scholarly journals Simvastatin Reduces Protection and Intestinal T Cell Responses Induced by a Norovirus P Particle Vaccine in Gnotobiotic Pigs

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 829
Author(s):  
Jacob Kocher ◽  
Tammy Bui Castellucci ◽  
Ke Wen ◽  
Guohua Li ◽  
Xingdong Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
T Cell Response ◽  
Post Inoculation ◽  
Cell Responses ◽  
Monophosphoryl Lipid A ◽  
Cell Immunity ◽  
Ifn Γ

Noroviruses (NoVs) are a leading cause of acute gastroenteritis worldwide. P particles are a potential vaccine candidate against NoV. Simvastatin is a cholesterol-reducing drug that is known to increase NoV infectivity. In this study, we examined simvastatin’s effects on P particle-induced protective efficacy and T-cell immunogenicity using the gnotobiotic pig model of human NoV infection and diarrhea. Pigs were intranasally inoculated with three doses (100 µg/dose) of GII.4/VA387-derived P particles together with monophosphoryl lipid A and chitosan adjuvants. Simvastatin-fed pigs received 8 mg/day orally for 11 days prior to challenge. A subset of pigs was orally challenged with 10 ID50 of a NoV GII.4/2006b variant at post-inoculation day (PID) 28 and monitored for 7 days post-challenge. Intestinal and systemic T cell responses were determined pre- and postchallenge. Simvastatin abolished the P particle’s protection and significantly increased diarrhea severity after NoV infection. Simvastatin decreased proliferation of virus-specific and non-specific CD8 T cells in duodenum and virus-specific CD4 and CD8 T cells in spleen and significantly reduced numbers of intestinal mononuclear cells in vaccinated pigs. Furthermore, simvastatin significantly decreased numbers of duodenal CD4+IFN-γ+, CD8+IFN-γ+ and regulatory T cells and total duodenal activated CD4+ and CD8+ T cells in vaccinated pigs pre-challenge at PID 28. Following challenge, simvastatin prevented the IFN-γ+ T cell response in spleen of vaccinated pigs. These results indicate that simvastatin abolished P particle vaccine-induced partial protection through, at least in part, impairing T cell immunity. The findings have specific implications for the development of preventive and therapeutic strategies against NoV gastroenteritis, especially for the elderly population who takes statin-type drugs.

The Response to Repeated Vaccination with PR1 and WT1 Peptides Admixed in Montanide Adjuvant Is Transient and Fails to Induce Sustained Anti-Leukemia Responses in Patients with Myeloid Malignancies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4096-4096
Author(s):  
Katayoun Rezvani ◽  
Agnes S. M. Yong ◽  
Stephan Mielke ◽  
Behnam Jafarpour ◽  
Bipin N. Savani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Response ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Gm Csf ◽  
Cell Responses ◽  
Ifn Γ

