Homing Molecules Research Articles

A comparison between cytokine- and bead-stimulated polyclonal T cells: the superiority of each and their possible complementary role.

Cytokine-induced killer (CIK) cells and T cells expanded by co-stimulation with beads presenting anti-CD3 and -CD28 antibodies are both polyclonal T cells under intensive laboratory and clinical studies, but there has not been any direct comparison between both. We compared the expansion, memory T cell subsets and cytotoxicity for T cells expanded in parallel by the two methods. Bead-stimulated T cells showed superior expansion as compared to CIK cells on D14 of culture. Bead-stimulated T cells consisted of a significantly higher CD4(+) subset and significantly lower CD8(+) subset as compared to CIK cells, as well as a higher proportion of less terminally differentiated T cells and a higher proportion of homing molecules. On the other hand, CIK cells exhibited significantly superior cytotoxicity against two myelomonocytic leukemia cell lines (THP-1 and U937) and two RCC cell lines (786.0 and CaKi-2). The cytotoxicity on D14 against THP-1 was 58.1% for CIK cells and 8.3% for bead-stimulated T cells at E:T of 10:1 (p<0.01). Cytotoxicity correlated positively with the proportion of the CD8 subset in the culture and was independent of NKG2D recognition of susceptible targets. Polyclonal T cells expanded by different methods exhibit different characteristics which may define the specific role of each in different clinical scenario. We postulate that the more potent CIK cells may offer short term benefit while bead-stimulated T cells may offer a more sustained immune response.

The Role of CMV Serostatus of the Donor in the Graft Lymphoid Composition and Prediction of Gvhd and CMV Reactivation

Background. Cytomegalovirus (CMV) is a widespread persistent β–herpesvirus, which can cause severe complications during primary infection or reactivation in immunocompromised patients, such as after allogeneic stem cell transplantation (alloSCT). Another major complication associated with alloSCT is graft-versus-host disease (GVHD). The pathogenesis of GVHD involves migration of the transplanted donor naïve T-cells into the secondary lymphoid organs in the recipient, which is mainly steered by CD62L and CCR7. As these homing molecules have been associated with both acute GVHD (aGVHD) and chronic GVHD (cGVHD), we studied whether the CMV serostatus of the donor affects the lymphoid composition of the graft product and whether this phenotype can predict CMV reactivation and GVHD.Methods. This single-center study included 77 donor-recipient pairs who underwent alloSCT. 64 pairs were HLA identical, 12 had 1 mismatch and 3 had 2 mismatch. 36 donors were related to their recipients. All recipients were followed at least for 100 (aGVHD) or 360 days (cGVHD) after transplantation. 43 donors were CMV-seropositive (CMVpos) and 34 were CMV-seronegative (CMVneg). 62 recipients were CMVpos, and 32 of them developed CMV reactivation, 25 aGVHD and 30 cGVHD. Samples from the graft product (donor) were phenotyped by flow cytometry (CD45, CD3, CD8, CD4, CD62L, CCR7) and both frequency (freq) and absolute number (abs) of each T-cell subpopulation were analyzed.Results. When the donors were divided based on their CMV serostatus, we observed that the grafts from CMVpos donors had a lower freq of naïve (CCR7+CD62L+) CD4+ T-cells (of lymphocytes p=0.06, of CD3 p=0.06, of CD4 p=0.07) and naïve CD8+ T-cells (of leukocytes p=0.03, of lymphocytes p=0.041, of CD3 p=0.011, of CD8 p=0.012) compared to CMVneg donors. Further, the abs of transplanted naïve CD8+ T-cells was significantly lower in the grafts from CMVpos donors (p=0.048). No differences were observed in T-cells (CD3+, CD4+, CD8+).We next studied if the CMV-serostatus and T-cell phenotype of the graft associates with GVHD. CMVpos donors whose recipients developed aGVHD had higher abs (p=0.05) and freq of naïve CD8+ T-cells (of lymphocytes p=0.08, of CD3 p=0.08, of CD8 p=0.11) compared to those without aGVHD. The same trend was observed with abs (p=0.11) and freq of CCR7+CD4+ T-cells (of leukocytes p=0.15). Similarly, those CMVpos donors whose recipients developed cGVHD had higher abs (p=0.05) and freq of CCR7+CD8+ T-cells (of leukocytes p=0.03, of lymphocytes p=0.06). Further, cGVHD patients who received the transplant from CMVpos donors were infused with a higher freq of CD3+ (of leukocytes p=0.03) and CD4+ T cells (of leukocytes p=0.04) than patients who received a graft from CMVpos donors but did not develop cGVHD. In contrast, CMVneg donors who developed aGVHD had a higher freq of CD3+ (p=0.018) and CD4+ T-cells (p=0.09), whereas no differences were seen in the T-cell subpopulations. Conversely, the abs (p=0.08) and freq of CCR7+CD4+ T-cells (of leukocytes p=0.11) were higher in those who later developed cGVHD.To study whether the graft lymphoid composition can be used to predict CMV reactivation, we analyzed the lymphoid composition in the graft product of those donors (both CMVpos and CMVneg) whose recipients were CMV seropositive but did not develop any form of GVHD (to avoid the influence of GVHD in the reactivation of CMV). Despite the low number of patients (CMV reactivation n=9, and no CMV reactivation n=13), we observed trends of higher portion of CD4+ T-cells (p=0.09 of lymphocytes, of CD3 p=0.20) and CCR7+CD4+ T-cells (of lymphocytes p=0.18, of CD4 p=0.16) in those grafts that were transplanted into CMV seropositive recipients who did not reactivate CMV.Conclusions. CMVpos donors whose recipient developed either aGVHD or cGVHD had a higher abs and freq of naïve CD8+ T-cells, which was not seen with CMVneg donors. This suggests that seropositivity sets the abs and freq of CD8 subpopulations near to a decisive cutoff for the development of GVHD. Conversely, other factors influences the development of GVHD in those patients whose donors were seronegative. In other words, seropositivity of the donor affects the graft composition and thus the risk of GVHD. Finally, our data indicate that a higher proportion of naïve or central memory CCR7+ CD4+ T cells in the donor graft could prevent CMV reactivation suggesting that graft composition affects also CMV reactivation. DisclosuresNo relevant conflicts of interest to declare.

