Assesment of Hypoderma infestation in a wild population of Cervus elaphus from mountains Atlantic ecosystems in southwestern Europe (Spain).

Hypodermosis in Cervus elaphus was studied in the Riaño Regional Hunting Reserve, Province of León, north-western Spain. One hundred and ten red deer were examined for the presence of warble fly larvae. They were analyzed by PCR analysis of the COI region of mt-DNA and identified as Hypoderma actaeon. The prevalence of larvae was 42.7% with a mean intensity of 12.5 ± 18 (range 1-80) warbles/deer infested. The distribution of larvae in the infested animals showed an aggregated/overdispersed pattern (aggregation index = 25.84), where the larvae are not randomly or uniformly distributed, but strongly aggregated among their hosts. Larvae were found in all three states. First and second-instars were observed mainly in the autumn until the end of winter (November-March) and third-instars in late winter until mid-spring (March-May). The adult animals and the males had a higher prevalence than the young and the females, finding statistically significant differences only according to the sex of the animals. Seasonal variations were observed in the prevalence with the highest number of infested animals in winter and autumn, but not in terms of the mean intensity of parasites. Additionally, we assessed the presence of anti-Hypoderma antibodies in serum by means of indirect ELISA tests, using a crude larval extract (CLE) and a purified fraction the hypodermin C (HyC) obtained from first instars of Spanish isolates of Hypoderma lineatum (cattle). These findings confirm that H. actaeon is widely distributed in northern Spain, and provide new information about its chronobiology in mountainous Atlantic ecosystems from southwestern Europe.

Contribution to the Knowledge of Gastrointestinal Nematodes in Roe Deer (Capreolus capreolus) from the Province of León, Spain: An Epidemiological and Molecular Study.

A study of gastrointestinal nematodes in roe deer was carried out in the regional hunting reserves of Riaño and Mampodre, Province of León, Spain, to provide information on their prevalence and intensity of infection in relation to the sampling areas, age of the animals, and body weight. Through a regulated necropsy of the animals, all of them harbored gastrointestinal nematodes in their digestive tract, with a mean intensity of parasitism of 638 ± 646.1 nematodes/infected animal. Eleven genera were found and 18 species of gastrointestinal nematodes were identified, three of them polymorphic: Trichostrongylus axei, Trichostrongylus vitrinus, Trichostrongylus capricola, Trichostrongylus colubriformis, Haemonchus contortus, Spiculopteragia spiculoptera/Spiculopteragia mathevossiani, Ostertagia leptospicularis/Ostertagia kolchida, Ostertagia (Grosspiculopteragia) occidentalis, Teladorsagia circumcincta/Teladorsagia trifurcate, Marshallagia marshalli, Nematodirus europaeus, Cooperia oncophora, Capillaria bovis, Oesophagostomum venulosum, and Trichuris ovis. All of them have already been cited in roe deer in Europe, but Marshallagia marshalli, Capillaria bovis, and Ostertagia (Grosspiculopteragia) occidentalis are reported for the first time in Spain in this host. The abomasum was the intestinal section, where the prevalence (98.9%) and mean intensity (x¯ = 370.7 ± 374.4 worms/roe deer; range 3-1762) were significantly higher, but no statistically significant differences were found when comparing the sampling areas and age of animals. The animals with lower body weight had a higher parasite load than those in better physical condition, finding, in this case, statistically significant differences (p = 0.0020). Seven genera and 14 species were identified. In the small intestine, 88% of the animals examined presented gastrointestinal nematodes, with an average intensity of x¯ = 131.7 ± 225.6 parasites/infected animal, ranging between 4-1254 worms. No statistically significant differences were found when the three parameters studied were compared. Four genera and seven species were identified. In the large intestine/cecum, 78.3% of the examined roe deer presented adult worms, with an average intensity of 6.3 ± 5.5 worms/infected animal; range 1-26 worms. Only statistically significant differences were observed when considering the mean intensity of parasitism and the sampling area (p = 0.0093). Two genera and two species were identified. Several of the species found in the study were studied molecularly, and with the sequences obtained compared with those deposited in GenBank, phylogenetic trees were prepared to determine their taxonomic status. Using coprological techniques, the existing correlation in the shedding of gastrointestinal nematode eggs in roe deer was investigated with that of semi-extensive sheep farms in the same study area to verify the existence of cross-transmission of these parasites between wild and domestic animals. The high values found in the studied parameters show that northern Spain is an area of high-intensity infection for roe deer.

