Mixed Chimeras Research Articles

Multiple Inflammatory Cytokines Converge To Regulate CD8+ T Cell Expansion and Function during Tuberculosis

The differentiation of effector CD8(+) T cells is a dynamically regulated process that varies during different infections and is influenced by the inflammatory milieu of the host. In this study, we define three signals regulating CD8(+) T cell responses during tuberculosis by focusing on cytokines known to affect disease outcome: IL-12, type I IFN, and IL-27. Using mixed bone marrow chimeras, we compared wild-type and cytokine receptor knockout CD8(+) T cells within the same mouse following aerosol infection with Mycobacterium tuberculosis. Four weeks postinfection, IL-12, type 1 IFN, and IL-27 were all required for efficient CD8(+) T cell expansion in the lungs. We next determined if these cytokines directly promote CD8(+) T cell priming or are required only for expansion in the lungs. Using retrogenic CD8(+) T cells specific for the M. tuberculosis Ag TB10.4 (EsxH), we observed that IL-12 is the dominant cytokine driving both CD8(+) T cell priming in the lymph node and expansion in the lungs; however, type I IFN and IL-27 have nonredundant roles supporting pulmonary CD8(+) T cell expansion. Thus, IL-12 is a major signal promoting priming in the lymph node, but a multitude of inflammatory signals converge in the lung to promote continued expansion. Furthermore, these cytokines regulate the differentiation and function of CD8(+) T cells during tuberculosis. These data demonstrate distinct and overlapping roles for each of the cytokines examined and underscore the complexity of CD8(+) T cell regulation during tuberculosis.

Anti-CD45 radioimmunotherapy without TBI before transplantation facilitates persistent haploidentical donor engraftment

AbstractMany patients with hematologic malignancies cannot tolerate hematopoietic cell transplantation (HCT), whereas others may not have a compatible human leukocyte antigen–matched donor. To overcome these limitations, we optimized a conditioning regimen employing anti-CD45 radioimmunotherapy (RIT) replacing total body irradiation (TBI) before haploidentical HCT in a murine model. Mice received 200 to 400 μCi 90Y-anti-CD45 antibody (30F11), with or without fludarabine (5 days starting day –8), with cyclophosphamide (CY; days –2 and +2) for graft-versus-host disease prophylaxis, and 1.5 × 107 haploidentical donor bone marrow cells (day 0). Haploidentical bone marrow transplantation (BMT) with 300 μCi 90Y-anti-CD45 RIT and CY, without TBI or fludarabine, led to mixed chimeras with 81.3 ± 10.6% mean donor origin CD8+ cells detected 1 month after BMT, and remained stable (85.5 ± 11% mean donor origin CD8+ cells) 6 months after haploidentical BMT. High chimerism levels were induced across multiple hematopoietic lineages 28 days after haploidentical BMT with 69.3 ± 14.1%, 75.6 ± 20.2%, and 88.5 ± 11.8% CD3+ T cells, B220+ B cells, and CD11b+ myeloid cells, respectively. Fifty percent of SJL leukemia-bearing mice treated with 400 μCi 90Y-DOTA-30F11, CY, and haploidentical BMT were cured and lived >200 days. Mice treated with 200 μCi 90Y-DOTA-30F11 had a median overall survival of 73 days, while untreated leukemic mice had a median overall survival of 34 days (P < .001, Mantel-Cox test). RIT-mediated haploidentical BMT without TBI may increase treatment options for aggressive hematologic malignancies.

