Screening copy number variations in 35 unsolved inherited retinal disease families.

The purpose of this study was to screen Copy Number Variations (CNVs) in 35 unsolved Inherited Retinal Dystrophy (IRD) families. Initially, next generation sequencing, including a specific Hereditary Eye Disease Enrichment Panel or Whole exome sequencing, was employed to screen (likely) pathogenic Single-nucleotide Variants (SNVs) and small Insertions and Deletions (indels) for these cases. All available SNVs and indels were further validated and co-segregation analyses were performed in available family members by Sanger sequencing. If not, after excluding deep intronic variants, Multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescence PCR (QF-PCR) and Sanger sequencing were employed to screen CNVs. We determined that 18 probands who had heterozygous SNVs/indels or whose parents were not consanguineous but had homozygous SNVs/indels in autosomal recessive IRDs genes had CNVs in another allele of these genes, 11 families had disease-causing hemizygous CNVs in X-linked IRD genes, 6 families had (likely) pathogenic heterozygous CNVs in PRPF31 gene. Of 35 families, 33 different CNVs in 16 IRD-associated genes were detected, with PRPF31, EYS and USH2A the most common disease-causing gene in CNVs. Twenty-six and 7 of them were deletion and duplication CNVs, respectively. Among them, 14 CNVs were first reported in this study. Our research indicates that CNVs contribute a lot to IRDs, and screening of CNVs substantially increases the diagnostic rate of IRD. Our results emphasize that MLPA and QF-PCR are ideal methods to validate CNVs, and the novel CNVs reported herein expand the mutational spectrums of IRDs.

Corneal deposits and nerve alterations in Bietti Corneoretinal Crystalline Dystrophy imaged using in vivo confocal microscopy.

PURPOSE: To evaluate the imaging features of corneal deposits and nerve alterations in Chinese patients with Bietti Corneoretinal Crystalline Dystrophy (BCD) using in vivo confocal microscopy (IVCM). METHODS: Twenty patients with BCD and 20 age- and sex-matched healthy controls were enrolled in this retrospective, observational study. Corneal deposits and sub-basal nerve plexus (SNP) were observed by IVCM. Parameters of SNP including total nerve density/number, main nerve trunk density/number, and branch nerve density/number were analyzed by Neuron J. RESULTS: Corneal deposits were observed in both eyes of all patients by IVCM. These crystals appeared as dot-shaped, needle-shaped, and rod-shaped hyperreflective bodies and were located not only in the sub-epithelium and stroma of cornea, but in endothelium which were not reported before. There was a decrease of total nerve density (P < 0.001), main nerve trunk density (P = 0.007), and branch nerve density (P = 0.001), in BCD compared to controls. The number of total nerves/frame (P = 0.001), main nerve trunks/frame (P = 0.005), and branch nerves/frame (P = 0.006) in BCD were lower than controls. CONCLUSION: New findings in locations of corneal crystals by IVCM expand the phenotype spectrum of BCD. Corneal deposits may be useful for diagnosis of BCD, especially ones without retinal deposits. Corneal nerve parameters were reduced in BCD, which may provide new insights to be further explored to contribute to our understanding of BCD. IVCM is a promising tool to evaluate corneal deposits and nerve alterations in BCD.

Treg suppression of immunity within inflamed allogeneic grafts.

CD4+Foxp3+ regulatory T cells (Tregs) restrain inflammation and immunity. However, the mechanisms underlying Treg suppressor function in inflamed nonlymphoid tissues remain largely unexplored. Here, we restricted immune responses to nonlymphoid tissues and used intravital microscopy to visualize Treg suppression of rejection by effector T cells (Teffs) within inflamed allogeneic islet transplants. Despite their elevated motility, Tregs preferentially contacted antigen-presenting cells (APCs) over Teffs. Interestingly, Tregs specifically targeted APCs that were extensively and simultaneously contacted by Teffs. In turn, Tregs decreased MHC-II expression on APCs and hindered Teff function. Last, we demonstrate that Treg suppressive function within inflamed allografts required ectonucleotidase CD73 activity, which generated the antiinflammatory adenosine. Consequently, CD73-/- Tregs exhibited fewer contacts with APCs within inflamed allografts compared with WT Tregs, but not in spleen. Overall, our findings demonstrate that Tregs suppress immunity within inflamed grafts through CD73 activity and suggest that Treg-APC direct contacts are central to this process.