Abstract Abstract 4096 Poster Board III-1031 We previously demonstrated the immunogenicity of a combined vaccine approach employing two leukemia-associated antigenic peptides, PR1 and WT1 (Rezvani Blood 2008). Eight patients with myeloid malignancies received one subcutaneous 0.3 mg and 0.5 mg dose each of PR1 and WT1 vaccines in Montanide adjuvant, with 100 μg of granulocyte-macrophage colony-stimulating factor (GM-CSF). CD8+ T-cell responses against PR1 or WT1 were detected in all patients as early as 1 week post-vaccination. However, responses were only sustained for 3-4 weeks. The emergence of PR1 or WT1-specific CD8+ T-cells was associated with a significant but transient reduction in minimal residual disease (MRD) as assessed by WT1 expression, suggesting a vaccine-induced anti-leukemia response. Conversely, loss of response was associated with reappearance of WT1 transcripts. We hypothesized that maintenance of sustained or at least repetitive responses may require frequent boost injections. We therefore initiated a phase 2 study of repeated vaccination with PR1 and WT1 peptides in patients with myeloid malignancies. Five patients with acute myeloid leukemia (AML) and 2 patients with myelodysplastic syndrome (MDS) were recruited to receive 6 injections at 2 week intervals of PR1 and WT1 in Montanide adjuvant, with GM-CSF as previously described. Six of 7 patients completed 6 courses of vaccination and follow-up as per protocol, to monitor toxicity and immunological responses. Responses to PR1 or WT1 vaccine were detected in all patients after only 1 dose of vaccine. However, additional boosting did not further increase the frequency of PR1 or WT1-specific CD8+ T-cell response. In 4/6 patients the vaccine-induced T-cell response was lost after the fourth dose and in all patients after the sixth dose of vaccine. To determine the functional avidity of the vaccine-induced CD8+ T-cell response, the response of CD8+ T-cells to stimulation with 2 concentrations of PR1 and WT1 peptides (0.1 and 10 μM) was measured by IC-IFN-γ staining. Vaccination led to preferential expansion of low avidity PR1 and WT1 specific CD8+ T-cell responses. Three patients (patients 4, 6 and 7) returned 3 months following the 6th dose of PR1 and WT1 peptide injections to receive a booster vaccine. Prior to vaccination we could not detect the presence of PR1 and WT1 specific CD8+ T-cells by direct ex-vivo tetramer and IC-IFN-γ assay or with 1-week cultured IFN-γ ELISPOT assay, suggesting that vaccination with PR1 and WT1 peptides in Montanide adjuvant does not induce memory CD8+ T-cell responses. This observation is in keeping with recent work in a murine model where the injection of minimal MHC class I binding peptides derived from self-antigens mixed with IFA adjuvant resulted in a transient effector CD8+ T cell response with subsequent deletion of these T cells and failure to induce CD8+ T cell memory (Bijker J Immunol 2007). This observation can be partly explained by the slow release of vaccine peptides from the IFA depot without systemic danger signals, leading to presentation of antigen in non-inflammatory lymph nodes by non-professional antigen presenting cells (APCs). An alternative explanation for the transient vaccine-induced immune response may be the lack of CD4+ T cell help. In summary these data support the immunogenicity of PR1 and WT1 peptide vaccines. However new approaches will be needed to induce long-term memory responses against leukemia antigens. To avoid tolerance induction we plan to eliminate Montanide adjuvant and use GM-CSF alone. Supported by observations that the in vivo survival of CD8+ T-effector cells against viral antigens are improved by CD4+ helper cells, we are currently attempting to induce long-lasting CD8+ T-cell responses to antigen by inducing CD8+ and CD4+ T-cell responses against class I and II epitopes of WT1 and PR1. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential Longevity of Memory CD4 and CD8 T Cells in a Cohort of the Mothers With a History of ZIKV Infection and Their Children

2021 ◽  
Vol 12 ◽  
Author(s):  
Jessica Badolato-Corrêa ◽  
Fabiana Rabe Carvalho ◽  
Iury Amancio Paiva ◽  
Débora Familiar-Macedo ◽  
Helver Gonçalves Dias ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
T Cell Immunity ◽  
Zikv Infection ◽  
Cell Responses ◽  
Cell Immunity ◽  
History Of ◽  
Ifn Γ

Background: Zika virus (ZIKV) infection causes for mild and self-limiting disease in healthy adults. In newborns, it can occasionally lead to a spectrum of malformations, the congenital Zika syndrome (CZS). Thus, little is known if mothers and babies with a history of ZIKV infection were able to develop long-lasting T-cell immunity. To these issues, we measure the prevalence of ZIKV T-cell immunity in a cohort of mothers infected to the ZIKV during pregnancy in the 2016–2017 Zika outbreak, who gave birth to infants affected by neurological complications or asymptomatic ones.Results: Twenty-one mothers and 18 children were tested for IFN-γ ELISpot and T-cell responses for flow cytometry assays in response to CD4 ZIKV and CD8 ZIKV megapools (CD4 ZIKV MP and CD8 ZIKV MP). IFN-γ ELISpot responses to ZIKV MPs showed an increased CD4 and CD8 T-cell responses in mothers compared to children. The degranulation activity and IFN-γ-producing CD4 T cells were detected in most mothers, and children, while in CD8 T-cells, low responses were detected in these study groups. The total Temra T cell subset is enriched for IFN-γ+ CD4 T cells after stimulation of CD4 ZIKV MP.Conclusion: Donors with a history of ZIKV infection demonstrated long-term CD4 T cell immunity to ZIKV CD4 MP. However, the same was not observed in CD8 T cells with the ZIKV CD8 MP. One possibility is that the cytotoxic and pro-inflammatory activities of CD8 T cells are markedly demonstrated in the early stages of infection, but less detected in the disease resolution phase, when the virus has already been eliminated. The responses of mothers' T cells to ZIKV MPs do not appear to be related to their children's clinical outcome. There was also no marked difference in the T cell responses to ZIKV MP between children affected or not with CZS. These data still need to be investigated, including the evaluation of the response of CD8 T cells to other ZIKV peptides.