Compartmentalization of simian immunodeficiency virus replication within secondary lymphoid tissues of rhesus macaques is linked to disease stage and inversely related to localization of virus-specific CTL.

We previously demonstrated that HIV replication is concentrated in lymph node B cell follicles during chronic infection and that HIV-specific CTL fail to accumulate in large numbers at those sites. It is unknown whether these observations can be generalized to other secondary lymphoid tissues or whether virus compartmentalization occurs in the absence of CTL. We evaluated these questions in SIVmac239-infected rhesus macaques by quantifying SIV RNA(+) cells and SIV-specific CTL in situ in spleen, lymph nodes, and intestinal tissues obtained at several stages of infection. During chronic asymptomatic infection prior to simian AIDS, SIV-producing cells were more concentrated in follicular (F) compared with extrafollicular (EF) regions of secondary lymphoid tissues. At day 14 of infection, when CTL have minimal impact on virus replication, there was no compartmentalization of SIV-producing cells. Virus compartmentalization was diminished in animals with simian AIDS, which often have low-frequency CTL responses. SIV-specific CTL were consistently more concentrated within EF regions of lymph node and spleen in chronically infected animals regardless of epitope specificity. Frequencies of SIV-specific CTL within F and EF compartments predicted SIV RNA(+) cells within these compartments in a mixed model. Few SIV-specific CTL expressed the F homing molecule CXCR5 in the absence of the EF retention molecule CCR7, possibly accounting for the paucity of F CTL. These findings bolster the hypothesis that B cell follicles are immune privileged sites and suggest that strategies to augment CTL in B cell follicles could lead to improved viral control and possibly a functional cure for HIV infection.

Gut-Homing Conventional Plasmablasts and CD27− Plasmablasts Elicited after a Short Time of Exposure to an Oral Live-Attenuated Shigella Vaccine Candidate in Humans

Currently, there is no licensed Shigella vaccine; however, various promising live-attenuated vaccine candidates have emerged, including CVD1208S (ΔguaBA, Δset, Δsen S. flexneri 2a), which was shown to be safe and immunogenic in Phase 1 clinical trials. Here, we report the immune responses elicited in an outpatient Phase 2 clinical trial in which subjects were vaccinated with CVD 1208S. Oral immunization with CVD 1208S elicited high anti-S. flexneri 2a LPS and IpaB antibody responses as well as an acute plasmablast (PB) infiltration in peripheral blood 7 days after immunization. PB sorted based on their expression of homing molecules confirmed that cells expressing integrin α4β7 alone or in combination with CD62L were responsible for antibody production (as measured by ELISpot). Furthermore, using high-color flow-cytometry, on day 7 after immunization, we observed the appearance of conventional PB (CPB, CD19dim CD20− CD27+high CD38+high CD3−), as well as a PB population that did not express CD27 (CD27− PB; pre-plasmablasts). The pattern of individual or simultaneous expression of homing markers (integrin α4β7, CD62L, CXCR3, and CXCR4) suggested that CPB cells homed preferentially to the inflamed gut mucosa. In contrast, ~50% CD27− PB cells appear to home to yet to be identified peripheral lymphoid organs or were in a transition state preceding integrin α4β7 upregulation. In sum, these observations demonstrate that strong immune responses, including distinct PB subsets with the potential to home to the gut and other secondary lymphoid organs, can be elicited after a short time of exposure to a shigella oral vaccine.

