Expression Of BTLA Research Articles

Usurpation of a lymphocyte costimulatory molecule as a novel melanoma cell survival pathway (P2187)

Abstract BTLA (B- and T- lymphocyte attenuator), binds to the TNF-R family member HVEM (herpes virus entry mediator). HVEM through its interaction with BTLA in trans or in cis has been shown to act as a pro-survival signal through activation of NF-κB. In contrast, signaling through BTLA following HVEM engagement has been found to induce a negative signaling pathway by activating SHP1/2 phosphatases. During an initial immunohistochemistry screen for the expression of BTLA in melanoma tumor sections, we unexpectedly found that a many tumor cells also exhibited a uniform and strong positive intracellular expression of BTLA. BTLA was expressed in both primary and metastatic melanoma cells with low to negative expression in benign nevi. PCR analysis also revealed that although most tumor lines expressed isoform 1 of BTLA, some lines expressed both isoform 1 and 2 suggesting BTLA by be secreted. Moreover, we also found that melanoma cells express both surface and intracellular HVEM. Intriguingly, significant pools of BTLA were found to be co-localized with intracellular HVEM in the melanoma cells. This co-localization suggests that tumor cells may use HVEM activation as a mechanism of cell survival. Blockade of BTLA or inhibition of BTLA or HVEM expression on melanoma cells may therefore be a novel approach to inhibit tumor cell survival as well as prevent negative signaling through BTLA in immune cells.

Intravenous transfer of MOG-pulsed, bone marrow-derived dendritic cells modulates expression of co-stimulatory molecules and cytokines by CD4+ T cells (P5217)

Abstract Dendritic cells (DCs) can induce immune tolerance in vivo; however, the mechanisms of DC-mediated tolerance have not been fully elucidated. Here we demonstrate that intravenous (i.v.) transfer of bone marrow-derived DCs pulsed with MOG peptide blocks the development of experimental autoimmune encephalomyelitis (EAE). Transfer of MOG-pulsed DCs leads to down-regulation of IL-17A and IFN-γ production, while the production of IL-10 is up-regulated. The numbers of CD4+IL-10+IFN-γ+ T cells and CD4+FoxP3+IL-10+ regulatory T cells are also increased after MOG-pulsed DC transfer. Intravenous transfer of MOG-pulsed DCs down-regulates protein expression of OX40, CD154 and CD28 on CD4+ T cells, whereas expression of CD152, CD80, PD-1, ICOS and BTLA is up-regulated. Our results indicate that DCs pulsed with MOG inhibit CD4+ T cell activity via modulation of co-stimulatory signaling.

Immune tolerance induced by intravenous transfer of immature dendritic cells via up-regulating numbers of suppressive IL-10+ IFN-γ+-producing CD4+ T cells

Dendritic cells (DCs) regulate immunity and immune tolerance in vivo. However, the mechanisms of DC-mediated tolerance have not been fully elucidated. Here, we demonstrate that intravenous (i.v.) transfer of bone marrow-derived DCs pulsed with myelin oligodendrocyte glycoprotein (MOG) peptide blocks the development of experimental autoimmune encephalomyelitis in C57BL/6J mice. i.v. transfer of MOG-pulsed DCs leads to the down-regulation of the production of IL-17A and IFN-γ and up-regulation of IL-10 secretion. The development of regulatory T cells (Tregs) is facilitated via up-regulation of FoxP3 expression and production of IL-10. The number of suppressive CD4(+)IL-10(+)IFN-γ(+) T cells is also improved. The expression of OX40, CD154, and CD28 is down-regulated, but the expression of CD152, CD80, PD-1, ICOS, and BTLA is up-regulated on CD4(+) T cells after i.v. transfer of immature DCs. The expression of CCR4, CCR5, and CCR7 on CD4(+) T cells is also improved. Our results suggest that immature DCs may induce tolerance via facilitating the development of CD4(+)FoxP3(+) Tregs and suppressive CD4(+)IL-10(+)IFN-γ(+) T cells in vivo.