T-cell–intrinsic Tif1α/Trim24 regulates IL-1R expression on TH2 cells and TH2 cell-mediated airway allergy

There is a paucity of new therapeutic targets to control allergic reactions and forestall the rising trend of allergic diseases. Although a variety of immune cells contribute to allergy, cytokine-secreting αβ(+)CD4(+) T-helper 2 (TH2) cells orchestrate the type-2-driven immune response in a large proportion of atopic asthmatics. To identify previously unidentified putative targets in pathogenic TH2 cells, we performed in silico analyses of recently published transcriptional data from a wide variety of pathogenic TH cells [Okoye IS, et al. (2014) Proc Natl Acad Sci USA 111(30):E3081-E3090] and identified that transcription intermediary factor 1 regulator-alpha (Tif1α)/tripartite motif-containing 24 (Trim24) was predicted to be active in house dust mite (HDM)- and helminth-elicited Il4(gfp+)αβ(+)CD4(+) TH2 cells but not in TH1, TH17, or Treg cells. Testing this prediction, we restricted Trim24 deficiency to T cells by using a mixed bone marrow chimera system and found that T-cell-intrinsic Trim24 is essential for HDM-mediated airway allergy and antihelminth immunity. Mechanistically, HDM-elicited Trim24(-/-) T cells have reduced expression of many TH2 cytokines and chemokines and were predicted to have compromised IL-1-regulated signaling. Following this prediction, we found that Trim24(-/-) T cells have reduced IL-1 receptor (IL-1R) expression, are refractory to IL-1β-mediated activation in vitro and in vivo, and fail to respond to IL-1β-exacerbated airway allergy. Collectively, these data identify a previously unappreciated Trim24-dependent requirement for IL-1R expression on TH2 cells and an important nonredundant role for T-cell-intrinsic Trim24 in TH2-mediated allergy and antihelminth immunity.

Unmanipulated, G-CSF Mobilized Peripheral Blood Stem Cells As Graft Source for Non-Myeloablative Haploidentical Transplantation: Fast Engraftment, No Evidence of Graft Rejection, and Low Incidence of Graft-Versus-Host Disease

Haploidentical bone marrow transplantation using non-myeloablative conditioning and high-dose, post-transplant cyclophosphamide (Cy) has been shown to be a feasible approach for patients lacking an HLA identical donor with acceptable rates of acute and chronic Graft-versus-Host-Disease (GVHD); in the attempt to reduce the associated risk of graft failure and relapse incidence we decided to apply the same conditioning regimen using unmanipulated mobilized peripheral blood stem cells (PBSC). From June 2010 to December 2014, 31 consecutive patients affected by high-risk hematological malignancies, median age 51 years (range 19-70) (3 Acute Myeloid Leukemia, 6 Acute Lymphoblastic Leukemia, 9 Non-Hodgkin Lymphoma, 5 Multiple Myeloma, 2 Myelodysplastic Syndrome, 6 Hodgkin Lymphoma), with no available HLA identical donor (neither related or unrelated) underwent peripheral stem cell transplant from a haploidentical family donor. Disease status at transplant was the following: 13 complete remission (5 patients in 1°CR, 6 patients in 2°CR, 2 patients in 3°CR), 9 partial