Neutrophils exhibit differential requirements for homing molecules in their lymphatic and blood trafficking into draining lymph nodes.

Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.

Interleukin-15 enhances cellular proliferation and upregulates CNS homing molecules in pre-B acute lymphoblastic leukemia

AbstractGenome-wide association studies have consistently implicated the interleukin-15 (IL-15) gene in acute lymphoblastic leukemia (ALL) biology, including associations with disease susceptibility, and increased risk of central nervous system (CNS) involvement. However, whether pre-B ALL blasts directly respond to IL-15 is unknown. Here, we show that most pre-B ALL primary samples and cell lines express IL-15 and components of its receptor and that primary pre-B ALL cells show increased growth in culture in response to IL-15. Investigation of mechanisms of action using IL-15–responsive SD-1 cells shows this growth advantage is maximal under low-serum conditions, mimicking those found in cerebrospinal fluid. IL-15 also upregulates PSGL-1 and CXCR3, molecules associated with CNS trafficking. Investigation of downstream signaling pathways indicates that IL-15 induces signal transducer and activator of transcription 5 (STAT5), extracellular signal-regulated kinase (ERK) 1/2, and to a lesser extent phosphatidylinositol 3-kinase (PI3K) and nuclear factor κB (NF-κB) phosphorylation. The IL-15–mediated growth advantage is abolished by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK), PI3K, and NF-κB inhibitors but preserved in the presence of STAT5 inhibition. Together, these observations provide a mechanistic link between increased levels of IL-15 expression and leukemogenesis, high-risk disease, and CNS relapse and suggest potential therapeutic targets.

Integrin α4β1 Is Necessary for CD4+ T Cell–Mediated Protection against Genital Chlamydia trachomatis Infection

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection in the United States and a significant health burden worldwide. Protection from Chlamydia infection in the genital mucosa is dependent on IFN-γ derived from CD4(+) Th1 cells. These CD4(+) T cells must home successfully to the genital tract to exert their effector function and decrease C. trachomatis burden. Although adhesion receptors expressed by CD4(+) T cells in the genital tract have been characterized, the integrin receptor required for Chlamydia-specific CD4(+) T cell-mediated protection has not been explored. In this study, we demonstrate that C. trachomatis infection of the upper genital tract results in recruitment of Chlamydia-specific CD4(+) T cells robustly expressing the integrin α4β1. Interfering with α4β1, but not α4β7, function resulted in defective CD4(+) T cell trafficking to the uterus and high bacterial load. We conclude that integrin α4β1 is necessary for CD4(+) T cell-mediated protection against C. trachomatis infection in the genital mucosa. By identifying homing molecules required for successful CD4(+) T cell trafficking to C. trachomatis-infected tissues, we will be better equipped to design vaccines that elicit sterilizing, long-lasting immunity without inducing immune pathologies in the upper genital tract.

CCR10 regulates intestinal immune homeostasis through regulating both Treg and Teff cells in the intestine (MUC2P.832)

Abstract Intestinal immune homeostasis is an active process of balanced tolerant responses towards the gut-resident commensals and other food-born stimulations. Failure of the balanced response could lead to inflammatory diseases and cancers. Understanding molecular mechanisms that differentially regulate the homeostatic vs. inflammatory activation of T cells are fundamentally important in the intestinal immunity. CCR10, through interaction with its mucosa-specific ligand CCL28, was previously found important in regulation of IgA antibody-producing B cells in intestines. On the other hand, CCR10 was suggested to be a skin- but not gut- homing molecule for T cells. Using a novel strain of CCR10-knockout/EGFP-knockin mouse, we found that CCR10 is specifically induced on activating T cells in healthy intestines in situ and is critically important in balanced maintenance and functions of Treg and Teff cells to promote the intestinal homeostasis. On the other hand, during intestinal immune activations associated with infection or other inflammatory stimulations, the induction of CCR10 on activated intestinal T cells were suppressed that allows the T cell activation along different lineages. Our findings represent the first demonstration that a chemokine receptor is specifically associated with the homeostatic status of the intestine and reveal that there exists an auto-promoting feedback circuit of intestinal homeostasis in which the CCR10/ligand axis are a critical molecular link.

