PINK1 deficiency alters muscle stem cell fate decision and muscle regenerative capacity.

Maintenance of mitochondrial function plays a crucial role in the regulation of muscle stem cell (MuSC), but the underlying mechanisms remain ill defined. In this study, we monitored mitophagy in MuSCS under various myogenic states and examined the role of PINK1 in maintaining regenerative capacity. Results indicate that quiescent MuSCs actively express mitophagy genes and exhibit a measurable mitophagy flux and prominent mitochondrial localization to autophagolysosomes, which become rapidly decreased during activation. Genetic disruption of Pink1 in mice reduces PARKIN recruitment to mitochondria and mitophagy in quiescent MuSCs, which is accompanied by premature activation/commitment at the expense of self-renewal and progressive loss of muscle regeneration, but unhindered proliferation and differentiation capacity. Results also show that impaired fate decisions in PINK1-deficient MuSCs can be restored by scavenging excess mitochondrial ROS. These data shed light on the regulation of mitophagy in MuSCs and position PINK1 as an important regulator of their mitochondrial properties and fate decisions.

Murine MHC-Deficient Nonobese Diabetic Mice Carrying Human HLA-DQ8 Develop Severe Myocarditis and Myositis in Response to Anti-PD-1 Immune Checkpoint Inhibitor Cancer Therapy.

Myocarditis has emerged as an immune-related adverse event of immune checkpoint inhibitor (ICI) cancer therapy associated with significant mortality. To ensure patients continue to safely benefit from life-saving cancer therapy, an understanding of fundamental immunological phenomena underlying ICI myocarditis is essential. We recently developed the NOD-cMHCI/II-/-.DQ8 mouse model that spontaneously develops myocarditis with lower mortality than observed in previous HLA-DQ8 NOD mouse strains. Our strain was rendered murine MHC class I and II deficient using CRISPR/Cas9 technology, making it a genetically clean platform for dissecting CD4+ T cell-mediated myocarditis in the absence of classically selected CD8+ T cells. These mice are highly susceptible to myocarditis and acute heart failure following anti-PD-1 ICI-induced treatment. Additionally, anti-PD-1 administration accelerates skeletal muscle myositis. Using histology, flow cytometry, adoptive transfers, and RNA sequencing analyses, we performed a thorough characterization of cardiac and skeletal muscle T cells, identifying shared and unique characteristics of both populations. Taken together, this report details a mouse model with features of a rare, but highly lethal clinical presentation of overlapping myocarditis and myositis following ICI therapy. This study sheds light on underlying immunological mechanisms in ICI myocarditis and provides the basis for further detailed analyses of diagnostic and therapeutic strategies.

HLA-DQ8 Supports Development of Insulitis Mediated by Insulin-Reactive Human TCR-Transgenic T Cells in Nonobese Diabetic Mice.

In an effort to improve HLA-"humanized" mouse models for type 1 diabetes (T1D) therapy development, we previously generated directly in the NOD strain CRISPR/Cas9-mediated deletions of various combinations of murine MHC genes. These new models improved upon previously available platforms by retaining ß2-microglobulin functionality in FcRn and nonclassical MHC class I formation. As proof of concept, we generated H2-Db/H2-Kd double knockout NOD mice expressing human HLA-A*0201 or HLA-B*3906 class I variants that both supported autoreactive diabetogenic CD8+ T cell responses. In this follow-up work, we now describe the creation of 10 new NOD-based mouse models expressing various combinations of HLA genes with and without chimeric transgenic human TCRs reactive to proinsulin/insulin. The new TCR-transgenic models develop differing levels of insulitis mediated by HLA-DQ8-restricted insulin-reactive T cells. Additionally, these transgenic T cells can transfer insulitis to newly developed NSG mice lacking classical murine MHC molecules, but expressing HLA-DQ8. These new models can be used to test potential therapeutics for a possible capacity to reduce islet infiltration or change the phenotype of T cells expressing type 1 diabetes patient-derived ß cell autoantigen-specific TCRs.

Nfkbid Overexpression in Nonobese Diabetic Mice Elicits Complete Type 1 Diabetes Resistance in Part Associated with Enhanced Thymic Deletion of Pathogenic CD8 T Cells and Increased Numbers and Activity of Regulatory T Cells.