Leukemia-Associated Antigen Specific T-Cell Responses Following Combined PR1 and WT1 Peptide Vaccination in Patients with Myeloid Malignancies.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 287-287 ◽  
Author(s):  
Katayoun Rezvani ◽  
Agnes S.M. Yong ◽  
Stephan Mielke ◽  
Bipin N. Savani ◽  
Laura Musse ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Post Vaccination ◽  
Myeloid Malignancies ◽  
Cell Responses ◽  
Ifn Γ

Abstract The graft-versus-leukemia (GVL) effect following allogeneic stem cell transplantation (SCT) is evidence that T lymphocytes can eradicate leukemia. The successful identification of a range of leukemia-associated antigens such as proteinase 3 (PR3) and Wilms tumour-1 (WT1) has stimulated efforts to induce leukemia-specific T-cell responses to these antigens using peptide vaccines. Here we describe the safety and immunogenicity of a combined vaccine of two leukemia-associated antigenic peptides, PR1 and WT1. Eight HLA-A*0201 positive patients with myeloid malignancies (2 myelodysplasia, 5 acute myeloid leukemia and 1 chronic myeloid leukemia) received one subcutaneous dose each of PR1 and WT1 vaccines in Montanide adjuvant, with granulocyte-macrophage colony-stimulating factor (GM-CSF). All patients completed 4 weeks follow-up to monitor toxicity and immunological responses. Toxicity was limited to grade 1–2. All remain alive at a median of 252 days (range 105–523). We analyzed the immunological response to vaccination using PR1/HLA-A*0201 and WT1/HLA-A*0201 tetrameric complexes and flow cytometry for intracellular interferon-gamma (IFN-γ) in samples obtained pre- and weekly post-vaccination. A significant CD8+ T-cell response to the vaccine was defined as the emergence of PR1 or WT1-specific CD8+ T-cells when the pre-study analysis was negative or a twofold increase in frequencies when responses were present pre-vaccination. Following vaccination, a significant CD8+ T-cell response to PR1 was seen in 7/8 patients (median 0.34%, range 0.04–0.48%), to WT1 in 5/8 patients (median 0.29%, range 0–0.42%) and to one or both antigens in 8/8 patients. Vaccine-induced CD8+ T-cells were seen as early as 1 week post-vaccination, produced IFN-γ and were preferentially expanded in the effector compartment (CD45RO+/-CD27−). Post-vaccination, there was a strong correlation between the emergence of PR1 or WT1+CD8+ T-cells and a reduction in WT1 mRNA expression, a marker of minimal residual disease, suggesting a vaccine-driven anti-leukemia effect. Loss of response was associated with reappearance of WT1 transcripts (P<0.01). Two patients with detectable CD8+ T-cell responses to PR1 who failed to have a reduction in MRD relapsed 3–6 months following completion of vaccination. This is the first demonstration that a combined PR1 and WT1 vaccine is immunogenic. Based on these results we have initiated a phase 2 study of repeated vaccination with PR1 and WT1 peptides in patients with myeloid malignancies.

scholarly journals Interleukin-18-Related Genes Are Induced during the Contraction Phase but Do Not Play Major Roles in Regulating the Dynamics or Function of the T-Cell Response to Listeria monocytogenes Infection

2009 ◽  
Vol 77 (5) ◽  
pp. 1894-1903 ◽  
Author(s):  
Jodie S. Haring ◽  
John T. Harty
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
T Cell Response ◽  
Interleukin 18 ◽  
Contraction Phase ◽  
Cell Responses ◽  
Ifn Γ ◽  
Kinetics Of