Epicutaneous Immunotherapy-Induced Regulatory T Cells Could Migrate To More Various Sites Of Allergen Exposure Compared To Sublingual Or Subcutaneous Immunotherapy In Mice Sensitized To Peanut

In mice, epicutaneous immunotherapy (EPIT) increases Foxp3+ regulatory T cells (Tregs) (Dioszeghy, J Immunol 2011). Tregs could display distinct efficacy based on their expression of homing molecules. The aim of this study was to evaluate homing receptors expression by Tregs induced during EPIT compared to sublingual (SLIT) or subcutaneous (SCIT) immunotherapy.

Melanoma stem cells and metastasis: mimicking hematopoietic cell trafficking?

Malignant melanoma is a highly metastatic cancer that bears responsibility for the majority of skin cancer-related deaths. Amidst the research efforts to better understand melanoma progression, there has been increasing evidence that hints at a role for a subpopulation of virulent cancer cells, termed malignant melanoma stem or initiating cells (MMICs), in metastasis formation. MMICs are characterized by their preferential ability to initiate and propagate tumor growth and their selective capacity for self-renewal and differentiation into less tumorigenic melanoma cells. The frequency of MMICs has been shown to correlate with poor clinical prognosis in melanoma. In addition, MMICs are enriched among circulating tumor cells in the peripheral blood of cancer patients, suggesting that MMICs may be a critical factor in the metastatic cascade. Although these links exist between MMICs and metastatic disease, the mechanisms by which MMICs may advance metastatic progression are only beginning to be elucidated. Recent studies have shown that MMICs express molecules critical for hematopoietic cell maintenance and trafficking, providing a possible explanation for how circulating MMICs could drive melanoma dissemination. We therefore propose that MMICs might fuel melanoma metastasis by exploiting homing mechanisms commonly utilized by hematopoietic cells. Here we review the biological properties of MMICs and the existing literature on their metastatic potential. We will discuss possible mechanisms by which MMICs might initiate metastases in the context of established knowledge of cancer stem cells in other cancers and of hematopoietic homing molecules, with a particular focus on selectins, integrins, chemokines and chemokine receptors known to be expressed by melanoma cells. Biological understanding of how these molecules might be utilized by MMICs to propel the metastatic cascade could critically impact the development of more effective therapies for advanced disease.

An Intrinsic Propensity of Murine Peritoneal B1b Cells to Switch to IgA in Presence of TGF-β and Retinoic Acid

AimsIn the present study we have investigated the comparative switching propensity of murine peritoneal and splenic B cell subpopulations to IgA in presence of retinoic acid (RA) and TGF-β.Methods and ResultsTo study the influence of RA and TGF-β on switching of B cell subpopulations to IgA, peritoneal (B1a, B1b and B2 cells) and splenic (B1a, marginal zone, and B2) B cells from normal BALB/c mice were FACS purified, cultured for 4 days in presence of RA and TGF-β and the number of IgA producing cells was determined by ELISPOT assay or FACS analysis. In presence of TGF-β, peritoneal B1b cells switched to IgA more potently than other peritoneal B cell subpopulations. When TGF-β was combined with retinoic acid (RA), switching to IgA was even more pronounced. Under these conditions, “innate” B cells like peritoneal and splenic B1 cells and MZ B cells produced IgA more readily than B2 cells. Additionally, high frequency of nucleotide exchanges indicating somatic hypermutation in VH regions was observed. Besides IgA induction, RA treatment of sorted PEC and splenic B cells led to expression of gut homing molecules - α4β7 and CCR9. Intraperitoneal transfer of RA-treated B1 cells into Rag1-/- recipients resulted in IgA in serum and gut lavage, most efficiently amongst B1b cell recipients.ConclusionPresent study demonstrates the differential and synergistic effect of RA and TGF-β on switching of different B cell subpopulations to IgA and establishes the prominence of peritoneal B1b cells in switching to IgA under the influence of these two factors. Our study extends our knowledge about the existing differences among B cell subpopulations with regards to IgA production and indicates towards their differential contribution to gut associated humoral immunity.

Vaginal Immunization to Elicit Primary T-Cell Activation and Dissemination

Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4+ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4+ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs.

