Sirolimus enhances the magnitude and quality of viral-specific CD8+ T-cell responses to vaccinia virus vaccination in rhesus macaques.

Sirolimus is a potent antiproliferative agent used clinically to prevent renal allograft rejection. However, little is known about the effects of maintenance immunosuppressive agents on the immune response to potentially protective vaccines. Here we show that sirolimus paradoxically increases the magnitude and quality of the CD8+ T-cell response to vaccinia vaccination in nonhuman primates, fostering more robust recall responses compared to untreated and tacrolimus-treated controls. Enhancement of both the central and effector memory compartments of the vaccinia-specific CD8+ T-cell response was observed. These data elucidate new mechanistic characteristics of sirolimus and suggest immune applications extending beyond its role as an immunosuppressant.

Engraftment of adult porcine islet xenografts in diabetic nonhuman primates through targeting of costimulation pathways.

Recent advances in human allogeneic islet transplantation have established beta-cell replacement therapy as a potentially viable treatment option for individuals afflicted with Type 1 diabetes. Two recent successes, one involving neonatal porcine islet xenografts transplanted into diabetic rhesus macaques treated with a costimulation blockade-based regimen and the other involving diabetic cynomolgus monkeys transplanted with adult porcine islet xenografts treated with an alternative multidrug immunosuppressive regimen have demonstrated the feasibility of porcine islet xenotransplantation in nonhuman primate models. In the current study, we assessed whether transplantation of adult porcine islet xenografts into pancreatectomized macaques, under the cover of a costimulation blockade-based immunosuppressive regimen (CD28 and CD154 blockade), could correct hyperglycemia. Our findings suggest that the adult porcine islets transplanted into rhesus macaques receiving a costimulation blockade-based regimen are not uniformly subject to hyperacute rejection, can engraft (2/5 recipients), and have the potential to provide sustained normoglycemia. These results provide further evidence to suggest that porcine islet xenotransplantation may be an attainable strategy to alleviate the islet supply crisis that is one of the principal obstacles to large-scale application of islet replacement therapy in the treatment of Type 1 diabetes.

Antigenic disparity impacts outcome of agonism but not blockade of costimulatory pathways in experimental transplant models.

Treatment regimens consisting of CTLA-4 Ig/anti-CD154 or agonistic anti-CD28/rapamycin have both been shown to prevent GVHD in fully allogeneic murine model systems. Using a transgenic approach to track the fate of host-reactive T cells in a minor antigen disparity model of GVHD, we found that while treatment with CTLA-4 Ig/anti-CD154 retained efficacy, the costimulation agonist anti-CD28 combined with rapamycin failed to prevent GVHD. Analysis of the host-reactive CD4(+) and CD8(+) T-cell responses revealed that in contrast to CTLA-4Ig/anti-CD154-treated recipients, host-reactive T cells in recipients treated with agonistic anti-CD28/rapamycin displayed enhanced and accelerated T-cell proliferation and failed to undergo activation-induced cell death. An increase in systemic levels of inflammatory cytokines was observed in the anti-CD28/rapamycin-treated recipients of the minor but not major antigen disparity bone marrow transplants. Our results demonstrate the intricacies of costimulatory receptor signaling pathways, and reveal how agonism of the CD28 pathway can have opposing outcomes depending on the degree of antigenic disparity.

Induction of chimerism in rhesus macaques through stem cell transplant and costimulation blockade-based immunosuppression.

