[Anti-inflammatory Strategies by Focusing on the Particularity of Ocular Immunity].

Particularity of ocular immunity is manifested by "Immune privilege". For example, it has been generally known that corneal transplantation is a typically successful organ transplantation compared with other organs. This immune privilege can be explained by "immune-suppressive ocular microenvironment" and "anterior chamber-associated immune deviation, ACAID". This review focused on molecular mechanisms of the "immune-suppressive ocular microenvironment" and "ACAID", so that possible anti-inflammatory strategies could be raised. Especially, in murine ACAID model, anti-inflammatory actions were induced probably through induction of Treg cells. As an anti-inflammatory strategy, anti-inflammatory Treg cells could be induced in vitro. Treg cells that are specifically responsive for a specific antigen can be induced by culturing spleen cells with the antigen and transforming growth factor-ß (TGF-ß). The induced Treg cells were activated by stimulation with the specific antigen. When the induced Treg cells were adoptively transferred to recipient mice, antigen-induced inflammation was effectively suppressed. The Treg cells may be able to be efficiently induced by eye-based mechanisms. Further analyses of mechanisms underlying the ocular immune privilege can be useful for development of new anti-inflammatory strategies on the eye basis.

Investigation of Cytotoxic T Lymphocyte Function during Allorejection in the Anterior Chamber of the Eye.

Cytotoxic T lymphocytes (CTL) are an essential part of our immune system by killing infected and malignant cells. To fully understand this process, it is necessary to study CTL function in the physiological setting of a living organism to account for their interplay with other immune cells like CD4+ T helper cells and macrophages. The anterior chamber of the eye (ACE), originally developed for diabetes research, is ideally suited for non-invasive and longitudinal in vivo imaging. We take advantage of the ACE window to observe immune responses, particularly allorejection of islets of Langerhans cells by CTLs. We follow the onset of the rejection after vascularization on islets until the end of the rejection process for about a month by repetitive two-photon microscopy. We find that CTLs show reduced migration on allogeneic islets in vivo compared to in vitro data, indicating CTL activation. Interestingly, the temporal infiltration pattern of T cells during rejection is precisely regulated, showing enrichment of CD4+ T helper cells on the islets before arrival of CD8+ CTLs. The adaptation of the ACE to immune responses enables the examination of the mechanism and regulation of CTL-mediated killing in vivo and to further investigate the killing in gene-deficient mice that resemble severe human immune diseases.

Immune Checkpoints Contribute Corneal Immune Privilege: Implications for Dry Eye Associated with Checkpoint Inhibitors.

The eye is provided with immune protection against pathogens in a manner that greatly reduces the threat of inflammation-induced vision loss. Immune-mediated inflammation and allograft rejection are greatly reduced in the eye, a phenomenon called 'immune privilege'. Corneal tissue has inherent immune privilege properties with underlying three mechanisms: (1) anatomical, cellular, and molecular barriers in the cornea; (2) an immunosuppressive microenvironment; and (3) tolerance related to regulatory T cells and anterior chamber-associated immune deviation. This review describes the molecular mechanisms of the immunosuppressive microenvironment and regulatory T cells in the cornea that have been elucidated from animal models of ocular inflammation, especially those involving corneal transplantation, it also provides an update on immune checkpoint molecules in corneal and systemic immune regulation, and its relevance for dry eye associated with checkpoint inhibitor therapy.

Corneal Nerve Ablation Abolishes Ocular Immune Privilege by Downregulating CD103 on T Regulatory Cells.

Purpose: Severing corneal nerves during orthotopic corneal transplantation elicits the elaboration of the neuropeptide substance P (SP), which induces the generation of CD11c+ contrasuppressor (CS) cells. CS cells disable T regulatory cells (Tregs) that are induced when antigens enter the anterior chamber (AC), either by direct injection or by orthotopic corneal transplantation. This study examined the crucial cell surface molecules on Tregs that are adversely affected by CS cells that are generated by severing corneal nerves. Methods: CS cells were induced by producing shallow 2.0-mm circular incisions in the corneal epithelium in BALB/c mice. CD8+ Tregs were generated by injecting ovalbumin into the AC. The effects of CS cells and SP on the expression and function of two cell surface molecules (CD103 and the receptor of interferon-γ) that are crucial for the induction and function of CD8+ Tregs were analyzed. Results: SP converted CD11c+, but not CD11c- , dendritic cells (DCs) to CS cells. Severing corneal nerves resulted in a 66% reduction in the expression of CD103 on CD8+ AC-associated immune deviation (ACAID) Tregs, and a 50% reduction in the interferon-γ receptor (IFN-γR). These effects could be mimicked in vitro by coculturing CS cells with CD8+ ACAID Tregs. Conclusions: The elaboration of SP in response to corneal nerve ablation converts CD11c+ DCs to CS cells. CS cells disable CD8+ ACAID Tregs by downregulating two crucial cell surface molecules, CD103 and IFN-γR, by an SP-dependent pathway. Blocking this pathway may provide a means of restoring ocular immune privilege in corneas subjected to corneal nerve injury.

