scholarly journals 596 Combining Bempegaldesleukin (CD122-preferential IL-2 pathway agonist) and NKTR-262 (TLR7/8 agonist) pairs local innate activation with systemic CD8+ T cell expansion to enhance anti-tumor immunity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A626-A626
Author(s):  
Annah Rolig ◽  
Daniel Rose ◽  
Grace Helen McGee ◽  
Saul Kivimae ◽  
Werner Rubas ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Cycle Phase ◽  
Tumor Cell Death ◽  
Specific Immunity

BackgroundTumor cell death caused by radiation therapy (RT) can trigger anti-tumor immune responses in part because dying cells release adjuvant factors that amplify and sustain DC and T cell responses. We previously demonstrated that bempegaldesleukin (BEMPEG:NKTR-214, a first-in-class CD122-preferential IL-2 pathway agonist), significantly enhanced the anti-tumor efficacy of RT through a T cell-dependent mechanism. Because RT can induce either immunogenic or tolerogenic cell death, depending on a multitude of factors (radiation dose, cell cycle phase, and tumor microenvironment), we hypothesized that providing a specific immunogenic adjuvant, like intratumoral NKTR-262, a novel toll-like receptor (TLR) 7/8 agonist, to the tumor site would further improve systemic tumor-specific immunity by promoting synergistic activation of local immunostimulatory innate immune responses. Therefore, we evaluated whether intratumoral NKTR-262, combined with systemic BEMPEG treatment would result in improved tumor-specific immunity and survival compared to BEMPEG combined with RT.MethodsTumor-bearing mice (CT26; EMT6) received BEMPEG (0.8 mg/kg; iv), RT (16 Gy x 1), and/or intratumoral NKTR-262 (0.5 mg/kg). Flow cytometry was used to evaluate CD4+ and CD8+ T cell activation status in the blood and tumor (7 days post-treatment). The contribution of specific immune subsets was determined by depletion of CD4+, CD8+, or NK cells. CD8+ T cell cytolytic activity was determined in vitro with an Incucyte assay. Data are representative of 1–2 independent experiments (n=5–14/group) and statistical significance was determined by 1-way ANOVA (p-value cut-off of 0.05).ResultsBEMPEG/NKTR-262 resulted in significantly improved survival compared to BEMPEG/RT. Both combination therapies were CD8+ T cell dependent. However, response to BEMPEG/NKTR-262 was characterized by a significant expansion of activated CD8+ T cells (GzmA+; Ki-67+; ICOS+; PD-1+) in the blood, which correlated with reduced tumor size (p<0.05). In the tumor, BEMPEG/NKTR-262 induced higher frequencies of GzmA+ CD8+ T cells exhibiting reduced expression of suppressive molecules (PD-1+, TIM-3+), compared to BEMPEG/RT. Additionally, CD8+ T cells isolated from BEMPEG/NKTR-262-treated tumors had greater cytolytic capacity than those from BEMPEG/RT-treated mice.ConclusionsCombining BEMPEG with NKTR-262 lead to a more robust expansion of activated CD8+ T cells compared to the BEMPEG/RT combination. Enhancement of the activated CD8+ T cell response in mice treated with NKTR-262 in combination with BEMPEG suggests that intratumoral TLR stimulation provides superior antigen presentation and costimulatory activity compared to RT. A clinical trial of BEMPEG/NKTR-262 for patients with metastatic solid tumors is in progress (NCT03435640).

scholarly journals TLR9 and Dendritic Cells Are Required for CD8+ T Cell Responses to the AAV Capsid

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 552-552 ◽  
Author(s):  
Geoffrey L. Rogers ◽  
Roland W Herzog
Keyword(s):  
Pattern Recognition ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Post Injection ◽  
Cell Responses