Cord Blood CD34+ Stem Cell Sialyl Lewis X Levels and E-Selectin Binding Are Predictive Of Engraftment In Mice: Functional Separation Of Stemness and Homing To Improve Engraftment

BackgroundCord blood (CB) is an increasingly used alternative to bone marrow and mobilized peripheral blood as a source of hematopoietic tissue for transplantation. However, the relatively low cell dose and significantly delayed engraftment when compared to BM and mPB remain significant hurdles. A deficit in the homing of CB hematopoietic progenitor and stem cells (HPSC) to the hematopoietic microenvironment due to suboptimal expression and/or activity of homing molecules is thought to be responsible in part for the delayed engraftment seen with CB in patients. Sialyl Lewis X (sLeX) bearing cells can bind E-selectin, and we have previously reported that ex vivo fucosylation of CB HPSCs with fucosyltransferase VI or VII enhance the rapidity and magnitude of engraftment in mice. Here we explore the engraftment potential of endogenous sLeX bearing CD34+ CB HPSCs to determine if physiologic levels of E-selectin binding predicts engraftment in murine xenografts. ApproachCB cells were sorted with CD34 and HECA-452 (anti-sLex) antibodies. sLeX-bearing CD34+ HPSCs (CD34+HECA+) and CD34+ HPSCs lacking sLeX (CD34+HECA-) were compared phenotypically for stem cell and differentiation markers, by gene expression profiling, E/P/L-selectin binding, colony-forming assays, and for multi-lineage engraftment into NOD-SCID-IL2Rg immune-deficient mice. ResultsCord blood CD34+HECA+ cells represent 10-20% of CB MNCs and show no significant phenotypic differences from the CD34+HECA- cells in stem cell (CD133, CD90 (Thy-1), CD117 (c-kit), CD143/BB9) and differentiation (CD38, CD33, CD14, CD3, CD19) markers. In agreement, similar percentages of CD34+CD38- and CD34+CD38+ CB cells were found to be HECA+ (18% and 15% respectively, p=0.38), showing no significant bias toward the more immature CD34+CD38- phenotype. mRNA-seq expression analysis revealed relatively few differences in gene expression patterns, although CD34+HECA+ cells express higher levels of the gamma globin genes HBG1 and HBG2, the components of fetal hemoglobin. As predicted, CD34+HECA+ cells demonstrated significantly increased ability to bind recombinant E-selectin in vitro, with no differences in P- and L-selectin binding. Importantly, colony forming assays revealed a small (30%) disadvantage to the CD34+HECA+ cells revealing that the CD34+HECA+ CB cells do not have enriched stem cells activity by CFU assay.However, CD34+HECA+ cells demonstrated significantly higher rate and magnitude of engraftment when compared to CD34+HECA- cells in three independent NSG experiments (Figure 1). Indeed bone marrow, peripheral blood and splenic levels of human hematopoietic cells were consistently 3-5-fold higher in CD34+HECA+ injected mice than in CD34+HECA- injected controls (Figure 2). Multi-lineage engraftment data reveals higher levels of myeloid (CD33+/CD14+), B-lymphocytes (CD19+/CD20+) and platelets (6-14-fold, CD41a+/CD61+) in CD34+HECA+ cells, but interestingly lower levels of T-lymphocytes (CD3+). Finally, secondary transplants had equal magnitude of engraftment, indicating no bias in short- versus long-term HSPCs. ConclusionsData presented here supports the hypothesis that endogenous sLex levels on CD34+ cells is associated with enhanced engraftment rapidity and magnitude but that this is not reflective of an enriched stem cell fraction. Rather it appears to be an indicator of homing to the bone marrow through E-selectin binding. Functional separation of stemness and homing supports the approach to improve CB transplantation through decoration of CB cells with sLex via ex vivo fucosylation (see clinical trial abstract by Popat and Shpall)▪ Disclosures:No relevant conflicts of interest to declare.

Eomesodermin Regulates The Early Activation Of Alloreactive CD4 T Cells and Is Critical For Both Gvh and GVL Responses