Type 1 diabetes (T1D) in both humans and NOD mice is caused by T cell-mediated autoimmune destruction of pancreatic ß cells. Increased frequency or activity of autoreactive T cells and failures of regulatory T cells (Tregs) to control these pathogenic effectors have both been implicated in T1D etiology. Due to the expression of MHC class I molecules on ß cells, CD8 T cells represent the ultimate effector population mediating T1D. Developing autoreactive CD8 T cells normally undergo extensive thymic negative selection, but this process is impaired in NOD mice and also likely T1D patients. Previous studies identified an allelic variant of Nfkbid, a NF-κB signal modulator, as a gene strongly contributing to defective thymic deletion of autoreactive CD8 T cells in NOD mice. These previous studies found ablation of Nfkbid in NOD mice using the clustered regularly interspaced short palindromic repeats system resulted in greater thymic deletion of pathogenic CD8 AI4 and NY8.3 TCR transgenic T cells but an unexpected acceleration of T1D onset. This acceleration was associated with reductions in the frequency of peripheral Tregs. In this article, we report transgenic overexpression of Nfkbid in NOD mice also paradoxically results in enhanced thymic deletion of autoreactive CD8 AI4 T cells. However, transgenic elevation of Nfkbid expression also increased the frequency and functional capacity of peripheral Tregs, in part contributing to the induction of complete T1D resistance. Thus, future identification of a pharmaceutical means to enhance Nfkbid expression might ultimately provide an effective T1D intervention approach.

T Cells from NOD-PerIg Mice Target Both Pancreatic and Neuronal Tissue.

It has become increasingly appreciated that autoimmune responses against neuronal components play an important role in type 1 diabetes (T1D) pathogenesis. In fact, a large proportion of islet-infiltrating B lymphocytes in the NOD mouse model of T1D produce Abs directed against the neuronal type III intermediate filament protein peripherin. NOD-PerIg mice are a previously developed BCR-transgenic model in which virtually all B lymphocytes express the H and L chain Ig molecules from the intra-islet-derived anti-peripherin-reactive hybridoma H280. NOD-PerIg mice have accelerated T1D development, and PerIg B lymphocytes actively proliferate within islets and expand cognitively interactive pathogenic T cells from a pool of naive precursors. We now report adoptively transferred T cells or whole splenocytes from NOD-PerIg mice expectedly induce T1D in NOD.scid recipients but, depending on the kinetics of disease development, can also elicit a peripheral neuritis (with secondary myositis). This neuritis was predominantly composed of CD4+ and CD8+ T cells. Ab depletion studies showed neuritis still developed in the absence of NOD-PerIg CD8+ T cells but required CD4+ T cells. Surprisingly, sciatic nerve-infiltrating CD4+ cells had an expansion of IFN-γ- and TNF-α- double-negative cells compared with those within both islets and spleen. Nerve and islet-infiltrating CD4+ T cells also differed by expression patterns of CD95, PD-1, and Tim-3. Further studies found transitory early B lymphocyte depletion delayed T1D onset in a portion of NOD-PerIg mice, allowing them to survive long enough to develop neuritis outside of the transfer setting. Together, this study presents a new model of peripherin-reactive B lymphocyte-dependent autoimmune neuritis.

A Hypermorphic Nfkbid Allele Contributes to Impaired Thymic Deletion of Autoreactive Diabetogenic CD8+ T Cells in NOD Mice.

In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8+ T cells recognizing pancreatic ß cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed that the common H2-Kd and/or H2-Db class I molecules expressed by this strain aberrantly lose the ability to mediate the thymic deletion of pathogenic CD8+ T cell responses through interactions with T1D susceptibility genes outside the MHC. A gene(s) mapping to proximal chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8+ T cells. Using an inducible model of thymic negative selection and mRNA transcript analyses, we initially identified an elevated Nfkbid expression variant as a likely NOD-proximal chromosome 7 region gene contributing to impaired thymic deletion of diabetogenic CD8+ T cells. CRISPR/Cas9-mediated genetic attenuation of Nfkbid expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8+ T cells. These results indicated that allelic variants of Nfkbid contribute to the efficiency of intrathymic deletion of diabetogenic CD8+ T cells. However, although enhancing thymic deletion of pathogenic CD8+ T cells, ablating Nfkbid expression surprisingly accelerated T1D onset that was associated with numeric decreases in both regulatory T and B lymphocytes in NOD mice.

HLA-B*39:06 Efficiently Mediates Type 1 Diabetes in a Mouse Model Incorporating Reduced Thymic Insulin Expression.

