direct effect of CD28 blockade on these cells, by blocking survival signals and effector functions. With the purpose of mimicking the events following T cell priming in the periphery, in vitro assays were performed. Cells were collected on day 7 post-immunization from dLN of EAU mice, incubated with PV1 and stimulated with the 161-180 IRBP peptide (mimicking the antigen re-encounter); these data were compared to cells without incubation with PV1. CD28 blockade with PV1 led to a decrease in CD4 + IFN-γ + T cell frequency, when compared to untreated cells (Fig 6A, 6B and 6C). T H 17 cell population and IL-2 production were not affected by PV1 treatment (Fig 6A, 6B and 6C), confirming that PV1 acts on primed T lymphocytes, therefore leading to lower IFN-γ production.

Discussion
In this study we show that the selective blockade of CD28 using a monovalent Fab fragment is effective in the treatment of experimental autoimmune uveitis acting directly on effector T cells, specifically dampening IFN-γ production with no induction of anergy or enhancement of T reg cell activity. These results are in line with previous data where complete anti-CD28 antibodies were employed [19,20,22], confirming this strategy as a valid therapeutic option for autoimmune uveitis. Moreover, PV1 human homologue, FR104, was shown to be effective in preventing renal allograft rejection ( [35]), and in the treatment of skin inflammation [36,37] and experimental autoimmune encephalomyelitis [38] in non-human primates. Nonetheless, a careful evaluation is opportune, to ensure no unexpected events will arise due to unforeseen interactions by the effector Fc portions of the antibody or cross-reactivity with CTLA-4 [27,33]. Different immunotherapies promote their suppressive effects by sequestering effector lymphocytes to the peripheral lymphoid organs and preventing migration of the pathogenic cells to inflamed tissues. That is the case for FTY720 [39] and the monoclonal antibody natalizumab [40,41]. CD28 is known to control circulation of auto-reactive T lymphocytes through IL-2inducible Tec kinase (ITK) signaling [42]. However, the lack of differences between untreated and PV1-treated groups regarding the total number of eye-infiltrating and dLN cells discards this explanation. Instead, the observed decrease in the ratio of effector/naïve eye-infiltrating T cells and the lower expression of CD25 and PD-1 in PV1-treated animals raises the possibility that PV1 is hindering the activation of effector T lymphocytes. In line with these findings, the decrease in overall activation status of lymphocytes found in peripheral lymphoid organs from mice treated with PV1, showed that CD28 blockade prevented the full activation of T CD4 + and T CD8 + lymphocytes. In accordance to the two-signal theory of T cell activation, CD28 engagement has been shown to promote expression of additional costimulatory and activation molecules and inhibit the expression of co-inhibitory molecules [43]. In particular, upregulation of CD25 is a key feature in CD28 signaling through the IL-2 pathway [44]. The CD28-IL-2 axis also has an important role on the induction and activity of PD-1, as IL-2 produced after CD28 engagement is responsible for the inhibition of PD-1 expression [45]. Thus, we expected that CD28 blockade by PV1 would generate inhibitory signals mediated by upregulation of coinhibitory molecules [33]. Our results, however, showed otherwise. Both costimulatory and coinhibitory molecules were downregulated in PV1-treated mice. As CD28 is constitutively expressed on T cells and one of the first costimulatory signals triggered upon T cell activation, blocking this pathway would stop the ensuing expression of surface molecules such as CD69, CD25, and PD-1, which would, in turn, direct immune responses to an arrested pattern.
Arrested T cell activity is achieved by several mechanisms, a major one being by way of regulatory T lymphocytes and another one by IL-2 deprivation [46], which dampens proliferation and survival of T lymphocytes causing anergy. Selective CD28 blockade mitigates EAU T reg lymphocytes are generated in the periphery in order to build and maintain peripheral tolerance and to control responses to external antigens [32]. Accordingly, incomplete activation of T cells, with lack of costimulatory signals is thought to result in either anergy or T reg generation [34]. In fact, the monovalent anti-CD28 antibody FR104 was shown to enhance T reg function [33] leading us to hypothesize that CD28 blockade with PV1 antibody would induce T reg lymphocytes, blocking T cell activation and decreasing EAU severity. Surprisingly, PV1-treated mice exhibited lower frequencies and absolute numbers of T reg in peripheral lymphoid organs, indicating a detrimental effect of PV1 on this T lymphocyte subpopulation. Although unexpected, these results are supported by previous findings that suggest an important role for CD28 in maintaining regulatory T cell homeostasis [47][48][49]. Still, these data need to be further explored, as this apparent T reg depletion could be actually the result of T reg migration to uveitic eyes and/or the enhancement of T reg activity, as previously observed in a kidney transplantation model using the CD28 selective blockade strategy [33].