Adipose-derived stem cells integrate into trabecular meshwork with glaucoma treatment potential.

The trabecular meshwork (TM) is an ocular tissue that maintains intraocular pressure (IOP) within a physiologic range. Glaucoma patients have reduced TM cellularity and, frequently, elevated IOP. To establish a stem cell-based approach to restoring TM function and normalizing IOP, human adipose-derived stem cells (ADSCs) were induced to differentiate to TM cells in vitro. These ADSC-TM cells displayed a TM cell-like genotypic profile, became phagocytic, and responded to dexamethasone stimulation, characteristic of TM cells. After transplantation into naive mouse eyes, ADSCs and ADSC-TM cells integrated into the TM tissue, expressed TM cell markers, and maintained normal IOP, outflow facility, and extracellular matrix. Cell migration and affinity results indicated that the chemokine pair CXCR4/SDF1 may play an important role in ADSC-TM cell homing. Our study demonstrates the possibility of applying autologous or allogeneic ADSCs and ADSC-TM cells as a potential treatment to restore TM structure and function in glaucoma.

What Makes Cornea Immunologically Unique and Privileged? Mechanistic Clues from a High-Resolution Proteomic Landscape of the Human Cornea.

Success rates of corneal transplantation are particularly high owing to its unique, innate immune privilege derived from a phenomenon known as Anterior Chamber-Associated Immune Deviation (ACAID). Of note, cornea is a transparent, avascular structure that acts as a barrier along with sclera to protect the eye and contributes to optical power. Molecular and systems biology mechanisms underlying ACAID and the immunologically unique and privileged status of cornea are not well known. We report here a global unbiased proteomic profiling of the human cornea and the identification of 4824 proteins, the largest catalog of human corneal proteins identified to date. Moreover, signaling pathway analysis revealed enrichment of spliceosome, phagosome, lysosome, and focal adhesion pathways, thereby demonstrating the protective functions of corneal proteins. We observed an enrichment of neutrophil-mediated immune response processes in the cornea as well as proteins belonging to the complement and ER-Phagosome pathways that are suggestive of active immune and inflammatory surveillance response. This study provides a key expression map of the corneal proteome repertoire that should enable future translational medicine studies on the pathological conditions of the cornea and the mechanisms by which cornea immunology are governed. Molecular mechanisms of corneal immune privilege have broad relevance to understand and anticipate graft rejection in the field of organ transplantation.

Post-Transplant Immune Tolerance in Rats Following Lymphocyte Injection Into the Anterior Chamber of the Eye.

OBJECTIVE: We aimed to compare the clinical and histopathological results of skin graft transplants between rats that had been injected with lymphocytes into the anterior chamber of the eye with those that had not. METHODS: A total of 16 Wistar albino, male rats were included in the study. Subjects were divided into 2 groups, namely a test group and a control group. Lymphocyte suspensions derived from the subjects of the control group were injected into the anterior chamber of the eye of each opposing subject of the test group. Also, an identical volume of physiological saline was injected into the anterior chamber of each subject in the control group to prevent bias. One week after this procedure, circular skin grafts of 1 cm in diameter were transplanted within the opposing groups. After a period of 1 week, transplanted graft tissues were excised to compare tissue healing. RESULTS: The occurence of granulation and reepithelialization was more evident in the test group (96% and 33%, respectively, vs 80% and 17% for the control group, respectively). On the other hand, it was determined that acute inflammation was more intense in the control group (77% vs 50% for the test group). CONCLUSION: We had created immune tolerance in rats through anterior chamber lymphocyte injection, which slowed down the rejection process. If this can be successfully implemented in practice, survival for transplant patients without long-term rejection will move closer to becoming a reality.

The efficacy of systemic and intravitreal infliximab treatments in an endotoxin-induced uveitis model.

