Identification of Naturally Processed Mumps Virus Epitopes by Mass Spectrometry: Confirmation of Multiple CD8+ T-Cell Responses in Mumps Patients

Abstract Background The re-emergence of mumps among vaccinated young adults has become a global issue. Besides waning of antibody responses, suboptimal induction of T-cell responses may reduce protection. In a recent study, we observed a dominant polyfunctional CD8+ T-cell response after natural mumps virus (MuV) infection that was not present after vaccination. Unraveling the MuV epitope repertoire can provide insight in the specificity, functionality, and breadth of the T-cell response against MuV. Methods Peptides were eluted from human leukocyte antigen (HLA) class I molecules of MuV-infected cells and characterized by advanced mass spectrometry. Selected identified MuV peptides were tested for in vitro and ex vivo immunogenicity. Results In this study, we identified a broad landscape of 83 CD8+ T-cell epitopes of MuV, 41 of which were confirmed based on synthetic peptide standards. For 6 epitopes, we showed induction of an HLA-A*02-restriced CD8+ T-cell response. Moreover, robust T-cell responses against 5 selected MuV epitopes could be detected in all tested mumps patients using peptide/HLA-A*02:01 dextramers. Conclusions The identified CD8+ T-cell epitopes will help to further characterize MuV-specific T-cell immunity after natural MuV infection or vaccination. These MuV epitopes may provide clues for a better understanding of, and possibly for preventing, mumps vaccine failure. We identified for the first time 41 mumps virus (MuV)-specific HLA-A*02 epitopes. For 6 epitopes, CD8+ T-cell responses were confirmed in T cells derived from several mumps cases, and MuV-specific CD8+ T cells could be identified by peptide/dextramer staining.

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

Journal of Virology ◽

10.1128/jvi.79.15.9419-9429.2005 ◽

2005 ◽

Vol 79 (15) ◽

pp. 9419-9429 ◽

Cited By ~ 29

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.

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Public T-cell epitopes shared among SARS-CoV-2 variants are presented on prevalent HLA class I alleles

10.1101/2021.10.13.463911 ◽

2021 ◽

Author(s):

Saskia Meyer ◽

Isaac Blaas ◽

Ravi Chand Bollineni ◽

Marina Delic-Sarac ◽

Trung T Tran ◽

...

Keyword(s):

T Cell ◽

Cell Response ◽

T Cell Responses ◽

Hla Class I ◽

Cd8 T Cell ◽

T Cell Response ◽

Class I ◽

T Cell Epitopes ◽

Low Frequencies ◽

Cell Responses

T-cell epitopes with broad population coverage may form the basis for a new generation of SARS-CoV-2 vaccines. However, published studies on immunoprevalence are limited by small test cohorts, low frequencies of antigen-specific cells and lack of data correlating eluted HLA ligands with T-cell responsiveness. Here, we investigate CD8 T-cell responses to 48 peptides eluted from prevalent HLA alleles, and an additional 84 predicted binders, in a large cohort of convalescents (n=83) and pre-pandemic control samples (n=19). We identify nine conserved SARS-CoV-2 specific epitopes restricted by four of the most prevalent HLA class I alleles in Caucasians, to which responding CD8 T cells are detected in 70-100% of convalescents expressing the relevant HLA allele, including two novel epitopes. We find a strong correlation between immunoprevalence and immunodominance. Using a new algorithm, we predict that a vaccine including these epitopes would induce a T cell response in 83% of Caucasians. Significance Statement: Vaccines that induce broad T-cell responses may boost immunity as protection from current vaccines against SARS-CoV-2 is waning. From a manufacturing standpoint, and to deliver the highest possible dose of the most immunogenic antigens, it is rational to limit the number of epitopes to those inducing the strongest immune responses in the highest proportion of individuals in a population. Our data show that the CD8 T cell response to SARS-CoV-2 is more focused than previously believed. We identify nine conserved SARS-CoV-2 specific CD8 T cell epitopes restricted by four of the most prevalent HLA class I alleles in Caucasians and demonstrate that seven of these are endogenously presented.

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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 ◽

10.1182/blood.v114.22.4096.4096 ◽

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.