Abstract CD8+ T cell responses to the adeno-associated virus (AAV) capsid have posed a significant barrier to transduction in clinical trials of AAV-mediated gene therapy for hemophilia B, as reactivation of a memory CTL response to the capsid is capable of eliminating transduced hepatocytes in the absence of immunosuppression. Recently, it has been suggested that innate immune responses induced by the toll-like receptor (TLR) pathway can influence the development of adaptive immune responses to AAV-mediated gene transfer. In particular, reports have implicated TLR2 (AAV capsid), TLR9 (AAV genome), and MyD88 (downstream signaling adaptor of both these TLRs). Herein, we have used a modified AAV2 with an insertion of the immunodominant MHC class I epitope of ovalbumin into the capsid (AAV2-SIINFEKL) to study the mechanism of CD8+ T cell responses to the AAV capsid. Using an H2-Kb-SIINFEKL tetramer reagent, we determined that anti-capsid CD8+ T cell responses depended on the TLR9-MyD88 pathway. While the frequency of circulating capsid-specific CD8+ T cells peaked around 7-10 days post-injection and subsided after about 21 days in wild type (WT) mice, tetramer-positive cells were not detected in TLR9-/- or MyD88-/- mice. The kinetics and magnitude of the response was unaltered in TLR2-/- mice. Mice deficient in STING, a downstream adaptor of multiple cytoplasmic DNA sensing pathways, also developed comparable capsid-specific CD8+ T cell frequencies to WT mice, suggesting that this is not a general effect of pattern recognition of DNA. Interestingly, the frequency of capsid-specific CD8+ T cells was not reduced in AP3-/- mice, which are deficient in type I IFN signaling downstream of TLR9. Adoptively transferred OVA-specific OT-1 T cells proliferated in WT but not TLR9-/- mice that received AAV2-SIINFEKL, confirming the importance of TLR9. The effect was antigen-specific, as OT-1 cells in WT mice that received AAV2 lacking SIINFEKL showed minimal proliferation comparable to TLR9-/- mice. In addition to pattern-recognition receptors, we also assessed the role of antigen-presenting cells in the CD8+ T cell response to capsid. The formation of capsid-specific CD8+ T cells was unaltered in mice that received gadolinium chloride to inactivate macrophages, or in B cell-deficient μMT mice. Depletion of B cells in WT mice prior to vector administration also failed to affect the anti-capsid CD8+ T cell response. However, transient depletion of dendritic cells (DCs) in CD11c-DTR mice resulted in a delayed development of capsid-specific CD8+ T cells. Seven days post-injection, DC-depleted mice had a significantly reduced frequency of tetramer-positive CD8+ T cells which recovered to normal by 10 days, likely due to the repopulation of DCs before the input capsid was completely cleared. Overall, our results show that TLR9 signaling, most likely in DCs, is required for the formation of de novo anti-capsid CD8+ T cell responses. Disclosures Herzog: Genzyme: AAV-FIX technology Patents & Royalties.

scholarly journals Direct and Indirect endocrine-mediated suppression of human endometrial CD8+T cell cytotoxicity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Z. Shen ◽  
M. Rodriguez-Garcia ◽  
M. V. Patel ◽  
C. R. Wira
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cytotoxic Activity ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Cd8 T Cell ◽  
Gynecological Cancers ◽  
Protective Strategies ◽  
T Cell Cytotoxicity

AbstractRegulation of endometrial (EM) CD8+T cells is essential for successful reproduction and protection against pathogens. Suppression of CD8+T cells is necessary for a tolerogenic environment that promotes implantation and pregnancy. However, the mechanisms regulating this process remain unclear. Sex hormones are known to control immune responses directly on immune cells and indirectly through the tissue environment. When the actions of estradiol (E2), progesterone (P) and TGFβ on EM CD8+T cells were evaluated, cytotoxic activity, perforin and granzymes were directly suppressed by E2 and TGFβ but not P. Moreover, incubation of polarized EM epithelial cells with P, but not E2, increased TGFβ secretion. These findings suggest that E2 acts directly on CD8+T cell to suppress cytotoxic activity while P acts indirectly through induction of TGFβ production. Understanding the mechanisms involved in regulating endometrial CD8+T cells is essential for optimizing reproductive success and developing protective strategies against genital infections and gynecological cancers.