Purpose: To compare the efficacy of systemic and intravitreal infliximab treatments in an experimental endotoxin-induced uveitis (EIU) model. Methods: Twenty-eight white New Zealand rabbits were equally divided into 4 groups. Group 1 received an intravitreal injection of 0.1 cc saline, group 2 received an intravitreal injection of 2 µg/0.1 cc lipopolysaccharide (LPS), group 3 received an intravitreal injection of 2 µg/0.1 cc LPS and 2 mg/0.1 cc infliximab, and group 4 received intravitreal injection of 2 µg/0.1 cc LPS and intravenous injection of 5 mg/kg infliximab. Clinical, biochemical (aqueous and vitreous humour protein levels and TNF-α concentrations), and histopathological evaluations were performed. Results: The clinical examination score was lower in group 4 than in group 2 (p = 0.006); but there was no significant difference between groups 2 and 3 (Bonferroni correction, p = 0.016). No statistically significant difference was found among groups 2, 3, and 4 for aqueous humour protein levels (p > 0.05). Significantly higher aqueous humour concentrations of TNF-α was measured in group 3 comparing to both group 1 and 4 (p = 0.003 and p = 0.002, respectively). No significant difference was found in vitreous protein levels or TNF-α concentrations among all study groups (Bonferroni correction, p = 0.026 and p = 0.101, respectively). Histopathological evaluation of the uveal tissue and anterior chamber reaction revealed the highest inflammation in group 3 (p < 0.001). In group 4, histopathological evaluation of uveal tissue was lower than in groups 2 and 3 (p < 0.001 and p = 0.001, respectively); whereas there was no difference in anterior chamber inflammation between groups 2 and 4 (p = 1.00). Conclusion: Intravitreal 2 mg/0.1 cc infliximab injection exacerbated inflammation in an EIU model; whereas systemic infliximab treatment at a dose of 5 mg/kg suppressed inflammation effectively and rapidly.

Anterior chamber associated immune deviation to cytosolic neural antigens avoids self-reactivity after optic nerve injury and polarizes the retinal environment to an anti-inflammatory profile.

It has been hypothesized that anterior chamber-associated immune deviation (ACAID) to neural antigens induced prior to central nervous system injury can inhibit self-reactivity and lessen secondary degeneration. This work evaluated the effect of ACAID induced to three neural tissue-derived extracts (whole extract, cytosolic extract, CE; or organelle-membrane extract) prior to optic nerve injury on retinal ganglion cell (RGC) survival. The results show that only ACAID to the CE increased RGC survival at 7 and14 days post-injury (dpi). This effect was achieved by retinal polarization towards an anti-inflammatory profile, driven by regulatory T cells and M2-type macrophages at 7 dpi.

Local release of rapamycin by microparticles delays islet rejection within the anterior chamber of the eye.

The anterior chamber of the eye (ACE) has emerged as a promising clinical islet transplantation site because of its multiple advantages over the conventional intra-hepatic portal site. This includes reduced surgical invasiveness and increased islet graft survival rate. It also allows for enhanced accessibility and monitoring of the islets. Although the ACE is initially an immuno-privileged site, this privilege is disrupted once the islet grafts are re-vascularized. Given that the ACE is a confined space, achieving graft immune tolerance through local immunosuppressive drug delivery is therefore feasible. Here, we show that islet rejection in the ACE of mice can be significantly suppressed through local delivery of rapamycin by carefully designed sustained-release microparticles. In this 30-day study, allogeneic islet grafts with blank microparticles were completely rejected 18 days post-transplantation into mice. Importantly, allogeneic islet grafts co-injected with rapamycin releasing microparticles into a different eye of the same recipient were preserved much longer, with some grafts surviving for more than 30 days. Hence, islet allograft survival was enhanced by a localized and prolonged delivery of an immunosuppressive drug. We envisage that this procedure will relieve diabetic transplant recipients from harsh systemic immune suppression, while achieving improved glycemic control and reduced insulin dependence.

CD4- and CD8-expressing cells found in the bovine and porcine anterior chamber of the eye.