ABSTRACT Proinflammatory cytokines, such as gamma interferon (IFN-γ), impact aspects of T-cell responses after infection, including expansion, contraction, and memory formation. Interleukin-18 (IL-18) functions as a proinflammatory cytokine by stimulating the production of IFN-γ from multiple cell types and accentuating the development of Th1 CD4 T-cell responses. Focused microarray analyses revealed upregulation of IL-18 and IL-18 receptor genes in CD8 T cells during the contraction phase. Based on these findings we investigated if and how signaling through the IL-18 receptor influences the development and kinetics of antigen (Ag)-specific CD8 and CD4 T-cell responses following infection. IL-18Rα−/− and IL-18−/− mice developed frequencies and total numbers of Ag-specific CD8 T cells after Listeria monocytogenes infection that were similar to those of wild-type C57BL/6 mice. The kinetics of expansion, contraction, and memory CD8 T-cell maintenance were also similar. When IL-18Rα deficiency was isolated to Ag-specific CD8 T cells, the kinetics of the expansion and contraction phases were also normal. These basic findings were confirmed by examining the response to vaccinia virus infection. In contrast, the expansion of Ag-specific CD4 T cells was slightly curtailed by the absence of IL-18Rα; however, contraction and the maintenance of memory were not altered. Importantly, both memory Ag-specific CD8 and CD4 T cells generated in the absence of IL-18Rα expanded appropriately after secondary antigen exposure and were protective, indicating that signaling through the IL-18 receptor is not required for normal T-cell response kinetics and survival of immunized mice challenged with a lethal L. monocytogenes infection.

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.

Ex-Vivo Characterization of Polyclonal Memory CD8+ T-Cell Responses to PRAME-Specific Peptides in Patients with Acute Leukemia and Chronic Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2910-2910
Author(s):  
Katayoun Rezvani ◽  
Agnes S. M. Yong ◽  
Abdul Tawab ◽  
Behnam Jafarpour ◽  
Rhoda Eniafe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
High Avidity ◽  
Cell Responses ◽  
Ifn Γ

Abstract PRAME (Preferentially expressed antigen of melanoma) is aberrantly expressed in hematological malignancies and may be a useful target for immunotherapy in leukemia. We studied CD8+ T-cell responses to four HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and healthy donors, using PRA300/HLA-A*0201 tetramer staining, intracellular cytokine (IC) assay and ex-vivo and cultured ELISPOT analysis. CD8+ T-cells recognizing PRAME peptides were detected directly ex-vivo in 4/10 ALL, 6/10 AML, 3/10 CML patients and 3/10 donors. The frequency of PRAME-specific CD8+ T-cells was greater in patients with AML, CML and ALL than in healthy controls. All peptides were immunogenic in patients, whilst PRA300 was the only immunogenic peptide in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to two or more PRAME epitopes (4/7 vs. 0/23 in individuals with low PRAME expression, P = 0.001), suggesting a PRAME-driven T-cell response. In 2 patients studied PRA300/HLA-A*0201+ CD8+T-cells were found to be a mixture of effector and central memory phenotypes. To determine the functional avidity of the PRAME T-cell response, the response of CD8+ T-cells to stimulation with 2 concentrations of peptide was measured by IC-IFN-γ staining. High-avidity CD8+ T-cells were defined as those capable of producing IFN-γ in response to the lower concentration of peptide (0.1μM), while low-avidity CD8+ T-cells were those that only produced IFN-γ in response to the higher concentration of peptide (10 μM). Both high and low-avidity CD8+ T-cell responses could be detected for all peptides tested (median 1.05, 0.90, 0.52, 0.40 high/lowavidity ratios for PRA100, PRA142, PRA300 and PRA425 respectively). In patients with high PRAME expression (&gt;0.001 PRAME/ABL) low-avidity CD8+ T-cell responses to PRAME peptides were more prominent than high-avidity responses, suggesting selective deletion of high-avidity T-cells. In contrast, in some patients with levels &lt;0.001 PRAME/ABL, we could detect the presence of high-avidity CD8+ T-cell responses to PRAME. PRAME-specific CD8+ T-cells were further characterized by IC staining for IL-2, IL-4 and IL-10 production and CD107a mobilization (as a marker of cytotoxicity). Following stimulation with the relevant PRAME peptide, there was no significant production of IL-2, IL-4 or IL-10, suggesting a Tc1 effector response but no significant CD107a mobilization was detected despite significant CD107a mobilization in the same patient in response to CMVpp65495. This finding suggests that patients with leukemia have a selective functional impairment of PRAME-specific CD8+ T-cells, consistent with PRAME-specific T cell exhaustion. However, PRAME-specific T-cells were readily expanded in the presence of cytokines in short-term cultures in-vitro to produce IFN-γ, suggesting that it may be possible to improve the functional capacity of PRAME-specific T-cells for therapeutic purposes. These results provide evidence for spontaneous T-cell reactivity against multiple epitopes of PRAME in ALL, AML and CML and support the usefulness of PRAME as a target for immunotherapy in leukemia. The predominance of low-avidity PRAME-specific CD8+ T-cells suggests that achievement of a state of minimal residual disease may be required prior to peptide vaccination to augment T-cell immune surveillance.