scholarly journals Type I Interferons Regulate the Magnitude and Functionality of Mouse Polyomavirus-Specific CD8 T Cells in a Virus Strain-Dependent Manner

2016 ◽  
Vol 90 (10) ◽  
pp. 5187-5199 ◽  
Author(s):  
Qingsong Qin ◽  
Shwetank ◽  
Elizabeth L. Frost ◽  
Saumya Maru ◽  
Aron E. Lukacher
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Response ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Single Amino Acid ◽  
Type I ◽  
Type I Ifns ◽  
Vp1 Capsid Protein

ABSTRACTMouse polyomavirus (MPyV) is a ubiquitous persistent natural mouse pathogen. A glutamic acid (E)-to-glycine (G) difference at position 91 of the VP1 capsid protein shifts the profile of tumors induced by MPyV from an epithelial to a mesenchymal cell origin. Here we asked if this tropism difference affects the MPyV-specific CD8 T cell response, which controls MPyV infection and tumorigenesis. Infection by the laboratory MPyV strain RA (VP1-91G) or a strain A2 mutant with an E-to-G substitution at VP1 residue 91 [A2(91G)] generated a markedly smaller virus-specific CD8 T cell response than that induced by A2(VP1-91E) infection. Mutant A2(91G)-infected mice showed a higher frequency of memory precursor (CD127hiKLRG1lo) CD8 T cells and a higher recall response than those of A2-infected mice. Using T cell receptor (TCR)-transgenic CD8 T cells and immunization with peptide-pulsed dendritic cells, we found that early bystander inflammation associated with A2 infection contributed to recruitment of the larger MPyV-specific CD8 T cell response. Beta interferon (IFN-β) transcripts were induced early during A2 or A2(91G) infections. IFN-β inhibited replication of A2 and A2(91G)in vitro. Using mice lacking IFN-αβ receptors (IFNAR−/−), we showed that type I IFNs played a role in controlling MPyV replicationin vivobut differentially affected the magnitude and functionality of virus-specific CD8 T cells recruited by A2 and A2(91G) viral infections. These data indicate that type I IFNs are involved in protection against MPyV infection and that their effect on the antiviral CD8 T cell response depends on capsid-mediated tropism properties of the MPyV strain.IMPORTANCEIsolates of the human polyomavirus JC virus from patients with the frequently fatal demyelinating brain disease progressive multifocal leukoencephalopathy (PML) carry single amino acid substitutions in the domain of the VP1 capsid protein that binds the sialic acid moiety of glycoprotein/glycolipid receptors on host cells. These VP1 mutations may alter neural cell tropism or enable escape from neutralizing antibodies. Changes in host cell tropism can affect recruitment of virus-specific CD8 T cells. Using mouse polyomavirus, we demonstrate that a single amino acid difference in VP1 known to shift viral tropism profoundly affects the quantity and quality of the anti-polyomavirus CD8 T cell response and its differentiation into memory cells. These findings raise the possibility that CD8 T cell responses to infections by human polyomaviruses may be influenced by VP1 mutations involving domains that engage host cell receptors.

scholarly journals Role of Thymic Output in Regulating CD8 T-Cell Homeostasis during Acute and Chronic Viral Infection

2005 ◽  
Vol 79 (15) ◽  
pp. 9419-9429 ◽  
Author(s):  
Nicole E. Miller ◽  
Jennifer R. Bonczyk ◽  
Yumi Nakayama ◽  
M. Suresh
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Response ◽  
Viral Infections ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Cell Responses ◽  
Lcmv Infection

