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CD200 is known as an immune checkpoint molecule that inhibits innate immune cell activation. Using a head and neck squamous cell carcinoma (HNSCC) model, we sought to determine whether localized delivery of adenovirus-expressing sCD200R1-Ig, the soluble extracellular domain of CD200R1, enhances antitumor immunity. Mouse-derived bone marrow cells and M1/M2-like macrophages were cocultured with tumor cells and analyzed for macrophage polarization. As an in vivo model, C57BL/6 mice were subcutaneously injected with MEER/CD200High cells, CD200-overexpressing mouse HNSCC cells. Adenovirus-expressing sCD200R1-Ig (Ad5sCD200R1) was designed, and its effect was tested. Components in the tumor-immune microenvironment (TIME) were quantified using flow cytometry. CD200 promoted tumor growth and induced the expression of immune-related genes, especially macrophage colony-stimulating factor (M-CSF). Interestingly, CD200 induced M2-like polarization both in vitro and in vivo. Consequently, CD200 recruited more regulatory T (Treg) cells and fewer CD8+ effector T cells. These effects were effectively abolished by local injection of Ad5sCD200R1. These protumor effects of CD200 were driven through the β-catenin/NF-κB/M-CSF axis. CD200 upregulated PD-L1, and the combined targeting of CD200 and PD-1 thus showed synergy. The immune checkpoint CD200 upregulated immune-related genes through β-catenin signaling, reprogrammed the TIME, and exerted protumor effects. Ad5sCD200R1 injection could be an effective targeted strategy to enhance antitumor immunoediting., Graphical abstract, The immune checkpoint CD200 upregulated immune-related genes through β-catenin signaling, reprogrammed the TIME, and exerted protumor effects. Adenovirus expressing sCD200R1-Ig, the soluble extracellular domain of CD200R1 (Ad5sCD200R1), was designed. M1/M2-like polarization by CD200 was abolished by sCD200R1-Ig through modulation of immune-related genes, especially M-CSF.

Significance This study reveals that the tryptophan-degrading reaction catalyzed by indoleamine 2,3-dioxygenase 1 (IDO1) is linked to reactive oxygen species (ROS) scavenging in Gr-1+CD11b+ myeloid cells. The IDO1-mediated ROS scavenging promotes myeloid-derived suppressor cell characteristics in Gr-1+CD11b+ cells, suppressing their differentiation into proinflammatory neutrophils. These results could explain the increased lethality in graft-versus-host disease as well as the enhanced proinflammatory and reduced regulatory T cell responses after transplantation of IDO1-deficient bone marrow cells. Our findings provide a mechanistic insight into the immune-modulatory roles of IDO1., Tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) also has an immunological function to suppress T cell activation in inflammatory circumstances, including graft-versus-host disease (GVHD), a fatal complication after allogeneic bone marrow transplantation (allo-BMT). Although the mononuclear cell expression of IDO1 has been associated with improved outcomes in GVHD, the underlying mechanisms remain unclear. Herein, we used IDO-deficient (Ido1−/−) BMT to understand why myeloid IDO limits the severity of GVHD. Hosts with Ido1−/− BM exhibited increased lethality, with enhanced proinflammatory and reduced regulatory T cell responses compared with wild type (WT) allo-BMT controls. Despite the comparable expression of the myeloid-derived suppressor cell (MDSC) mediators, arginase-1, inducible nitric oxide synthase, and interleukin 10, Ido1−/− Gr-1+CD11b+ cells from allo-BMT or in vitro BM culture showed compromised immune-suppressive functions and were skewed toward the Ly6ClowLy6Ghi subset, compared with the WT counterparts. Importantly, Ido1−/−Gr-1+CD11b+ cells exhibited elevated levels of reactive oxygen species (ROS) and neutrophil numbers. These characteristics were rescued by human IDO1 with intact heme-binding and catalytic activities and were recapitulated by the treatment of WT cells with the IDO1 inhibitor L1-methyl tryptophan. ROS scavenging by N-acetylcysteine reverted the Ido1−/−Gr-1+CD11b+ composition and function to an MDSC state, as well as improved the survival of GVHD hosts with Ido1−/− BM. In summary, myeloid-derived IDO1 enhances GVHD survival by regulating ROS levels and limiting the ability of Gr-1+CD11b+ MDSCs to differentiate into proinflammatory neutrophils. Our findings provide a mechanistic insight into the immune-regulatory roles of the metabolic enzyme IDO1.