Despite increasing insights into its immunobiology, graft vs host disease (GVHD) remains a major obstacle for successful allogeneic hematopoietic stem/progenitor cell transplantation (allo-HCT). Separation of GVHD from graft vs. leukemia/lymphoma (GVL) responses also remains an elusive goal for allo-HSCT. Efforts to delineate the transcriptional networks regulating T cell differentiation post-HCT have suggested that multiple transcription factors may be involved in the regulation of alloreactive helper T (Th) cells and GVHD. However, conflicting data have emerged regarding the role of Th1 and Th17 pathways, and it remains unclear which transcription factors mediate the early activation of alloreactive T cells necessary for subsequent GVHD development. The T-box transcription factor eomesodermin (Eomes) cooperates with T-bet to regulate CD8 T cell cytotoxic function, IFNy production, and memory cell formation. Recently, a role for Eomes in CD4 Th cell polarization has been described as well.In order to evaluate the role of Eomes in T cell function in the context of allo-HCT, we used a MHC-disparate mouse model (C57BL/6 into BALB/c) with T cell depleted donor bone marrow (TCD-BM) and wild-type (WT) or Eomes knock out (KO) donor T cells. Recipients were conditioned with lethal total body irradiation. Eomes deficiency in donor T cells led to a significant reduction in GVHD mortality (Fig 1, p<.001), morbidity (p<.001), and intestinal pathology (p<.05, colon). Notably, Eomes KO T cells exerted significantly less GVHD mortality than T-bet KO T cells (Fig 1, p<.001). Given the reduced gastrointestinal (GI) GVHD observed with Eomes KO T cells, we next analyzed the expression of homing molecules important for T cell migration to the GI tract. Consistent with reduced GI GVHD, we detected reduced expression of α4β7 integrin on Eomes KO donor CD8 T cells one week post-HCT. We also observed an increase in the proportion and absolute numbers of Foxp3+ regulatory T cells, as well as a decrease in expression of T-bet in mesenteric lymph nodes (MLNs). Moreover, we found decreased production of IFNy by Eomes KO donor CD4 T cells two weeks (spleen and MLN, p<.001) and three weeks (spleen, p<.01) post-HCT without a comcomitant increase in IL-17. We also found increased IL-4 production by Eomes KO CD4 T cells two weeks post-HCT (MLN, p<.05), indicating a shift from Th1 to Th2 polarization in the absence of Eomes. Strikingly, one of the greatest differences we observed between WT and Eomes KO donor T cells was impaired early activation of CD4 T cells; Eomes deficiency was associated with reduced proliferation (p<.001), reduced expression of CD25 (p<.001, spleen; p<.001, MLN), and increased expression of CD62L (p<.01, spleen; p<.001, MLN) in CD4 T cells within the first 72 hours post-HCT (Fig 2).In order to determine if Eomes was important for T cell-mediated GVL responses, we performed allo-HCT in the presence of A20 lymphoma cells. Despite the reduction in GVHD mortality as described above, A20 tumor challenge led to increased mortality in recipients of Eomes KO T cells, indicating that Eomes was also critical for effective GVL function. Given the importance of Eomes in early alloactivation of CD4 T cells, we evaluated if the impaired GVL function was due to an intrinsic CD8 defect or lack of CD4 help. B6 TCD-BM was transplanted into BALB/c recipients along with either WT or Eomes KO CD4 or CD8 T cells. Eomes deficiency in both CD4 and CD8 T cells again led to significant mortality, but HCT with Eomes KO CD4 T cells and WT CD8 T cells led to the greatest survival due to less GVHD and intact GVL (Fig 3), suggesting that Eomes is essential for intrinsic CD8 function during GVL, but not for CD4 help.In summary, we identified distinct requirements for Eomes in CD4 versus CD8 T cells in the context of allo-HCT. Eomes regulated multiple aspects of CD4 T cell function following allo-HCT, including early activation, cytokine production, and gut trafficking. The multifacted functions of Eomes in CD4 T cells likely explain its requirement for GVHD. In contrast, Eomes deficiency in CD8 T cells led to impaired GVL, consistent with its established importance for cytotoxic CD8 T cell differentiation. To our knowledge, this is one of the first descriptions of a transcription factor necessary for effective GVL capacity. Our results suggest that selective manipulation of Eomes function in T cell subsets may be useful for both limiting GVHD and enhancing GVL. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

NK-Cells In Dasatinib-Treated Chronic Myeloid Leukemia Patients Display a Unique Phenotype Associated With Cytotoxic Potential