A strategy for producing high-level hematopoietic chimerism after non-myeloablative conditioning has been established in the rhesus macaque. This strategy relies on hematopoietic stem cell transplantation after induction with a non-myeloablative dose of busulfan and blockade of the IL2-receptor in the setting of mTOR inhibition with sirolimus and combined CD28/CD154 costimulation blockade. Hematopoietic stem cells derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level chimerism. Mean peripheral blood peak donor chimerism was 81% with a median chimerism duration of 145 days. Additional immune modulation strategies, such as pre-transplant CD8 depletion, donor-specific transfusion, recipient thymectomy or peritransplant deoxyspergualin treatment did not improve the level or durability of chimerism. Recipient immunologic assessment suggested that chimerism occurred amidst donor-specific down-regulation of alloreactive T cells, and the reappearance of vigorous T-mediated alloreactivity accompanied rejection of the transplants. Furthermore, viral reactivation constituted a significant transplant-related toxicity and may have negatively impacted the ability to achieve indefinite survival of transplanted stem cells. Nevertheless, this chimerism-induction regimen induced amongst the longest-lived stem cell chimerism reported to date for non-human primates and thus represents a platform upon which to evaluate emerging tolerance-induction strategies.

Transplant tolerance in non-human primates: progress, current challenges and unmet needs.

Given the significant morbidity associated with current post-transplant immunosuppressive regimens, induction of immune tolerance continues to be an important goal of clinical organ transplantation. While many strategies for inducing tolerance have been successfully applied in murine models, significant barriers are faced when translating these approaches to the clinic. This has necessitated pre-clinical studies in the more closely related model system, the non-human primates (NHP). In this review, we will discuss the four most prominent strategies for inducing transplantation tolerance and highlight their relative success and shortcomings in NHP. These strategies are: (1) T-cell costimulation blockade (2) mixed chimerism induction (3) T-cell depletion and (4) tolerance induction through regulatory T-cells. After discussing the progress that has been made with each of these strategies, we will identify this field's most pressing unmet needs and discuss how we may best overcome the resulting barriers to tolerance induction.

A mouse model for polyomavirus-associated nephropathy of kidney transplants.

Polyomavirus-associated nephropathy is an important cause of dysfunction and failure of renal transplants. BK virus is an ubiquitous human polyoma virus that persistently infects the kidney. This otherwise silent infection can reactivate in immunosuppressed individuals, resulting in renal complications. Because polyoma viruses are highly species-specific, we developed a mouse polyoma virus-renal transplant model in order to investigate the pathogenesis of polyomavirus-associated nephropathy. Using this model, we found that polyoma virus preferentially replicates in the allogeneic kidney grafts, accelerating graft failure; thus, this animal model is able to mimic the polyomavirus-associated nephropathy seen in human renal transplant patients. Acute polyoma virus infection of mouse allograft recipients augmented the alloreactive CD8+ T-cell response, while maintaining the anti-viral CD8+ T-cell response. In addition to the known virus-induced cytopathology, these findings demonstrate a potential role for an enhanced anti-donor T-cell response in the pathogenesis of polyomavirus-associated nephropathy.

Bystander activation involving T lymphocytes in herpetic stromal keratitis.

Herpes simplex virus infection of mouse corneas can lead to the development of an immunopathological lesion, termed herpetic stromal keratitis (HSK). Such lesions also occur in TCR-transgenic mice backcrossed to SCID (TgSCID) that are unable to mount detectable HSV-specific immune responses. The present study demonstrates that lesion expression in such mice depends on continuous viral replication, whereas in immunocompetent mice, lesions occurred even if virus replication was terminated at 4 days after infection. The continuous replication in TgSCID mice was considered necessary to produce an activating stimulus to CD4(+) T cells that invade the cornea. Lesions in TgSCID were resistant to control by cyclosporin A, but were inhibited by treatment with rapamycin. This result was interpreted to indicate that T cell activation involved a non-TCR-mediated cytokine-driven bystander mechanism. Bystander activation was also shown to play a role in HSK lesions in immunocompetent mice. Accordingly, in immunocompetent DO11.10 mice, lesions were dominated by KJ1.26(+) OVA-specific CD4(+) T cells that were unreactive with HSV. In addition, KJ1.26(+) HSV nonimmune cells parked in ocularly infected BALB/c mice were demonstrable in HSK lesions. These results provide insight for the choice of new strategies to manage HSK, an important cause of human blindness.

Bystander activation of CD4+ T cells accounts for herpetic ocular lesions.