response (PR), 1 stable disease (SD), 7 progressive disease (PD), and a patient with myelodysplastic syndrome who received the transplant as upfront therapy. Conditioning regimen consisted of cyclophosphamide, fludarabine and TBI followed by infusion of haploidentical PBSC; post-transplant immunosuppression consisted of high-dose Cy on days +3 and +4, tacrolimus and micofenolate mofetile from day +5.A median of 5.7x106 (range 3.8-13.7) CD34+ cells/kg was infused with a median of 2.8x108 (range 0.3-5.4) CD3+ cells/kg. Patients readily engrafted with a median time to absolute neutrophil count ≥500/µL of 17.5 days (range 14-28) in 28/31 patients and a median time to platelet ≥20.000/µL of 21 days (range 11-60) in 27/31 evaluable patients. Three patients died of infection before engraftment, another patient showed myeloid engraftment but never recovered platelet counts and prematurely died of cytomegalovirus (CMV) disease.At a median follow-up of 366 days (16-1682), 16 patients are alive (55%): 13 patients are in CR, the other 3, all affected by Multiple Myeloma, have progressed and are undergoing other treatments, with a cumulative incidence of relapse of 33%. Fifteen patients have died; causes of death included progressive disease in 9 patients (60%) and infections in the remaining 6, with a cumulative transplant related mortality of 18%. CMV reactivation occurred in 15 of 27 patients at risk, at a median time of 34 days (range 22-55) after transplant, resolving with preemptive therapy in 14 patients and causing a fatal infection in 1 heavily pretreated patient.Grade I-II acute GvHD (aGvHD) occurred in 6/28 evaluable patients, with 1-year cumulative incidence of grade I-II aGvHD of 21% and no incidence of grade III-IV GvHD. Mild chronic GvHD (cGvHD) was observed in 3/27 evaluable patients with 1-year cumulative incidence of cGvHD of 11%. Achievement of mixed donor chimerism was rapid: 22/28 evaluable patients showed a CD3+ chimerism >50% by day +28. At day + 84, in 22/26 evaluable patients CD3+ cells chimerism was >90%, while 4/26 showed still a mixed donor chimerism: withdrawal of immunosuppression increased CD3+ chimerism in one patient to 90% at day +360; one patient developed hemolytic anemia and he is under immunosuppression, still a mixed chimera at day +401 while in CR; the other two died from infection at +251 and +361 never reaching the full donor chimerism. No graft failure was observed.Our data show that haploidentical non-myeloablative peripheral blood hematopoietic cell transplantation with high-dose post-transplant Cy is a feasible for patients lacking an HLA identical donor. The use of unmanipulated PBSC with the infusion of a greater number of CD3+ cells allows a rapid and sustained engraftment, reduces the risk of graft failure, does not appear to increase the risk of GVHD with a low/moderate relapse risk. Although the incidence of major infection was low, CMV reactivation remains a critical issues and further studies are needed to clarify recovery of CMV immunity and to reduce the overall treatment related toxicity. DisclosuresNo relevant conflicts of interest to declare.