Novel variants in PDE6A and PDE6B genes and its phenotypes in patients with retinitis pigmentosa in Chinese families.

BACKGROUND: Retinitis pigmentosa (RP) is a genetically heterogeneous disease with 89 causative genes identified to date. However, only approximately 60% of RP cases genetically solved to date, predicating that many novel disease-causing variants are yet to be identified. The purpose of this study is to identify novel variants in PDE6A and PDE6B genes and present its phenotypes in patients with retinitis pigmentosa in Chinese families. METHODS: Five retinitis pigmentosa patients with PDE6A variants and three with PDE6B variants were identified through a hereditary eye disease enrichment panel (HEDEP), all patients' medical and ophthalmic histories were collected, and ophthalmological examinations were performed, followed by an analysis of the possible causative variants. Sanger sequencing was used to verify the variants. RESULTS: We identified 20 variants in eight patients: 16 of them were identified in either PDE6A or PDE6B in a compound heterozygous state. Additional four heterozygous variants were identified in the genes ADGRA3, CA4, OPTN, RHO. Two novel genetic changes in PDE6A were identified (c.1246G > A and c.1747 T > A), three novel genetic changes in PDE6B were identified (c.401 T > C, c.2293G > C and c.1610-1612del), out of the novel identified variants one was most probably non-pathogenic (c.2293G > C), all other novel variants are pathogenic. Additional variant was identified in CA4 and RHO, which can cause ADRP (c.243G > A, c.688G > A). In addition, a novel variant in ADGRA3 was identified (c.921-1G > A). CONCLUSIONS: This study reveals novel and known variants in PDE6A and PDE6B genes in Chinese families with autosomal recessive RP, and expands the clinical and genetic findings of photoreceptor-specific enzyme deficiencies.

Genotype and Ocular Phenotype in Sixteen Chinese Patients with Bietti Corneoretinal Crystalline Dystrophy.

OBJECTIVE: To investigate CYP4V2 gene variants and ocular clinical characteristics of Bietti corneoretinal crystalline dystrophy in China so as to provide more references for genotype and phenotype of BCD. METHODS: Sixteen Chinese probands were recruited in Beijing Tongren Hospital in a retrospective study. All patients underwent CYP4V2 gene detection and ophthalmic clinical examinations. RESULTS: CYP4V2 gene variants were detected in all patients. Eight variants were identified, and the most common one was c.802-8_810del17bpinsGC. Onset age of BCD was from 12 to 44 years, and the first symptoms mostly were decreased visual acuity or night blindness. Corneal crystalline depositions were observed in all patients and were found not only in epithelium and superficial stroma near the limbus but also in corneal endothelium. OCT showed atrophy of RPE in all patients, outer retinal tubulation in ten patients, macular edema in four patients, macular hole in three patients with one accompanied with retinal detachment, and choroidal neovascularization in one patient. CONCLUSION: CYP4V2 gene variants were detected in all patients consistent with the genetic locus homogeneity of BCD, and c.802-8_810del17bpinsGC was the most common mutation. Corneal crystalline depositions were observed in all patients, which may be features of BCD and helpful for the diagnosis of BCD patients, especially those in the advanced stage without typical fundus crystalline depositions or without gene detection. However, considerable phenotypic variability was detected. Corneal crystalline deposits were observed not only in epithelium and superficial stroma but also in endothelium, which has not been reported before. This may provide further evidence for the variable phenotypic expression between affected individuals.

Innate Allorecognition and Memory in Transplantation.