PURPOSE: Stromal keratitis is an immunopathologic consequence of herpes simplex virus (HSV) infection of the cornea. The lesion is immunopathologic, but the identities of molecules that drive the reaction remain unresolved. To exclude viral antigen recognition as a necessary step in the disease process, ocular HSV infection was followed in Tg-RAG mice (OVA-TCR transgenic mice crossed to RAG2-deficient mice) whose limited T-cell repertoire did not include immune responsiveness to HSV. METHODS: Mice with T-cell specificity to OVA peptide (Tg-RAG mice) as well as control DO11.10 and BALB/c mice were infected with HSV on the scarified cornea and subjected to clinical, histologic, and immunologic analysis. To evaluate involvement of OVA-specific CD4+ T cells in lesion development in Tg-RAG mice, monoclonal antibody to CD4+ T cells was used for in vivo CD4+ T-cell depletion. RESULTS: Tg-RAG mice were capable of eliciting ocular lesions in the absence of detectable reactivity to viral antigens. Lesion manifestation in Tg-RAG mice was CD4+ T-cell dependent and the cellular infiltrates and their inflammatory products in the HSV-infected cornea were comparable to similarly infected BALB/c and DO11.10 mice. CONCLUSIONS: The authors conclude that mechanisms other than viral antigen recognition, and hence molecular mimicry, are at play and are sufficient to cause HSV-induced stromal keratitis. The data imply a significant role for non-virus-specific CD4+ T cells that could become activated by an inflammatory milieu consisting of enhanced accessory molecules and proinflammatory cytokines in the cornea.

Bystander activation of CD4(+) T cells can represent an exclusive means of immunopathology in a virus infection.

Herpetic stromal keratitis (HSK) is an immunopathological lesion involving herpes simplex virus (HSV) infection and CD4(+) T cells of the Th1 phenotype, but the nature of the target antigens which drive HSK remains uncertain. In the present report we show that ovalbumin TCR-transgenic mice backcrossed to SCID mice unable to recognize HSV show clinical signs of HSK but die of viral encephalitis before the lesions become severe. However, passive transfer of anti-HSV serum at 24 h clears virus and affords protection from both HSK lesions and death. Adoptive transfer of CD8(+) T cells at 72 h usually conferred protection but animals developed severe corneal pathology by 3 weeks post infection. At this time viral antigens were not demonstrable in the cornea and the T cells in the inflammatory lesions were CD4(+)KJ1-26.1 idiotype positive, i. e. OVA peptide specific. These results indicate bystander activation of CD4(+) T cells in a virus-induced inflammatory milieu. This mechanism of immunoinflammation may represent an important component of any lesion which involves CD4(+) T cells.

Virus-induced immunoinflammatory lesions in the absence of viral antigen recognition.

Herpetic stromal keratitis (HSK) is a CD4+ T cell-controlled immunopathologic lesion in the eye that results from infection with herpes simplex virus (HSV). Target Ags involved in HSK remain undefined. In this study, we determined if HSK could be induced in animals genetically incapable of generating HSV Ag-specific CD4+ T cells. Mice bearing transgenic TCR specific to OVA peptide 323-339 (DO11.10) were crossed to SCID mice whose offspring (Tg-SCID) possessed CD4+ T cells, >98% of which expressed the OVA peptide-specific TCR. HSV infection of Tg-SCID mice was lethal, and mice failed to generate detectable T cell responses even after repeated immunization with a mutant avirulent virus (AN-1). Immunization with AN-1 virus followed by ocular challenge with HSV resulted in ocular inflammation before encephalitis, in contrast to the protection conferred in the control BALB/c and DO11.10 mice. These results indicate that clinical HSK may not require viral Ag recognition by CD4+ T cells and that T cells of irrelevant specificity can be recruited, activated, and driven into effector function in the HSV-infected cornea. This is suggested to represent a bystander activation effect resulting from the presence of proinflammatory mediators resulting from HSV replication.

Control of herpetic stromal keratitis using CTLA4Ig fusion protein.