Sirolimus-based regimen promotes inhibitory costimulatory signal of HVEM/BTLA/CD160/LIGHT pathway in allo-renal recipients

HVEM/BTLA/CD160/LIGHT pathway is a very special costimulatory molecule system which can regulate T-cell immune responses by activating both inflammatory and inhibitory signalings. The regulatory effect of Sirolimus on HVME costimulatory system in allo-renal recipients has not been reported. In this study, we analyzed the expression of HVEM, BTLA, CD160 and LIGHT on circulating T cell subgroups and the expression of HVEM on CD4+ Tregs by flow cytometry and also the pre-dose concentration of Sirolimus by automatic analyzer. Both the allo-renal recipients receiving Sirolimus immunosuppressive regimen and health volunteers were included. The expression of both BTLA and CD160 on T cells increased significantly while the expression of LIGHT on T cells decreased significantly in allo-renal recipients receiving Sirolimus regimen (p<0.05). The expression of HVEM on T cells and CD4+ T-cell subgroup decreased (p<0.05) while that on CD8+ T-cell subgroup remained roughly normal (p>0.05).The expression of HVEM on CD4+ Tregs increased significantly (p<0.05) in allo-renal recipients receiving Sirolimus regimen (p<0.05). Though regulating the expression of HVEM/BTLA/CD160/LIGHT costimulatory system, Sirolimus-based regimen promotes inhibitory costimulatory signal in T cells and enhances the function of CD4+ Tregs in allo-renal recipients, which are in benefit of the control of transplant rejection as well as the induction and maintenance of transplant tolerance.

The upregulation of the inhibitory receptors BTLA and PD-1 defines a novel subset of dysfunctional tumor antigen-specific CD8+ T cells (127.19)

Abstract Cytotoxic T cells present at the periphery or at tumor sites recognize tumor epitopes presented by melanoma cells but fail to induce tumor rejection in melanoma patients. Inhibitory receptors including PD-1 and Tim-3 expressed by spontaneous CD8+ T cells directed against the cancer-germline antigen NY-ESO-1 play a role in promoting tumor-induced T cell dysfunction. In the present study, we show that BTLA is upregulated on spontaneous NY-ESO-1-specific CD8+ T cells and that BTLA-expressing PD-1+Tim-3- CD8+ T cells represent the largest subset of NY-ESO-1-specific CD8+ T cells in melanoma patients. These cells were partially dysfunctional, producing less IFN-γ than BTLA- T cells, but more IFN-γ, TNF and IL-2 than the highly dysfunctional subset expressing all three receptors. Unlike PD-1, BTLA expression by NY-ESO-1-specific CD8+ T cells does not increase with higher T cell dysfunction or upon cognate antigen stimulation, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Finally, BTLA blockade adds to PD-1 and Tim-3 blockades, enhancing NY-ESO-1-specific CD8+ T cell expansion, proliferation and cytokine production. Collectively, our findings support the targeting of BTLA, PD-1 and Tim-3 pathways to reverse tumor-induced T cell dysfunction in patients with advanced melanoma.

Abstract 5407: The upregulation of BTLA and PD-1 defines a novel subset of dysfunctional tumor antigen-specific CD8+ T cells

Abstract Cytotoxic T cells present at the periphery or at tumor sites recognize tumor epitopes presented by melanoma cells but fail to induce tumor rejection in melanoma patients. Inhibitory receptors including PD-1 and Tim-3 expressed by spontaneous CD8+ T cells directed against the cancer-germline antigen NY-ESO-1 play a role in promoting tumor-induced T cell dysfunction. In the present study, we show that BTLA is upregulated on spontaneous NY-ESO-1-specific CD8+ T cells and that BTLA+PD-1+Tim-3− CD8+ T cells represent the largest subset of NY-ESO-1-specific CD8+ T cells in melanoma patients. BTLA+PD-1+Tim-3− CD8+ T cells are partially dysfunctional, producing less IFN-γ than BTLA−PD-1+Tim-3− and BTLA−PD-1−Tim-3− but more IFN-γ, TNF and IL-2 than highly dysfunctional BTLA+PD-1+Tim-3+ NY-ESO-1-specific CD8+ T cells. Unlike PD-1, BTLA expression by NY-ESO-1-specific CD8+ T cells does not increase with higher T cell dysfunction or upon cognate antigen stimulation, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Finally, BTLA pathway blockade adds to PD-1 and Tim-3 blockades, enhancing NY-ESO-1-specific CD8+ T cell expansion, proliferation and cytokine production. Collectively, our findings support the targeting of BTLA, PD-1 and Tim-3 pathways to reverse tumor-induced T cell dysfunction in patients with advanced melanoma. This work was supported by the National Institutes of Health/National Cancer Institute grants R01CA90360 and R01CA157467 (to H.M. Zarour), an award from the Melanoma Skin Spore grant NCI P50CA121973 (to H.M. Zarour) and a grant from the Cancer Research Institute (to H.M. Zarour). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5407. doi:1538-7445.AM2012-5407

