CD28 between tolerance and autoimmunity: the side effects of animal models

Introduction

Shifting the balance toward restoration of immune tolerance could represent an important goal of the ongoing research in autoimmunity. Promising therapeutic strategies would be aimed to concomitantly dampen pathogenic inflammatory T-cell responses and induce/expand suppressive regulatory T (Treg) cells. Since its discovery in 1980^1,2 and based on the high homology between rodent (mouse and rat) and human CD28^3,4, several in vivo animal models have been generated for understanding the role of CD28 in T-lymphocyte activation and differentiation. CD28 is constitutively expressed on both naïve and activated T cells. By binding its ligands B7.1/CD80 or B7.2/CD86 on the surface of professional antigen-presenting cells (APCs), through a MYPPPY motif within its extracellular immunoglobulin (Ig)-V-like domain^5,6 and to B7-H2 through a region outside the MYPPPY motif⁷, CD28 delivers signals, which lower the T-cell receptor (TCR) activation threshold, thus leading to optimal cytokine production, cell cycle progression, and survival⁸. Furthermore, in the human system, CD28 is able to emanate TCR-independent autonomous signals, which account for its critical role in regulating pro-inflammatory cytokine/chemokine production and T-cell survival^9,10. Finally, pre-clinical mouse models also showed a paradoxical function of CD28 in the development and homeostasis of CD4⁺CD25⁺ Treg cells^11–13. Treg cells are negative regulators of T-cell signaling and contribute to T-cell anergy and to the maintenance of self-tolerance by suppressing autoreactive T cells¹⁴. Therefore, CD28 can either reduce or enhance the susceptibility to autoimmune diseases by altering T-cell effector and Treg cell compartments. However, the translation of knowledge from pre-clinical mouse models led to the development of CD28 signaling modulators that often failed when applied in clinical trials. Here, we discuss CD28 regulatory functions in mouse models of autoimmune diseases by showing the success and failure of pre-clinical studies when translated to humans.

CD28 role in autoimmune diseases: from animal models to human clinical trials

Owing to the high conservation between Mus musculus and Homo sapiens, mice represent the favorite experimental models used by immunologists. Most of the data on the pivotal role of CD28 in regulating tolerance and susceptibility to autoimmunity derive from mouse models, which have been extensively used for clarifying the pathogenic mechanisms of several autoimmune diseases as well as for identifying molecular targets to translate in clinical trials. These efforts led to the development of soluble CTLA-4-binding domain linked to the Fc region of Ig (CTLA-4Ig) able to efficiently bind B7 molecules (with a 20-fold higher affinity compared with the CD28Ig) and to block CD28/B7 interaction through the removal of its ligands from APC, a process known as trans-endocytosis, and by directly interfering with T/APC interaction¹⁵. The success obtained in animal models led to many pre-clinical and clinical trials in order to assess the potential use of CTLA-4Ig to ameliorate the onset, progression, and clinical course of human autoimmune diseases¹³. However, despite the positive results gained in non-human organisms, data from CTLA-4Ig clinical trials showed discrepant results that, for brevity, are discussed below for two major autoimmune diseases: rheumatoid arthritis (RA) and multiple sclerosis (MS).

CD28 in the regulation of tolerance: spotlight on regulatory T cell functions

Despite the pivotal role of CD28 in favoring the proliferation, differentiation, and functions of conventional T cells, increasing evidence accumulated during the last two decades highlighted a critical function of CD28 in promoting the homeostasis and suppressor function of Treg cells. Depending on the context, CD28 can deliver either pro-inflammatory or anti-inflammatory signals. Indeed, CD28 is required both for efficient generation of Treg cells in the thymus and for Treg cell peripheral homeostasis, as shown by the initial demonstration that mice deficient in CD28 or CD80/CD86 exhibit a strong reduction of thymic Treg cells and develop diabetes in a non-obese diabetic (NOD) background³³. More recent data on conditional deletion of CD28 in FOXP3⁺ Treg cells showed a 25–30% decrease of thymic Treg cells, whereas the percentage of Treg cells in lymph nodes and spleens was unaffected, thus indicating that CD28 influences the cell number and turnover of thymic, but not peripheral, Treg cells³⁴. However, all mice developed signs of systemic autoimmunity, such as lymphadenopathy and splenomegaly, that could be prevented by supplementation with CD28-sufficient Treg cells. Moreover, they showed accumulation of activated T cells in the skin and liver and failed to suppress induced colitis and EAE³⁴. A more detailed characterization of the skin disease in these animals revealed that, in the absence of CD28, Treg cells failed to mature and differentiate from a quiescent/central to an effector phenotype that is characterized by the downregulation of the CCR7 chemokine receptor (lymphoid retention) and by the expression of the chemokine receptors required for skin homing, such as CCR6³⁵. More recently, the same group showed that CD28 was also essential for the numbers and function of follicular Treg cells, whose loss in a CD28-deficient mouse caused increased germinal center B cells and Ab production³⁶. Similarly, Franckaert et al. found that CD28 deficiency in Treg cells caused a severe autoimmune syndrome as a result of impaired Treg cell proliferation and functions³⁷. These data strongly suggest a role for CD28 in maintaining the homeostasis of both thymic and peripheral Treg cells and in sustaining their suppressive functions necessary to maintain immune tolerance in vivo. However, other studies displayed discordant results. Vahl et al. showed that the differentiation and maintenance of effector Treg cells as well as their suppressive functions were severely compromised by TCR ablation in mature Treg cells³⁸. Similar results were obtained by Levine et al.³⁹, thus suggesting a critical role for continuous TCR signals in maintaining the suppressive function of Treg cells in vivo. Data from Dilek et al. showed that, in human Treg cells, blockade of CD28 interaction with CD80 or CD86 prolongs Treg cell/APC contacts and calcium mobilization without affecting cell motility⁴⁰. In contrast, in a mouse model, CD28 interaction with CD80 is critical for stopping motility and forming symmetrical immunological synapse in the presence of antigen⁴¹. Finally, recent data from Kishore et al. showed a crucial role for CD28 signals in inducing the migration of Treg cells and for their redistribution from lymphoid tissues⁴². This scenario was further complicated following the discovery, by the Hünig research group, of a class of CD28 superagonistic Abs (CD28SAs).