Excellent Engraftment and Long-Term Survival in Patients with Severe Aplastic Anemia (SAA) Undergoing Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) with Haplo-Identical CD34+ Cells Combined with a Single Umbilical Cord Blood Unit

For SAA patients (pts) lacking an HLA identical donor, outcomes of HSCT using single or dual unrelated cord blood (UCB) transplants or haplo-identical donors have been associated with high graft failure rates and poor survival. We investigated whether co-infusion of a single UCB unit with CD34+ selected cells from a haplo-identical relative following a highly immunosuppressive conditioning could improve transplant outcome for this high-risk population.Methods: Eligible pts for HSCT were required to be between the ages of 4-55, have a diagnosis of SAA or SAA evolved to MDS, be unresponsive to immunosuppressive therapy, have severe neutropenia (ANC<500 cells/µL), lack an HLA-matched donor, have an available haploidentical family member, and at least one ≥ 4/6 HLA-matched UCB unit with a minimum TNC dose ≥1.5x107 cells/kg. Pts received conditioning with cyclophosphamide (120 mg/kg), fludarabine (125 mg/m2), equine-ATG (160 mg/kg) and 200 cGy of total body irradiation. On Day 0, pts received a CD34-selected (Miltenyi CliniMacs) G-CSF mobilized haplo-donor allograft combined with a single UCB. Tacrolimus and MMF were used for GVHD prophylaxis. Results: 21 pts (median age: 19.6 yrs, range: 4.5-48.6) including 14 with SAA and 7 with SAA that evolved to early stage MDS (< 5% blasts) underwent haplo-cord transplant. 14 pts received 4/6, and seven pts received a 5/6 HLA-matched UCB unit. UCB units contained a median 3.77x107 TNCs/kg (range: 1.96-6.93), a median 1.54x105 CD34+ cells/kg (range 0.46-3.45), and the haplo-graft contained a median 3.2x106 CD34+ cells/kg (range: 3.0-4.1) and a median 0.95x103 CD3+ cells/kg (range 0.3-5.04). All 21 pts engrafted, which was sustained and associated with transfusion independence in 20/21 pts. At a median follow-up of 3 yrs (1116 days, range: 226-2572), 19/21 pts survive for an overall survival rate of 90%. 2 pts died at day 414 and 402 post-HSCT from viral related complications (CMV pneumonitis and limbic encephalopathy). Neutrophil and platelet recovery occurred at a median 10 (range: 9-22), and 30 (range: 16-129) days respectively. Cord myeloid engraftment (cord ANC>500 cells/µl, calculated from chimerism data) occurred in 18/21 pts at a median 42 days while 3/21 did not achieve a cord ANC>500 but had haplo-donor engraftment which was sustained in 2 cases. The cumulative incidence of grade II-IV acute GVHD (aGVHD) was 28.6%, with only 1 pt (4.8%) developing grade III and no cases of grade IV or steroid refractory aGVHD. The cumulative incidence of chronic GVHD (cGVHD) was 47.8%, with 37% mild, 11% moderate, and no severe cGVHD. Engraftment profiles showed 16 achieved full cord chimerism in all cell lineages, 2 remained mixed haplo-cord chimeras, and 2 failed to have UCB engraftment but had sustained 100% haplo-donor myeloid chimerism. Higher degrees of HLA matching between recipient and UCB unit were associated with faster rates of cord engraftment (P =0.012) and the achievement of full cord chimerism (P= 0.004). Among the 16 pts who had lower degrees of HLA matching (≤5/8 HLA alleles) between the recipient and UCB unit, 9 received grafts with KIR ligand incompatibility in the haplo vs. cord direction ( defined as the presence of a KIR ligand in the haplo-donor graft that was absent in the UCB unit at HLA epitopes Bw4, HLA-C Groups 1 & 2, HLA-A3, and HLA-A11); KIR ligand incompatibility had a negative impact on cord myeloid engraftment as 6/9 (67%) of these pts failed to achieve full cord myeloid chimerism by day 400 and 5/9 (56%) pts failed to achieve a cord ANC>500 by day 250 post-HSCT. In contrast, cord myeloid engraftment occurred quicker in the 12 pts who did not have haplo vs. cord KIR ligand incompatibility including 7 pts with lower degrees of HLA matching (≤5/8 HLA alleles between the recipient and UCB) and 5 pts with higher degrees of HLA matching, where the median times to achieve a cord ANC>500 and full cord myeloid chimerism was 42 days (range: 28-58) and 60 days (range: 14-230) respectively ( both P<0.01 compared to the pts that received grafts with KIR ligand incompatibility). Conclusion: Transplantation of haplo-identical CD34+ cells with a single UCB unit in neutropenic pts with SAA and SAA evolved to MDS was associated with rapid neutrophil recovery, durable donor engraftment and excellent long-term survival. These results suggest that haplo-cord HSCT is an effective treatment option associated with excellent survival in pts with SAA who lack an HLA-matched donor. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mixed chimerism renders residual host dendritic cells incapable of alloimmunization of the marrow donor in the canine model of allogeneic marrow transplantation.

This study tested whether an alloimmune response can occur in the marrow donor when infused or injected with leukocytes from their mixed chimeric transplant recipient. Two mixed chimeras were produced after conditioning with three Gray total body irradiation, donor marrow infusion, and post-grafting immunosuppression. The marrow donors were then repeatedly infused and injected with leukocytes from their respective chimeric recipient. A donor lymphocyte infusion (DLI) into their mixed chimeras had no effect, even after the experiments were repeated. The presence of blood dendritic cells (DCs) of recipient origin was confirmed in chimeric recipients, as well as the presence of microchimerism in the marrow donors. Donor sensitization did occur following placement of a recipient skin graft that was confirmed following DLI into recipients that changed the mixed chimeras into full donor chimeras. These observations suggest that mixed chimerism renders recipient peripheral blood DCs incapable of inducing a donor T cell response.