Type 1 diabetes (T1D) is characterized by T cell-mediated destruction of the insulin-producing ß cells of the pancreatic islets. Among the loci associated with T1D risk, those most predisposing are found in the MHC region. HLA-B*39:06 is the most predisposing class I MHC allele and is associated with an early age of onset. To establish an NOD mouse model for the study of HLA-B*39:06, we expressed it in the absence of murine class I MHC. HLA-B*39:06 was able to mediate the development of CD8 T cells, support lymphocytic infiltration of the islets, and confer T1D susceptibility. Because reduced thymic insulin expression is associated with impaired immunological tolerance to insulin and increased T1D risk in patients, we incorporated this in our model as well, finding that HLA-B*39:06-transgenic NOD mice with reduced thymic insulin expression have an earlier age of disease onset and a higher overall prevalence as compared with littermates with typical thymic insulin expression. This was despite virtually indistinguishable blood insulin levels, T cell subset percentages, and TCR Vß family usage, confirming that reduced thymic insulin expression does not impact T cell development on a global scale. Rather, it will facilitate the thymic escape of insulin-reactive HLA-B*39:06-restricted T cells, which participate in ß cell destruction. We also found that in mice expressing either HLA-B*39:06 or HLA-A*02:01 in the absence of murine class I MHC, HLA transgene identity alters TCR Vß usage by CD8 T cells, demonstrating that some TCR Vß families have a preference for particular class I MHC alleles.

Improved Murine MHC-Deficient HLA Transgenic NOD Mouse Models for Type 1 Diabetes Therapy Development.

Improved mouse models for type 1 diabetes (T1D) therapy development are needed. T1D susceptibility is restored to normally resistant NOD.ß2m-/- mice transgenically expressing human disease-associated HLA-A*02:01 or HLA-B*39:06 class I molecules in place of their murine counterparts. T1D is dependent on pathogenic CD8+ T-cell responses mediated by these human class I variants. NOD.ß2m-/--A2.1 mice were previously used to identify ß-cell autoantigens presented by this human class I variant to pathogenic CD8+ T cells and for testing therapies to attenuate such effectors. However, NOD.ß2m-/- mice also lack nonclassical MHC I family members, including FcRn, required for antigen presentation, and maintenance of serum IgG and albumin, precluding therapies dependent on these molecules. Hence, we used CRISPR/Cas9 to directly ablate the NOD H2-Kd and H2-Db classical class I variants either individually or in tandem (cMHCI-/-). Ablation of the H2-Ag7 class II variant in the latter stock created NOD mice totally lacking in classical murine MHC expression (cMHCI/II-/-). NOD-cMHCI-/- mice retained nonclassical MHC I molecule expression and FcRn activity. Transgenic expression of HLA-A2 or -B39 restored pathogenic CD8+ T-cell development and T1D susceptibility to NOD-cMHCI-/- mice. These next-generation HLA-humanized NOD models may provide improved platforms for T1D therapy development.

MHC-mismatched mixed chimerism restores peripheral tolerance of noncross-reactive autoreactive T cells in NOD mice.

Autoimmune type 1 diabetes (T1D) and other autoimmune diseases are associated with particular MHC haplotypes and expansion of autoreactive T cells. Induction of MHC-mismatched but not -matched mixed chimerism by hematopoietic cell transplantation effectively reverses autoimmunity in diabetic nonobese diabetic (NOD) mice, even those with established diabetes. As expected, MHC-mismatched mixed chimerism mediates deletion in the thymus of host-type autoreactive T cells that have T-cell receptor (TCR) recognizing (cross-reacting with) donor-type antigen presenting cells (APCs), which have come to reside in the thymus. However, how MHC-mismatched mixed chimerism tolerizes host autoreactive T cells that recognize only self-MHC-peptide complexes remains unknown. Here, using NOD.Rag1-/-BDC2.5 or NOD.Rag1-/-BDC12-4.1 mice that have only noncross-reactive transgenic autoreactive T cells, we show that induction of MHC-mismatched but not -matched mixed chimerism restores immune tolerance of peripheral noncross-reactive autoreactive T cells. MHC-mismatched mixed chimerism results in increased percentages of both donor- and host-type Foxp3+ Treg cells and up-regulated expression of programmed death-ligand 1 (PD-L1) by host-type plasmacytoid dendritic cells (pDCs). Furthermore, adoptive transfer experiments showed that engraftment of donor-type dendritic cells (DCs) and expansion of donor-type Treg cells are required for tolerizing the noncross-reactive autoreactive T cells in the periphery, which are in association with up-regulation of host-type DC expression of PD-L1 and increased percentage of host-type Treg cells. Thus, induction of MHC-mismatched mixed chimerism may establish a peripheral tolerogenic DC and Treg network that actively tolerizes autoreactive T cells, even those with no TCR recognition of the donor APCs.