BackgroundDasatinib is a second-generation tyrosine kinase inhibitor (TKI), which is used successfully in the treatment of chronic myeloid leukemia (CML). Dasatinib has two unique features when compared to other TKIs (imatinib, nilotinib); first, dasatinib has a significantly shorter half-life in the plasma and second, dasatinib inhibits a wider spectrum of kinases, including several kinases known to be important in the function of the immune system (src, tec, and syk families), which are not affected by the other TKIs. Interestingly, it has been recently shown that both short-term exposure to dasatinib in vivo and long-term treatment with dasatinib improves NK-cell cytotoxicity, however, the mechanisms are not known.To study the improved cytotoxicity observed in dasatinib-treated patients, we aimed to perform a complete NK-cell phenotyping in these patients. Finally, our goal is to correlate NK-cell phenotype with NK-cell cytotoxicity, and to study the possible correlation between phenotypical changes, NK-cell function and clinical outcome. MethodsThis study included 19 dasatinib-treated (DA) CML patients, both first-line (n=7) and second-line (n=12). To investigate the specificity of the immunomodulatory effects of dasatinib, a control group of 9 CML patients treated with imatinib (IM) and another group of 12 healthy donors (HD) were included. Peripheral blood samples obtained before the patients took their daily drug dose were phenotyped with a comprehensive 8-color flow cytometry panel (total 32 antibodies, table 1).To study the correlation between phenotypical changes and NK-cell cytotoxicity, we performed a standard CD107 degranulation assay. Mononuclear cells were incubated for 6 hours in the presence of the target cell line K562 and a CD107 antibody. CD107 positive NK-cells were then phenotyped with the same panel of 32 antibodies. ResultsAll results are summarized in table 1. In brief, DA- and IM-treated CML patients and HD had equal proportions of NK-cells (CD3negCD56+) of total lymphocytes. Regarding trafficking molecules, NK-cells in both DA- and IM-patients had a lower frequency of the chemokine receptor CCR7 when compared to HD. This suggests a reduction in the NK-cell population that is able to migrate to lymph nodes, and is likely caused by the disease or TKIs in general. Moreover, DA-treatment specifically decreases the expression of the homing molecule CD62L in NK-cells.In addition, NK-cells in DA-patients, when compared to IM and HD, expressed less CD11b and significantly more often CD11c and HLA-DR, which reproduce the immunophenotypic changes that typically occurs in recently activated NK-cells and has been shown to associate with improved clinical benefits. Conversely, increased expression of CD57 together with a lower frequency of CD27 and CD28 were observed in both groups of patients and were similar to those typically observed in conditions of chronic NK-cell stimulation.In contrast, DA-patients had a lower frequency of most of the studied NK-receptors (Nkp30, Nkp46, NKG2D, CD94, CD161, KIR2DL1/S1) when compared to IM and HD. This suggests that NK-cells in DA-treated patients have a more mature phenotype, which is caused by the treatment. ConclusionsNK-cells in TKI-treated CML patients display a mature phenotype, which is often observed after chronic stimulation suggesting that TKIs have immunomodulatory effects on NK-cells or the disease itself causes the changes. Interestingly, NK-cells in DA-treated patients express a highly differentiated phenotype characterized by high expression of CD57, and decreased expression of Nkp30, Nkp46 and CD161. Similar changes were not seen in IM-patients or HD. It is possible that NK-cells expressing this phenotype might also represent those NK-cells that have previously been driven into clonal expansion by encounters with pathogens because of the specific immunomodulatory effects of dasatinib. This phenotype of highly mature NK-cells, which is associated with high cytolytic potential, could be responsible for the previously described enhanced NK-cytotoxicity caused by dasatinib. In accordance, our preliminary results suggest that these unique phenotypic changes observed in DA-treated patients correlates with the cytotoxic potential. Studies to correlate these results with therapy outcome are ongoing. [Display omitted] Disclosures:Garcia:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Research Funding. Steegmann:Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Peptide Vaccination in Montanide Adjuvant Induces and GM-CSF Increases CXCR3 and Cutaneous Lymphocyte Antigen Expression by Tumor Antigen–Specific CD8 T Cells

T cell infiltration of melanoma is associated with enhanced clinical efficacy and is a desirable endpoint of immunotherapeutic vaccination. Infiltration is regulated, in part, by chemokine receptors and selectin ligands on the surface of tumor-specific lymphocytes. Therefore, we investigated the expression of two homing molecules--CXCR3 and CLA - on vaccine-induced CD8 T cells, in the context of a clinical trial of a melanoma-specific peptide vaccine. Both CXCR3 and CLA have been associated with T cell infiltration of melanoma. We demonstrate that a single subcutaneous/intradermal administration of peptide vaccine in Montanide adjuvant induces tumor-specific CD8 T cells that are predominantly positive for CXCR3, with a subpopulation of CXCR3(+)CLA(+) cells. Addition of GM-CSF significantly enhances CXCR3 expression and increases the proportion of CLA-expressing cells. Concurrent with CXCR3 and CLA expression, vaccine-induced CD8 cells express high levels of Tbet, IFN-γ, and IL-12Rβ1. Collectively, these studies demonstrate that peptide vaccination in adjuvant induces CD8 T cells with a phenotype that may support infiltration of melanoma.