Over the past few decades, we have witnessed a decline in the rates of acute rejection without significant improvement in chronic rejection. Current treatment strategies principally target the adaptive immune response and not the innate response. Therefore, better understanding of innate immunity in transplantation and how to target it is highly desirable. Here, we review the latest advances in innate immunity in transplantation focusing on the roles and mechanisms of innate allorecognition and memory in myeloid cells. These novel concepts could explain why alloimmune response do not abate over time and shed light on new molecular pathways that can be interrupted to prevent or treat chronic rejection.

PIRs mediate innate myeloid cell memory to nonself MHC molecules.

Immunological memory specific to previously encountered antigens is a cardinal feature of adaptive lymphoid cells. However, it is unknown whether innate myeloid cells retain memory of prior antigenic stimulation and respond to it more vigorously on subsequent encounters. In this work, we show that murine monocytes and macrophages acquire memory specific to major histocompatibility complex I (MHC-I) antigens, and we identify A-type paired immunoglobulin-like receptors (PIR-As) as the MHC-I receptors necessary for the memory response. We demonstrate that deleting PIR-A in the recipient or blocking PIR-A binding to donor MHC-I molecules blocks memory and attenuates kidney and heart allograft rejection. Thus, innate myeloid cells acquire alloantigen-specific memory that can be targeted to improve transplant outcomes.

Cross-dressed dendritic cells sustain effector T cell responses in islet and kidney allografts.

Activation of host T cells that mediate allograft rejection is a 2-step process. The first occurs in secondary lymphoid organs where T cells encounter alloantigens presented by host DCs and differentiate to effectors. Antigen presentation at these sites occurs principally via transfer of intact, donor MHC-peptide complexes from graft cells to host DCs (cross-dressing) or by uptake and processing of donor antigens into allopeptides bound to self-MHC molecules (indirect presentation). The second step takes place in the graft, where effector T cells reengage with host DCs before causing rejection. How host DCs present alloantigens to T cells in the graft is not known. Using mouse islet and kidney transplantation models, imaging cytometry, and 2-photon intravital microscopy, we demonstrate extensive cross-dressing of intragraft host DCs with donor MHC-peptide complexes that occurred early after transplantation, whereas host DCs presenting donor antigen via the indirect pathway were rare. Cross-dressed DCs stably engaged TCR-transgenic effector CD8+ T cells that recognized donor antigen and were sufficient for sustaining acute rejection. In the chronic kidney rejection model, cross-dressing declined over time but was still conspicuous 8 weeks after transplantation. We conclude that cross-dressing of host DCs with donor MHC molecules is a major antigen presentation pathway driving effector T cell responses within allografts.

Novel mutations of RPGR in Chinese families with X-linked retinitis pigmentosa.

BACKGROUND: RP (retinitis pigmentosa) is a group of hereditary retinal degenerative diseases. XLRP is a relatively severe subtype of RP. Thus, it is necessary to identify genes and mutations in patients who present with X-linked retinitis pigmentosa. METHODS: Genomic DNA was extracted from peripheral blood. The coding regions and intron-exon boundaries of the retinitis pigmentosa GTPase regulator (RPGR) and RP2 genes were amplified by PCR and then sequenced directly. Ophthalmic examinations were performed to identify affected individuals from two families and to characterize the phenotype of the disease. RESULTS: Mutation screening demonstrated two novel nonsense mutations (c.1541C > G; p.S514X and c.2833G > T; p.E945X) in the RPGR gene. The clinical manifestation of family 1 with mutations in exon 13 was mild. Genotype-phenotype correlation analysis suggested that patients with mutations close to the downstream region of ORF15 in family 2 manifested an early loss of cone function. Family 2 carried a nonsense mutation in ORF15 that appeared to have a semi-dominant pattern of inheritance. All male patients and two female carriers in family 2 manifested pathological myopia (PM), indicating that there may be a distinctive X-linked genotype-phenotype correlation between RP and PM. CONCLUSIONS: We identified two novel mutations of the RPGR gene, which broadens the spectrum of RPGR mutations and the phenotypic spectrum of the disease in Chinese families.

Donor SIRPα polymorphism modulates the innate immune response to allogeneic grafts.

