scholarly journals 3O A pre-existing inflammatory immune microenvironment predicts the clinical and immunological response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 peptide vaccination

2019 ◽  
Vol 30 (Supplement_11) ◽  
Author(s):  
Z Abdulrahman ◽  
N F C C de Miranda ◽  
M I E van Poelgeest ◽  
S H van der Burg
Keyword(s):  
T Cells ◽  
Clinical Response ◽  
Cd8 T Cells ◽  
Multispectral Imaging ◽  
Imaging System ◽  
Conflicts Of Interest ◽  
Human Papilloma Virus Type ◽  
Therapeutic Vaccination ◽  
High Grade ◽  
Immune Microenvironment

Abstract Background Vulvar High-grade Squamous Intraepithelial Lesion (vHSIL) is predominantly induced by high-risk Human Papilloma Virus type 16 (HPV16). In two independent trials, therapeutic vaccination against the oncoproteins of HPV16 with synthetic long peptides (SLP) resulted in vHSIL regression in about half of the patients after 12 months. Several studies have shown that the immune microenvironment influences therapy outcome. Therefore, a thorough investigation of the vHSIL immune microenvironment before and after SLP vaccination was performed, and its impact on clinical response was studied. Methods Two novel multiplex immunofluorescence panels were designed for formalin-fixed paraffin-embedded tissue, one for T cells (CD3, CD8, FoxP3, Tim3, Tbet, PD-1, DAPI) and one for myeloid cells (CD14, CD33, CD68, CD163, CD11c, PD-L1, DAPI). Pre- and 3 months post-vaccination biopsies of 29 patients and 27 healthy vulva excisions were stained, scanned with the Vectra multispectral imaging system, and automatically phenotyped and counted with inForm advanced image analysis software. Results A pre-existing pro-inflammatory TME, marked by high numbers of CD4 and CD8 T cells expressing Tbet and/or PD-1 as well as CD14+ inflammatory macrophages, is a strong predictor for good clinical response. A clear stepwise increase in pre-vaccination infiltrating Tbet+, CD4+, CD8+ T cells and CD14+ macrophages, and decrease in Foxp3+ Tregs was observed as response increased from non to partial to complete response. Moreover, the pre-vaccination immune microenvironment of complete responders resembled healthy vulva. Vaccination further increased infiltrating CD4+ and Tbet+ T cells and CD14+ macrophages and decreased FoxP3+ Tregs in the complete and partial responders, but not in the non responders. Conclusion Clinical responsiveness to therapeutic HPV16 SLP vaccination requires a pre-existing inflamed type 1 immune contexture in vHSIL. Hence, only patients with an inflamed TME should be selected for monotherapy by therapeutic vaccination, since this strategy is incapable of creating an inflamed TME in patients where this is absent. Legal entity responsible for the study The authors. Funding Leiden University Medical Center. Disclosure S.H. van der Burg: Advisory / Consultancy: ISA Pharmaceuticals. All other authors have declared no conflicts of interest.

scholarly journals Pre-existing inflammatory immune microenvironment predicts the clinical response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 vaccination

2020 ◽  
Vol 8 (1) ◽  
pp. e000563 ◽  
Author(s):  
Ziena Abdulrahman ◽  
Noel de Miranda ◽  
Edith M G van Esch ◽  
Peggy J de Vos van Steenwijk ◽  
Hans W Nijman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clinical Response ◽  
Myeloid Cells ◽  
Cell Infiltration ◽  
Therapeutic Vaccination ◽  
High Grade ◽  
Immune Microenvironment ◽  
Immune Infiltration ◽  
Hla Dr