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LB-18. Broad and Prevalent SARS-CoV-2 CD8+ T Cell Response in Recovered COVID-19 Individuals Demonstrates Kinetics of Early Differentiation

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa515.1915 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S852-S853

Author(s):

Hassen Kared ◽

Evan Bloch ◽

Andrew Redd ◽

Alessandra Nardin ◽

Hermi Sumatoh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cell Response ◽

T Cell Responses ◽

Cd8 T Cell ◽

T Cell Response ◽

Peptide Epitopes ◽

Cell Responses ◽

Candidate Peptide

Abstract Background Understanding the diversity, breadth, magnitude, and functional profile of the T cell response against SARS-CoV-2 in recovered COVID-19 individuals is critical to evaluate the contribution of T cells to produce a potentially protective immune response. Methods We used a multiplexed peptide-MHC tetramer approach to screen a total of 408 SARS-CoV-2 candidate peptide epitopes for CD8+ T cell recognition in a cohort of 30 individuals recovered from COVID-19. The peptides spanned the whole viral genome and were restricted to six prevalent HLA alleles; T cells were simultaneously characterized by a 28-marker phenotypic panel. The evolution of the SARS-CoV-2 T cell responses was then statistically modeled against time from diagnosis, and in relation to humoral and inflammatory response. Workflow for Study. A multiplexed peptide-MHC tetramer approach was used to screen SARS-CoV-2 candidate peptide epitopes in a cohort of 30 COVID-19 recovered patients across 6 prevalent HLA alleles, and T cells profiled with a 28-marker phenotypic panel. Multiplex tetramer screen. One representative COVID-19 recovered patient and one healthy donor were screened for HLA- relevant SARS-CoV-2 epitopes, as well as epitopes for CMV, EBV, Influenza, Adenovirus and MART-1. Shown are the frequencies of tetramer-positive CD8 T cells from 2 technical replicates per subject. Results Almost all individuals screened showed a T cell response against SARS-CoV-2 (29/30): 132 SARS-CoV-2-specific CD8+ T cells hits were detected, corresponding to 52 unique reactive epitopes. Twelve of the 52 unique SARS-CoV-2-specific epitopes were recognized by more than 40% of the individuals screened, indicating high prevalence in the subjects. Importantly, these CD8+ T cell responses were directed against both structural and non-structural viral proteins, with the highest magnitude against nucleocapsid derived peptides, but without any antigen-driven bias in the phenotype of specific T cells. Overall, SARS-CoV-2 T cells showed specific states of differentiation (stem-cell memory and transitional memory), which differed from those of MART-1, influenza, CMV and EBV-specific T cells. UMAP visualization revealed a phenotypic profile of SARS-CoV-2-specific CD8 T cells in COVID-19 convalescent donors that is distinct from other viral specificities, such as influenza, CMV, EBV and Adenovirus. SARS-CoV-2 epitope screening revealed CD8+ T cell responses directed against both structural and non-structural viral proteins, with the highest magnitude response against nucleocapsid derived peptides Conclusion The kinetics modeling demonstrates a dynamic, evolving immune response characterized by a time-dependent decrease in overall inflammation, increase in neutralizing antibody titer, and progressive differentiation of a broad SARS-CoV-2 CD8 T cell response. It could be desirable to aim at recapitulating the hallmarks of this robust CD8 T cell response in the design of protective COVID-19 vaccines. Disclosures Hassen Kared, PhD, ImmunoScape (Shareholder) Alessandra Nardin, DvM, ImmunoScape (Shareholder) Hermi Sumatoh, BSc, Dip MTech, ImmunoScape (Shareholder) Faris Kairi, BSc, ImmunoScape (Shareholder) Daniel Carbajo, PhD, ImmunoScape (Shareholder) Brian Abel, PhD, MBA, ImmunoScape (Shareholder) Evan Newell, PhD, ImmunoScape (Shareholder)

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Analysis of CD8+T cell response during the 2013–2016 Ebola epidemic in West Africa

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806200115 ◽

2018 ◽

Vol 115 (32) ◽

pp. E7578-E7586 ◽

Cited By ~ 28

Author(s):