Transient BAFF Blockade Inhibits Type 1 Diabetes Development in Nonobese Diabetic Mice by Enriching Immunoregulatory B Lymphocytes Sensitive to Deletion by Anti-CD20 Cotherapy.

In NOD mice and also likely humans, B lymphocytes play an important role as APC-expanding autoreactive T cell responses ultimately causing type 1 diabetes (T1D). Currently, humans at high future T1D risk can only be identified at late prodromal stages of disease indicated by markers such as insulin autoantibodies. When commenced in already insulin autoantibody+ NOD mice, continuous BAFFR-Fc treatment alone or in combination with anti-CD20 (designated combo therapy) inhibited T1D development. Despite eliciting broader B lymphocyte depletion, continuous combo therapy afforded no greater T1D protection than did BAFFR-Fc alone. As previously observed, late disease stage-initiated anti-CD20 monotherapy did not inhibit T1D, and in this study was additionally found to be associated with development of drug-blocking Abs. Promisingly, NOD mice given transient late disease stage BAFFR-Fc monotherapy were rendered T1D resistant. However, combo treatment abrogated the protective effect of transient BAFFR-Fc monotherapy. NOD mice receiving transient BAFF blockade were characterized by an enrichment of regulatory B lymphocytes that inhibit T1D development through IL-10 production, but this population is sensitive to deletion by anti-CD20 treatment. B lymphocytes from transient BAFFR-Fc-treated mice suppressed T cell proliferation to a greater extent than did those from controls. Proportions of B lymphocytes expressing CD73, an ecto-enzyme operating in a pathway converting proinflammatory ATP to anti-inflammatory adenosine, were also temporarily increased by transient BAFFR-Fc treatment, but not anti-CD20 therapy. These collective studies indicate transient BAFFR-Fc-mediated B lymphocyte depletion elicits long-term T1D protection by enriching regulatory B lymphocytes that are deleted by anti-CD20 cotherapy.

Antibiotic-associated Manipulation of the Gut Microbiota and Phenotypic Restoration in NOD Mice.

Segmented filamentous bacterium (SFB) a gram-positive, anaerobic, and intestinal commensal organism directly influences the development of Th17 helper cells in the small intestine of mice. In NOD mice, SFB colonization interferes with the development of type 1 diabetes (T1D), a T-cell-mediated autoimmune disease, suggesting that SFB may influence Th17 cells to inhibit Th1 populations associated with the anti-ß-cell immune response. This effect is a serious concern for investigators who use NOD mice for diabetes research because the expected incidence of disease decreases markedly when they are colonized by SFB. A room housing mice for T1D studies at The Jackson Laboratory was determined by fecal PCR testing to have widespread SFB colonization of multiple NOD strains after a steady decline in the incidence of T1D was noted. Rederivation of all NOD-related mouse strains was not feasible; therefore an alternative treatment using antibiotics to eliminate SFB from colonized mice was undertaken. After antibiotic treatment, soiled bedding from NOD mouse strains housed in SFB-free high-health-status production barrier rooms was used to reintroduce the gastrointestinal microbiota. Over the past 16 mo since treating the mice and disinfecting the mouse room, regular PCR testing has shown that no additional SFB colonization of mice has occurred, and the expected incidence of T1D has been reestablished in the offspring of treated mice.

Genetic and Small Molecule Disruption of the AID/RAD51 Axis Similarly Protects Nonobese Diabetic Mice from Type 1 Diabetes through Expansion of Regulatory B Lymphocytes.