BackgroundVulvar high-grade squamous intraepithelial lesion (vHSIL) is predominantly induced by high-risk human papilloma virus type 16 (HPV16). In two independent trials, therapeutic vaccination against the HPV16 E6 and E7 oncoproteins resulted in objective partial and complete responses (PRs/CRs) in half of the patients with HPV16+vHSIL at 12-month follow-up. Here, the prevaccination and postvaccination vHSIL immune microenvironment in relation to the vaccine-induced clinical response was investigated.MethodsTwo novel seven-color multiplex immunofluorescence panels to identify T cells (CD3, CD8, Foxp3, Tim3, Tbet, PD-1, DAPI) and myeloid cells (CD14, CD33, CD68, CD163, CD11c, PD-L1, DAPI) were designed and fully optimized for formalin-fixed paraffin-embedded tissue. 29 prevaccination and 24 postvaccination biopsies of patients with vHSIL, and 27 healthy vulva excisions, were stained, scanned with the Vectra multispectral imaging system, and automatically phenotyped and counted using inForm advanced image analysis software.ResultsHealthy vulvar tissue is strongly infiltrated by CD4 and CD8 T cells expressing Tbet and/or PD-1 and CD14+HLA-DR+inflammatory myeloid cells. The presence of such a coordinated pre-existing proinflammatory microenvironment in HPV16+vHSIL is associated with CR after vaccination. In partial responders, a disconnection between T cell and CD14+myeloid cell infiltration was observed, whereas clinical non-responders displayed overall lower immune cell infiltration. Vaccination improved the coordination of local immunity, reflected by increased numbers of CD4+Tbet+T cells and HLA-DR+CD14+expressing myeloid cells in patients with a PR or CR, but not in patients with no response. CD8+T cell infiltration was not increased after vaccination.ConclusionA prevaccination inflamed type 1 immune contexture is required for stronger vaccine-induced immune infiltration and is associated with better clinical response. Therapeutic vaccination did not overtly increase immune infiltration of cold lesions.

747 Evaluation of immune microenvironment of primary lung cancer and synchronous liver metastasis with multispectral imaging system

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A795-A795
Author(s):  
Hyeonbin Cho ◽  
Jae-Hwan Kim ◽  
Ji-Hyun Kim
Keyword(s):  
T Cells ◽  
Liver Metastasis ◽  
Multispectral Imaging ◽  
Immune Cell ◽  
Imaging System ◽  
Cytotoxic T Cells ◽  
Primary Lung Cancer ◽  
Helper T Cells ◽  
Synchronous Liver Metastasis ◽  
Immune Microenvironment

BackgroundCancer immunotherapy (CIT) has substantially improved the survival of cancer patients. However, according to recent studies, liver metastasis was reported to predict worse outcomes for CIT. The main objective of the study is to evaluate the differences in the immune microenvironment (IME) between the primary lung cancer (PL) and synchronous liver metastasis (LM) using a multispectral imaging system.MethodsSix immune markers (CD4, CD8, CTLA-4, granzyme B (GZB), Foxp3 and PD-L1) were analyzed using a multiplex IHC system and inForm program (Akoya) on paired lung-liver samples of 10 patients. Cells were categorized into tumor nest and stroma, and cell counts per unit area were measured for comparison.ResultsThe number of tumor-infiltrating cytotoxic T cells (TIL) in PL (262.5 cells/mm2) was higher than that of LM (113.3 cells/mm2). Additionally, the ratio between the number of TIL and non-TIL was greater in PL (0.31) compared to that of LM (0.26). A similar trend appeared for Helper T cells and regulatory T cells (Treg), as PL consisted of higher numbers of T cells (791.8 Helper T cells/mm2, 195.7 Treg/mm2) than LM (626.3 Helper T cells/mm2, 121.3 Treg/mm2). However, cytotoxic T cells exhibiting GZB+ and CTLA-4- were fewer in PL (140.2 cells/mm2) than in LM (203.3 cells/mm2), and the ratio is 0.69. The mean number of GZB+ TIL in PL (32.5 cells/mm2) was lower than in LM (35.3 cells/mm2), and their proportions among total TIL counts were 0.12 and 0.31, respectively. In PL, GZB+: GZB- ratio is 0.16 while the ratio is 1.91 for LM. A fewer number of TILs exhibiting GZB suggests that PL has lower efficiency in immune response than LM. Another crucial checkpoint receptor that inhibits immune response, CTLA-4, was more prevalent in PL, with CTLA-4+: CTLA-4- ratio in Treg being 0.36 in PL, compared to 0.11 in LM. The tumor proportion score (TPS) of PD-L1 was higher in PL than LM (40.0 vs. 6.6).ConclusionsIn our study, we showed the differences in the numbers of TIL or regulatory T cells and expressions of immune checkpoint receptors (PD-L1, CTLA-4), which significantly influence outcomes for CIT. The study is ongoing to confirm different IME between the PL and LM groups in a larger tumor cohort.ReferencesPeng, Jianhong, et al., Immune Cell Infiltration in the Microenvironment of Liver Oligometastasis from Colorectal Cancer: Intratumoural CD8/CD3 Ratio Is a Valuable Prognostic Index for Patients Undergoing Liver Metastasectomy. Cancers 2019 Dec; 11(12): 1922. https://doi.org/10.3390/cancers11121922Tumeh, Paul C., et al., Liver Metastasis and treatment outcome with Anti-PD-1 monoclonal antibody in patients with melanoma and NSCLC. Cancer Immunol Res 2017 May; 5(5): 417–424. doi: 10.1158/2326-6066.CIR-16-0325Parra, E.R., Immune Cell Profiling in Cancer Using Multiplex Immunofluorescence and Digital Analysis Approaches; Streckfus, C.F., Ed.; IntechOpen: London, UK, 2018; pp. 1–13. doi: 10.5772/intechopen.80380Ribas, A., Hu-Lieskovan, S., What does PD-L1 positive or negative mean?. The Journal of Experimental Medicine 2016;213(13):2835–2840. https://doi.org/10.1084/jem.20161462