Saori Sakabe ◽

Brian M. Sullivan ◽

Jessica N. Hartnett ◽

Refugio Robles-Sikisaka ◽

Karthik Gangavarapu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

West Africa ◽

Cell Response ◽

Acute Infection ◽

T Cell Responses ◽

T Cell Response ◽

Cell Mediated Immunity ◽

T Cell Epitopes ◽

Cell Responses

The recent Ebola epidemic exemplified the importance of understanding and controlling emerging infections. Despite the importance of T cells in clearing virus during acute infection, little is known about Ebola-specific CD8+T cell responses. We investigated immune responses of individuals infected with Ebola virus (EBOV) during the 2013–2016 West Africa epidemic in Sierra Leone, where the majority of the >28,000 EBOV disease (EVD) cases occurred. We examined T cell memory responses to seven of the eight Ebola proteins (GP, sGP, NP, VP24, VP30, VP35, and VP40) and associated HLA expression in survivors. Of the 30 subjects included in our analysis, CD8+T cells from 26 survivors responded to at least one EBOV antigen. A minority, 10 of 26 responders (38%), made CD8+T cell responses to the viral GP or sGP. In contrast, 25 of the 26 responders (96%) made response to viral NP, 77% to VP24 (20 of 26), 69% to VP40 (18 of 26), 42% (11 of 26) to VP35, with no response to VP30. Individuals making CD8+T cells to EBOV VP24, VP35, and VP40 also made CD8+T cells to NP, but rarely to GP. We identified 34 CD8+T cell epitopes for Ebola. Our data indicate the immunodominance of the EBOV NP-specific T cell response and suggest that its inclusion in a vaccine along with the EBOV GP would best mimic survivor responses and help boost cell-mediated immunity during vaccination.

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Cross Priming of Transgene Product-Specific CD8+ T Cells in Hepatic AAV Gene Transfer Depends on IL-1 Receptor and XCR1+ Dendritic Cells but Not TLR9

Blood ◽

10.1182/blood-2020-142926 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 2-3

Author(s):