Mice devoid of T, B, and natural killer (NK) cells distinguish between self and allogeneic nonself despite the absence of an adaptive immune system. When challenged with an allograft, they mount an innate response characterized by accumulation of mature, monocyte-derived dendritic cells (DCs) that produce interleukin-12 and present antigen to T cells. However, the molecular mechanisms by which the innate immune system detects allogeneic nonself to generate these DCs are not known. To address this question, we studied the innate response of Rag2-/- γc-/- mice, which lack T, B, and NK cells, to grafts from allogeneic donors. By positional cloning, we identified that donor polymorphism in the gene encoding signal regulatory protein α (SIRPα) is a key modulator of the recipient's innate allorecognition response. Donors that differed from the recipient in one or both Sirpa alleles elicited an innate alloresponse. The response was mediated by binding of donor SIRPα to recipient CD47 and was modulated by the strength of the SIRPα-CD47 interaction. Therefore, sensing SIRPα polymorphism by CD47 provides a molecular mechanism by which the innate immune system distinguishes between self and allogeneic nonself independently of T, B, and NK cells.

CD8+ Effector T Cell Migration to Pancreatic Islet Grafts Is Dependent on Cognate Antigen Presentation by Donor Graft Cells.

Pancreatic islet transplantation is a promising therapy for diabetes, but acute rejection of the islets by host effector T cells has hindered clinical application. In this study, we addressed the mechanisms of CD8(+) effector T cell migration to islet grafts because interrupting this step is key to preventing rejection. We found that effector T cell migration to revascularized islet transplants in mice is dependent on non-self Ag recognition rather than signaling via Gαi-coupled chemokine receptors. Presentation of non-self Ag by donor cells was necessary for migration, whereas Ag presentation by recipient cells was dispensable. We also observed that deficiency of SKAP1, an immune cell adaptor downstream of the TCR and important for integrin activation, prolongs allograft survival but does not reduce effector T cell migration to the graft. Therefore, effector T cell migration to transplanted islets is Ag driven, not chemokine driven, but SKAP1 does not play a critical role in this process.

Non-self recognition by monocytes initiates allograft rejection.

Maturation of T cell-activating APCs directly links innate and adaptive immunity and is typically triggered by microbial infection. Transplantation of allografts, which are sterile, generates strong T cell responses; however, it is unclear how grafts induce APC maturation in the absence of microbial-derived signals. A widely accepted hypothesis is that dying cells in the graft release "danger" molecules that induce APC maturation and initiate the adaptive alloimmune response. Here, we demonstrated that danger signals associated with dying cells are not sufficient to initiate alloimmunity, but that recognition of allogeneic non-self by the innate immune system is required. In WT as well as in T cell-, B cell-, and innate lymphoid cell-deficient mice, allogeneic grafts elicited persistent differentiation of monocytes into mature DCs that expressed IL-12 and stimulated T cell proliferation and IFN-γ production. In contrast, syngeneic grafts in the same mice elicited transient and less pronounced differentiation of monocytes into DCs, which neither expressed IL-12 nor stimulated IFN-γ production. In a model in which T cell recognition is restricted to a single foreign antigen on the graft, rejection occurred only if the allogeneic non-self signal was also sensed by the host's innate immune system. These findings underscore the importance of innate recognition of allogeneic non-self by monocytes in initiating graft rejection.

Promoting long-term survival of insulin-producing cell grafts that differentiate from adipose tissue-derived stem cells to cure type 1 diabetes.