ABSTRACT Although it is well documented that CD8 T cells play a critical role in controlling chronic viral infections, the mechanisms underlying the regulation of CD8 T-cell responses are not well understood. Using the mouse model of an acute and chronic lymphocytic choriomeningitis virus (LCMV) infection, we have examined the relative importance of peripheral T cells and thymic emigrants in the elicitation and maintenance of CD8 T-cell responses. Virus-specific CD8 T-cell responses were compared between mice that were either sham thymectomized or thymectomized (Thx) at ∼6 weeks of age. In an acute LCMV infection, thymic deficiency did not affect either the primary expansion of CD8 T cells or the proliferative renewal and maintenance of virus-specific lymphoid and nonlymphoid memory CD8 T cells. Following a chronic LCMV infection, in Thx mice, although the initial expansion of CD8 T cells was normal, the contraction phase of the CD8 T-cell response was exaggerated, which led to a transient but striking CD8 T-cell deficit on day 30 postinfection. However, the virus-specific CD8 T-cell response in Thx mice rebounded quickly and was maintained at normal levels thereafter, which indicated that the peripheral T-cell repertoire is quite robust and capable of sustaining an effective CD8 T-cell response in the absence of thymic output during a chronic LCMV infection. Taken together, these findings should further our understanding of the regulation of CD8 T-cell homeostasis in acute and chronic viral infections and might have implications in the development of immunotherapy.

scholarly journals Serum-derived exosomes promote CD8+ T cells to overexpress PD-1, affecting the prognosis of hypopharyngeal carcinoma

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Qian Gao ◽  
Hui-Ting Liu ◽  
Yu-Qin Xu ◽  
Lin Zhang ◽  
Yuan-Ru Liu ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Programmed Cell Death ◽  
Cd8 T Cells ◽  
Poor Prognosis ◽  
Cell Function ◽  
Immune Escape ◽  
Cd8 T Cell ◽  
Serum Exosomes

Abstract Background Hypopharyngeal cancer (HPC) is associated with a poor prognosis and a high recurrence rate. Immune escape is one of the reasons for the poor prognosis of malignant tumors. Programmed cell death ligand 1 (PD-L1) and programmed cell death-1 (PD-1) have been shown to play important roles in immune escape. However, the role of PD-1/PD-L1 in HPC remains unclear. In this experiment, we investigated the effect of exosomes from HPC patient serum on CD8+ T cell function and PD-1/PD-L1 expression and, thus, on prognosis. We hope to provide guidance for the identification of new targets for HPC immunotherapy. Methods PD-1 and CD8 expression in 71 HPC tissues and 16 paracarcinoma tissues was detected by immunohistochemistry. Concurrently, the clinicopathological data of the patients were obtained to conduct correlation analysis. Exosomes were isolated from serum and then identified by Western blotting (WB), transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). Flow cytometry was used to assess the activity of CD8+ T cells after exosome stimulation. The effects of exosomes on the ability of CD8+ T cells to kill FaDu cells were assessed by CCK-8 assay. The expression of IL-10 and TGF-β1 was measured by enzyme-linked immunosorbent assay (ELISA). PD-L1 expression in HPC tissue samples was evaluated by immunohistochemistry, and the relationship between PD-1/PD-L1 expression and prognosis was investigated with patient specimens. Results PD-1 expression was significantly upregulated on CD8+ T cells in tumor tissues compared with those in normal tissues. The overall survival (OS) and disease-free survival (DFS) of PD-1-overexpressing patients were decreased. Serum exosomes from patients can elevate PD-1 expression on CD8+ T cells and suppress their killing capacity and secretory function. The rate of positive PD-L1 expression was increased in HPC tissues compared with paracancerous tissues. The DFS and OS of the PD-1(+)-PD-L1(+) group were significantly lower than those of the PD-1(−)-PD-L1(−) group. Conclusion Our findings indicate that serum exosomes from HPC patients can inhibit CD8+ T cell function and that the PD-1-PD-L1 pathway plays an important role in the immune escape of HPC. Exosomes combined with immunotherapy may guide the treatment of patients with advanced disease in the future.