BTLA expression contributes to septic morbidity and mortality by inducing innate inflammatory cell dysfunction

A proper innate inflammatory response is essential for prevention of the systemic inflammation associated with sepsis. BTLA is an immune-regulatory receptor demonstrated to be expressed not only on adaptive immune populations and have potent inhibitory effects on CD4(+) T cells but is also expressed on innate cell populations (CD11c(+) and CD11b(+) cells) and has been shown to diminish pathogen clearance following bacterial and parasite infection. The role of BTLA in sepsis and the mechanisms by which BTLA alters pathogen clearance, however, have not been addressed clearly. Here, we show that following acute experimental sepsis induction in mice (CLP), the number of infiltrating BTLA- and HVEM (the ligand for BTLA)-expressing macrophages, inflammatory monocytes, mature and immature DCs, and neutrophils increased in the peritoneum compared with sham surgery, suggesting that a high level of HVEM:BTLA interactions occurs between these cells at the site of septic insult. Given this, we evaluated BTLA(-/-) mice, 24 h post-CLP, and observed a marked increase in the degree of activation on these cell populations, as well as a reduction in peritoneal bacterial burden and IL-10 induction, and most importantly, BTLA(-/-) mice exhibited a higher rate of survival and protection from organ injury when compared with WT mice. Such changes were not restricted to experimental mice, as circulating BTLA+ and HVEM+ monocytes and HVEM+ granulocytes were increased in septic ICU patients, supporting a role for BTLA and/or HVEM as potential, novel diagnostic markers of innate immune response/status and as therapeutic targets of sepsis.

CD8+ T Cells Specific for Tumor Antigens Can Be Rendered Dysfunctional by the Tumor Microenvironment through Upregulation of the Inhibitory Receptors BTLA and PD-1

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.

BTLA Expression is Increased on Circulating CD4+ T-cells in Trauma Patients. a Potential Mechanism for Trauma Induced Immunosuppression

BTLA Expression is Increased on Circulating CD4+ T-cells in Trauma Patients. a Potential Mechanism for Trauma Induced Immunosuppression

BTLA (C272) expression on CD4+ T cells in Behçet patients

To investigate the peripheral blood levels of B and T lymphocyte attenuator (BTLA/CD272) on T cells and its role in Behçet disease (BD), and to define the role of inhibitory receptors on BD. Materials and methods: Included in this study were 25 patients with BD and 20 healthy control subjects, matched for age and sex. Results: CD4+CD272+ (P < 0.001) and CD3+CD272+ (P < 0.001) T cell levels were significantly higher in BD patients (active and remission) compared to the healthy controls. However, there was no difference between the active patients and those in remission in terms of CD4+CD272+ T cells, while CD3+CD272+ T cell levels were significantly higher in active-phase BD patients compared to patients in the remission phase (P < 0.05). Conclusion: Further functional studies could be implemented to investigate the effectiveness of the present research parameter in the diagnosis or follow-up of certain diseases similar to BD and with obscure etiology, as well as its contribution to immune dysregulation of BD. BTLA can additionally modulate T cell response in BD on different inflammatory areas and it can be upregulated in BD because of T cell activation. The results of the present study indicate that BTLA expression on CD4+ Th (T helper) cells might play a limiting role on the inflammation at peripheral sites or organs in BD.