BACKGROUND: Insulin-producing cell clusters (IPCCs) have recently been generated in vitro from adipose tissue-derived stem cells (ASCs) to circumvent islet shortage. However, it is unknown how long they can survive upon transplantation, whether they are eventually rejected by recipients, and how their long-term survival can be induced to permanently cure type 1 diabetes. IPCC graft survival is critical for their clinical application and this issue must be systematically addressed prior to their in-depth clinical trials. METHODOLOGY/PRINCIPAL FINDINGS: Here we found that IPCC grafts that differentiated from murine ASCs in vitro, unlike their freshly isolated islet counterparts, did not survive long-term in syngeneic mice, suggesting that ASC-derived IPCCs have intrinsic survival disadvantage over freshly isolated islets. Indeed, ß cells retrieved from IPCC syngrafts underwent faster apoptosis than their islet counterparts. However, blocking both Fas and TNF receptor death pathways inhibited their apoptosis and restored their long-term survival in syngeneic recipients. Furthermore, blocking CD40-CD154 costimulation and Fas/TNF signaling induced long-term IPCC allograft survival in overwhelming majority of recipients. Importantly, Fas-deficient IPCC allografts exhibited certain immune privilege and enjoyed long-term survival in diabetic NOD mice in the presence of CD28/CD40 joint blockade while their islet counterparts failed to do so. CONCLUSIONS/SIGNIFICANCE: Long-term survival of ASC-derived IPCC syngeneic grafts requires blocking Fas and TNF death pathways, whereas blocking both death pathways and CD28/CD40 costimulation is needed for long-term IPCC allograft survival in diabetic NOD mice. Our studies have important clinical implications for treating type 1 diabetes via ASC-derived IPCC transplantation.

Cutting edge: programmed death-1 defines CD8+CD122+ T cells as regulatory versus memory T cells.

Recent convincing data have shown that naturally occurring CD8(+)CD122(+) T cells are also regulatory T cells. Paradoxically, CD8(+)CD122(+) T cells have been well described as memory T cells. Given their critical role in tolerance versus long-term immunity, it is important to reconcile this profound dichotomy. In this study, we reported that CD8(+)CD122(+) T cells contain both programmed death-1 (PD-1)(-) and PD-1(+) populations. It was CD8(+)CD122(+)PD-1(+) T cells, but not their PD-1(-) counterparts, that suppressed T cell responses in vitro and in vivo. This suppression was largely dependent on their production of IL-10. Moreover, the costimulatory signaling of both CD28 and PD-1 is required for their optimal IL-10 production. In contrast, Ag-specific CD8(+)CD122(+)PD-1(-) T cells were bona fide memory T cells. Thus, CD8(+)CD122(+) T cells can be either regulatory T or memory T cells, depending on their PD-1 expression and Ag specificity. This study reconciles previously contradictory findings and has important implications for tolerance induction.

Manipulating IL-2 availability amid presentation of donor MHC antigens suppresses murine alloimmune responses by inducing regulatory T cells.

BACKGROUND: Major histocompatibility complex (MHC) antigens are important for alloimmune responses as well as immune tolerance. Previous studies have shown that presentation of donor MHC antigens by donor-specific transfusion prior to or upon transplantation promotes transplant tolerance induced by other agents. However, it is unclear whether presentation of donor MHC antigens by DNA vaccination induces long-term allograft survival. METHODOLOGY/PRINCIPAL FINDINGS: We investigated whether presentation of MHC class-II and/or class-I donor antigens by DNA vaccination suppresses alloimmune responses and promotes long-term allograft acceptance. We initially found that presentation of both MHC donor antigens by DNA vaccination itself prior to transplantation fails to significantly prolong islet allograft survival in otherwise untreated mice. However, islet allograft survival was significantly prolonged when MHC class-II DNA vaccination was accompanied with IL-2 administration (MHCII + IL-2) while MHC class-I DNA vaccination was followed by IL-2 and subsequent neutralizing anti-IL-2 treatments (MHCI + IL-2/anti-IL-2). Especially, this protocol promoted long-term allograft survival in the majority of recipients (57%) when combined with low doses of rapamycin post-transplantation. Importantly, MHCII + IL-2 induced FoxP3+ Treg cells in both spleens and grafts and suppressed graft-infiltrating CD4+ cell proliferation, whereas MHCI + IL-2/anti-IL-2 mainly inhibited graft-infiltrating CD8+ cell proliferation and donor-specific CTL activity. The combined protocol plus rapamycin treatment further reduced both CD4+ and CD8+ T cell proliferation as well as donor-specific CTL activity but spared FoxP3+ Treg cells. Depleting CD25+ Treg cells or adoptive transfer of pre-sensitized CD8+ T cells abolished this long-term allograft survival. CONCLUSIONS/SIGNIFICANCE: Manipulating IL-2 availability during presentation of MHC class-II and class-I donor antigens by DNA vaccination pre-transplantation induces Treg cells, suppresses alloimmune responses and promotes long-term allograft survival.