scholarly journals Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge

2018 ◽  
Author(s):  
Xiaoyan Zheng ◽  
Jennifer Dora Oduro ◽  
Julia Désirée Boehme ◽  
Lisa Borkner ◽  
Thomas Ebensen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Response ◽  
Influenza A ◽  
Clinical Medicine ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Vaccine Vector ◽  
Protective Capacity

Cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.

scholarly journals Fas-mediated apoptosis of CD4+ and CD8+ T cells from human immunodeficiency virus-infected persons: differential in vitro preventive effect of cytokines and protease antagonists

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 4959-4966 ◽  
Author(s):  
J Estaquier ◽  
M Tanaka ◽  
T Suda ◽  
S Nagata ◽  
P Golstein ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Cd4 T Cell ◽  
Death Process ◽  
T Cell Apoptosis ◽  
Soluble Fas ◽  
Immunodeficiency Syndrome

Human immunodeficiency syndrome (HIV) infection leads to a progressive loss of T-cell-mediated immunity associated with T-cell apoptosis. We report here that CD4+ and CD8+ T cells from HIV-1-infected persons are sensitive to Fas (CD95/APO-1)-mediated death induced either by an agonistic anti-Fas antibody or by the physiologic soluble Fas ligand, although showing no sensitivity to tumor necrosis factor alpha-induced death. CD4+ and CD8+ T-cell apoptosis induced by Fas ligation was enhanced by inhibitors of protein synthesis and was prevented either by a soluble Fas receptor decoy or an antagonistic anti-Fas antibody. Fas- mediated apoptosis could also be prevented in a CD4+ or CD8+ T-cell- type manner (1) by several protease antagonists, suggesting the involvement of the interleukin-1beta (IL-1beta)-converting enzyme (ICE)- related cysteine protease in CD4+ T-cell death and of both a CPP32- related cysteine protease and a calpain protease in CD8+ T-cell death; and (2) by three cytokines, IL-2, IL-12, and IL-10, that exerted their effects through a mechanism that required de novo protein synthesis. Finally, T-cell receptor (TCR)-induced apoptosis of CD4+ T cells from HIV-infected persons involved a Fas-mediated death process, whereas TCR stimulation of CD8+ T cells led to a different Fas-independent death process. These findings suggest that Fas-mediated T-cell death is involved in acquired immunodeficiency syndrome (AIDS) pathogenesis and that modulation of Fas-mediated signaling may represent a target for new therapeutic strategies aimed at the prevention of CD4+ T-cell death in AIDS.

scholarly journals Chimeric Yellow Fever/Dengue Virus as a Candidate Dengue Vaccine: Quantitation of the Dengue Virus-Specific CD8 T-Cell Response

2000 ◽  
Vol 74 (17) ◽  
pp. 8094-8101 ◽  
Author(s):  
Robbert G. van der Most ◽  
Kaja Murali-Krishna ◽  
Rafi Ahmed ◽  
James H. Strauss
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dengue Virus ◽  
Yellow Fever ◽  
Cd8 T Cells ◽  
Cell Response ◽  
Protective Efficacy ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
E Proteins

ABSTRACT We have constructed a chimeric yellow fever/dengue (YF/DEN) virus, which expresses the premembrane (prM) and envelope (E) genes from DEN type 2 (DEN-2) virus in a YF virus (YFV-17D) genetic background. Immunization of BALB/c mice with this chimeric virus induced a CD8 T-cell response specific for the DEN-2 virus prM and E proteins. This response protected YF/DEN virus-immunized mice against lethal dengue encephalitis. Control mice immunized with the parental YFV-17D were not protected against DEN-2 virus challenge, indicating that protection was mediated by the DEN-2 virus prM- and E-specific immune responses. YF/DEN vaccine-primed CD8 T cells expanded and were efficiently recruited into the central nervous systems of DEN-2 virus challenged mice. At 5 days after challenge, 3 to 4% of CD8 T cells in the spleen were specific for the prM and E proteins, and 34% of CD8 T cells in the central nervous system recognized these proteins. Depletion of either CD4 or CD8 T cells, or both, strongly reduced the protective efficacy of the YF/DEN virus, stressing the key role of the antiviral T-cell response.