B lymphocytes play a key role in type 1 diabetes (T1D) development by serving as a subset of APCs preferentially supporting the expansion of autoreactive pathogenic T cells. As a result of their pathogenic importance, B lymphocyte-targeted therapies have received considerable interest as potential T1D interventions. Unfortunately, the B lymphocyte-directed T1D interventions tested to date failed to halt ß cell demise. IgG autoantibodies marking humans at future risk for T1D indicate that B lymphocytes producing them have undergone the affinity-maturation processes of class switch recombination and, possibly, somatic hypermutation. This study found that CRISPR/Cas9-mediated ablation of the activation-induced cytidine deaminase gene required for class switch recombination/somatic hypermutation induction inhibits T1D development in the NOD mouse model. The activation-induced cytidine deaminase protein induces genome-wide DNA breaks that, if not repaired through RAD51-mediated homologous recombination, result in B lymphocyte death. Treatment with the RAD51 inhibitor 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid also strongly inhibited T1D development in NOD mice. The genetic and small molecule-targeting approaches expanded CD73+ B lymphocytes that exert regulatory activity suppressing diabetogenic T cell responses. Hence, an initial CRISPR/Cas9-mediated genetic modification approach has identified the AID/RAD51 axis as a target for a potentially clinically translatable pharmacological approach that can block T1D development by converting B lymphocytes to a disease-inhibitory CD73+ regulatory state.

Interferon-γ Limits Diabetogenic CD8+ T-Cell Effector Responses in Type 1 Diabetes.

Type 1 diabetes development in the NOD mouse model is widely reported to be dependent on high-level production by autoreactive CD4+ and CD8+ T cells of interferon-γ (IFN-γ), generally considered a proinflammatory cytokine. However, IFN-γ can also participate in tolerance-induction pathways, indicating it is not solely proinflammatory. This study addresses how IFN-γ can suppress activation of diabetogenic CD8+ T cells. CD8+ T cells transgenically expressing the diabetogenic AI4 T-cell receptor adoptively transferred disease to otherwise unmanipulated NOD.IFN-γnull , but not standard NOD, mice. AI4 T cells only underwent vigorous intrasplenic proliferation in NOD.IFN-γnull recipients. Disease-protective IFN-γ could be derived from any lymphocyte source and suppressed diabetogenic CD8+ T-cell responses both directly and through an intermediary nonlymphoid cell population. Suppression was not dependent on regulatory T cells, but was associated with increased inhibitory STAT1 to STAT4 expression levels in pathogenic AI4 T cells. Importantly, IFN-γ exposure during activation reduced the cytotoxicity of human-origin type 1 diabetes-relevant autoreactive CD8+ T cells. Collectively, these results indicate that rather than marking the most proinflammatory lymphocytes in diabetes development, IFN-γ production could represent an attempted limitation of pathogenic CD8+ T-cell activation. Thus, great care should be taken when designing possible diabetic intervention approaches modulating IFN-γ production.

B-lymphocytes expressing an Ig specificity recognizing the pancreatic ß-cell autoantigen peripherin are potent contributors to type 1 diabetes development in NOD mice.

While the autoimmune destruction of pancreatic ß-cells underlying type 1 diabetes (1D) development is ultimately mediated by T-cells in NOD mice and also likely humans, B-lymphocytes play an additional key pathogenic role. It appears expression of plasma membrane bound immunoglobulin (Ig) molecules that efficiently capture ß-cell antigens allows autoreactive B-lymphocytes bypassing normal tolerance induction processes to be the subset of antigen presenting cells most efficiently activating diabetogenic T-cells. NOD mice transgenically expressing Ig molecules recognizing antigens that are (insulin) or not (hen egg lysozyme; HEL) expressed by ß-cells have proven useful in dissecting the developmental basis of diabetogenic B-lymphocytes. However, these transgenic Ig specificities were originally selected for their ability to recognize insulin or HEL as foreign, rather than autoantigens. Thus, we generated and characterized NOD mice transgenically expressing an Ig molecule representative of a large proportion of naturally occurring islet-infiltrating B-lymphocytes in NOD mice recognizing the neuronal antigen peripherin. Transgenic peripherin autoreactive B-lymphocytes infiltrate NOD pancreatic islets, acquire an activated proliferative phenotype, and potently support accelerated T1D development. These results support the concept of neuronal autoimmunity as a pathogenic feature of T1D, and targeting such responses could ultimately provide an effective disease intervention approach.

MHC-mismatched mixed chimerism mediates thymic deletion of cross-reactive autoreactive T cells and prevents insulitis in nonobese diabetic mice.