Interaction of programmed death-1 and programmed death-1 ligand-1 contributes to testicular immune privilege.

BACKGROUND: Immune responses are tempered in immunologically privileged sites including the testis. Previous studies have shown that islet transplantation in the testis significantly prolongs islet allograft survival. However, mechanisms underlying testicular immune privilege and intratesticular allograft survival remain unclear. METHODS: Allogeneic murine islets were transplanted in the testis. Programmed death-1 ligand-1 (PD-L1) expression was detected by immunohistochemstry and real-time polymerase chain reaction. Infiltrating T-cell proliferation was measured by bromodeoxyuridine uptakes, whereas their apoptosis was quantified by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling methods. Transgenic T cells were used to track allospecific memory T-cell generation. RESULTS: We found that programmed death-1 (PD-1):PD-L1 negative costimulation is essential for prolonged survival of intratesticular islet allografts, as blocking PD-L1 or PD-1, but not PD-L2 and cytotoxic T-lymphocyte antigen 4, abrogated long-term survival of intratesticular islet allografts. As controls, blocking PD-1 or PD-L1 did not significantly accelerate the acute rejection of islet allografts transplanted under the renal capsule, a conventional islet-grafting site. We also found for the first time that PD-L1 is constitutively expressed mainly by spermatocytes and spermatids in seminiferous tubules of the testis. Moreover, infiltrating T cells underwent less vigorous proliferation but faster apoptosis in the testis than in the kidney. Blocking PD-1:PD-L1 costimulation largely abolished the suppression of T-cell proliferation and acceleration of T-cell apoptosis. Importantly, testicular immune privilege significantly suppressed the generation and proliferation of donor-specific memory CD8 T cells. CONCLUSIONS: The constitutive expression of PD-L1 in the testis is an important mechanism underlying testicular immune privilege and long-term survival of intratesticular islet allografts.

The role of tryptophan catabolism in acquisition and effector function of memory T cells.

PURPOSE OF REVIEW: Indoleamine 2,3-dioxygenase (IDO) catalyzes tryptophan and suppresses adaptive immunity by inhibiting T-cell proliferation and promoting their apoptosis. This article reviews the impact of IDO on cellular immunity as well as alloimmunity and particularly discusses the role of tryptophan catabolism in the generation and function of allospecific memory T cells, as the latter pose a long-term threat to allograft survival. RECENT FINDINGS: IDO catalyzes tryptophan and suppresses T-cell proliferation while tryptophan metabolites induce T-cell apoptosis. IDO, therefore, suppresses adaptive immunity. Recent studies have shown that IDO overexpression suppresses allograft rejection and autoimmune diseases. Particularly, our new study has shown that IDO is capable of inhibiting the generation and function of allospecific central memory CD8+ T cells, however it does not impair effector memory CD8+ T-cell function. SUMMARY: IDO has several immunosuppressive properties that make it a potential candidate for use in transplantation. An effective method to deliver IDO in vivo, however, is lacking. Moreover, IDO alone is insufficient for inducing long-term allograft survival, as high expression of IDO in the graft fails to prevent acute rejection. Further studies are warranted to search for drugs that increase IDO expression in vivo or to explore strategies of prolonging the half life of IDO.

The role for monocyte chemoattractant protein-1 in the generation and function of memory CD8+ T cells.