Genetic deletion of HVEM in a leukemia B cell line promotes a preferential increase of PD-1- stem cell-like T cells over PD-1+ T cells curbing tumor progression.

Introduction: A high frequency of mutations affecting the gene encoding Herpes Virus Entry Mediator (HVEM, TNFRSF14) is a common clinical finding in a wide variety of human tumors, including those of hematological origin. Methods: We have addressed how HVEM expression on A20 leukemia cells influences tumor survival and its involvement in the modulation of the anti-tumor immune responses in a parental into F1 mouse tumor model of hybrid resistance by knocking-out HVEM expression. HVEM WT or HVEM KO leukemia cells were then injected intravenously into semiallogeneic F1 recipients and the extent of tumor dissemination was evaluated. Results: The loss of HVEM expression on A20 leukemia cells led to a significant increase of lymphoid and myeloid tumor cell infiltration curbing tumor progression. NK cells and to a lesser extent NKT cells and monocytes were the predominant innate populations contributing to the global increase of immune infiltrates in HVEM KO tumors compared to that present in HVEM KO tumors. In the overall increase of the adaptive T cell immune infiltrates, the stem cell-like PD-1- T cells progenitors and the effector T cell populations derived from them were more prominently present than terminally differentiated PD-1+ T cells. Conclusions: These results suggest that the PD-1- T cell subpopulation is likely to be a more relevant contributor to tumor rejection than the PD-1+ T cell subpopulation. These findings highlight the role of co-inhibitory signals delivered by HVEM upon engagement of BTLA on T cells and NK cells, placing HVEM/BTLA interaction in the spotlight as a novel immune checkpoint for the reinforcement of the anti-tumor responses in malignancies of hematopoietic origin.

Identification of Hypoderma actaeon (Diptera: Oestridae) in red deer (Cervus elaphus) from northern Spain: Microscopy study and molecular analysis.

Hypoderma spp. larvae were observed subcutaneously in the dorsal and lumbar regions of red deer (Cervus elaphus) hunted in the province of León (northwestern Spain) causing a myiasis. They were removed and initially classified by their size, shape, color, and location under the skin into the three larval stages that parasitize these animals. The morphological characteristics of the first and second-instar are described and from the features of the third-instar the species was identified as Hypoderma actaeon. To accurately identify this species, five isolates of genomic DNA from the third-instar, two from the second-instar and two from first-instar of H. actaeon were analyzed by PCR analysis of the COI region of mt-DNA. The results confirmed that the examined samples exactly matched with H. actaeon. This study has shown the morphological identification of the three larval stages of H. actaeon and, for the first time, the first and second-instar larvae have been molecularly characterized. Finally, identification of only H. actaeon suggests that this species is the only affecting red deer in the Iberian Peninsula.

Differential Engraftment of Parental A20 PD-L1 WT and PD-L1 KO Leukemia Cells in Semiallogeneic Recipients in the Context of PD-L1/PD-1 Interaction and NK Cell-Mediated Hybrid Resistance.

The contribution of natural killer (NK) cells to tumor rejection in the context of programmed death-ligand 1/programmed death 1 (PD-L1/PD-1) blockade is a matter of intense debate. To elucidate the role of PD-L1 expression on tumor cells and the functional consequences of engaging PD-1 receptor on cytotoxic cells, PD-L1 expression was genetically inactivated and WT or PD-L1-deficient parental tumor cells were adoptively transferred intravenously into F1 recipients. The engraftment of PD-L1-deficient A20 tumor cells in the spleen and liver of F1 recipients was impaired compared with A20 PD-L1 WT tumor counterparts. To elucidate the mechanism responsible for this differential tumor engraftment and determine the relevance of the role of the PD-L1/PD-1 pathway in the interplay of tumor cells/NK cells, a short-term competitive tumor implantation assay in the peritoneal cavity of semiallogeneic F1 recipients was designed. The results presented herein showed that NK cells killed target tumor cells with similar efficiency regardless of PD-L1 expression, whereas PD-L1 expression on A20 tumor cells conferred significant tumor protection against rejection by CD8 T cells confirming the role of the co-inhibitory receptor PD-1 in the modulation of their cytotoxic activity. In summary, PD-L1 expression on A20 leukemia tumor cells modulates CD8 T-cell-mediated responses to tumor-specific antigens but does not contribute to inhibit NK cell-mediated hybrid resistance, which correlates with the inability to detect PD-1 expression on NK cells neither under steady-state conditions nor under inflammatory conditions.