scholarly journals Radiotherapy, Temozolomide, and Antiprogrammed Cell Death Protein 1 Treatments Modulate the Immune Microenvironment in Experimental High-Grade Glioma

Neurosurgery ◽  
2020 ◽  
Author(s):  
Matteo Riva ◽  
Roxanne Wouters ◽  
Edmond Sterpin ◽  
Roberto Giovannoni ◽  
Louis Boon ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Cd8 T Cells ◽  
Checkpoint Inhibitors ◽  
Statistical Significance ◽  
Suppressor Cells ◽  
High Grade ◽  
Immune Microenvironment ◽  
The Impact ◽  
Cell Death Protein

Abstract BACKGROUND The lack of immune synergy with conventional chemoradiation could explain the failure of checkpoint inhibitors in current clinical trials for high-grade gliomas (HGGs). OBJECTIVE To analyze the impact of radiotherapy (RT), Temozolomide (TMZ) and antiprogrammed cell death protein 1 (αPD1) (as single or combined treatments) on the immune microenvironment of experimental HGGs. METHODS Mice harboring neurosphere /CT-2A HGGs received RT (4 Gy, single dose), TMZ (50 mg/kg, 4 doses) and αPD1 (100 μg, 3 doses) as monotherapies or combinations. The influence on survival, tumor volume, and tumor-infiltrating immune cells was analyzed. RESULTS RT increased total T cells (P = .0159) and cluster of differentiation (CD)8+ T cells (P = .0078) compared to TMZ. Lymphocyte subpopulations resulting from TMZ or αPD1 treatment were comparable with those of controls. RT reduced M2 tumor-associated macrophages/microglia (P = .0019) and monocytic myeloid derived suppressor cells (mMDSCs, P = .0003) compared to controls. The effect on mMDSC was also seen following TMZ and αPD1 treatment, although less pronounced (P = .0439 and P = .0538, respectively). Combining RT with TMZ reduced CD8+ T cells (P = .0145) compared to RT alone. Adding αPD1 partially mitigated this effect as shown by the increased CD8+ T cells/Tregs ratio, even if this result failed to reach statistical significance (P = .0973). Changing the combination sequence of RT, TMZ, and αPD1 did not alter survival nor the immune effects. CONCLUSION RT, TMZ, and αPD1 modify the immune microenvironment of HGG. The combination of RT with TMZ induces a strong immune suppression which cannot be effectively counteracted by αPD1.

scholarly journals Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence

2020 ◽  
Vol 25 (4) ◽  
pp. 417-432
Author(s):  
Hidetoshi Mori ◽  
Jennifer Bolen ◽  
Louis Schuetter ◽  
Pierre Massion ◽  
Clifford C. Hoyt ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Multispectral Imaging ◽  
Imaging System ◽  
Predictive Biomarkers ◽  
Cell Types ◽  
Immune Microenvironment ◽  
Tumor Immune Microenvironment ◽  
Manual Processing ◽  
Cell Densities ◽  
Immune Profiling

AbstractMultiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment.