scholarly journals Asian Elephant T Cell Responses to Elephant Endotheliotropic Herpesvirus

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Angela Fuery ◽  
Ann M. Leen ◽  
Rongsheng Peng ◽  
Matthew C. Wong ◽  
Hao Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activity ◽  
T Cell Responses ◽  
T Cell Response ◽  
Asian Elephant ◽  
Hemorrhagic Disease ◽  
Asian Elephants ◽  
Cell Responses ◽  
Ifn Γ

ABSTRACTElephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants, an endangered species. One hypothesis to explain this vulnerability of some juvenile elephants is that they fail to mount an effective T cell response to the virus. To our knowledge, there have been no studies of Asian elephant T cell responses to EEHV. To address this deficiency, we validated the gamma interferon (IFN-γ) enzyme-linked immunospot assay for tracking antigen-directed T cell activity by monitoring rabies-specific responses in vaccinated elephants. In addition, we generated monoclonal antibodies to Asian elephant CD4 and CD8 to facilitate phenotypic T cell profiling. Using these tools, we screened healthy elephants with a history of EEHV infection for reactivity against nine EEHV proteins whose counterparts in other herpesviruses are known to induce T cell responses in their natural hosts. We identified glycoprotein B (gB) and the putative regulatory protein E40 as the most immunogenic T cell targets (IFN-γ responses in five of seven elephants), followed by the major capsid protein (IFN-γ responses in three of seven elephants). We also observed that IFN-γ responses were largely from CD4+T cells. We detected no activity against the predicted major immediate early (E44) and large tegument (E34) proteins, both immunodominant T cell targets in humans latently infected with cytomegalovirus. These studies identified EEHV-specific T cells in Asian elephants for the first time, lending insight into the T cell priming that might be required to protect against EEHV disease, and will guide the design of effective vaccine strategies.IMPORTANCEEndangered Asian elephants are facing many threats, including lethal hemorrhagic disease from elephant endotheliotropic herpesvirus (EEHV). EEHV usually establishes chronic, benign infections in mature Asian elephants but can be lethal to juvenile elephants in captivity and the wild. It is the leading cause of death in captive Asian elephants in North America and Europe. Despite the availability of sensitive tests and protocols for treating EEHV-associated illness, these measures are not always effective. The best line of defense would be a preventative vaccine. We interrogated normal healthy elephants previously infected with EEHV for T cell responses to nine EEHV proteins predicted to induce cellular immune responses. Three proteins elicited IFN-γ responses, suggesting their potential usefulness as vaccine candidates. Our work is the first to describe T cell responses to a member of the proposed fourth subfamily of mammalian herpesviruses, theDeltaherpesvirinae, within a host species in the clade Afrotheria. An EEHV vaccine would greatly contribute to the health care of Asian and African elephants that are also susceptible to this disease.

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 EXTH-51. GENETICALLY STABLE POLIOVIRUS VECTOR PLATFORM FOR DIPG IMMUNOTHERAPY

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi93-vi93
Author(s):  
Matthias Gromeier ◽  
Mubeen Mosaheb ◽  
Elena Dobrikova ◽  
Michael Brown ◽  
Darell Bigner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Viral Vector ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Anatomical Location ◽  
Cell Responses ◽  
Cell Immunity

Abstract Options for the immunotherapy of diffuse intrinsic pontine glioma (DIPG), due to its anatomical location and inherent therapy resistance, are limited. The histone 3.3(K27M) mutation in ~80% of such tumors offers a unique opportunity for immunotherapy intervention, as it defines a high affinity, HLA-A2-restricted tumor neoantigen that spontaneously elicits CD8+ T cell responses in DIPG patients. Immunizing against the H3.3(K27M) signature in the clinic has been challenging, as conventional approaches (i.e. peptide-conjugates administered with adjuvants) lack the costimulatory signals known to drive CD8+ effector T cell responses. Therefore, we built on a viral vector approach for engaging innate immune responses to virus infection specifically in antigen presenting cells. Viruses naturally engage innate immunity, induce antigen presentation, and mediate CD8 T cell priming against foreign antigens. Polioviruses can provide a context optimal for generating antigen-specific CD8 T cells, as they have natural tropism for dendritic cells, preeminent inducers of CD8 T cell immunity; elicit Th1-promoting inflammation; and lack interference with innate or adaptive immunity. However, notorious genetic instability and underlying neuropathogenicity has hampered poliovirus-based vector applications. We devised a strategy based on the polio:rhinovirus chimera PVSRIPO, devoid of viral neuropathogenicity after intracerebral inoculation in human subjects, for stable expression of exogenous antigens. PVSRIPO vectors infect, activate, and induce epitope presentation in DCs in vitro; recruit and activate DCs with Th1-dominant cytokine profiles at the injection site in vivo. They efficiently prime tumor antigen-specific CD8 T cells in vivo, induce CD8 T cell migration to the tumor site, delay tumor growth and enhance survival in syngeneic rodent tumor models. We are preparing a prototype PVSRIPO-derived vector delivering the H3.3(K27M) signature for clinical investigation.