Herpetic stromal keratitis (HSK) is an immunoinflammatory lesion in the cornea of the eye set off by infection with herpes simplex virus (HSV). The disease appears to be orchestrated by CD4+ T cells of the Th1 phenotype but the identity of target antigens involved in HSK remains unknown. In this proposal, we investigated if the inhibition of T cell activation with the fusion protein CTLA4Ig would abrogate the disease process when administered systemically. BALB/c mice infected with HSV-1 (RE strain) by corneal scarification were injected intraperitoneally on a single occasion with CTLA4Ig or L6 control (IgG Fc) given on day 2, day 5, or day 8 postinfection. Lesions in CTLA4Ig-treated mice showed markedly reduced severity judged by both slit lamp biomicroscopy and histopathology if treated on day 2 or day 5. Treated animals also expressed minimal HSV-specific splenic T cell and humoral antibody responses. Judged by the profile of T cell and IgG subset responses, inhibition by CTLA4Ig appeared more directly on the HSV-specific Th1 response, correlating with the known role of such cells in HSK. Delay of treatment until the time of disease onset (day 8) had marginal or negligible effects. The results indicate that blockade of coreceptor interaction between T cells and antigen-presenting cells during the induction phase of immune response significantly impairs onset and severity of herpetic stromal keratitis.

On the essential involvement of neutrophils in the immunopathologic disease: herpetic stromal keratitis.

Corneal infection with herpes simplex virus-1 in immunocompetent mice induces an immunopathologic response termed herpetic stromal keratitis (HSK). The earliest sign of disease is neutrophil infiltration, which lasts for 48 to 72 h and then disappears. However, a secondary neutrophil infiltration, this time more massive, occurs, beginning 8 to 9 days postinfection, a time in which HSK becomes clinically evident. The role of neutrophils in HSK expression was investigated by eliminating such cells using a specific mAb (RB6-8C5). In neutrophil-depleted immunocompetent mice, virus replicated more abundantly, but no effects on HSK expression were observed, possibly because sustained neutropenia could not be maintained. However, using a severe combined immunodeficient mouse model, in which HSK does not occur unless given adoptive transfer of CD4+ T cells, the effects of neutrophil depletion were more pronounced. There were significantly less incidence and severity of HSK in CD4+ T cell-reconstituted severe combined immunodeficient mice that were depleted of neutrophils as compared with controls. Neutrophil-depleted mice displayed moderate to severe periocular skin lesions, progressively became cachetic, and developed signs of encephalitis. Virus was recovered at higher titers and for longer periods from eyes of neutrophil-depleted animals. Brain virus titers were also significantly higher on day 12 postinfection as compared with control animals. These results suggest that herpes simplex virus infection of the cornea rapidly invokes recruitment of neutrophils that may aid in viral clearance, and that neutrophils directly or indirectly serve as agonists in perpetuating a CD4+ T cell-mediated inflammatory reaction.

Herpes simplex virus type 1-mediated up-regulation of IL-12 (p40) mRNA expression. Implications in immunopathogenesis and protection.

In this report we investigated whether induction of IL-12 occurs in response to herpes simplex virus (HSV) infection of the mouse eye, which may serve to regulate the nature of the subsequent immune response. The data show early induction and continued maintenance of IL-12 (p40) mRNA in the cornea and draining lymph node upon ocular infection with HSV. Using a very sensitive radioimmunoassay technique, IL-12 (p40) protein also was detected in the cornea upon ocular HSV infection. Unfractionated splenocytes and enriched populations of dendritic cells, macrophages, and neutrophils all responded to HSV infection in vitro by up-regulating the expression of IL-12 (p40) mRNA. However, cultured corneal cells failed to generate IL-12 (p40) upon exposure to HSV. The data indicate that inflammatory cells that infiltrate the cornea in response to HSV-1 infection are the main source of IL-12 in the eye. Our results are consistent with the hypothesis that IL-12 induction in the eye acts as a triggering event that biases HSV-specific immunity to a type 1 T cell response, which, in the environment of the eye, culminates in an immunopathologic disease.