PD-1+ T Cell Subsets in Follicular Lymphoma Tumor Microenvironment and Their Implications for Prognosis and Therapy

Abstract 2648 Background:Programmed death (PD)-1, a coinhibitory receptor expressed by effector T cells (Teffs) is highly expressed on intratumoral T cells (mean 61%, range 34–86% for CD4+ T cells and mean 44%, range 31–69% for CD8+ T cells) in follicular lymphoma (FL), a finding associated with impaired ability to recognize autologous tumor (Nattamai et al, ASH 2007). Hence, PD-1 expression would be expected to confer an unfavorable prognosis in FL. However, correlation of PD-1 with clinical outcome in FL has been inconsistent with two studies showing favorable (Carreras et al, J Clin Oncol 2009; Wahlin et al, Clin Cancer Res 2010) and one study showing unfavorable (Richendollar et al, Hum Pathol 2011) outcome. While differences in method of analysis and type of treatment may explain the disparate results, a more complex model may be necessary to understand the prognostic impact of PD-1 in FL as PD-1 is expressed not only on antitumor Teffs but also on protumor follicular helper T cells (Tfh) and regulatory T cells (Tregs). Methods:To determine the nature of PD-1+ T cells in FL we performed comprehensive genomic and immunologic studies. By flow cytometry, we observed that the intratumoral CD4+ T cells in FL may be categorized into 3 subsets based on PD-1 expression - PD-1 high (PD-1hi), intermediate (PD-1int), and low (PD-1lo). The intratumoral CD8+ T cells consisted of PD-1int and PD-1lo subsets. The 3 CD4+ T cell subsets were FACSorted from FL tumors (n=3) and whole genome gene expression profiling (GEP) was performed. T cell subsets sorted similarly from tonsils served as controls for reactive follicular hyperplasia (FH) (n=3). Differentially expressed genes in GEP studies were confirmed at the mRNA level by real-time PCR (n=5) and at the protein level by flow cytometry when antibodies were available (n=5–10). Results:Our results suggested that CD4+PD-1hi T cells are Tfh cells (CXCR5hiBcl6hi ICOShiCD40LhiSAPhiPRDM1loIL-4hiIL-21hi); the CD4+PD-1int T cells consisted of a mixture of activated Teffs (CD45RO+CD45RA−) including Th1 (Tbet+IFNg+), Th2 (IL-10+), and Th17 cells (RORc+IL-17+), and Tregs (Foxp3+CD25hiCD127lo); and the CD4+PD-1lo T cells consisted of a mixture of activated Teffs (CD45RO+CD45RA− but IFNg−IL-4−IL-10−IL-17−), Tregs, and naïve T cells (CD45RO−CD45RA+CCR7+). Although these subsets were present in both FL and FH, there were important differences. IL-4 expression was significantly higher in Tfh in FL vs. FH and may play a role in the pathogenesis of FL. IL-17 expression was low and expression of coinhibitory molecules BTLA and CD200 was high in CD4+PD-1int T cells in FL vs. FH. BTLA and CD200 were also increased in CD8+PD-1int T cells in FL vs. FH. However, other coinhibitory molecules (LAG-3, Tim-3, CD160, CTLA-4, CD244, KLRG1) were not significantly different between FL and FH. CD4+PD-1int T cells also had higher expression of BATF, a transcription factor associated with T cell exhaustion in FL vs. FH. Together, these results suggest that the CD4+PD-1int T cells in FL may be in a state of T cell exhaustion whereas the CD4+PD-1int T cells in FH may represent recently activated Teffs. Consistent with this, blocking PD-1 with anti-PD-1 blocking antibody significantly enhanced proliferation and the production of Th1 (IFNg, TNFa) but not Th2 (IL-4, IL-5, IL-10, IL-13) cytokines by intratumoral CD4+ and CD8+ T cells in response to stimulation with autologous FL tumor cells (n=3). As expected, Tregs were increased in number in FL vs. FH and were present in the PD-1int and PD-1lo T cell subsets. We found 74% (range 40–97%) of FL Tregs expressed PD-1. Among the CD4+PD-1lo and CD8+PD-1lo T cells, there were more activated Teffs and fewer naïve T cells in FL vs. FH. Conclusions:Our results suggest that the PD-1+ T cells in FL are comprised of a mixture of antitumor Teffs and protumor Tfh and Tregs. The prognostic impact of PD-1+ T cells in FL may dependent on the relative frequency of these subsets as ligation of PD-1 may produce favorable (inhibition of protumor Tfh and Tregs) or unfavorable (inhibition of antitumor Teffs) outcomes by inhibiting or promoting tumor growth, respectively. Conversely, our results imply that agents that block PD-1/PD-ligand pathway may have the opposite effect on these T cell subsets and enumeration of the intratumoral PD-1+ T cell subsets may serve as biomarker to predict response to these agents in FL and possibly other B-cell malignancies. Disclosures:Dong:GSK: Consultancy; Genentech: Honoraria; Tempero: Consultancy; Ono: Consultancy; AnaptysBio: Consultancy. Neelapu:Cure Tech Ltd: Research Funding.