Memory T cells are resistant to the conventional costimulatory blockade and therefore impede tolerance induction. However, their migratory, survival, and functional requirements for chemokines are not well understood. We herein examine the role for MCP-1 or CCL2 in the generation, migration, and function of memory CD8+ T cells. We found that overall generation of both central memory (TCM) and effector memory (TEM) CD8+ T cells was severely impaired in the absence of MCP-1. Importantly, the survival of TEM, but not TCM, CD8+ cells was reduced without MCP-1, whereas the homeostatic proliferation of TCM, but not TEM, CD8+ cells was weakened in MCP-1-/- mice. However, once they were generated in the absence of MCP-1, in vitro function of both subsets of memory cells remained intact as determined by their proliferation and IFN-gamma production. Interestingly, the migration of TCM, but not TEM, CD8+ cells to inflammatory sites was significantly delayed without MCP-1, whereas both subsets of memory cells underwent comparable expansion and apoptosis with or without MCP-1 during the effector phase. Moreover, the function to eliminate a graft of TCM, but not TEM, CD8+ cells was impaired without MCP-1. Thus, this study demonstrates that MCP-1 plays an important role in not only migration but also generation and survival of memory T cells. This finding provides new insight into the requirement of chemokines for the generation, survival, and function of differential subsets of memory T cells and may have clinic implications for tolerance induction.

Bystander central memory but not effector memory CD8+ T cells suppress allograft rejection.

Memory T cells respond faster and more vigorously than their naive counterparts and are critical for adaptive immunity. However, it is unknown whether and how memory T cells react in the face of irrelevant Ags. It is generally accepted that bystander memory T cells are neutral in immune responsiveness. In this study, we present the first evidence that bystander central memory (TCM), but not effector memory (TEM), CD8+ T cells suppress allograft rejection as well as T cell proliferation in the draining lymph nodes (DLN) of recipient mice. Both bystander TCM and naive T cells, but fewer TEM cells, migrated to DLN, whereas TCM cells exhibited faster turnover than their naive counterparts, suggesting that bystander TCM cells have an advantage over their naive counterparts in suppression. However, bystander TEM cells migrated to inflammatory graft sites, but not DLN, and yet failed to exert their suppression. These findings indicate that bystander memory T cells need to migrate to lymph nodes to exert their suppression by inhibiting responder T cell activation or homeostatic proliferation. Moreover, the suppression mediated by bystander TCM cells was largely dependent on IL-15, as IL-15 was required for their homeostatic proliferation and TCM-mediated suppression of allograft rejection. This suppression also required the presence of TGFbeta1, as TCM cells expressed TGFbeta1 while neutralizing TGFbeta1 abolished their suppression. Thus, bystander TCM, but not TEM, CD8+ T cells are potent suppressors rather than bystanders. This new finding will have an impact on cellular immunology and may have clinic implications for tolerance induction.

Suppression of memory CD8 T cell generation and function by tryptophan catabolism.

Dendritic cell-derived indoleamine 2,3-dioxygenase (IDO) suppresses naive T cell proliferation and induces their apoptosis by catalyzing tryptophan, and hence is essential for the maintenance of peripheral tolerance. However, it is not known whether memory T cells are subject to the regulation by IDO-mediated tryptophan catabolism, as memory T cells respond more rapidly and vigorously than their naive counterparts and are resistant to conventional costimulatory blockade. In this study, we present the evidence that memory CD8+ T cells are susceptible to tryptophan catabolism mediated by IDO. We found that overexpression of IDO in vivo attenuated the generation of both central memory CD8+ T cells (T(CM)) and effector memory CD8+ T cells (T(EM)) while suppressing IDO activity promoted their generation. Moreover, IDO overexpression suppressed the effector function of T(CM) cells or T(CM) cell-mediated allograft rejection as well as their proliferation in vivo. Interestingly, T(CM) cells were resistant to apoptosis induced by tryptophan catabolism. However, IDO overexpression did not suppress the effector function of T(EM) cells or T(EM) cell-mediated allograft rejection, suggesting that T(EM) cells, unlike T(CM) cells, do not require tryptophan for their effector function once they are generated. This study provides insight into the mechanisms underlying the differential regulation of memory T cell responsiveness and has clinical implications for vaccination or tolerance induction.