IntroductionChimeric antigen receptor (CAR) T-cells have been developed recently, producing impressive outcomes in patients with hematologic malignancies. However, there is no standardized method for cell trafficking and in vivo CAR T-cell monitoring. We assessed the feasibility of real-time in vivo89Zr-p-Isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS, DFO) labeled CAR T-cell trafficking using positron emission tomography (PET).ResultsThe 89Zr-DFO radiolabeling efficiency of Jurkat/CAR and human peripheral blood mononuclear cells (hPBMC)/CAR T-cells was 70–79%, and cell radiolabeling activity was 98.1–103.6 kBq/106 cells. Cell viability after radiolabeling was >95%. Compared with unlabeled cells, cell proliferation was not significantly different during the early period after injection; however, the proliferative capacity decreased over time (p = 0.02, day 7 after labeling). IL-2 or IFN-γ secretion was not significantly different between unlabeled and labeled CAR T-cells. PET/magnetic resonance images in the xenograft model showed that most of the 89Zr-DFO-labeled Jurkat/CAR T-cells were distributed in the lung (24.4% ± 3.4%ID) and liver (22.9% ± 5.6%ID) by 1 hour after injection. The cells gradually migrated from lung to the liver and spleen by day 1, and remained stably until day 7 (on day 7: lung 3.9% ± 0.3%ID, liver 36.4% ± 2.7%ID, spleen 1.4% ± 0.3%ID). No significant accumulation of labeled cells was identified in tumors. A similar pattern was observed in ex vivo biodistributions on day 7 (lung 3.0% ± 1.0%ID, liver 19.8% ± 2.2%ID, spleen 2.3% ± 1.7%ID). 89Zr-DFO-labeled hPBMC/CAR T-cells showed the similar distribution on serial PET images as Jurkat/CAR T-cells. The distribution of CAR T-cells was cross-confirmed by flow cytometry, Alu polymerase chain reaction, and immunohistochemistry.ConclusionUsing PET imaging of 89Zr-DFO-labeled CAR T-cells, real time in vivo cell trafficking is feasible. It can be used to investigate cellular kinetics, initial in vivo biodistribution, and the safety profile in future CAR T-cell development.

To understand the role of myeloid differentiation factor 88 (MyD88) expressed by donor bone marrow (BM) in the pathophysiology of graft-vs.-host disease (GVHD), we investigated the effects of transplantation of MyD88-deficient T cell-depleted BM (MyD88KO TCD-BM) on the severity of GVHD. Transplantation with MyD88KO TCD-BM aggravated GVHD; serious gut damage was evident, with high infiltration of T cells into the intestines of recipients and markedly reduced expansion of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs). MDSCs from MyD88KO mice were defective in inducing donor T-cell apoptosis and inhibiting T-cell proliferation. Supplementation of transplanted mice with MDSCs from wild-type mice, but not MyD88KO mice, attenuated GVHD severity with reduced intestinal T-cell infiltration in MyD88KO TCD-BM recipients. Pretreatment of BM donors with lipopolysaccharide to increase MDSC levels and MyD88 transcription in the TCD-BM transplant alleviated GVHD severity and intestinal T-cell infiltration. The T cell/MDSC ratios were correlated with intestinal GVHD severity in both animal models and human patients. This study indicates that MyD88-dependent MDSC expansion from donor BM is critical for protection against fatal intestinal GVHD.

Graft-versus-host disease (GVHD), a life-threatening complication after bone marrow transplantation (BMT), is induced by activation of alloreactive donor T cells. Our previous study demonstrated that transplantation of myeloid differentiation factor 88 (MyD88)-deficient knockout (KO) bone marrow (BM) resulted in aggravation of GVHD. Here, to understand the cellular mechanism, we performed longitudinal in vivo imaging and flow cytometric analyses followed by transcriptome and functional examination of donor MyD88-KO BM progenies in GVHD hosts, using a major histocompatibility complex-matched but minor histocompatibility antigen-mismatched C57BL/6→BALB.B model. In GVHD hosts with MyD88-KO BMT, donor BM-derived CD11b+Gr-1+ cells were found to undergo cell death, a fate significantly different from the explosive expansion shown by the wild type (WT) counterparts, and also from the moderate expansion of the WT or MyD88-KO BM-derived cells in non-GVHD hosts. It was also revealed that MyD88-KO CD11b+Gr-1+ cells preferred differentiation into CD11c+ dendritic cells (DCs) to expansion as myeloid-derived suppressor cells in GVHD hosts or in high inflammatory in vitro conditions. These CD11c+ DCs comprised the majority of MyD88-KO CD11b+Gr-1+ apoptotic cells in GVHD hosts. Their ability to cross-present alloantigens of host origin contributed to the enhancement of T cell alloreactivity, causing GVHD aggravation and eventually death through the killing function of activated T cells. These results provide insights into the roles of MyD88 in myelopoiesis of donor BM and the protective effects in GVHD hosts, helpful information for development of a strategy to control GVHD.