An encephalomyelitis-specific locus on chromosome 16 in mouse controls disease development and expression of immune-regulatory genes

A locus on mouse chromosome 16 was found to control experimental autoimmune encephalomyelitis (EAE) in studies using congenic mice. Genes within the congenic region control encephalomyelitis but not arthritis, indicating the presence of genes in this region involved in central nervous system (CNS) specific mechanisms. Flow cytometry analyses of expression of two candidate genes within the linked locus, Cd200 and Btla, demonstrated a significantly lower expression of CD200 on CD4 + T cells and higher expression of BTLA on B cells from the congenic mice. These results suggest that genes within this mouse chromosome 16 locus specifically control EAE development possibly through immune-regulatory cell-surface molecules.

Human sepsis induces a novel cell exhaustion phenotype in the lung characterized by T cell expression of PD-1 &amp; BTLA and HVEM expression on airway epithelium (99.9)

Abstract Approximately 200,000 people die annually in the US from sepsis. Sepsis is characterized by an acute “cytokine storm” followed by an immune suppressed state. While apoptosis of innate and adaptive immune cells has been demonstrated, the mechanism for immunosuppression in sepsis is poorly defined. In this study, we analyzed the function and phenotype of cells isolated from lung and spleen of septic patients collected within 120 minutes of death and compared these to cells similarly isolated from non-septic patients. Post-stimulation, splenocytes from septic patients had dramatically reduced secretion of the cytokines IFNγ, TNFα, IL-6 and IL-10. Expression of activating and inhibitory receptors was examined by flow cytometry. Splenic T cells had decreased expression of the IL-7 receptor and elevated expression of CD69. Both lung and splenic T cells had increased expression of the inhibitory receptor PD-1 as well as increased expression of its ligands PDL1 and PDL2 on dendritic cells. BTLA but not CTLA4 expression was detected on the majority of lung T cells from septic and control patients. Interestingly HVEM, the ligand for BTLA, was detected by immunohistochemistry on airway epithelial cells of lungs from septic but not control patients, providing a receptor:ligand interaction with the potential to mediate local immune suppression. These data are consistent with a state of immune cell exhaustion, and provide a novel mechanism for local immunosuppression in the lung.

B and T Lymphocyte Attenuator Is Highly Expressed on CMV-Specific T Cells during Infection and Regulates Their Function

B and T lymphocyte attenuator (BTLA), like its relative programmed cell death-1 (PD-1), is a receptor that negatively regulates murine T cell activation. However, its expression and function on human T cells is currently unknown. We report in this study on the expression of BTLA in human T cell subsets as well as its regulation on virus-specific T cells during primary human CMV infection. BTLA is expressed on human CD4(+) T cells during different stages of differentiation, whereas on CD8(+) T cells, it is found on naive T cells and is progressively downregulated in memory and differentiated effector-type cells. During primary CMV infection, BTLA was highly induced on CMV-specific CD8(+) T cells immediately following their differentiation from naive cells. After control of CMV infection, BTLA expression went down on memory CD8(+) cells. Engagement of BTLA by mAbs blocked CD3/CD28-mediated T cell proliferation and Th1 and Th2 cytokine secretion. Finally, in vitro blockade of the BTLA pathway augmented, as efficient as anti-PD-1 mAbs, allogeneic as well as CMV-specific CD8(+) T cell proliferation. Thus, our results suggest that, like PD-1, BTLA provides a potential target for enhancing the functional capacity of CTLs in viral infections.