The aim of the present study was to investigate whether the anterior chamber constitutes part of the normal migratory pathway of CD4+ and CD8+ lymphocytes in cattle and swine. The cells obtained from aqueous humor of cows and pigs were stained for CD4 and CD8 receptors, and subsequently analyzed with flow cytometry. The mean percentage of CD4+CD8-, CD4-CD8+ and CD4+CD8+ cells within the total lymphocyte population of the bovine anterior chamber was, respectively, 17.88, 12.64 and 27.26%. In turn, the mean values of these parameters in pigs were 1.77, 38.48 and 17.45, respectively. Among bovine and porcine CD4+CD8+ cells prevalent were those displaying CD4lowCD8low and CD4lowCD8high phenotypes, respectively. The results suggest that the anterior chamber in cattle and swine is an element in the normal migratory pathway of CD4+, CD8+ and CD4+CD8+ cells. Furthermore, the contribution of these subsets in the anterior chamber lymphocyte population can differ considerably between animal species.

Induction of Contrasuppressor Cells and Loss of Immune Privilege Produced by Corneal Nerve Ablation.

Purpose: Severing of corneal nerves in preparation of corneal transplantation abolishes immune privilege of subsequent corneal transplants placed into either eye: a phenomenon termed sympathetic loss of immune privilege (SLIP). SLIP is due to the disabling of T regulatory cells (Tregs) by CD11c+ contrasuppressor (CS) cells. This study characterized the induction, function, and manipulation of CS cell activity and the effect of these cells on Tregs induced by anterior chamber-associated immune deviation (ACAID). Methods: CS cells were induced using a 2.0-mm trephine to score the corneal epithelium. CD11c+ CS cells were evaluated by adoptive transfer and by their capacity to disable CD8+ ACAID Tregs in local adoptive transfer (LAT) of suppression assays. CD11c+ cells were deleted from the ocular surface by subconjunctival injection of clodronate-containing liposomes. Results: CD11c+ CS cell were radiosenstive and long lived. As few as 1000 CS cells blocked the suppressive activity of previously generated CD8+ ACAID Tregs, indicating that CS cells act at the efferent arm of the immune response. Depletion of resident CD11c+ cells at the ocular surface prevented the generation of CS cells. Conclusions: Corneal nerve injury that occurs during keratoplasty converts ocular surface CD11c+ cells into CS cells that block CD8+ Tregs, which are induced by introducing antigens into the anterior chamber (i.e., ACAID Tregs). Depletion of CD11c+ cells at the ocular surface prevents the generation of CS cells and may be a useful strategy for preventing SLIP and enhancing the survival of second corneal transplants.

Anterior chamber associated immune deviation used as a neuroprotective strategy in rats with spinal cord injury.

The inflammatory response is probably one of the main destructive events occurring after spinal cord injury (SCI). Its progression depends mostly on the autoimmune response developed against neural constituents. Therefore, modulation or inhibition of this self-reactive reaction could help to reduce tissue destruction. Anterior chamber associated immune deviation (ACAID) is a phenomenon that induces immune-tolerance to antigens injected into the eye´s anterior chamber, provoking the reduction of such immune response. In the light of this notion, induction of ACAID to neural constituents could be used as a potential prophylactic therapy to promote neuroprotection. In order to evaluate this approach, three experiments were performed. In the first one, the capability to induce ACAID of the spinal cord extract (SCE) and the myelin basic protein (MBP) was evaluated. Using the delayed type hypersensibility assay (DTH) we demonstrated that both, SCE and MBP were capable of inducing ACAID. In the second experiment we evaluated the effect of SCE-induced ACAID on neurological and morphological recovery after SCI. In the results, there was a significant improvement of motor recovery, nociceptive hypersensitivity and motoneuron survival in rats with SCE-induced ACAID. Moreover, ACAID also up-regulated the expression of genes encoding for anti-inflammatory cytokines and FoxP3 but down-regulated those for pro-inflamatory cytokines. Finally, in the third experiment, the effect of a more simple and practical strategy was evaluated: MBP-induced ACAID, we also found significant neurological and morphological outcomes. In the present study we demonstrate that the induction of ACAID against neural antigens in rats, promotes neuroprotection after SCI.

Effect of Corneal Nerve Ablation on Immune Tolerance Induced by Corneal Allografts, Oral Immunization, or Anterior Chamber Injection of Antigens.