Identification of the AAV2 Capsid CD8+ T Cell Epitope in C57BL/6 Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3188-3188
Author(s):  
Denise E. Sabatino ◽  
Federico Mingozzi ◽  
Haifeng Chen ◽  
Peter Colosi ◽  
Hildegund C.J. Ertl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Capsid Protein ◽  
Cd8 T Cells ◽  
Cell Epitope ◽  
T Cell Response ◽  
Cd8 Cells ◽  
Media Control ◽  
Ifn Γ

Abstract Recently, a clinical trial for adeno-associated virus serotype 2 (AAV2) mediated liver directed gene transfer of human Factor IX to subjects with severe hemophilia B revealed that two patients developed transient asymptomatic transaminitis following vector administration. Immunology studies in the second patient demonstrated a transient T cell response to AAV2 capsid peptides suggesting that the immune response to the AAV capsid may be related to the transient transaminitis. We hypothesized that the observations made in the human subjects were due to a CD8 T cell response to AAV2 capsid protein. Preclinical studies in mice and dogs, which are not naturally infected by wild type AAV2 viruses, did not predict these findings in the clinical study. Thus, we developed a mouse model in which we were able to mimic this phenomenon (Blood 102:493a). In an effort to further characterize the immune responses to AAV2 capsid proteins in this mouse model, we identified the T cell epitope in the AAV capsid protein recognized by murine C57Bl/6 CD8 T cells. A peptide library of AAV2 VP1 capsid peptides (n=145) that were synthesized as 15mers overlapping by 10 amino acids were divided into 6 pools each containing 24–25 peptides. C57Bl/6 mice were immunized intramuscularly with an adenovirus expressing AAV2 capsid protein. Nine days later the spleen was harvested and intracellular cytokine staining (ICS) was used to assess release of IFN-γ from CD8 T cells in response to 6 AAV2 capsid peptide pools. ICS demonstrated CD8 cells from mice immunized with Ad-AAV2 produced IFN-γ (3.5% of the CD8 cells) in response to Pool F (amino acid 119–145) while no IFN-γ release in CD8 cells was detected with Pool A to E (mean 0.28%±0.25%) compared to the media control (0.16%). This detection of IFN-γ release from CD8 T cells indicates a specific proliferation to a peptide(s) within this peptide pool (Pool F). A matrix approach was used to further define which peptide(s) contained the immunodominant epitope. Eleven small peptide pools of Pool F were created in which each peptide was represented in 2 pools. ICS of splenocytes from immunized (Ad-AAV2 capsid) C57Bl/6 mice demonstrated IFN-γ response from CD8 cells to 3 of the matrix pools corresponding to peptide 140 (PEIQYTSNYNKSVNV) and 141 (TSNYNKSVNVDFTVD) compared with media controls. To determine the exact peptide sequence that binds to the MHC Class I molecule, 9 amino acid peptides (n=7) were created that overlap peptide 140 and 141. Peptide SNYNKSVNV showed positive staining for both CD8 and IFN- γ(3.2%) compared with the six other peptides (0.14%±0.08%), media control (0.08%) and mice that were not immunized (0.11%). This epitope lies in the C terminus of the AAV2 VP1 capsid protein. Current studies using strains of mice with different MHC H2 haplotypes will allow us to determine which of the C57Bl/6 MHC alleles the epitope binds. These findings will provide us with a powerful tool for assessing immune responses to AAV capsid in the context of gene therapy. Specifically, they will allow us to determine how long immunologically detectable capsid sequences persist in an animal injected with AAV vectors. This in turn will provide a basis for a clinical study in which subjects are transiently immunosuppressed, from the time of vector injection until capsid epitopes are no longer detectable by the immune system.

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