SLAM-associated protein (SAP)-deficiency impedes the function of T follicular helper cells in germinal center formation (99.3)

Abstract Mutations affecting SAP lead to XLP, an immune disorder with impaired antibody responses. SAP-/- mice show defective formation of germinal centers (GC), due to a defect in T:B cell interactions. T follicular helper cells (Tfh) are important for providing help to B cells and GC formation. To investigate the role of SAP in Tfh cells for GC formation, we have developed a modified protocol to generate “Tfh-like” cells in vitro, resulting in IL-21 producing cells with elevated expression of hallmark Tfh markers CXCR5, ICOS, PD-1, BTLA, and SLAM family receptors CD84 and Ly108, along with enhanced levels of Bcl6 transcripts. Cell transfer of control Tfh-like cells into SAP-/- hosts can rescue GC formation upon immunization significantly better than polarized Th1, Th2 or Th17 cells, suggesting these cells act as functional Tfh cells in vivo. In vitro differentiation of SAP-/- cells is similar to control cells, yet, upon cell transfer, SAP-/- Tfh-like cells are unable to support GC formation. Interestingly, differentiation of Tfh-like cells is not terminal, as they can be repolarized to produce other distinct Th cell cytokines. Similarly, other Th cell populations can be repolarized to Tfh-like cells. Analyses of epigenetic modifications of transcription factors also supports plasticity between these Th cell lineages. These findings provide further insight into Tfh cell biology and suggest that SAP is critical for Tfh cell function in GC formation and immune homeostasis.

IL-17 plays differential role in primary verse memory immune responses and recognition of cellular verse soluble antigens (87.30)

Abstract IL-17 is a proinflammatory cytokine that has been linked to many autoimmune related diseases such as rheumatoid arthritis, asthma, lupus, and multiple sclerosis. Recent studies have indicated that IL-17 may also play central role in driving the development of germinal center-derived autoantibody and Th response in an autoimmune disease model setting. In addition, activated and memory T cells can both produce IL-17. In this report, we found that IL-17 differentially affected primary versus memory immune response to cellular versus soluble antigen. Briefly, we initiated primary or memory immune response with SRBC or OVA and blocked IL-17 using anti-IL-17 antibody (Amgen). We found that blockade of IL-17 did not affect OVA-induced primary or memory response, nor did it affect SRBC-induced memory response; however, the blockade inhibited SRBC-induced primary response. SRBC-induced hemagglutination, DTH and specific T cell proliferation were inhibited; serum IFN-gamma, IL-12 and SRBC-specific IgG1 were decreased; numbers of germinal centers and IgG1-producing B cells in the germinal center were decreased; IL-17R knockout-CD4 T cells showed a decreased expression of BTLA and ICOS; however, percentages of CD4, CD8, CD3 and CD19 subsets in the spleen did not change with the antibody blockade. Our report suggests a differential role of IL-17 on T-B, T-DC cell interaction or Ig class switch in response to cellular versus soluble antigens, and primary versus memory immune response.