The impact of CD160 deficiency on alloreactive CD8 T cell responses and allograft rejection.

CD160 is a member of the immunoglobulin superfamily with a pattern of expression mainly restricted to cytotoxic cells. To assess the functional relevance of the HVEM/CD160 signaling pathway in allogeneic cytotoxic responses, exon 2 of the CD160 gene was targeted by CRISPR/Cas9 to generate CD160 deficient mice. Next, we evaluated the impact of CD160 deficiency in the course of an alloreactive response. To that aim, parental donor WT (wild-type) or CD160 KO (knock-out) T cells were adoptively transferred into non-irradiated semiallogeneic F1 recipients, in which donor alloreactive CD160 KO CD4 T cells and CD8 T cells clonally expanded less vigorously than in WT T cell counterparts. This differential proliferative response rate at the early phase of T cell expansion influenced the course of CD8 T cell differentiation and the composition of the effector T cell pool that led to a significant decreased of the memory precursor effector cells (MPECs) / short-lived effector cells (SLECs) ratio in CD160 KO CD8 T cells compared to WT CD8 T cells. Despite these differences in T cell proliferation and differentiation, allogeneic MHC class I mismatched (bm1) skin allograft survival in CD160 KO recipients was comparable to that of WT recipients. However, the administration of CTLA-4.Ig showed an enhanced survival trend of bm1 skin allografts in CD160 KO with respect to WT recipients. Finally, CD160 deficient NK cells were as proficient as CD160 WT NK cells in rejecting allogeneic cellular allografts or MHC class I deficient tumor cells. CD160 may represent a CD28 alternative costimulatory molecule for the modulation of allogeneic CD8 T cell responses either in combination with costimulation blockade or by direct targeting of alloreactive CD8 T cells that upregulate CD160 expression in response to alloantigen stimulation.

The Role of TNFR2 and DR3 in the In Vivo Expansion of Tregs in T Cell Depleting Transplantation Regimens.

Regulatory T cells (Tregs) are essential for the maintenance of tolerance to self and non-self through cell-intrinsic and cell-extrinsic mechanisms. Peripheral Tregs survival and clonal expansion largely depend on IL-2 and access to co-stimulatory signals such as CD28. Engagement of tumor necrosis factor receptor (TNFR) superfamily members, in particular TNFR2 and DR3, contribute to promote peripheral Tregs expansion and sustain their survival. This property can be leveraged to enhance tolerance to allogeneic transplants by tipping the balance of Tregs over conventional T cells during the course of immune reconstitution. This is of particular interest in peri-transplant tolerance induction protocols in which T cell depletion is applied to reduce the frequency of alloreactive T cells or in conditioning regimens that allow allogeneic bone marrow transplantation. These conditioning regimens are being implemented to limit long-term side effects of continuous immunosuppression and facilitate the establishment of a state of donor-specific tolerance. Lymphopenia-induced homeostatic proliferation in response to cytoreductive conditioning is a window of opportunity to enhance preferential expansion of Tregs during homeostatic proliferation that can be potentiated by agonist stimulation of TNFR.

Critical role of PD-L1 expression on non-tumor cells rather than on tumor cells for effective anti-PD-L1 immunotherapy in a transplantable mouse hematopoietic tumor model.

