B and T Lymphocyte Attenuator (BTLA)/Herpes Virus Entry Mediator (HVEM) Interaction: Attenuating Virus Specific CD8+T Cell Function In HTLV-1 Infection
Authors: Chibueze Ezinne Chioma , Yoshimitsu Makoto , Arima Naomichi 
Published: October 2019

Background and Aims
Human T-lymphotropic virus type 1(HTLV-1) infection is a retroviral infection with varied manifestations, either clinically asymptomatic carriers (AC) or symptomatic with about 1-5% developing the hematological manifestation, Adult T cell lymphoma/leukemia (ATLL). The host response to virus infections is mediated by CD8+ T cell function which in turn is partly mediated by expression of inhibitory molecules. Expression of these inhibitory molecules is a feature of dysfunctional CD8+T cells. CD8+ T cells from HTLV-1 infected individuals have been shown to be dysfunctional, in part due to expression of inhibitory molecules. One of such molecules is BTLA (B and T lymphocyte attenuator), a member of the CD28 co-signaling family of receptors. BTLA has been shown to interact with HVEM (herpes virus entry mediator) and to have varied functions on CD8+T cell function; however, its role in virus specific CD8+T cells in HTLV-1 infection has not been addressed.

Methods
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll centrifugation and cryopreserved until use. For staining, cells were incubated with fluorochrome-conjugated antibodies against BTLA, CD4, CD8, and HVEM. We compared the ex-vivo expression of BTLA on CD8+ T cells (ATLL, n=14; AC, n= 9; Healthy Individuals, n=11) and virus specific CD8+T cells corresponding to prevalent immune-dominant epitopes HLA tetramers HLA-A*0201 (Tax 11-19) or *2402 (Tax 301-309) obtained from HTLV-1 infected patients (ATLL, n=6; AC, n=6). We analyzed the expression of HVEM on CD4+ T cells; to determine the effect of addition of HVEM antibodies on CD8+ T lymphocyte function, PBMCs were cultured with and without Tax peptide and/or anti-HVEM monoclonal antibody in the presence of monensin and surface degranulation of CD107a measured in in-vitro assays. Quantitation was done using FACS Calibur flow-cytometer and analyzed with Flowjo software. Differences were analyzed by Mann-Whitney U test and pairs by Wilcoxon matched pairs test using GraphPad Prism (6.01). p-values <0.05 were considered significant.

Results
We observed a significantly lower expression of BTLA on CD8+T cells from HTLV-1 infected (Asymptomatic carriers (AC)) and Adult T cell leukemia/lymphoma (ATLL)) compared to healthy individuals (AC =17.8 ± 4.8(mean ± standard error), n=9; ATLL= 15.9 ± 4.2, n= 14; Healthy individuals = 33.4 ± 4.9, n=9; (p ATLL: HI <0.05; AC: HI <0.05)). We also observed a down-regulation of BTLA expression on HTLV-1 specific CD8+T cells in both ATLL and asymptomatic carriers. HVEM (herpes-virus entry mediator) was also expressed on CD4+ T cells but down regulated on CD4+ T cells from ATLL in comparison to asymptomatic carriers( ATLL= 60.9 ±4.7(n=11); AC =88.3 ± 2.2 (n=12); HI= 84.07± 2.5; p ATLL: HI= <0.05). Addition of HVEM monoclonal antibodies in the presence of tax stimulation resulted in an improvement in CD8+ T cell function as measured by surface degranulation of CD107a compared to tax stimulation alone( tax stimulation(26.8 ± 4.4) : tax stimulation + HVEM antibody addition( 39.4±3.7); p< 0.05).

Conclusion
Our findings indicate a possible involvement of BTLA/HVEM signaling in the regulation of immune response to HTLV-1 infection and suggests an involvement in the exhaustion/attenuation of HTLV-1- specific CD8+ T cells with possible extension to therapeutic interventions in the treatment of HTLV-1 infection.

Disclosures:
No relevant conflicts of interest to declare.