Effect of BTLA-HVEMinhibitory pathway blockade on DCs in vitro and in vivo

：Objective To explore theeffect of blocking BTLA-HVEM (herpesvirus entry mediator-B and T lymphocyte attenuator)pathway on dendritic cell function and the related immunological mechanisms. MethodsMurine BTLA extracellular domain eukaryotic expression vector psBTLA was constructed bygene recombination and transfected CHO by Lipofection method. Mouse bone marrow cells wereinduced to differentiate into DCs by GM-CSF plus IL-4. Expression of BTLA and HVEM on DCswas detected after HSPT0-TC-1 peptide complex stimulation by FACS. Expression of BT-1 andsecretion of IL-12 were detected after HSP70-TC-1 peptide complex plus psBTLA transfectedCHO culture supernatant stimulation on DCs. Pretreated DCs co-cultured with the samegenetic background mouse splenocytes and lymphocytes proliferation and cytokine secretionwere detected. Effect of psBTLA gene transfer in vivo on BT-1 expression of DCs and tumorgrowth on tumor-bearing mice was detected. Results Extracellular domain of murine BTLA wassuccessfully constructed, psBTLA stable transfection CHO cells were obtained andexpression of BTLA extracellular domain(sBTLA) was detected the in its culturesupernatant. BTLA and HVEM expression of DCs were increased after stimulation by theantigen peptide complex. When DCs were treated with antigen peptide complex plus culturesupernatant containing sBTLA, B7-1 expression and IL-12 secretion were increased.Co-cultured with splenocytes, lymphocytes proliferation and cytokine secretion, such asIL-2 and IFN-γ,,were also increased. Gene transfection with psBTLA in vivo promoted B7-1 expression on DCsand inhibited cervical cancer cells growth. Conclusion Blockade of BTLA-HVEM inhibitorypathway with sBTLA can further improve DCs function, activation of lymphocytes and promoteantitumor immune response. Key words: BTLA; HVEM; Dendritic cell; Costimulatory molecule

Identification of Escape Mechanisms Involved in the Absence of Viral Control Despite Activation of Both Innate (natural killer and gammadelta T cells) and Virus-Specific T Cells in Children with CMV or EBV Disease Following Cord Blood Transplantation.

Abstract 2659Poster Board II-635 Introduction:Cord blood transplantation (CBT) are being more and more used both for children and adults. However, one of the major limitation remains the low virus- CTL (cytotoxic T lymphocyte) response associated with the delayed immune recovery. Because the large majority of cord blood lymphocytes are naïve and so are not able to rapid clonal expansion, the monitoring of patients undergoing CBT provides a unique opportunity to 1) better understand the development of innate and adaptive human immune system and 2) identify the immune parameters associated to the viral response especially innate immunity (NK and gammadelta T cells) as well as T cells. Specifically, we have analysed their status of activation, function, and the expression of receptors (PD-1, 2B4, BTLA) recently demonstrated to be associated to immune escape. Materials and Methods:We investigated children with severe CMV or EBV disease following CBT and compared them both with children who controlled viral-reactivation following CBT or allogenic bone marrow transplantation (BMT) and with healthy donors. All patients received myeloablative conditioning including ATG and achieved full donor chimerism. Moreover, none of the selected patients received corticosteroid at the time of disease. CMV and EBV-specific CD8 T cells were investigated using HLA class I pentamers or tetramers together with extensive phenotype analyses by multi parametric flow cytometry and functional assay were performed using IFN gamma ELISPOT assay. Results:Kinetic of immune recovery showed a dramatic increase of CD8 but no CD4 T cells in patients with CMV or EBV disease. All CD8 T cells were highly activated (CD69high, CD57 high, PD-1 high, BTLAlow, 2B4 high, CD28 high). Virus-specific CTL were present in variable proportion. The major population of EBV-CTL were differentiated in CD45RA-CD27-CD28- late effector memory (EM) with almost no effector memory CD45RA+ cells (TEMRA) nor EBV specific activated naïve T cell. This is in sharp contrast with EBV-CTL of either: 1) healthy volunteers with controlled viral infection, who were mainly in a CD45RA-CD27+CD28+ Central Memory (CM) and CD45RA-CD27+CD28- early EM stage of differentiation; 2) patients with EBV infection during post transplantation immunosuppression who have activated naïve and TEMRA EBV-CTL. CMV-CTL were also mainly in late EM but no TEMRA differentiation stages in contrast to healthy volunteers and patients who controlled their CMV infection after CBT. Virus- CTL were highly activated (High expression of 2B4, CD28, PD-1, DR) with an overexpression of inhibitory receptors as PD-1 and 2B4 but no BTLA.The T cell response against viruses was highly variable in regard to different virus and patients as analysed by IFNg ELISPOT assay. CD4 T cells although low (<50 /mm3) were differentiated. The innate effectors gamma-delta T cells and NK cells were highly activated. Gamma-delta T cells belonged mainly to VD2- subset with a late EM and EMRA differentiation stage. VD2- subset were activated (CD69high, CD57high, CD8high) with low expression of BTLA. Their status was identical to CBT recipients with controlled viral infection. Similarly, NK cells were activated as determined by NKG2A, NKG2C, NKp30 and NKp46 expression. In addition, they were functional as demonstrated by the expression of CD57, which is associated to perforin and granzyme content. Conclusion:Thus, these reports illustrate a full activation of innate effectors as well as virus-CTL during viral disease after CBT, despite lack of viral clearance. We have identified at least three mechanisms that could explain this apparent immune failure:1.Profound CD4 T cell deficiency2.Altered pattern of virus-specific CD8 T cells differentiation associated with overexpression of inhibitory receptors such as PD-1 and 2B4 but no BTLA3.Deficiency in the generation of naive virus-specific CD8 T cells. Perspectives:These data support for the first time the hypothesis that after CBT, the immune system although quantitatively insufficient, is highly activated and so that in-vitro expansion of patient self-lymphocytes or means to expand CD4 and CD8 T cells pools are a potential way to cure patients suffering from uncontrolled viral replication after CBT together with blockade of inhibitory pathways. Disclosures:No relevant conflicts of interest to declare.