Sandeep Kumar ◽

Moanaro Biswas ◽

Annie R Pineros ◽

Ype P De Jong ◽

Roland W Herzog

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Cd8 T Cells ◽

Cell Response ◽

Adaptor Protein ◽

T Cell Responses ◽

Cd8 T Cell ◽

T Cell Response ◽

Cell Responses

Introduction: Adeno-associated virus (AAV) mediated gene transfer is currently evaluated in multiple Phase I/II and Phase III studies for the treatment of hemophilia. However, immune responses to both the AAV capsid and encoded transgene remain major impediments to clinical translation. Several studies have implicated innate immune sensors such as Toll-like receptors (TLR) and their downstream adaptor molecule MyD88 in sensing viral structures. TLR9-MyD88 signaling has been linked to cross-priming of CD8+ T cell responses to capsid and also to transgene product-specific CD8+ T cell responses. However, little is known about other signaling pathways that may lead to immune activation. Previously, our lab has shown that while liver gene transfer is capable of inducing immunological tolerance to AAV encoded transgene products, vector dose and design play a critical role. For instance, low hepatic gene expression levels may elicit a CD8+ T cell response to the AAV encoded transgene, resulting in loss of the model antigen ovalbumin (OVA) in C57BL/6 mice or of FIX expression in hemophilia B mice. We investigated innate immune sensing pathways that may play a role in initiating transgene specific CD8+ T cell response in the hepatic microenvironment. Further, we determined the contribution of hepatic antigen presenting cells (APC) by selectively depleting/neutralizing APCs and evaluating their effect on presentation of transgene product-derived antigen following AAV8-OVA liver gene delivery. Methods: Wild-type (WT) C57BL6 and specific innate sensing knockout mice on the C57BL6 background were intravenously (IV) injected with a predetermined immunogenic dose (1x109vg) of hepatotropic AAV8-OVA vector (Mol Ther 25:880, 2017). PBMCs were quantified at 4 weeks for OVA-specific CD8+ T cells using a class I MHC tetramer. Hepatic APC types [Kupffer cells, neutrophils, CD103+ dendritic cell (DC), CD11c+ DC, XCR1+ DC] involved in transgene specific CD8+ T cell activation were selectively depleted/inactivated by pre-treatment with gadolinium chloride (GdCl3), Ly6G, CD103 antibody respectively, or by administering diphtheria toxin (DT) to CD11c-DTR and XCR1-DTR mice. This was followed by intravenous administration of AAV8-OVA and CellTrace violet labeled OT-1 cells. Results: Similar to WT mice, TLR9-/-, TLR2-/-, TRIF-/-, IFNaR-/- and MDA5-/- mice developed a CD8+ T cell response indicating that these sensors do not play a role in transgene specific CD8+ T cells response. Interestingly, adaptor protein MyD88-/- mice did not elicit CD8+ T cell response to OVA, implying a MyD88 dependent but TLR9 independent response. Since MyD88 is an essential adaptor protein not only for TLR but also for interleukin-1 (IL-1) signaling pathways, we next analyzed IL-1R-/- mice. Similar to MyD88-/- mice, IL-1R-/- mice did not show OVA specific CD8+ T cells (p=0.006, 0.007 respectively), indicating that transgene-specific adaptive responses are mediated by IL-1R/MyD88 signaling. Kupffer cells and DCs are principal APCs in liver and infiltrating neutrophils could also act as APCs under inflammatory conditions in liver microenvironment. Using proliferation of OT-I cells as readout we tested if any of these cell types are required for presentation to transgene specific CD8+ T cells. In naïve control, GdCl3 treated and a-Ly6G antibody treated mice, OT-I cell proliferation reached 60%, 65% and 48% on average, respectively. Depletion of CD11c DCs substantially reduced the proliferation of OT-I cells to ~6% (p<0.0001) indicating a critical role for DCs in mediating transgene specific CD8+ T cell responses. Since XCR1+ DCs are the major cross-presenting DCs and hepatic resident CD103+ DCs are shown to have intrinsically enhanced capacity to process and present antigen to naïve CD8+ T cells, we further sought to assess if any of these DCs plays a role in activation of transgene specific CD8+ T cells. Neutralization of CD103+ DCs reduced OT-I proliferation to 39% (p=0.01) whereas depletion of XCR1+ DCs reduced the proliferation to ~20% (p<0.0001) indicating a major role for XCR1+ DCs. Conclusions: In summary, we uncovered a novel-signaling pathway that can activate CD8+ T cell responses during AAV gene transfer independent of TLR9 sensing. The IL-1R/MyD88 pathway drives activation of transgene specific CD8+ T cell, and XCR1+ DCs are critically involved in cross-presenting transgene product-derived antigen to CD8+ T cells. Disclosures Herzog: Takeda Pharmaceuticals: Patents & Royalties.

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CD4+ T helper cells use CD154–CD40 interactions to counteract T reg cell–mediated suppression of CD8+ T cell responses to influenza

Journal of Experimental Medicine ◽

10.1084/jem.20130097 ◽

2013 ◽

Vol 210 (8) ◽

pp. 1591-1601 ◽

Cited By ~ 28

Author(s):

André Ballesteros-Tato ◽

Beatriz León ◽

Frances E. Lund ◽

Troy D. Randall

Keyword(s):

T Cells ◽

T Cell ◽

Cell Response ◽

T Helper Cells ◽

Cd4 T Cells ◽

T Cell Responses ◽

Cd8 T Cell ◽

T Cell Response ◽

T Helper ◽

Cell Responses

CD4+ T cells promote CD8+ T cell priming by licensing dendritic cells (DCs) via CD40–CD154 interactions. However, the initial requirement for CD40 signaling may be replaced by the direct activation of DCs by pathogen-derived signals. Nevertheless, CD40–CD154 interactions are often required for optimal CD8+ T cell responses to pathogens for unknown reasons. Here we show that CD40 signaling is required to prevent the premature contraction of the influenza-specific CD8+ T cell response. CD40 is required on DCs but not on B cells or T cells, whereas CD154 is required on CD4+ T cells but not CD8+ T cells, NKT cells, or DCs. Paradoxically, even though CD154-expressing CD4+ T cells are required for robust CD8+ T cell responses, primary CD8+ T cell responses are apparently normal in the absence of CD4+ T cells. We resolved this paradox by showing that the interaction of CD40-bearing DCs with CD154-expressing CD4+ T cells precludes regulatory T cell (T reg cell)–mediated suppression and prevents premature contraction of the influenza-specific CD8+ T cell response. Thus, CD4+ T helper cells are not required for robust CD8+ T cell responses to influenza when T reg cells are absent.