Successful treatment of murine autoimmune cholangitis by parabiosis: Implications for hematopoietic therapy

There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor β receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4−/−Tg).Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN-γ produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4−/−Tg mice with established disease at 8–9 weeks of age with C57BL/6 control mice. Such parabiotic “twins” had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4−/−Tg and CD8−/− mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8+ T effector cells in the presence of wild type CD4 cells was noted. In conclusion, “correcting” the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis.

The T Cell Repertoire-Diversifying Enzyme TSSP Contributes to Thymic Selection of Diabetogenic CD4 T Cell Specificities Reactive to ChgA and IAPP Autoantigens.

Multiple studies highlighted the overtly self-reactive T cell repertoire in the diabetes-prone NOD mouse. This autoreactivity has primarily been linked to defects in apoptosis induction during central tolerance. Previous studies suggested that thymus-specific serine protease (TSSP), a putative serine protease expressed by cortical thymic epithelial cells and thymic dendritic cells, may edit the repertoire of self-peptides presented by MHC class II molecules and shapes the self-reactive CD4 T cell repertoire. To gain further insight into the role of TSSP in the selection of self-reactive CD4 T cells by endogenous self-Ags, we examined the development of thymocytes expressing distinct diabetogenic TCRs sharing common specificity in a thymic environment lacking TSSP. Using mixed bone marrow chimeras, we evaluated the effect of TSSP deficiency confined to different thymic stromal cells on the differentiation of thymocytes expressing the chromogranin A-reactive BDC-2.5 and BDC-10.1 TCRs or the islet amyloid polypeptide-reactive TCR BDC-6.9 and BDC-5.2.9. We found that TSSP deficiency resulted in deficient positive selection and induced deletion of the BDC-6.9 and BDC-10.1 TCRs, but it did not affect the differentiation of the BDC-2.5 and BDC-5.2.9 TCRs. Hence, TSSP has a subtle role in the generation of self-peptide ligands directing diabetogenic CD4 T cell development. These results provide additional evidence for TSSP activity as a novel mechanism promoting autoreactive CD4 T cell development/accumulation in the NOD mouse.

Foxo1 Is a T Cell-Intrinsic Inhibitor of the RORγt-Th17 Program.

An uncontrolled exaggerated Th17 response can drive the onset of autoimmune and inflammatory diseases. In this study, we show that, in T cells, Foxo1 is a negative regulator of the Th17 program. Using mixed bone marrow chimeras and Foxo1-deficient mice, we demonstrate that this control is effective in vivo, as well as in vitro during differentiation assays of naive T cells with specific inhibitor of Foxo1 or inhibitors of the PI3K/Akt pathway acting upstream of Foxo1. Consistently, expressing this transcription factor in T cells strongly decreases Th17 generation in vitro as well as transcription of both IL-17A and IL-23R RORγt-target genes. Finally, at the molecular level, we demonstrate that Foxo1 forms a complex with RORγt via its DNA binding domain to inhibit RORγt activity. We conclude that Foxo1 is a direct antagonist of the RORγt-Th17 program acting in a T cell-intrinsic manner.

Prenatal Allospecific NK Cell Tolerance Hinges on Instructive Allorecognition through the Activating Receptor during Development.

Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire toward tolerance or immunity. Specifically, the effect on NK cell education arising from developmental corecognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hematopoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically hostile NK cells that express an allospecific activating receptor without coexpressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the bone marrow during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual nondeleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype, but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell-allospecific education.

Rapid Functional Decline of Activated and Memory Graft-versus-Host–Reactive T Cells Encountering Host Antigens in the Absence of Inflammation