Acute graft-versus-host-disease (GVHD) is characterized by selective damage to the liver, the skin, and the gastrointestinal tract. Following allogeneic hematopoietic stem cell transplantation, donor bone marrow (BM) cells repopulate the immune system of the recipient. We previously demonstrated that the acute intestinal GVHD (iGVHD) mortality rate was higher in MyD88-deficient BM recipients than that in the control BM recipients. In the present study, the role of MyD88 (expressed by donor BM) in the pathophysiology of hepatic GVHD (hGVHD) was examined. Unlike iGVHD, transplantation with MyD88-deficient T-cell depleted (TCD) BM attenuated hGVHD severity and was associated with low infiltration of T cells into the liver of the recipients. Moreover, GVHD hosts, transplanted with MyD88-deficient TCD BM, exhibited markedly reduced expansion of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) in the liver. Adoptive injection of the MDSC from wild type mice, but not MyD88-deficient mice, enhanced hepatic T cell infiltration in the MyD88-deficient TCD BM recipients. Pre-treatment of BM donors with LPS increased MDSC levels in the liver of allogeneic wild type BM recipients. In conclusion, hGVHD and iGVHD may occur through various mechanisms based on the presence of MyD88 in the non-T cell compartment of the allograft.

Whether hematopoietic cell-restricted distribution of antigens affects the degree of thymic negative selection has not been investigated in detail. Here, we show that T cells specific for hematopoietic cell-restricted antigens (HRA) are not completely deleted in the thymus, using the mouse minor histocompatibility antigen H60, the expression of which is restricted to hematopoietic cells. As a result, low avidity T cells escape from thymic deletion. This incomplete thymic deletion occurs to the T cells developing de novo in the thymus of H60-positive recipients in H60-mismatched bone marrow transplantation (BMT). H60-specific thymic deletion escapee CD8+ T cells exhibit effector differentiation potentials in the periphery and contribute to graft-versus-leukemia effects in the recipients of H60-mismatched BMT, regressing H60+ hematological tumors. These results provide information essential for understanding thymic negative selection and developing a strategy to treat hematological tumors., Antigen distribution patterns affect thymic negative selection. Here, the authors show that incomplete thymic negative selection occurs to T cells for hematopoietic cell-restricted antigen H60 in mice, driving graft-versus-leukemia after H60-mismatched bone marrow transplantation.

CD8+ T cells activated without CD4+ T-cell help are impaired in memory expansion. To understand the underlying cellular mechanism, here we track the dynamics of helper-deficient CD8+ T-cell response to a minor histocompatibility antigen by phenotypic and in vivo imaging analyses. Helper-deficient CD8+ T cells show reduced burst expansion, rapid peripheral egress, delayed antigen clearance and continuous activation, and are eventually exhausted. Contrary to the general consensus that CD4 help encodes memory programmes in CD8+ T cells and helper-deficient CD8+ T cells are abortive, these cells can differentiate into effectors and memory precursors. Importantly, accelerating antigen clearance or simply increasing the burst effector size enables generation of memory cells by CD8+ T cells, regardless of CD4 help. These results suggest that the memory programme is CD8+ T-cell-intrinsic, and provide insight into the role of CD4 help in CD8+ T-cell responses., Persistent antigen stimulation can cause exhaustion and unresponsiveness of CD8 cells, impairing the immune response. Here the authors show that increasing the number of CD8 cells, decreasing the antigen load or providing CD4 help can overcome the exhaustion and establish a memory response.

To understand the cellular mechanism underlying the therapeutic effects exerted by hematopoietic stem cell transplantation in the repair of tissue damage, we investigated the in vivo dynamics of bone marrow (BM) lineage-negative (Lin−) cells transplanted into mice with hyper sensitivity dermatitis. Longitudinal in vivo imaging and flow cytometry analyses revealed that Lin− cells home directly to inflamed skin within 6 h, where they undergo extensive expansion with the peak on day 14 post-transplantation and preferential differentiation into CD11b+Ly6GintLy6C+ cells by day 7. Cells with phenotypic profiles of neutrophils, macrophages and DCs appeared in inflamed skin on day 14. Progenies of transplanted Lin− cells showed similar kinetics of expansion and myeloid differentiation in BM. However, differentiation into CD11b+Ly6GintLy6C+ cells in the inflamed skin on day 7 was more skewed toward CD115+ cells (≥60%) with immune suppressive function and higher expression levels of iNOS, arginase and IL-10, compared with those in the BM. Transplantation of Lin− cells reduced the levels of Cd3 transcript and CD4+/CD8+ cells in inflamed skin. These results demonstrate differentiation of transplanted Lin− cells into myeloid-derived suppressor cells in inflamed skin to be the basis of the alleviation of skin inflammation after Lin− cell transplantation.