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AAV Vector Dose Determines TLR9 Dependence of CD8+ T Cell Response to Transgene Product

Blood ◽

10.1182/blood-2020-141054 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 3-3

Author(s):

Ning Li ◽

Thais Bertolini ◽

Roland W Herzog

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Immune Responses ◽

Cell Response ◽

T Cell Responses ◽

Cd8 T Cell ◽

T Cell Response ◽

Innate Immune ◽

Cell Responses

Adeno-associated viral (AAV) vectors are currently evaluated in multiple Phase III clinical trial for the treatment of hemophilia and neuromuscular disorders. A major concern is the potential for immune responses. Viral vectors are initially sensed by the innate immune system, which shapes subsequent adaptive immune responses. Particularly, toll-like receptors (TLRs) have been reported as major sensors of pathogens during innate immune response. TLRs recognize pathogen-associated molecular patterns (PAMPs). Our previous studies found that cross-priming of AAV capsid-specific CD8+ T cells depended on TLR9-MyD88 pathway. TLR9 is an endosomal DNA receptor that responds most potently to unmethylated CpG motifs as found in bacterial and viral DNA. Similarly, others documented TLR9-dependent CD8+ T cell responses against non-secreted transgene products such as LacZ and hemagglutinin upon muscle-directed AAV gene transfer. Similarly, we published that CD8+ T cell responses to a secreted ovalbumin (ova) transgene product were substantially reduced (although not entirely eliminated) upon muscle gene transfer in TLR9-deficient mice [J Innate Immun. 7:302-14]. For those studies, we had used a self-complementary scAAV genomes, which we found to more strongly activate TLR9 than conventional single-stranded ssAAV vectors. Here, we performed intramuscular injections of 3 doses of ssAAV1-CMV-ova vector (2X1010, 2X1011 and1X1012 vg) in wild-type (WT), TLR9-/-, or MYD88-/- C57BL/6 mice. Using MHC tetramer (H2-Kb -SIINFEKL), ova-specific CD8+ T cell frequencies were monitored in peripheral blood for up to 6 weeks. As expected from prior studies, TLR9-/- mice showed a substantially reduced response (1.2% tetramer+ of CD8) at the low dose when compared to WT (12% tetramer+ of CD8) animals (p<0.0001, n=5/group). To our surprise, CD8+ T cell responses were similar in TLR9-/- and WT mice at the 2 higher doses. TLR9-/- mice displayed 16% and 3.3% tetramer+ of CD8 frequencies at the median and the high doses, respectively; which was comparable to WT mice, where 15% and 4.8% tetramer+ of CD8 frequencies were observed (n=5/group). Therefore, sensing of the AAV genome by TLR9 is more critical for the CD8+ T cell response to the secreted transgene product at lower vector doses (possibly related to the lower levels of transgene expression). Interestingly, transgene product-specific CD8+ T cell responses were much reduced in MyD88-/- mice, in which 0.2% and 1.7% tetramer+ of CD8 frequencies were found for low and median doses. Therefore, an alternative signaling pathway that includes the MyD88 adaptor molecule likely exists that is more critical than TLR9 above a certain level of expression. The reduced strength of the CD8+ T cell response seen at the highest vector dose compared to the medium dose may be explained by a transient increase in FoxP3+ Treg and in PD-1+ T cells that we observed 1 week after gene transfer and that was significantly greater at the highest vector dose. In related experiments, we performed intramuscular gene transfer using a ssAAV1-EF1a-FIX vector in hemophilia B mice (C3H/HeJ F9-/-, 1x1011 vg/mouse). Here, we used either a vector with native sequences or with an expression cassette that was entirely devoid of CpG motifs (and there stimulates TLR9 less effectively). CpG depletion did not have substantial effects on antibody formation against human FIX or the viral capsid. However, CD8+ T cell infiltrates in skeletal muscle were markedly reduced but not entirely eliminated when tissue sections were examined 1 month after gene transfer. In conclusion, TLR9 signaling is one important factor in the activation of transgene product-specific CD8+ T cells in AAV gene transfer, but other pathways exist that may be more critical depending on vector dose or levels of expression. Disclosures Herzog: Takeda Pharmaceuticals: Patents & Royalties.