452 Combination treatment using KISIMATM protein-based cancer vaccine and systemic STING agonist results in profound modulation of tumor microenvironment and improved tumor control

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A479-A479
Author(s):  
Matteo Rossi ◽  
Elodie Belnoue ◽  
Susanna Carboni ◽  
Wilma Besson-Di Berardino ◽  
Erika Riva ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Tumor Microenvironment ◽  
Combination Therapy ◽  
Cancer Vaccine ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Tumor Control ◽  
Therapeutic Vaccination ◽  
Potent Tumor

BackgroundKISIMATM platform allows the development of protein-based cancer vaccines able to induce a potent, tumor-specific CD8 and CD4 T cells response. While the cell penetrating peptide and the Anaxa portions confer, respectively, the cell delivery and self-adjuvanticity properties, the multiantigenic domain allows the targeting of different cancer antigens, resulting in anti-tumoral efficacy in different murine models.1 The first clinical candidate developed from KISIMATM is currently tested, together with anti-PD-1 blockade, in a phase I study in metastatic colorectal cancer patients. Stimulator of interferon genes agonists (STINGa) were shown to induce a potent type I interferon response in preclinical studies. The intratumoral administration of STINGa, to promote tumor inflammation, was shown to result in a protective spontaneous immune response in several murine tumor models. However, the encouraging preclinical results are not supported by recent clinical data, challenging the efficacy of unspecific monotherapy.As it is more and more clear that an effective cancer immunotherapy will require the combination of different treatment strategies, we investigate here the efficacy of combining KISIMATM cancer vaccine with STINGa treatment.MethodsMice were vaccinated with subcutaneous (s.c.) injection of KISIMATM vaccine combined with s.c. administration of STINGa. Safety and immunogenicity were assessed by measuring temperature, serum cytokines and the peripheral antigen-specific response. Anti-tumoral efficacy as well as in depth monitoring of TILs and tumor microenvironment modulation were assessed following therapeutic vaccination in a HPV16 E6 and E7 expressing TC-1 cold tumor model.ResultsCombination treatment was well tolerated and promoted the development of circulating antigen-specific CD8 T cells. In TC-1 tumor bearing mice, KISIMATM therapeutic vaccination resulted in the infiltration of both antigen-specific CD8 and CD4 T cells within the tumor, as well as a switch of tumor associated macrophages polarization toward the more inflammatory type 1. Combination therapy further increased the tumor microenvironment modulation induced by KISIMATM vaccine, promoting the polarization of inflammatory Thelper 1 CD4 T cells and increasing the effector function of antigen-specific CD8 T cells. The profound modulation of the tumor microenvironment induced by combination therapy enhanced the beneficial effect of KISIMATM vaccination, resulting in a prolonged tumor control.ConclusionsCombination of KISIMATM cancer vaccine with systemic STINGa treatment induces the development of a potent, tumor-specific immune response resulting in a profound modulation of the TME. As check-point inhibitor (CPI) therapy is ineffective on poorly infiltrated tumors, combination with therapies able to highly enhance tumor infiltration by T cells could expand CPI indications.Ethics ApprovalThe study was approved by the Canton of Geneva Ethic Board, under the license number GE165/19ReferenceBelnoue E, et al. Targeting self and neo-epitopes with a modular self-adjuvanting cancer vaccine. JCI Insight 2019. 4:11.

A Crucial Role for Antigen Presenting Cells in Donor CD4+CD25+ Regulatory T Cell Mediated Suppression of Experimental Gvhd.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 688-688
Author(s):  
Isao Tawara ◽  
Tomomi Toubai ◽  
Chelsea Malter ◽  
Yaping Sun ◽  
Evelyn Nieves ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Antigen Presenting Cells ◽  
Class Ii ◽  
Indoleamine 2 ◽  
Effector Phase ◽  
Antigen Presenting