Inflammation in the priming host environment has critical effects on the graft-versus-host (GVH) responses mediated by naive donor T cells. However, it is unclear how a quiescent or inflammatory environment impacts the activity of GVH-reactive primed T and memory cells. We show in this article that GVH-reactive primed donor T cells generated in irradiated recipients had diminished ability compared with naive T cells to increase donor chimerism when transferred to quiescent mixed allogeneic chimeras. GVH-reactive primed T cells showed marked loss of cytotoxic function and activation, and delayed but not decreased proliferation or accumulation in lymphoid tissues when transferred to quiescent mixed chimeras compared with freshly irradiated secondary recipients. Primed CD4 and CD8 T cells provided mutual help to sustain these functions in both subsets. CD8 help for CD4 cells was largely IFN-γ dependent. TLR stimulation after transfer of GVH-reactive primed T cells to mixed chimeras restored their cytotoxic effector function and permitted the generation of more effective T cell memory in association with reduced PD-1 expression on CD4 memory cells. Our data indicate that an inflammatory host environment is required for the maintenance of GVH-reactive primed T cell functions and the generation of memory T cells that can rapidly acquire effector functions. These findings have important implications for graft-versus-host disease and T cell-mediated immunotherapies.

IL-15 receptor α signaling constrains the development of IL-17–producing γδ T cells

The development and homeostasis of γδ T cells is highly dependent on distinct cytokine networks. Here we examine the role of IL-15 and its unique receptor, IL-15Rα, in the development of IL-17-producing γδ (γδ-17) T cells. Phenotypic analysis has shown that CD44(high) γδ-17 cells express IL-15Rα and the common gamma chain (CD132), yet lack the IL-2/15Rβ chain (CD122). Surprisingly, we found an enlarged population of γδ-17 cells in the peripheral and mesenteric lymph nodes of adult IL-15Rα KO mice, but not of IL-15 KO mice. The generation of mixed chimeras from neonatal thymocytes indicated that cell-intrinsic IL-15Rα expression was required to limit IL-17 production by γδ T cells. γδ-17 cells also were increased in the peripheral lymph nodes of transgenic knock-in mice, where the IL-15Rα intracellular signaling domain was replaced with the intracellular portion of the IL-2Rα chain (that lacks signaling capacity). Finally, an analysis of neonatal thymi revealed that the CD44(lo/int) precursors of γδ-17 cells, which also expressed IL-15Rα, were increased in newborn mice deficient in IL-15Rα signaling, but not in IL-15 itself. Thus, these findings demonstrate that signaling through IL-15Rα regulates the development of γδ-17 cells early in ontogeny, with long-term effects on their peripheral homeostasis in the adult.

Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

IFN-α/β receptor signaling promotes regulatory T cell development and function under stress conditions.

Type I IFNs are a family of cytokines with antiviral and immunomodulatory properties. Although the antiviral effects of IFNs are well characterized, their immunomodulatory properties are less clear. To specifically address the effects of type I IFNs on T regulatory cells (Tregs), we studied mixed bone marrow chimeras between wild-type and IFN-α/β receptor (IFNAR) knockout (KO) mice, and heterozygous female mice expressing a Treg-specific deletion of the IFNAR. In these two models, IFNAR signaling promotes the development of the Treg lineage in the thymus and their survival in the periphery. IFNAR KO Tregs had a higher expression of the proapoptotic gene Bim and higher frequency of active caspase-positive cells. IFNAR KO Tregs from chimeric mice displayed a more naive phenotype, accompanied by lower levels of CD25 and phosphorylated STAT5. Therefore, in Tregs, IFNAR signaling may directly or indirectly affect phosphorylation of STAT5. In mixed chimeras with Scurfy fetal liver, Tregs derived from IFNAR KO bone marrow were unable to control T effector cell activation and tissue inflammation. Under stress conditions or in a competitive environment, IFNAR signaling may be required to maintain Treg homeostasis and function.

Local antigen and inflammation in the lung induce the preferential establishment and maintenance of CXCR6 and CD49a-expressing antigen-specific CD8 TRM cells in the lung parenchyma and airways (MUC1P.900)