The expression of PD-L1 on tumor cells or within the tumor microenvironment has been associated with good prognosis and sustained clinical responses in immunotherapeutic regimens based on PD-L1/PD-1/CD80 immune checkpoint blockade. To look into the current controversy in cancer immunotherapy of the relative importance of PD-L1 expression on tumor cells versus non-tumor cells of the tumor microenvironment, a hematological mouse tumor model was chosen. By combining a genetic CRISPR/Cas9 and immunotherapeutic approach and using a syngeneic hematopoietic transplantable tumor model (E.G7-cOVA tumor cells), we demonstrated that dual blockade of PD-L1 interaction with PD-1 and CD80 enhanced anti-tumor immune responses that either delayed tumor growth or led to its complete eradication. PD-L1 expression on non-tumor cells of the tumor microenvironment was required for the promotion of tumor immune escape and its blockade elicited potent anti-tumor responses to PD-L1 WT and to PD-L1-deficient tumor cells. PD-L1+ tumors implanted in PD-L1-deficient mice exhibited delayed tumor growth independently of PD-L1 blockade. These findings emphasize that PD-L1 expression on non-tumor cells plays a major role in this tumor model. These observations should turn our attention to the tumor microenvironment in hematological malignancies because of its unappreciated contribution to create a conditioned niche for the tumor to grow and evade the anti-tumor immune response.

CD160 serves as a negative regulator of NKT cells in acute hepatic injury.

CD160 and BTLA both bind to herpes virus entry mediator. Although a negative regulatory function of BTLA in natural killer T (NKT) cell activation has been reported, whether CD160 is also involved is unclear. By analyzing CD160-/- mice and mixed bone marrow chimeras, we show that CD160 is not essential for NKT cell development. However, CD160-/- mice exhibit severe liver injury after in vivo challenge with α-galactosylceramide (α-GalCer). Moreover, CD160-/- mice are more susceptible to Concanavalin A challenge, and display elevated serum AST and ALT levels, hyperactivation of NKT cells, and enhanced IFN-γ, TNF, and IL-4 production. Lastly, inhibition of BTLA by anti-BTLA mAb aggravates α-GalCer-induced hepatic injury in CD160-/- mice, suggesting that both CD160 and BTLA serve as non-overlapping negative regulators of NKT cells. Our data thus implicate CD160 as a co-inhibitory receptor that delivers antigen-dependent signals in NKT cells to dampen cytokine production during early innate immune activation.

Correction to: T follicular helper expansion and humoral-mediated rejection are independent of the HVEM/BTLA pathway.

In this article, one of the grating agencies requested us to incorporate the information, Spanish Government and co-funded by European Union ERDF/ESF, "Investing in your future", in the acknowledgments section. The correct acknowledgement is as follows: "This work has been supported by grants of the Spanish Ministry of Health (Fondo de Investigaciones Sanitarias, PI13/00029, Spanish Government and co-funded by European Union ERDF/ESF, "Investing in your future"), Department of Education of Castilla and Leon Regional Government (Grant# LE093U13) and Mutua Madrileña Foundation (Basic research grants 2012) to J.I.R.B.; by Miguel Servet National Program (Ministry of National Health) CP12/03063 and by Gerencia Regional de Salud GRS963/A/2014 to M.L.R.G. We are particularly grateful to Mr. Leonides Alaiz for outstanding animal husbandry." The authors regret the errors.

Downregulation of BTLA on NKT Cells Promotes Tumor Immune Control in a Mouse Model of Mammary Carcinoma.

Natural Killer T cells (NKT cells) are emerging as critical regulators of pro- and anti-tumor immunity, both at baseline and in therapeutic settings. While type I NKT cells can promote anti-tumor immunity, their activity in the tumor microenvironment may be limited by negative regulators such as inhibitory immune checkpoints. We observed dominant expression of B- and T-lymphocyte attenuator (BTLA) on type I NKT cells in polyoma middle T oncogene-driven (PyMT) murine autochthonous mammary tumors. Other immune checkpoint receptors, such as programmed cell death 1 (PD-1) were equally distributed among T cell populations. Interference with BTLA using neutralizing antibodies limited tumor growth and pulmonary metastasis in the PyMT model in a therapeutic setting, correlating with an increase in type I NKT cells and expression of cytotoxic marker genes. While therapeutic application of an anti-PD-1 antibody increased the number of CD8+ cytotoxic T cells and elevated IL-12 expression, tumor control was not established. Expression of ZBTB16, the lineage-determining transcription factor of type I NKT cells, was correlated with a favorable patient prognosis in the METABRIC dataset, and BTLA levels were instrumental to further distinguish prognosis in patents with high ZBTB16 expression. Taken together, these data support a role of BTLA on type I NKT cells in limiting anti-tumor immunity.

T follicular helper expansion and humoral-mediated rejection are independent of the HVEM/BTLA pathway.