Graft-Versus-Leukemia Antigen CML66 Elicits Coordinated B and T Cell Immunity After Donor Lymphocyte Infusion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2449-2449
Author(s):  
Wandi Zhang ◽  
Jaewon Choi ◽  
Wanyong Zeng ◽  
Edwin P Alyea ◽  
Christine Canning ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Cell Clone ◽  
T Cell Responses ◽  
Cell Responses ◽  
Cell Immunity ◽  
T Cell Clone

Abstract Abstract 2449 Poster Board II-426 Donor lymphocyte infusion (DLI) is a highly effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic hematopoietic stem cell transplantation (HSCT), in which 70-80% patients achieve curative responses. Despite its efficacy and established clinical use, the mechanism by which DLI achieves anti-tumor immunity remains incompletely understood. CML66 was discovered as an overexpressed nonpolymorphic CML-associated antigen that elicited a high-titer (>1:50,000) antibody response in a patient who received CD4+ DLI for relapsed CML. CML66 is preferentially expressed on myeloid leukemia progenitor cells and the development of CML66 antibodies temporally correlated with attainment of molecular remission. Since CD4+ T cell responses are involved in generating both antigen-specific B cell and CD8+T cell responses, we queried whether development of potent B cell immunity was also associated with the development of cytolytic T cell immunity against CML66. To detect specific T cell responses, post-DLI PBMC from this patient were screened for reactivity against pools of overlapping 15- to 18-mer peptides encoding the entire protein sequence of CML66. Patient CD8+ T cells were found to be reactive against a single CML66-derived peptide pool, and specifically an 8 amino acid peptide (HDVDALLW), beginning at residue 459. This T cell epitope is positioned in a different region of the protein compared to the B cell epitope (at residues 198-217: GFYVSLEWVTISKKNQDNK). A peptide-specific T cell clone was isolated, whose MHC class I molecule binding was restricted to HLA B*4403. The CML66-reactive T cell clone recognized the HLA-B4403-expressing CD34+ AML cell line MUTZ-3, providing evidence that this epitope is naturally processed and presented by myeloid leukemia cells. Functional cytotoxic T cell responses to CML66 appeared to precede the appearance of antigen-specific antibody responses. While CML66-specific plasma antibody was only detectable starting 2-3 months after DLI, reactivity to the HLA-B4403-restricted epitope by peripheral blood-derived CD8+ T cells was detected by IFNγ ELISpot as early as one month following DLI. To sensitively detect CD8+ CML66-specific T cells in vivo, we sequenced the CDR3 region specific to the CML66-specific clone, and designed clone-specific quantitative PCR primers. Nested PCR of RNA directly extracted from patient PBMC revealed only minimal detection of the CML66-specific T cell clone prior to DLI, but then expansion between 1 to 6 months after DLI. Similar analysis of marrow-derived RNA also revealed the presence of CML66-specific T cell clone in patient marrow following DLI. Moreover, this clone was already detected in patient marrow prior to DLI, suggesting that marrow is a reservoir of leukemia-specific T cells following hematopoietic stem cell transplantation. Our studies provide the first description of coordinated adaptive immunity developing against a nonpolymorphic leukemia-associated antigen, in close temporal correlation with the attainment of long-lasting immune-mediated clinical remission after allogeneic HSCT. The detected cytolytic T cell response was associated with the secretion of high-titer IgG antibody specific for a distinct epitope in the same target protein. In this setting, our results indicate that infusion of CD4+ donor cells resulted in the rapid activation and expansion of pre-existing marrow-resident leukemia-specific CD8+ T cells, followed by a cascade of antigen-specific immune responses detectable in the periphery. Disclosures: No relevant conflicts of interest to declare.

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