Abstract Acting as sentinels capable of quickly organizing a secondary immune response upon pathogen challenge, resident memory CD8 T (TRM) populations are initially primed by microenvironment cues and APC licensing following acute infection of peripheral tissues. Despite their capacity for heterologous protection against influenza virus (flu) challenge, scant knowledge exists as to factors necessary to establish and maintain airway and lung parenchymal (LP) CD8 TRM cells. In contrast to mechanisms described for other tissues, lung CD8 TRM cell establishment required cognate antigen recognition once cells were recruited to the lung by local inflammation. Furthermore, this combination of local antigen and inflammation formed long-lasting TRM populations in the LP and airways that highly express the chemokine receptor CXCR6 and adhesion molecule CD49a. During a primary intranasal (IN) flu infection, CXCR6 is preferentially expressed on virus-specific CD8 T cells in the airways and LP by day 7 post-infection. Selectively, LP CD8 TRM cells highly express CXCR6, while systemically circulating flu-specific cells achieve only an intermediate expression level. Finally, use of mixed bone marrow chimeras show that wild-type virus-specific memory CD8 T cells predominate over CXCR6-/- cells in the airway and LP after IN infection with Sendai or flu virus. Thus, our data show that local antigen and inflammation coupled with CXCR6 expression is key to establish CD8 TRM cells in the LP and airways.

Role of miR-27 in controlling T cell immunity (IRM15P.605)

Abstract miR-23~27~24 clusters are highly enriched in regulatory cells while expressed at low levels in conventional T cells. However, despite a selective expression pattern of miR-23~27~24 clusters in T cells, to date, studies of this miRNA family have primarily focused on their role in tumorigenesis. Here, we show miR-23~27~24 clusters play a pivotal role in regulating T cell immunity. Particularly, our results have demonstrated that the majority of miR-23~27~24 cluster-dependent phenotypes could be attributed to a single member of this miRNA family, miR-27. Mice with T cell-specific overexpression of miR-27 spontaneously developed autoimmune phenotypes. On the other hand, in vitro polarization studies revealed that T cells with miR-27 overexpression exhibited impaired differentiation and effector function of multiple T helper cell lineages. Finally, our mixed bone marrow chimeras studies further demonstrated that miR-27 could control different T cell responses through either a T cell-intrinsic or -extrinsic manner. Collectively, Our results identify a new "immune regulatory" miRNA that can play multi-faceted roles in regulating T cell immunity.

MiR-146a as a negative regulator in controlling humoral immunity (IRM10P.612)

Abstract The germinal center (GC) reaction is the hallmark of B cell-mediated immune responses to T cell-dependent antigens Recently, a specialized immune population of follicular helper T (Tfh) cells has emerged as a central player in driving GC B cell response and promoting humoral immunity. It has been shown that many genes that are critically involved in the generation of GC B cells also play an indispensable role in the differentiation of Tfh cell population from naive T cells. Here, we show miR-146a is highly upregulated in both Tfh and GC B cells. Our analysis of mixed bone marrow chimeras as well as mice with cell-type-specific deletion of miR-146a demonstrated that elevated levels of miR-146a in both Tfh and B cells are pivotal to control corresponding responses during GC reactions in a cell intrinsic manner. Moreover, deletion of both miR-146a and its paralog, miR-146b resulted in further enhance phenotypes, suggesting that both miR-146 family members contribute to the negative regulation of humoral immunity. Finally, combining transcriptional profiling and biochemical approaches including HITS-CLIP (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation), several miR-146a targets with potential roles in controlling Tfh and GC B cell responses have been identified. In sum, our study identifies a miRNA family highly enriched in GC cell subsets with the capacity to control GC reaction by limiting differentiation of Tfh and GC B cells.

Nedd4 family interacting proteins activate ubiquitylation pathways to limit ERK signaling following TCR engagement (IRM7P.702)