The blockade of CD28 signaling pathway can also induce anergy in T lymphocytes, which could be, in turn, responsible for the immune suppression observed in PV1-treated mice. In experimental models of heart transplantion and autoimmune encephalomyelitis use of PV1-IgG3 or mAb-PV1 resulted in a decrease in IL-2 production of treated animals [19,20] suggesting anergy is one of the mechanisms of action of this monovalent antibody. However, in the present study no differences between PV1-treated and untreated groups were found regarding IL-2 production, either after specific or unspecific stimulatory conditions. The decrease of EAU severity observed in PV1-treated mice apparently is T reg independent and is not achieved through anergy induction. Thus, the decrease of IFN-γ-or IL-17-producing cells and disruption of T helper lymphocyte effector functions could explain the effect of PV1 on EAU pathogenesis [50][51][52][53][54]. Costimulatory molecules can induce different cytokines leading to differential polarization of T cells [55,56] and blockade of B7.1 and B7.2 results in decreased IFN-γ production and EAU severity [22]. In addition, in a heart transplant model, PV1-IgG3 treatment led to a reduction of IFN-γ mRNA levels, whilst enhancing graft survival [19]. Moreover, Poirier and colleagues [36] observed that FR104 pre-treated leukocytes produced less IFN-γ after antigen restimulation. These findings are in line with our results, as the observed dampening on IFN-γ production and decreased frequencies of T H 1 cells explains the effectiveness of PV1 on mitigating EAU, whilst the remainder of IL-17-producing T lymphocytes would explain the residual disease found in some mice. Although we cannot exclude that PV1 acts also in the reactivation of T H 17 cells, our in vitro findings, using specific antigen restimulation of dLN cells pre-treated with PV1, point to a T H 1 directed mechanism, as we did not find differences in the T H 17 populations after restimulation. Thus, the decrease in T H 17 cell numbers observed in vivo could be due to the overall suppression observed in CD4 + T lymphocytes. Also, it is important to note that Ville and colleagues [35] also found no differences in T H 17 cells after FR104 treatment in a renal allograft model. As the time window for PV1 treatment is less than 2 weeks in the present study, and usually in the EAU mouse model it takes at least 6 weeks to the lesions decrease in number and severity [13] it seems unlikely that PV1 blockade acts promoting the recovery of the ocular lesions. Altogether, our data suggests that PV1 blockade acts by suppressing effector T cell responses in general, but mainly in antigen-specific T H 1 lymphocytes rendering these cells unable to exert their effector functions upon antigen re-encounter in the target organ, similar to what was found after FR104 treatment in non-human primate models of skin inflammation [36] and autoimmunity [38].

Conclusion
Here we show that specific CD28 blockade, is a promising strategy for treating autoimmune uveitis, and that PV1 is a useful tool for dissecting the cellular events involved in this phenomenon. In an EAU mice model, PV1 suppressive effects were directed towards T H 1 lymphocytes. Improvement of the disease occurred without the desired enhancement of regulatory T cell function raising interesting issues on the roles of costimulatory molecules in the presence of a targeted CD28 blockade. In particular, the understanding of the effects of CD28 signaling on T reg is crucial, as most of modern immunomodulatory strategies aim to regulate this T lymphocyte subpopulation activity. Moreover, knowing the precise mechanisms of action of mPEG PV1-Fab' would help to improve future advances in this field, and specific and alternative treatments for autoimmune uveitis. OSE Immunotherapeutics provided support in the form of salaries for author B.V., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The other authors have no financial conflicts of interest.