Myeloid differentiation 88 (MyD88) is an adaptor molecule in toll-like receptor-associated signaling. To examine the effect of MyD88-deficiency in myeloid cell differentiation under inflammatory condition, we transplanted bone marrow (BM) cells isolated from MyD88-deficient C57BL/6 (B6) mice together with B6 T cells into MHC-matched allogeneic BALB.B strain mice. This transplantation induced more serious graft-versus-host disease in the BALB.B BM recipients, compared with the GVHD induced in the control BALB.B mice which received wild type B6 BM and T cells. The aggravation of GVHD was associated with impaired expansion of CD11b+Gr1+ myeloid derived suppressor cells (MDSCs) from the MyD88-deficient BM cells during the GVHD development. Instead of MDSC expansion, myeloid cells from MyD88-deficient BM cells showed enhanced apoptosis and dendritic cell (DC) differentiation, which resulted in enhancement of activation of allo-reactive B6 T cells. These results provide information helpful for the understanding of the role of MyD88 in myeloid cell differentiation and development of strategy to reduce the GVHD severity.

An immune check point molecule PD-1 is associated with CD8 T cell exhaustion in tumor-infiltrating and chronic infection situations. Previously, we have demonstrated that CD8 T cells specific for a transplantation antigen, minor histocompatibility antigen H60, are exhausted when primed under CD4 helper-deficient conditions, showing PD-1hi phenotype, and the exhaustion is ascribed to inefficient antigen-clearance. We examined whether the helpless CD8 T cells could respond to re-challenge with H60 in absence of PD-1-mediated signaling using PD-1-deficient mice. Upon re-challenge with H60 of the PD-1-deficient mice, which had been primed against H60 under helper-deficient conditions, the H60-specific CD8 T cells expanded vigorously, exhibiting a memory-like response. Despite the restored proliferative potential to the antigen-re-encounter, the H60-specific PD-1-deficient CD8 T cells did not generate CD44hiCD6Lhicentral memory T cells under helper-deficient priming condition. They exhibited an exhausted status, with an increased CXCR3+KLRG1+ Tint cell fraction. When transferred to naïve hosts, PD-1-deficient helpless CD8 T cells showed reduced generation of central memory cells compared to their wild type counterparts. These results suggest that PD-1 signaling influences fate of effector CD8 T cells and provide information regarding CD8 T cell memory generation.

Myeloid differentiation 88 (MyD88), an adaptor molecule in TLR-associated signaling, has recently been known to play a role in expansion/activation of CD11b+Gr-1+ myeloid-derived suppressor cells. To understand role of MyD88 expressed by donor-originating myeloid cells in GVHD development, we investigated dynamics of T cell-depleted bone marrow (TCD-BM) cells and gene-expressions of CD11b+Gr-1+ cells originated from Myd88-/- donor mice after transplantation into allogeneic BALB.B hosts with WT T cells. The allogeneic recipients of Myd88-/- TCD-BM and WT T cells showed aggravated GVHD, with significant loss of CD11b+Gr-1+ population, compared with BALB.B recipients of WT TCD-BM and T cells. Impaired expansion of Myd88-/- TCD-BM cells in BALB.B GVHD hosts was confirmed via in vivo tracking analysis. However, their expansion was intact in syngeneic C57BL/6 recipients, indicating that impaired CD11b+Gr-1+ cell expansion from Myd88-/- TCD-BM cells was specific to GVHD circumstance. In transcriptome analysis, CD11b+Gr-1+ cells of MyD88-/- TCD-BM origin exhibited higher expression of genes related to apoptosis and antigen-presentation than did those of WT TCD-BM origin. These results suggest that impaired expansion of the CD11b+Gr-1+ cells from Myd88-/- TCD-BM cells in allogeneic hosts is related to enhanced apoptosis in combination with enhanced differentiation into DC-like cells in the presence of activated allo-reactive T cells, and provide insight into the control of GVHD.