Purpose: Severing corneal nerves during corneal transplantation does not affect first corneal transplants, but abolishes immune privilege of subsequent corneal allografts. This abrogation of immune privilege is attributable to the disabling of T regulatory cells (T regs) induced by corneal transplantation. The goal of this study was to determine if severing corneal nerves induces the development of contrasuppressor (CS) cells, which disable T regs that impair other forms of immune tolerance. Methods: Effect of corneal nerve ablation on immune tolerance was assessed in four forms of immune tolerance: anterior chamber-associated immune deviation (ACAID); oral tolerance; corneal transplantation, and intravenously (IV) induced immune tolerance. T regulatory cell activity was assessed by adoptive transfer and by local adoptive transfer (LAT) of suppression assays. Results: Corneal nerve ablation prevented ACAID and oral tolerance, but did not affect IV-induced immune tolerance. Contrasuppressor cells blocked the action of T regs that were generated by anterior chamber injection, oral tolerance, or orthotopic corneal transplantation. The neuropeptide substance P (SP) was crucial for contrasuppressor activity as CS cells could not be induced in SP-/- mice and the SP receptor inhibitor, Spantide II, prevented the expression of CS cell activity in vivo. Contrasuppressor cells expressed CD11c surface marker that identifies dendritic cells (DC). Conclusions: The loss of immune privilege produced by corneal nerve ablation following corneal transplantation extends beyond the eye and also affects immune tolerance induced through mucosal surfaces and appears to be mediated by a novel cell population of CD11c+ CS cells that disables T regs.

Allogeneic Sensitization and Tolerance Induction After Corneal Endothelial Cell Transplantation in Mice.

PURPOSE: We evaluated the allogeneic response after corneal endothelial cell transplantation in the anterior chamber (AC) in a new mouse model by examining the acquisition of a delayed-type hypersensitivity (DTH) response, induction of allogeneic AC-associated immune deviation (ACAID), and acquisition of delayed transplantation tolerance. METHOD: The corneal eyecups from C57BL/6 mice were prepared. The epithelial layer was detached with EDTA solution and treated with trypsin to release mouse-derived primary corneal endothelial cells (mpCECs). The mpCECs (1 × 104 cells) were transplanted into the AC of the eye or subcutaneously (SC) into the neck of BALB/c mice. In the mouse model of endothelial cell transplantation, the endothelial cells in a 2-mm central area of the cornea were eliminated by cryoinjury. The mpCEC transplant model was evaluated by measuring allogeneic cell survival and corneal thickness. The allospecific DTH response and ACAID induction were evaluated 1 week after transplantation. The long-term transplantation tolerance was evaluated by observing a secondary penetrating keratoplasty (PKP) performed on the same donor C57BL/6 mice. RESULTS: The SC injection of mpCECs induced a DTH response, whereas the AC injection induced ACAID. However, eyes inflamed by cryoinjury showed neither the DTH response nor ACAID following AC injection. The mpCECs survived for at least 1 week after injection. Penetrating keratoplasty allografts at 8 weeks after mpCEC transplantation survived indefinitely (100%). CONCLUSIONS: The mpCECs display low allogenicity in the AC and are capable of inducing allogeneic tolerance. Corneal endothelial cell transplantation into the AC may represent a safe technique for allogeneic transplantation.

Natural Killer T Cells Contribute to Neutrophil Recruitment and Ocular Tissue Damage in a Model of Intraocular Tumor Rejection.

PURPOSE: Immune privilege of the eye protects the nonregenerative ocular tissues from innate and adaptive immune-mediated inflammation. In the case of intraocular tumors, immune privilege can be arrested to allow for immune-mediated rejection. Activation of innate immune cells can contribute to necrosis of the intraocular tumor and bystander ocular tissue. Identifying the cellular components of the innate immune system that contribute to ocular destruction, but are not needed for tumor rejection, provides insights into the immunopathological sequelae in intraocular tumor rejection. METHODS: Wild-type (WT), Jα18 knockout (KO) mice lacking type I natural killer T (NKT) cells, and CD1d KO mice lacking all NKT cells, were used to identify the role of type II NKT cells in intraocular tumor rejection immunopathology. RESULTS: CD1d KO mice had significantly lowered rates of necrotic eye destruction during tumor rejection compared to WT or Jα18 KO mice. Transcriptome and protein analyses revealed that CD1d KO mice had significantly lower expression of CXCL3 compared to WT or Jα18 KO mice, and this was associated with decreased neutrophil recruitment. The presence of type II NKT cells in WT or Jα18 KO mice led to increased CXCL3, which attracted neutrophils to the intraocular tumor and culminated in destruction of the eye. CONCLUSIONS: We found that type II NKT cells are critical in initiating a damaging inflammatory antitumor response involving the recruitment of neutrophils that compromises the integrity of the eye. Loss of type II NKT cells or depleting neutrophils allows for a productive intraocular tumor response that converts the rejection phenotype to preserve the eye.