Type 1 diabetic NOD mice have defects in both thymic negative selection and peripheral regulation of autoreactive T cells, and induction of mixed chimerism can effectively reverse these defects. Our recent studies suggest that MHC-mismatched mixed chimerism mediates negative selection of autoreactive thymocytes in wild-type NOD and TCR-transgenic NOD.Rag1(+/+).BDC2.5 mice. However, it remains unknown how mismatched I-A(b) MHC class II can mediate deletion of autoreactive T cells positively selected by I-A(g7). In the present study, we directly tested the hypothesis that mismatched MHC class II in mixed chimeras mediates deletion of cross-reactive autoreactive thymocytes. We first identify that transgenic BDC2.5 T cells from NOD.Rag1(+/+).BDC2.5 but not NOD.Rag1(-/-).BDC2.5 mice possess cross-reactive TCRs with endogenous TCRα-chains; MHC-mismatched H-2(b) but not matched H-2(g7) mixed chimerism mediates thymic deletion of the cross-reactive transgenic T cells in NOD.Rag1(+/+).BDC2.5 mice. Second, by transplanting T cell-depleted (TCD) bone marrow (BM) cells from NOD.Rag1(+/+).BDC2.5 or NOD.Rag1(-/-).BDC2.5 mice into lethally irradiated MHC-mismatched H-2(b) C57BL/6 or MHC-matched congenic B6.H-2(g7) recipients, we demonstrate that NOD.Rag1(+/+).BDC2.5 BM-derived cross-reactive transgenic T cells, but not NOD.Rag1(-/-).BDC2.5 BM-derived non-cross-reactive transgenic T cells, can be positively selected in MHC-mismatched H-2(b) thymus. Third, by cotransplanting NOD.Rag1(+/+).BDC2.5 TCD BM cells with BM cells from MHC-mismatched T cell-deficient C57BL/6 mice into lethally irradiated MHC-matched B6.H-2(g7) recipients, we demonstrate that thymic deletion of the cross-reactive transgenic T cells is dependent on MHC-mismatched donor BM-derived APCs but not on donor BM-derived T cells. Taken together, our studies indicate that MHC-mismatched mixed chimerism can mediate thymic deletion of cross-reactive autoreactive T cells that express more than one TCR.

B7H1/CD80 interaction augments PD-1-dependent T cell apoptosis and ameliorates graft-versus-host disease.

Interactions of B7H1 (programmed death ligand 1 [PD-L1]) with its two ligands, PD-1 and CD80, on T cells play a pivotal role in controlling T cell activation, proliferation, anergy, and apoptosis. However, the interactions between the two pathways remain unknown. Using an alloimmune response model of graft-versus-host disease (GVHD), we report in this study that: 1) Comparison of proliferation and apoptosis of wild-type (WT) and PD-1(-/-)CD4(+) conventional T (Tcon) cells in WT and B7H1(-/-) recipients revealed that B7H1/CD80 interaction per se augments T cell proliferation, and this interaction augments T cell apoptosis mediated by B7H1/PD-1 interaction. This observation was recapitulated in an in vitro MLR assay. 2) Specific blockade of the B7H1/CD80 axis by anti-B7H1 mAb reduces WT-alloreactive Tcon cell proliferation, IL-2 production, expression of PD-1, and apoptosis, resulting in worsening GVHD. In contrast, specific blockade of B7H1/CD80 interaction reduces donor PD-1(-/-) Tcon cell proliferation without an impact on apoptosis, resulting in ameliorating GVHD. 3) B7H1 fused to an Ig Fc domain (B7H1-Ig), when produced in vivo by hydrodynamic injection of B7H1-Ig plasmid, ameliorates GVHD by augmenting proliferation and apoptosis of WT- alloreactive Tcon cells. Conversely, B7H1-Ig treatment has no impact on apoptosis but augments PD-1(-/-) T cell proliferation and worsens GVHD. These results indicate that B7H1/CD80 interaction augments Tcon cell proliferation, IL-2 production, and expression of PD-1, which leads to increased apoptosis mediated by the B7H1/PD-1 pathway. Additionally, by engaging both PD-1 and CD80, B7H1-Ig can be a powerful therapeutic reagent for downregulating the T cell immune response.

MHC-mismatched chimerism is required for induction of transplantation tolerance in autoimmune nonobese diabetic recipients.