In rodent models, Hünig et al. found that CD28SAbs, by binding the laterally exposed C”D loop of the Ig-like domain of CD28 in a parallel manner, were able to expand Treg cells without any pro-inflammatory responses^43,44. The same group showed that in vivo treatment of EAE mice with CD28SAbs protected them from the disease⁴⁵. This discovery led to a plethora of pre-clinical experiments in mouse models of RA, MS, Guillain–Barré syndrome, and type 1 diabetes (T1D) in order to evaluate the potential use of these CD28SAbs to ameliorate the clinical course of human autoimmune diseases^46,47. The promising results obtained from these experimental models led to the generation of a fully humanized CD28SAb, named TGN1412, which, in March 2006, was injected in six healthy young men. Surprisingly, the phase I clinical trial turned into a catastrophe because all volunteers experienced a rapid and massive cytokine release syndrome⁴⁸. The ability of human CD28 stimulation to expand Treg cells has been supported by data showing that agonistic Abs⁴⁹ and the natural ligands B7.1/CD80 and B7.2/CD86, in the presence of recombinant human IL-2, mediate ex vivo expansion of human Treg cells⁵⁰. Other studies showed that human CD28 stimulation by either natural ligands or agonistic or superagonistic Abs induced a strong increase in pro-inflammatory cytokine production in CD4⁺ T lymphocytes from either healthy donors or patients with RR MS or T1D^9,10. Such a pro-inflammatory signature of human CD28 should be taken into account when stimulating T cells in vivo, as re-empathized by TGN1412 administration to a humanized mouse model in which it induced strong lymphopenia, pro-inflammatory cytokine production, and death within 2–6 hours⁵¹. Thus, although more recent data from Tabares et al. showed that low doses of CD28SAbs increased the number of activated Treg cells without affecting pro-inflammatory cytokine production⁵² and no detectable inflammatory cytokines were found in the plasma of healthy volunteers in a new phase I trial⁵³, CD28SAbs must be used with great caution. For instance, two recent studies showed that effector T cells from patients with MS may also acquire resistance to Treg cell suppressive mechanisms in an IL-6 receptor-dependent manner^54,55 and CD28 stimulation of peripheral CD4⁺ T cells from patients with RR MS strongly upregulates IL-6 production⁹. Moreover, the non-physiologic activation by CD28SAbs fails to induce PD-1 on the cell surface, thus leading to the loss of a crucial negative feedback conferred by the PD-1/PD-L1 interaction⁵⁶ that represents a key checkpoint of immune response by effecting its negative regulation mainly on CD28⁵⁷.

Conclusions

All these data suggest that, despite many similarities, a divergent evolution of about 65 million years may have generated significant differences between humans and mice that, if not taken into account, could determine new “errors in translation”. CD28 has a pivotal role in the orchestration of the immune response that makes it a precious target for the treatment of immune-based diseases, but caution is needed to translate experimental results from mice to humans because differences in CD28 functions and signaling capability might determine dramatic effects. For instance, our recent identification of a single amino acid variant within the cytoplasmic tail of human and rodent CD28 (P²¹² in human versus A²¹⁰ in rodent) as a critical residue for human CD28 pro-inflammatory and signaling functions⁵⁸ raises the question of whether or not rodents can be used as a model for the study of CD28-mediated functions and for the safety of new therapeutic approaches. Thus, new efforts to develop better in vivo and in vitro systems are required to take advantage of the great potential retained in the co-stimulatory pathway and to provide novel insights into CD28 biology and implications for therapies.