Identification of a resident T‐cell memory core transcriptional signature

Identification of a resident T‐cell memory core transcriptional signature

Virus-associated activation of innate immunity induces rapid disruption of Peyer's patches in mice

AbstractEarly in the course of infection, detection of pathogen-associated molecular patterns by innate immune receptors can shape the subsequent adaptive immune response. Here we investigate the influence of virus-associated innate immune activation on lymphocyte distribution in secondary lymphoid organs. We show for the first time that virus infection of mice induces rapid disruption of the Peyer's patches but not of other secondary lymphoid organs. The observed effect was not dependent on an active infectious process, but due to innate immune activation and could be mimicked by virus-associated molecular patterns such as the synthetic double-stranded RNA poly(I:C). Profound histomorphologic changes in Peyer's patches were associated with depletion of organ cellularity, most prominent among the B-cell subset. We demonstrate that the disruption is entirely dependent on type I interferon (IFN). At the cellular level, we show that virus-associated immune activation by IFN-α blocks B-cell trafficking to the Peyer's patches by downregulating expression of the homing molecule α4β7-integrin. In summary, our data identify a mechanism that results in type I IFN–dependent rapid but reversible disruption of intestinal lymphoid organs during systemic viral immune activation. We propose that such rerouted lymphocyte trafficking may impact the development of B-cell immunity to systemic viral pathogens.

Distinct Immunomodulatory and Migratory Mechanisms Underpin the Therapeutic Potential of Human Mesenchymal Stem Cells in Autoimmune Demyelination

Mesenchymal stem cells (MSCs) are efficacious in a variety of intractable diseases. While bone marrow (BM)-derived MSCs (BM-MSCs) have been widely investigated, MSCs from other tissue sources have also been shown to be effective in several autoimmune and inflammatory disorders. In the present study, we simultaneously assessed the therapeutic efficacy of human BM-MSCs, as well as MSCs isolated from adipose tissue (Ad-MSCs) and umbilical cord Wharton's jelly (UC-MSCs), in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Prior to in vivo experiments, we characterized the phenotype and function of all three MSC types. We show that BM-MSCs were more efficient at suppressing the in vitro proliferation of mitogen or antigen-stimulated T-cell responses compared to Ad-MSCs and UC-MSCs. Notably BM-MSCs induced the differential expression of cytokines from normal and stimulated T-cells. Paradoxically, intravenous transplantation of BM-MSCs into C57Bl/6 mice with chronic progressive EAE had a negligible effect on the disease course, even when multiple MSC injections were administered over a number of time points. In contrast, Ad-MSCs had the most significant impact on clinical and pathological disease outcomes in chronic progressive and relapsing-remitting EAE models. In vivo tracking studies revealed that Ad-MSCs were able to migrate to the central nervous system (CNS), a property that most likely correlated with their broader expression of homing molecules, while BM-MSCs were not detected in this anatomic region. Collectively, this comparative investigation demonstrates that transplanted Ad-MSCs play a significant role in tissue repair processes by virtue of their ability to suppress inflammation coupled with their enhanced ability to home to the injured CNS. Given the access and relatively ease for harvesting adipose tissue, these data further implicate Ad-MSCs as a cell therapeutic that may be used to treat MS patients.

TLR Activation Excludes Circulating Naive CD8+ T Cells from Gut-Associated Lymphoid Organs in Mice

The trafficking of effector T cells is tightly regulated by the expression of site-specific sets of homing molecules. In contrast, naive T cells are generally assumed to express a uniform pattern of homing molecules and to follow a random distribution within the blood and secondary lymphoid organs. In this study, we demonstrate that systemic infection fundamentally modifies the trafficking of circulating naive CD8(+) T cells. We show that on naive CD8(+) T cells, the constitutive expression of the integrin α4β7 that effects their entry into GALT is downregulated following infection of mice with Salmonella typhimurium. We further show that this downregulation is dependent on TLR signaling, and that the TLR-activated naive CD8(+) T cells are blocked from entering GALT. This contrasts strongly with Ag-experienced effector T cells, for which TLR costimulation in the GALT potently upregulates α4β7 and enhances trafficking to intestinal tissues. Thus, TLR activation leads to opposite effects on migration of naive and effector CD8(+) T cells. Our data identify a mechanism that excludes noncognate CD8(+) T cells from selected immune compartments during TLR-induced systemic inflammation.