Abstract Abstract 688 Several lines of evidence show that donor derived mature CD4+CD25+Foxp3+ regulatory T cells (Tregs) suppress experimental GVHD. The mechanism of GVHD suppression by donor Tregs is, however, not well understood. Recent observations have brought in a renewed focus on the role of professional antigen presenting cells (APCs) in the induction and maintenance of GVHD by alloreactive T cell effectors (Teffs). But the role of APCs in modulating the responses of Tregs after allogeneic BMT is not known. We first tested the requirement of host APCs in Treg mediated regulation of GVHD. We utilized a clinically relevant CD8+ T cell dependent MHC matched but miHA disparate C3H.SW (H-2b) → wild type (wt) or Class II deficient Abb (II-/-) B6 (H-2b) model of GVHD because host APCs and target tissues from the Abb animals do not express class II and as such donor CD4+CD25+ Tregs will not directly interact with the host tissues while alloreactive CD8+ T cells could still respond to miHA allo-antigens presented by the intact class I on host APCs. The recipient Abb (II-/-) and wt B6 animals were lethally irradiated and transplanted with 2 × 105 CD8+ T cells along with or without CD4+CD25+ Tregs at 1:2 ratio from either syngeneic B6 or allogeneic C3H.SW animals. The wt recipients that received Tregs showed significantly better survival compared with the wt animals that did not receive any Tregs (P< 0.01) while the class II-/- animals showed similar GVHD mortality regardless of Treg infusion (P>0.8). To confirm whether the lack of Treg mediated protection was only due to the absence of interaction with host type APCs and also to exclude the possibility of development of Tregs from the infused BM we thymectomized wt B6 animals and then generated [B6 B6] controls and the [Abb B6] chimeras. These chimeric animals were used as recipients in a second BMT and transplanted with CD8+ Teffs and Tregs from allogeneic C3H.SW mice. Tregs reduced GVHD mortality in the [B6 B6] (P<0.01) but not in the [Abb B6] animals (P>0.7). We next evaluated whether host APC expression of allo-antigens alone was sufficient for Treg mediated GVHD protection in the absence of class II expression on target tissues by generating [B6 B6] and [B6 Abb] chimeras and found that Tregs demonstrated equivalent GVHD protection even when the class II allo-antigens were expressed only on the host APCs. Mechanistic studies demonstrated that Tregs significantly inhibited the expansion of CD8+ Teffs on days +10 and 17 after BMT in the spleens of the WT recipients (P<0.05) but not in the class II-/- animals. However, infused Tregs demonstrated reduced expansion in the class II-/- animals only early after BMT (on day +10) but was equivalent at later time-point (days 17 and 29) to the WT recipients. We further determined the mechanisms by which host APCs might contribute to Treg mediated protection. To this end we used IL-10-/-, indoleamine 2, 3 dioxygenase (IDO)-/- deficient animals and generated [IL-10-/- B6] and [IDO-/- B6] animals as recipients. Tregs mitigated GVHD mortality regardless of the ability of the host APCs to express IL-10 or IDO. We next determined whether Tregs suppressed Teffs in their activation phase at the level of their interaction with host APCs or in the effector phase. C3H.SW CD8+ T cells were primed (both in vivo and ex vivo with B6 allo-antigens) and then infused into the [β2mg-/- B6] animals such that pre-activated CD8 Teffs would still be able to initiate GVHD without the need for host APCs for their activation. Infusion of donor Tregs into [β2mg-/- B6] animals that were transplanted with the pre-activated Teffs mitigated GVHD severity demonstrating that Tregs, once activated by host APCs, were capable of suppressing Teff cells in their effector phase. Collectively our data show (a) host APCs are critical (b) expression of allo-antigens on host target tissues is not obligatory (c) host derived IL-10 and IDO are not critical for Treg mediated GVHD protection and (d) Tregs can mitigate GVHD by suppressing alloreactive Teffs in the effector phase even after they have been activated. Disclosures: No relevant conflicts of interest to declare.

Immune Characteristics Associated with Lymphomatoid Granulomatosis and Outcome Following Treatment with Interferon-Alpha

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 963-963 ◽  
Author(s):  
Kieron Dunleavy ◽  
Pratip Chattopadhyay ◽  
Junichi Kawada ◽  
Sara Calattini ◽  
Emma Gostick ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Complete Remission ◽  
Interferon Alpha ◽  
Cd8 T Cells ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Lymphomatoid Granulomatosis ◽  
Grade Iii