The molecular pathways contributing to humoral-mediated allograft rejection are poorly defined. In this study, we assessed the role of the herpesvirus entry mediator/B- and T-lymphocyte attenuator (HVEM/BTLA) signalling pathway in the context of antibody-mediated allograft rejection. An experimental setting was designed to elucidate whether the blockade of HVEM/BTLA interactions could modulate de novo induction of host antidonor-specific antibodies during the course of graft rejection. To test this hypothesis, fully allogeneic major histocompatibility complex-mismatched skin grafts were transplanted onto the right flank of recipient mice that were treated with isotype control, anti-CD40L or modulatory antibodies of the HVEM/BTLA signalling pathway. The frequencies of CD4 T follicular helper (Tfh) cells (B220-, CD4+ CXCR5+ PD-1high), extrafollicular helper cells (B220-, CD4+ CXCR5- PD-1+ and PD-1-) and germinal centre (GC) B cells (B220+Fas+ GL7+) were analysed by flow cytometry in draining and non-draining lymph nodes at day 10 post transplantation during the acute phase of graft rejection. The host antidonor isotype-specific humoral immune response was also assessed. Whereas blockade of the CD40/CD40L pathway was highly effective in preventing the allogeneic humoral immune response, antibody-mediated blockade of the HVEM/BTLA-interacting pathway affected neither the expansion of Tfh cells nor the expansion of GC B cells. Consequently, the course of the host antidonor antibody-mediated response proceeded normally, without detectable evidence of impaired development. In summary, these data indicate that HVEM/BTLA interactions are dispensable for the formation of de novo host antidonor isotype-specific antibodies in transplantation.

Modulation of cytotoxic responses by targeting CD160 prolongs skin graft survival across major histocompatibility class I barrier.

CD160 is a glycosylphosphatidylinositol-anchored protein of the immunoglobulin superfamily. It exhibits a pattern of expression coincident in humans and mice that is mainly restricted to cytotoxic cells and to all intestinal intraepithelial T lymphocytes. B- and T-lymphocyte attenuator (BTLA) and CD160 interact with cysteine-rich domain 1 of the extracellular region of Herpesvirus entry mediator (HVEM). CD160 engagement by HVEM can deliver inhibitory signals to a small subset of human CD4 T cells and attenuate its proliferation and cytokine secretion, but can also costimulate natural killer cells or intraepithelial lymphocytes. In turn, CD160 and BTLA can also function as agonist ligands being capable of costimulating T cells through membrane HVEM. Based on the restricted pattern of CD160 expression in cytotoxic cells, we postulated that CD160 may represent a suitable target for immune intervention in the setting of transplantation to modulate allogeneic cytotoxic responses. We demonstrated that in vivo administration of anti-CD160 antibody in combination with anti-CD40 L antibody to limit CD4 T-cell help modulated cytotoxic responses in a major histocompatibility complex class I mismatched model of allogeneic skin graft transplantation (bm1 donor to C57BL/6 recipient) and significantly prolonged graft survival. The implementation of this strategy in transplantation may reinforce current immunosuppression protocols and contribute to a better control of CD8 T-cell responses.

Immunotherapeutic targeting of LIGHT/LTßR/HVEM pathway fully recapitulates the reduced cytotoxic phenotype of LIGHT-deficient T cells.

Tumor necrosis factor (TNF)/TNF receptor (TNFR) superfamily members play essential roles in the development of the different phases of the immune response. Mouse LIGHT (TNFSF14) is a type II transmembrane protein with a C-terminus extracellular TNF homology domain (THD) that assembles in homotrimers and regulates the course of the immune responses by signaling through 2 receptors, the herpes virus entry mediator (HVEM, TNFRSF14) and the lymphotoxin ß receptor (LTßR, TNFRSF3). LIGHT is a membrane-bound protein transiently expressed on activated T cells, natural killer (NK) cells and immature dendritic cells that can be proteolytically cleaved by a metalloprotease and released to the extracellular milieu. The immunotherapeutic potential of LIGHT blockade was evaluated in vivo. Administration of an antagonist of LIGHT interaction with its receptors attenuated the course of graft-versus-host reaction and recapitulated the reduced cytotoxic activity of LIGHT-deficient T cells adoptively transferred into non-irradiated semiallogeneic recipients. The lack of LIGHT expression on donor T cells or blockade of LIGHT interaction with its receptors slowed down the rate of T cell proliferation and decreased the frequency of precursor alloreactive T cells, retarding T cell differentiation toward effector T cells. The blockade of LIGHT/LTßR/HVEM pathway was associated with delayed downregulation of interleukin-7Rα and delayed upregulation of inducible costimulatory molecule expression on donor alloreactive CD8 T cells that are typical features of impaired T cell differentiation. These results expose the relevance of LIGHT/LTßR/HVEM interaction for the potential therapeutic control of the allogeneic immune responses mediated by alloreactive CD8 T cells that can contribute to prolong allograft survival.