In nonautoimmune recipients, induction of mixed and complete chimerism with hematopoietic progenitor cells from MHC (HLA)-matched or -mismatched donors are effective approaches for induction of organ transplantation immune tolerance in both animal models and patients. But it is still unclear whether this is the case in autoimmune recipients. With the autoimmune diabetic NOD mouse model, we report that, although mixed and complete MHC-mismatched chimerism provide immune tolerance to donor-type islet and skin transplants, neither mixed nor complete MHC-matched chimerism does. The MHC-mismatched chimerism not only tolerizes the de novo developed, but also the residual pre-existing host-type T cells in a mismatched MHC class II-dependent manner. In the MHC-mismatched chimeras, the residual host-type peripheral T cells appear to be anergic with upregulation of PD-1 and downregulation of IL-7Rα. Conversely, in the MHC-matched chimeras, the residual host-type peripheral T cells manifest both alloreactivity and autoreactivity; they not only mediate insulitis and sialitis in the recipient, but also reject allogeneic donor-type islet and skin grafts. Interestingly, transgenic autoreactive BDC2.5 T cells from Rag1(+/+), but not from Rag1(-/-), NOD mice show alloreactivity and mediate both insulitis and rejection of allografts. Taken together, MHC-mismatched, but not MHC-matched, chimerism can effectively provide transplantation immune tolerance in autoimmune recipients.

Administration of anti-CD20 mAb is highly effective in preventing but ineffective in treating chronic graft-versus-host disease while preserving strong graft-versus-leukemia effects.

Chronic graft-versus-host disease (cGVHD) is an autoimmune-like syndrome, and donor B cells play important roles in augmenting its pathogenesis. B cell-depleting anti-CD20 mAb has been administered before or after cGVHD onset for preventing or treating cGVHD in the clinic. Although administration before onset appeared to be more effective, the effect is variable and sometimes minimal. Here, we used 2 mouse cGVHD models to evaluate the preventive and therapeutic effect of anti-CD20 mAb. With the model of DBA/2 donor to MHC-matched BALB/c recipient, 1 intravenous injection of anti-CD20 mAb (40 mg/kg) the following day or on day 7 after hematopoietic cell transplantation when serum autoantibodies were undetectable effectively prevented induction of cGVHD and preserved a strong graft-versus-leukemia (GVL) effect. The separation of GVL effect from GVHD was associated with a significant reduction of donor CD4(+) T cell proliferation and expansion and protection of host thymic medullary epithelial cells. Anti-CD20 mAb administration also prevented expansion of donor T cells and induction of cGVHD in another mouse model of C57BL/6 donor to MHC-mismatched BALB/c recipients. In contrast, administration of anti-CD20 mAb after GVHD onset was not able to effectively deplete donor B cells or ameliorate cGVHD in either model. These results indicate that administration of anti-CD20 mAb before signs of cGVHD can prevent induction of autoimmune-like cGVHD while preserving a GVL effect; there is little effect if administered after cGVHD onset. This provides new insights into clinical prevention and therapy of cGVHD with B cell-depleting reagents.

Depletion of host CCR7(+) dendritic cells prevented donor T cell tissue tropism in anti-CD3-conditioned recipients.

We reported previously that anti-CD3 mAb treatment before hematopoietic cell transplantation (HCT) prevented graft-versus-host disease (GVHD) and preserved graft-versus-leukemia (GVL) effects in mice. These effects were associated with downregulated donor T cell expression of tissue-specific homing and chemokine receptors, marked reduction of donor T cell migration into GVHD target tissues, and deletion of CD103(+) dendritic cells (DCs) in mesenteric lymph nodes (MLN). MLN CD103(+) DCs and peripheral lymph node (PLN) DCs include CCR7(+) and CCR7(-) subsets, but the role of these DC subsets in regulating donor T cell expression of homing and chemokine receptors remain unclear. Here, we show that recipient CCR7(+), but not CCR7(-), DCs in MLN induced donor T cell expression of gut-specific homing and chemokine receptors in a retinoid acid-dependent manner. CCR7 regulated activated DC migration from tissue to draining lymph node, but it was not required for the ability of DCs to induce donor T cell expression of tissue-specific homing and chemokine receptors. Finally, anti-CD3 treatment depleted CCR7(+) but not CCR7(-) DCs by inducing sequential expansion and apoptosis of CCR7(+) DCs in MLN and PLN. Apoptosis of CCR7(+) DCs was associated with DC upregulation of Fas expression and natural killer cell but not T, B, or dendritic cell upregulation of FasL expression in the lymph nodes. These results suggest that depletion of CCR7(+) host-type DCs, with subsequent inhibition of donor T cell migration into GVHD target tissues, can be an effective approach in prevention of acute GVHD and preservation of GVL effects.