Abstract Abstract 963 Lymphomatoid granulomatosis (LYG) is a rare angiocentric/angiodestructive EBV+ B-cell lymphoproliferative disorder. LYG has a spectrum of clinical aggressiveness and histological grading. Grading relates to the number of EBV-positive B-cells with grade I /II being usually polyclonal or oligoclonal and grade III monoclonal. Historical outcomes of patients treated with steroids and/or chemotherapy have been poor with median survivals of 14 months. We have shown that LYG is associated with reduced CD8+ and CD4+ T-cells, and hypothesized that patients have defective immune surveillance of EBV+ B-cells. We are investigating the use of interferon-alpha (IFα) for grade I/II disease and have characterized the maturation, exhaustion, and homeostatic potential of bulk and antigen-specific CD8 T-cells. Patients with grade III disease are treated with DA-EPOCH-R. Characteristics of 53 patients on study include male sex 68%; median age (range) 46 (17-67) and median ECOG P.S. 1 (0-3). Disease sites include lung 98%, CNS 38%, kidney 15%, skin 17% and liver 19%. LYG grades are I –30%, II-26% and III-44%. Prior treatment was none –28%, chemotherapy+/− R-34% and steroids alone – 40% of patients. Herein, we report the outcome of patients with grade I/II LYG treated with IFα. IFα was commenced at 7.5 MIU TIW and dose escalated until best response and then continued for 1 year. Of 31 patients with grade I/II LYG treated with IFα, 28 were evaluable for response. Of these, 17 (60%) achieved a complete remission and 6 (21%) patients progressed with grade III disease and received chemotherapy. Of 10 patients with CNS disease, 9 achieved a CR with IFα. At a median follow-up time of 5 years, the progression-free survival of grade I/II LYG was 56%. The median time to remission was 9 months (3-40) and median IFα dose was 20 MIUs (7-40). Median EBV viral loads at study entry were 18 copies/106 genome equivalents (0-22727) (normal<200). We looked at T-cell kinetics in patients who achieved complete remission and observed statistically significant recovery in both CD4 (p=0.034) and CD8 p=0.034) cells after interferonα. We were interested in further elucidating T-cell function and used polychromatic flow cytometry to characterize CD8 T-cells in the peripheral blood of patients before and after IFα. In 17 patient samples, cells were stained with peptide-MHC I (pMHCI) multimers directed against T-cells specific for epitopes from latent and lytic EBV proteins along with antibodies defining CD8 sub-populations. Influenza or cytomegalovirus-specific pMHCI multimers were controls. We observed no difference in the frequency of EBV specific CD8 T-cells in the blood of LYG patients compared to controls. However, CD27 and PD1 expression appeared to be altered in the bulk CD8+ T-cells and in selected EBV-specific populations in LYG patients; these changes were marginally significant. Following completion of IFα, expression of PD-1, CD27 and CD127 were at normal levels. Evidence from some LYG patients suggests that IL2 production by EBV-specific T-cells is lost during LYG, and normalized after therapy. Our results suggest that LYG, an EBV-associated disease, may arise in the setting of a global deficit in CD8 T-cells with selected defects in EBV-specific immunity that resolve with successful therapy. Disclosures: No relevant conflicts of interest to declare.

Anti-Thymocyte Globulin (ATG) Differentially Depletes naïve and Memory T Cells and Permits Memory-Type Regulatory T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4670-4670
Author(s):  
Chang-Qing Xia ◽  
Anna Chernatynskaya ◽  
Clive Wasserfall ◽  
Benjamin Looney ◽  
Suigui Wan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Memory T Cells ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Hematopoietic Stem

Abstract Abstract 4670 Anti-thymocyte globulin (ATG) has been used in clinic for the treatment of allograft rejection and autoimmune diseases. However, its mechanism of action is not fully understood. To our knowledge, how ATG therapy affects naïve and memory T cells has not been well investigated. In this study, we have employed nonobese diabetic mouse model to investigate how administration of anti-thymocyte globulin (ATG) affects memory and naïve T cells as well as CD4+CD25+Foxp3+ regulatory T cells in peripheral blood and lymphoid organs; We also investigate how ATG therapy affects antigen-experienced T cells. Kinetic studies of peripheral blood CD4+ and CD8+ T cells post-ATG therapy shows that both populations decline to their lowest levels at day 3, while CD4+ T cells return to normal levels more rapidly than CD8+ T cells. We find that ATG therapy fails to eliminate antigen-primed T cells, which is consistent with the results that ATG therapy preferentially depletes naïve T cells relative to memory T cells. CD4+ T cell responses post-ATG therapy skew to T helper type 2 (Th2) and IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) are less sensitive to ATG depletion and remain at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory-like immunophenotype is significantly increased in ATG-treated animals, which might play an important role in controlling effector T cells post ATG therapy. In summary, ATG therapy may modulate antigen-specific immune responses through modulation of naïve and memory T cell pools and more importantly through driving T cell subsets with regulatory activities. This study provides important data for guiding ATG therapy in allogenieic hematopoietic stem cell transplantation and other immune-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Pre-Existing Anti-FVIII Immunity Alters Therapeutic Platelet-Targeted FVIII Engraftment in the System Preconditioned with Busulfan Alone through Cytotoxic CD8 T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 444-444
Author(s):  
Weiqing Jing ◽  
Christina Baumgartner ◽  
Feng Xue ◽  
Jocelyn A. Schroeder ◽  
Qizhen Shi
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Cd8 T Cells ◽  
Hemophilia A ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Specific Response ◽  
Hematopoietic Stem ◽  
Fviii Inhibitor