Therapeutic blockade of LIGHT interaction with herpesvirus entry mediator and lymphotoxin ß receptor attenuates in vivo cytotoxic allogeneic responses.

BACKGROUND: Tumor necrosis factor/tumor necrosis factor receptor superfamily members conform a group of molecular interaction pathways of essential relevance during the process of T-cell activation and differentiation toward effector cells and particularly for the maintenance phase of the immune response. Specific blockade of these interacting pathways, such as CD40-CD40L, contributes to modulate the deleterious outcome of allogeneic immune responses. We postulated that antagonizing the interaction of LIGHT expression on activated T cells with its receptors, herpesvirus entry mediator and lymphotoxin ß receptor, may decrease T cell-mediated allogeneic responses. METHODS: A flow cytometry competition assay was designed to identify anti-LIGHT monoclonal antibodies capable to prevent the interaction of mouse LIGHT with its receptors expressed on transfected cells. An antibody with the desired specificity was evaluated in a short-term in vivo allogeneic cytotoxic assay and tested for its ability to detect endogenous mouse LIGHT. RESULTS: We provide evidence for the first time that in mice, as previously described in humans, LIGHT protein is rapidly and transiently expressed after T-cell activation, and this expression was stronger on CD8 T cells than on CD4 T cells. Two anti-LIGHT antibodies prevented interactions of mouse LIGHT with its two known receptors, herpesvirus entry mediator and lymphotoxin ß receptor. In vivo administration of anti-LIGHT antibody (clone 10F12) ameliorated host antidonor short-term cytotoxic response in wild type B6 mice, although to a lesser extent than that observed in LIGHT-deficient mice. CONCLUSION: The therapeutic targeting of LIGHT may contribute to achieve a better control of cytotoxic responses refractory to current immunosuppressive drugs in transplantation.

ITIM-dependent negative signaling pathways for the control of cell-mediated xenogeneic immune responses.

Xenotransplantation is an innovative field of research with the potential to provide us with an alternative source of organs to face the severe shortage of human organ donors. For several reasons, pigs have been chosen as the most suitable source of organs and tissues for transplantation in humans. However, porcine xenografts undergo cellular immune responses representing a major barrier to their acceptance and normal functioning. Innate and adaptive xenogeneic immunity is mediated by both the recognition of xenogeneic tissue antigens and the lack of inhibition due to molecular cross-species incompatibilities of regulatory pathways. Therefore, the delivery of immunoreceptor tyrosine-based inhibitory motif (ITIM)-dependent and related negative signals to control innate (NK cells, macrophages) and adaptive T and B cells might overcome cell-mediated xenogeneic immunity. The proof of this concept has already been achieved in vitro by the transgenic overexpression of human ligands of several inhibitory receptors in porcine cells resulting in their resistance against xenoreactivity. Consequently, several transgenic pigs expressing tissue-specific human ligands of inhibitory coreceptors (HLA-E, CD47) or soluble competitors of costimulation (belatacept) have already been generated. The development of these robust and innovative approaches to modulate human anti-pig cellular immune responses, complementary to conventional immunosuppression, will help to achieve long-term xenograft survival. In this review, we will focus on the current strategies to enhance negative signaling pathways for the regulation of undesirable cell-mediated xenoreactive immune responses.

Selective blockade of herpesvirus entry mediator-B and T lymphocyte attenuator pathway ameliorates acute graft-versus-host reaction.