Abstract Ubiquitylation tunes signaling pathways in stimulated T cells to regulate activation and function. We have found that, in vitro, Nedd4-family interacting protein 1 (Ndfip1) and Ndfip2 activate Nedd4-family catalytic E3 ligases with known roles in T cells. Ndfip1 negatively regulates T cell activation and Th2 polarization, but the in vivo role of Ndfip2 is unknown. We generated Ndfip2-/- mice and found that Ndfip2 is not a prominent negative regulator of T cell activation or Th2 polarization. However, loss of Ndfip2 exacerbates the inflammatory Ndfip1-/- phenotype, suggesting that Ndfip2 dampens inflammation. We generated mixed fetal liver chimeras and found that Ndfip1/Ndfip2 deficiency leads to T cell intrinsic hyperactivity. To identify signaling pathways affected by loss of Ndfips, we used semiquantitative proteomics to compare stimulated control and knockout CD4+ T cells. Pathway analysis revealed increased MEK1 activity in knockout cells, which was confirmed by observing increased ERK phosphorylation in stimulated Ndfip1/Ndfip2 deficient CD4+ T cells. To identify aberrant ubqiuitylation of proteins upstream of ERK, we have taken a quantitative proteomic approach, using Tandem Ubiquitin Binding Entities (TUBEs) to enrich for polyubiquitylated proteins in isotope labeled control and Ndfip deficient CD4+ T cells. This approach has yielded several potential targets of Ndfip-dependent ubiquitylation in CD4+ T cells that are currently under investigation.

Control of T cell proliferation, differentiation, and effector function by the adaptor protein BCAP (IRC8P.449)

Abstract The PI3K network is required for a robust effector T cell response. However, the molecular mechanisms involved in regulating PI3K function during T cell activation are poorly understood. The adaptor protein B Cell Adaptor for PI3K (BCAP) has previously been detected in B cells and myeloid cells, and there is evidence suggesting that BCAP is involved in the regulation of PI3K signaling pathways in these cell populations. We have generated novel data demonstrating the role of BCAP in ensuring optimal T cell activation, proliferation and differentiation. BCAP is not expressed in naïve T cells but is detected in effector memory T cell populations, and can be rapidly induced in naïve T cells upon T cell activation in vitro. BCAP deficient T cells are hypo-proliferative, and have attenuated signaling cascades downstream of PI3K in vitro. To study the role of BCAP in T cells in vivo, we infected wild type and BCAP deficient mice with the pathogen Lymphocytic Choriomeningitis Virus (LCMV). At 7 days post infection, notable skewing in the effector CD8+ T cell populations was observed, with an increased frequency of KLRG1loCD127hi memory precursor cells (MPCs) at the expense of KLRG1hiCD127lo terminal effector cells (TECs). Finally, use of mixed bone marrow chimeras showed that wild type T cells have a significant competitive advantage over BCAP deficient T cells in vivo. Collectively, these data suggest that BCAP may have a non-redundant role in determining T cell fate in vivo.

Tumor progression locus 2 inhibits regulatory T cell development and immunosuppressive functions (LYM8P.640)

Abstract Regulatory T cells (Tregs) are a specialized subset of immunosuppressive T cells that function to maintain peripheral tolerance. We previously demonstrated that the serine-threonine kinase, tumor progression locus 2 (Tpl2, also designated Cot/Map3k8), regulates TCR signaling and inflammatory cytokine secretion in CD4+ T cells. Herein, we demonstrate that Tpl2 is preferentially expressed by Tregs and regulates their development and functions. Tpl2-/- mice exhibited increased proportions of thymic natural Tregs (nTregs) as well as peripheral splenic Tregs, in vivo. Enhanced Treg development was due to a T cell autonomous defect, since Treg development from tpl2-/- naïve T cells cultured in vitro was similarly enhanced, and peripheral Treg proportions were also increased within the tpl2-/- donor compartment of mixed bone marrow chimeras. This defect depended upon TCR signal strength and was enhanced at low antigen concentrations. Importantly, tpl2-/- Tregs had increased expression of the immunosupressive cytokines, IL-10 and IL-35, and provided better protection than wild-type Tregs in vivo in a T cell transfer model of colitis. These results demonstrate that Tpl2 has an important physiological role in limiting Foxp3 expression, Treg development and functions. Therefore, Tpl2 inhibition could potentially be used to deviate pathological immune responses in a variety of autoimmune diseases towards a protective, tolerogenic response through preferential Treg induction.