Indoleamine 2,3-dioxygenase-1 (IDO-1) is a tryptophan metabolic enzyme expressed by various types of cells including dendritic cells, myeloid-derived suppressor cells, and intestinal epithelial cells. The enzyme is also well known for immune suppressive function, while the underlying mechanism is still obscure. To understand cellular mechanism underlying the suppressive role of IDO-1, especially expressed by donor-derived myeloid cells in generation of graft-versus-host disease (GVHD), we transplanted T cell-depleted (TCD) BM cells from Ido-1-/- mice together with wild type (WT) T cells on C57BL/6 (B6) background into MHC-matched allogeneic BALB.B mice. Compared with BALB.B hosts that received WT TCD-BM and T cells, those of Ido-1-/- TCD-BM and WT T cells showed exacerbated GVHD. T cell frequencies in blood and target organs, and their productions of effector cytokines (IFN-γ- and IL-17) were enhanced in the recipients of Ido-1-/- TCD-BM, compared with those of WT TCD-BM. Generation of CD11b+Gr-1+ cells, especially of Ly6GhiLy6lo cells, was also enhanced in the formers. The CD11b+Gr-1+ cells originated from Ido-1-/- TCD-BM exerted lower suppressive activity over proliferation and cytokine production of co-incubated allo-reactive T cells. Proportions of CD244- neutrophils within the Ly6GhiLy6lo population of Ido-1-/- TCD-BM origin were significantly higher than that of WT TCD-BM. These results demonstrate implication of IDO-1 function in neutrophil generation.

Minor histocompatibility antigens are MHC-bound peptides and contribute to the generation of allo-responses after allogeneic transplantation. H60 is a dominant minor H antigen that induces a strong CD8 T-cell response in MHC-matched allogeneic transplantation settings. Here, we report establishment of a TCR transgenic mouse line named J15, wherein T cells express TCRs specific for H60 in complex with H-2K(b), and different fates of the thymocytes expressing J15 TCRs in various thymic antigenic environments. Thymocytes expressing the J15 TCRs were positively selected and differentiated into CD8(+) single positive (SP) cells in the thymus of C57BL/6 mice, wherein the cognate antigen H60 is not expressed. However, thymocytes were negatively selected in thymus tissue where H60 was transgenically expressed under the control of the actin promoter, with double-positive stages of cells being deleted. Despite the ability of the H60H peptide (LTFHYRNL) variant to induce cytotoxic activity from H60-specific CTL lines at ~50% of the activity induced by normal H60 peptides (LTFNYRNL), J15-expressing thymocytes were positively selected in the thymus where the variant H60H was transgenically expressed. These results demonstrate that a single amino-acid change in the H60 epitope peptide influences the fate of thymocytes expressing the cognate TCR.

To elucidate the fundamental role of CD4 help in CD8 T cell response, the dynamics of the CD8 T cells participating in the help-deficient response was tracked longitudinally via in vivo imaging and phenotypic analyses. Help-deficient CD8 T cells underwent intact effector differentiation with fast kinetics, albeit insignificant burst expansion. However, they failed to effectively eliminate the antigen, which rendered them to be activated continuously, defective in memory cell differentiation, and exhausted eventually. Although PD-1-deficiency rescued the exhaustion, it could not restore the memory differentiation. Instead, simply increasing the numbers of effector CD8 T cells or accelerating antigen clearance enabled efficient memory generation even in the absence of help. These results demonstrate that memory program is intrinsic to CD8 T cells, not encoded by CD4 help, and provide insight into the role of CD4 help, permitting a better understanding of CD8 T cell differentiation and immunity.

MHC complexes with peptides from minor histocompatibility (H) antigens which are naturally occurring polymorphic proteins are recognized by T cells. The mismatches at the Minor H loci induce allo-reactive T cell activation, leading to graft-rejection, graft-versus-host disease, or graft-versus-leukemia under MHC-matched transplantation circumstances. T cell response specific for H60 has been characterized as a dominant. In order to investigate the effect of antigenic expression pattern on the outcome of bone marrow transplantation, we generated a TCR transgenic mouse (named J15), of which specificity is H60 peptide in complexes with H2-Kb, with TCR α and β genes were originated a H60-specific CTL clone. The thymocytes in the J15 mice mostly developed to CD8 T lineage and showed proliferation in response to H60-stimulation. The thymocytes bearing J15 TCR were deleted in the mice where H60 are expressed in hematopoietic cell-restricted pattern and ubiquitously, that is in the F1 mice after crossing of J15 to H60 congenic (J15TgH60C/-) or to H60 transgenic mouse (J15TgAct-H60Tg), respectively. But the deletion timing was different during the double negative stages between the two F1 mice. The different deletion timing was observed when the bone marrow chimeras were generated with H60 congenic mice and with Act-H60Tg mice as hosts into which bone marrow from the J15 were transferred. These results demonstrate that antigen expression pattern influences the thymic selection timing.