ACAID as a potential therapeutic approach to modulate inflammation in neurodegenerative diseases.

The progressive loss of neurons and inflammation characterizes neurodegenerative diseases. Although the etiology, progression and outcome of different neurodegenerative diseases are varied, they share chronic inflammation maintained largely by central nervous system (CNS)-derived antigens recognized by T cells. Inflammation can be beneficial by recruiting immune cells to kill pathogens or to clear cell debris resulting from the primary insult. However, chronic inflammation exacerbates and perpetuates tissue damage. An increasing number of therapies that attempt to modulate neuroinflammation have been developed. However, so far none has succeeded in decreasing the secondary damage associated with chronic inflammation. A potential strategy to modulate the immune system is related to the induction of tolerance to CNS antigens. In this line, it is our hypothesis that this could be accomplished by using anterior chamber associated immune deviation (ACAID) as a strategy. Thus, we review current knowledge regarding some neurodegenerative diseases and the associated immune response that causes inflammation. In addition, we discuss further our hypothesis of the possible usefulness of ACAID as a therapeutic strategy to ameliorate damage to the CNS.

Corneal Immunosuppressive Mechanisms, Anterior Chamber-Associated Immune Deviation (ACAID) and Their Role in Allograft Rejection.

Corneal transplantation is the most frequently performed transplant procedure in humans. Human leukocyte antigen matching, while imperative for other types of organ transplants, is usually not performed before cornea transplantation. With the use of topical steroid immunosuppressants, which are subsequently tailed off to almost zero, most corneal transplants will not be rejected in recipients with low risk of graft rejection. This phenomenon has been described as immune privilege by Medawar many years ago. However, this immune privilege is relative and can be easily eroded, e.g. by postoperative nonspecific inflammation or other causes of corneal or ocular inflammation. Interestingly, corneas that are at high risk of rejection have a higher failure rate than other organs. Considerable progress has been made in recent years to provide a better understanding of corneal immune privilege. This chapter will review current knowledge on ocular immunosuppressive mechanisms including anterior chamber-associated immune deviation and discuss their role(s) in corneal allograft rejection. Ultimately, this evolving information will be of benefit in developing therapeutic strategies to prevent corneal transplant rejection.

Immune tolerance elicited via unique ocular and oral routes.

Immune tolerance can be induced by numerous methods. This review article aims to draw lines of similarity and contrast between two unique models of immune tolerance, namely Anterior Chamber Associated Immune Deviation (ACAID) and Nickel-induced oral tolerance. ACAID is an immune tolerance model that leads to the generation of CD4(+) T regulatory cells and CD8(+) T regulatory cells in the periphery after the injection of an antigen into the anterior chamber of the eye. Nickel-induced oral tolerance is another immune tolerance model that is induced by the contact allergen Nickel and leads to the generation of Nickel-specific CD4(+) CD25(+) T regulatory cells after oral exposure. The goal of comparing different models of immune tolerance is to identify which mechanisms are universal and which mechanisms are model-specific. The knowledge of such mechanisms would allow scientists and clinicians to better intervene in different immune deregulation scenarios.

Eye-mediated immune tolerance to Type II collagen in arthritis-prone strains of mice.

Type II collagen (CII) is a cartilage structural protein that plays important roles in joint function, arthritis and ageing. In studying the ability of CII to induce eye-mediated specific immune tolerance, we have recently proven that CII is capable of inducing anterior chamber-associated immune deviation (ACAID) in Balb/c mice. Here, we study the ability of CII to induce eye-mediated immune tolerance in strains of mice that are prone to the induction of rheumatoid arthritis. Thus, we hypothesized that CII induces ACAID in DBA/1 mice and in C57BL/6 mice through the AC route (direct injection) or the intravenous route (adoptive transfer of in vitro-generated CII-specific ACAID macrophages or of CII-specific in vitro-generated T regulatory cells). Specific immune tolerance induction was assessed using both delayed-type hypersensitivity (DTH) and local adoptive transfer (LAT) assays. Results indicated the ability of CII to generate CII-specific ACAID-mediated immune tolerance in vivo and in vitro in both DBA/1 mice and C57BL/6 mice. These findings could be beneficial in studies of immune tolerance induction using CII.