Abstract The development of anti-FVIII inhibitory antibodies (inhibitors) is a significant problem in FVIII protein replacement therapy in hemophilia A (HA). We have developed a platelet-targeted FVIII gene therapy approach, in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8) and demonstrated that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in FVIII null mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed. Since busulfan, an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell (HSC) transplantation preparative regimens in humans, we evaluated the efficacy of busulfan conditioning regimens in 2bF8 gene therapy. We found that busulfan conditioning alone resulted in sustained therapeutic levels of platelet-FVIII expression in FVIII null mice that received 2bF8-transduced HSCs in the non-inhibitor model but not in the inhibitor model. In the current study, we explored the mechanism of platelet FVIII loss upon busulfan conditioning in the FVIII inhibitor model. FVIII null mice were immunized with recombinant human FVIII (rhF8) to induce anti-FVIII inhibitor development to establish the inhibitor model. Once the inhibitor titers were confirmed, animals received busulfan preconditioning at the dose of 50 mg/kg followed by transplantation of either whole bone marrow or Sca-1 + cells from 2bF8 transgenic (2bF8 Tg) mice. After 4 weeks of bone marrow reconstitution, platelet-FVIII expression levels in recipients transplanted with 2bF8 Tg whole bone marrow cells were 7.19±8.59 mU/10 8 platelets (n=5), which were significantly higher than those obtained from animals transplanted with 2bF8 Tg Sca-1 cells (0.55±1.02 mU/10 8 platelets [n=15]). The differences in platelet-FVIII expression between the whole bone marrow and Sca-1 groups were maintained during the study period for 6 months. When CD8 T cells were depleted in addition to busulfan preconditioning, platelet-FVIII expression was significantly enhanced in rhF8-primed recipients that received 2bF8 Tg Sca-1 cells (2.14±2.25 mU/10 8 platelets [n=8]) and sustained during the study period. We then explored which subset of cells from 2bF8 Tg mice could activate rhF8-primed CD8 T cells using the mouse IFNγ ELISpot assay. rhF8-primed CD8 T cells were stimulated with platelets, Sca-1 + cells, or megakaryocytes sorted from either 2bF8 Tg or FVIII null mice. We found that CD8 T cells from rhF8-primed FVIII null mice were efficiently activated by Sca-1 + cells from 2bF8 Tg mice and secreted IFNγ but not by platelets or megakaryocytes. These results suggest that 2bF8 Tg-Sca-1 + cells could be a potential target for rhF8-primed CD8 T cells. As a control, Sca-1 + cells from FVIII null mice did not activate rhF8-primed CD8 T cells, suggesting that IFNγ production from rhF8-primed CD8 T cells stimulated with 2bF8 Tg-Sca-1 + cells was a FVIII-specific response. To explore whether the elimination of platelet-FVIII expression in the inhibitor model relies on antibody-dependent cellular cytotoxicity (ADCC), we transplanted 2bF8 Tg-Sca-1 + cells into rhF8-primed B-cell deficient μMT mice preconditioned with busulfan. We found that no platelet-FVIII was detected in μMT recipients even though they did not produce anti-FVIII antibodies, suggesting that the loss of platelet-FVIII expression in the inhibitor model is not mediated by the ADCC pathway. In summary, our studies demonstrate that pre-existing anti-FVIII immunity can alter the engraftment of 2bF8-genetically-manipulated Sca-1 + hematopoietic stem/progenitor cells via the cytotoxic CD8 T-cell killing pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet-targeted gene therapy in hemophilia A with pre-existing immunity. Disclosures No relevant conflicts of interest to declare.

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