The cosignaling network mediated by the herpesvirus entry mediator (HVEM; TNFRSF14) functions as a dual directional system that involves proinflammatory ligand, lymphotoxin that exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT; TNFSF14), and the inhibitory Ig family member B and T lymphocyte attenuator (BTLA). To dissect the differential contributions of HVEM/BTLA and HVEM/LIGHT interactions, topographically-specific, competitive, and nonblocking anti-HVEM Abs that inhibit BTLA binding, but not LIGHT, were developed. We demonstrate that a BTLA-specific competitor attenuated the course of acute graft-versus-host reaction in a murine F(1) transfer semiallogeneic model. Selective HVEM/BTLA blockade did not inhibit donor T cell infiltration into graft-versus-host reaction target organs, but decreased the functional activity of the alloreactive T cells. These results highlight the critical role of HVEM/BTLA pathway in the control of the allogeneic immune response and identify a new therapeutic target for transplantation and autoimmune diseases.

B- and T-lymphocyte attenuator targeting protects against the acute phase of graft versus host reaction by inhibiting donor anti-host cytotoxicity.

BACKGROUND: B- and T-lymphocyte attenuator (BTLA) functions as a coinhibitory/costimulatory molecule that belongs to the immunoglobulin superfamily and exhibits a pattern of expression restricted to the hematopoietic compartment. Engagement of BTLA by its ligand, herpes virus entry mediator (HVEM), delivers negative signals to T cells, whereas engagement of HVEM receptor on T cells by surface BTLA expressed on other immune cells costimulates T activation. Previous work has reported that parental donor BTLA knock-out or HVEM knock-out T cells adoptively transferred into nonirradiated F1 recipient mice survived poorly, and the rejection of host hematopoietic cells was attenuated compared with F1 recipients receiving wild-type T cells. METHODS: Parent into nonirradiated immunocompetent F1 murine model of acute graft versus host reaction, which is induced with the adoptive transfer of splenocytes from donor B6 mice (H-2(b)) into F1 recipients (BALB/c×B6, H-2(d/b)), was used as an experimental approach to test the therapeutic effect of targeting BTLA during the course of an allogeneic immune response. RESULTS: We herein provide evidence that administration of an anti-BTLA monoclonal antibody leads to significant reduction of donor anti-host allogeneic immune response against bone marrow and thymus during the acute phase of graft versus host reaction in a parent into nonirradiated F1 murine model of alloreactivity. Anti-BTLA protection against donor anti-host hematopoietic cell rejection correlated with impaired anti-host cytotoxic T-lymphocyte activity than reduction in T-cell number infiltrating host tissues. CONCLUSIONS: These findings place BTLA receptor as a potential immunoregulatory target for the modulation of cytotoxic T-lymphocyte-mediated alloresponses.

Flt3L-mobilized dendritic cells bearing H2-Kbm1 apoptotic cells do not induce cross-tolerance to CD8+ T cells across a class I MHC mismatched barrier.

Tolerization of allogeneic CD8(+) T cells is still a pending issue in the field of transplantation research to achieve long-term survival. To test whether dendritic cells (DC) bearing allogeneic major histocompatibility complex (MHC) class I mismatched apoptotic cells could induce cross-tolerance to alloreactive CD8(+) T cells, the following experimental strategy was devised. Rag2/γ(c) KO B6 mice were treated with Fms-like tyrosine kinase 3 ligand (Flt3L)-transduced B16 melanoma cells to drive a rapid expansion and mobilization of DC in vivo. Of all DC populations expanded, splenic CD11c(+) CD103(+) CD8α(+) DC were selectively involved in the process of antigen clearance of X-ray irradiated apoptotic thymocytes in vivo. Considering that CD11c(+) CD103(+) CD8α(+) DC selectively take up apoptotic cells and that they are highly specialized in cross-presenting antigen to CD8(+) T cells, we investigated whether B6 mice adoptively transferred with Flt3L-derived DC loaded with donor-derived apoptotic thymocytes could induce tolerance to bm1 skin allografts. Our findings on host anti-donor alloresponse, as revealed by skin allograft survival and cytotoxic T lymphocyte assays, indicated that the administration of syngeneic DC presenting K(bm1) donor-derived allopeptides through the indirect pathway of antigen presentation was not sufficient to induce cross-tolerance to alloreactive CD8(+) T cells responding to bm1 alloantigens in a murine model of skin allograft transplantation across an MHC class I mismatched barrier.