Activation-induced accumulation of B and T lymphocyte attenuator at the immunological synapse in CD4+ T cells

BTLA, a recently cloned coreceptor expressed on lymphocytes, negatively regulates cell activation by recruiting SHP-1/SHP-2. However, the mechanisms that regulate the intracellular localization of BTLA and its trafficking to the cell surface in T cells are still unknown. To determine the mechanisms that regulate the expression of BTLA on the surface of T cells, we examined the subcellular localization of BTLA in mouse T cells in a steady state, as well as upon activation by using a confocal laser-scanning microscopy. We found that BTLA was localized mainly in the Golgi apparatus and secretory lysosomes in resting CD4(+) T cells. We also found that intracellular BTLA was translocated to the cell surface and accumulated at the immunological synapse upon TCR stimulation. Furthermore, we found that the BTLA-HVEM interaction was required for the association of BTLA with lipid rafts. These results indicate that the surface expression of BTLA and its accumulation at the immunological synapse are tightly regulated by TCR and HVEM stimulation to deliver efficient inhibitory signals in the regulation of CD4(+) T cell activation.

Detection of protein on BTLA low cells and in vivo antibody-mediated down-modulation of BTLA on lymphoid and myeloid cells of C57BL/6 and BALB/c BTLA allelic variants

B- and T-lymphocyte attenuator (BTLA, CD272) is a polymorphic molecule belonging to the Ig superfamily (SF) that attenuates BCR and TCR-mediated signaling, and thereby functions as a negative regulator of lymphocyte activation. Herein, we report an anti-murine BTLA mAb (clone 4G12b) that remarkably detects protein expression on BTLA low cells such as naïve CD4 + cells, CD8 + T cells, dendritic cells (DC), as well as in NKT cells and for the first time, we found BTLA expression on DX5 dim and DX5 bright subsets of non-T NK cells in both C57BL/6 (B6) and BALB/c BTLA allelic variants. Anti-BTLA 4G12b mAb binds to an overlapping epitope to that recognized by anti-BTLA 6A6 mAb, but in contrast to the concept widely accepted of blocking activity of 6A6 mAb, surprisingly neither 4G12b nor 6A6 mAbs impeded murine HVEM–mIgG 2a.Fc recombinant fusion protein from interacting with BTLA-expressing cells. Lastly, in vivo administration of anti-BTLA 4G12b mAb induced a profound and lasting down-modulation of BTLA expression that led to BTLA receptor internalization with the potential utility of shutting down BTLA expression at any stage during the course of the immune response in both B6 and BALB/c strains of mice .