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Analysis of EBV-Specific T Cell Responses in Transplant Recipients with PTLD.

Blood ◽

10.1182/blood.v106.11.1915.1915 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1915-1915

Author(s):

Kathrin Sebelin ◽

Antje Meier ◽

Matthias Papp-Vary ◽

Stefan Oertel ◽

Antonio Pezzutto ◽

...

Keyword(s):

T Cells ◽

Viral Load ◽

T Cell ◽

Cell Response ◽

Low Frequency ◽

T Cell Responses ◽

Cd8 T Cell ◽

T Cell Response ◽

Cd4 T Cell ◽

Cell Responses

Abstract EBV causes a chronic infection in >95 % of the population and despite its strong growth transforming capacity the majority of EBV infected individuals remain asymptomatic. In contrary, in immunosuppressed patients (pts) the risk of EBV reactivation and development of posttransplant lymphoproliferative disease (PTLD) is high. This is assumed to be due to a defective T cell response. Here we analyzed the EBV-specific CD8 and CD4 T cell response to different EBV latent and lytic antigens in pts with newly diagnosed PTLD. A prospective study of 10 pts after solid organ transplantation at time of diagnosis of PTLD was performed. EBV-specific CD8 T cells were examined by flow cytometric analysis using HLA-A2, HLA-B7 and HLA-B8 restricted tetramers incorporating BMLF1 (lytic), EBNA3 and LMP2 (both latent)-derived peptides. Staining was done in conjunction with mAbs against CD8 and CCR7. The ability of CD8 T cells to produce IFN-γ in response to the same EBV-derived peptides was measured by cytokine secretion assay. In healthy, EBV+ donors, we previously have found a consistent CD4 T cell response to the latent EBV antigen EBNA1. Therefore, EBNA1-specific CD4 T cell responses were monitored for IFN-g / IL-4 secretion after protein stimulation. T cell analysis was combined with EBV-DNA quantiation by real time PCR. We found EBV-specific CD8 T cell responses at low frequency in most pts with PTLD (8/10). Half of the pts showed low frequency EBNA1 specific CD4+ T cell responses. All pts with an EBNA1 specific CD4 T cell response showed an EBV-specific CD8 T cell response. In 2/10 pts we found no EBV-specific CD4 and CD8 T cell responses and both pts died under initial therapy. EBV-viral load was found to inversely correlate to absolute CD4 T cell counts. In comparison to healthy normal donors, no significant differences in EBV-specific T cell response could be observed. However, pts EBV-specific T cells were decreased in comparison to pts with high EBV viral load after TX and no PTLD as well as in comparison to pts with infectious mononucleosis. These results indicate that impairment of EBV-specific T cells is not due to clonal depletion, but rather seems to be due to impaired functional activation and expansion. We therefore conclude that pts with PTLD have an inadequatly low EBV-specific T cell responses which correlates to a low absolute CD4 T cell count. We propose a combined immunomonitoring of EBV viral load, absolute CD4 T cell count and EBV-specific T cell enumeration in pts at risk for development of PTLD. Further studies are needed to evaluate the role of EBV-specific T cell monitoring in immunosuppressed pts for prediction of PTLD and the potential usefulness of T cell monitoring as a prognostic marker in PTLD.

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CD8+ T cell responses in convalescent COVID-19 individuals target epitopes from the entire SARS-CoV-2 proteome and show kinetics of early differentiation

10.1101/2020.10.08.330688 ◽

2020 ◽

Cited By ~ 1

Author(s):

Hassen Kared ◽

Andrew D Redd ◽

Evan M Bloch ◽

Tania S. Bonny ◽

Hermi Sumatoh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Response ◽

Inflammatory Responses ◽

Cell Epitope ◽

T Cell Responses ◽

Cd8 T Cell ◽

T Cell Response ◽

Cross Sectional ◽

Cell Responses

AbstractCharacterization of the T cell response in individuals who recover from SARS-CoV-2 infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 COVID-19 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis, humoral and inflammatory responses. 132 distinct SARS-CoV-2-specific CD8+ T cell epitope responses across six different HLAs were detected, corresponding to 52 unique reactivities. T cell responses were directed against several structural and non-structural virus proteins. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem-cell and transitional memory states, subsets, which may be key to developing durable protection.

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