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 ◽  
...  

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.

2005 ◽  
Vol 79 (15) ◽  
pp. 9419-9429 ◽  
Author(s):  
Nicole E. Miller ◽  
Jennifer R. Bonczyk ◽  
Yumi Nakayama ◽  
M. Suresh

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.


2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S852-S853
Author(s):  
Hassen Kared ◽  
Evan Bloch ◽  
Andrew Redd ◽  
Alessandra Nardin ◽  
Hermi Sumatoh ◽  
...  

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)


Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 2-3
Author(s):  
Sandeep Kumar ◽  
Moanaro Biswas ◽  
Annie R Pineros ◽  
Ype P De Jong ◽  
Roland W Herzog

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.


Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 287-287 ◽  
Author(s):  
Katayoun Rezvani ◽  
Agnes S.M. Yong ◽  
Stephan Mielke ◽  
Bipin N. Savani ◽  
Laura Musse ◽  
...  

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.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1974-1974
Author(s):  
Suzanne M. McGoldrick ◽  
Abraham Guerrero ◽  
Tori N. Yamamoto ◽  
Colleen Delaney ◽  
Stanley R. Riddell

Abstract Abstract 1974 Cytomegalovirus (CMV) is a major infectious complication in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) and has been linked to deficiencies of virus-specific T cell immunity. Compared to bone marrow or peripheral blood stem cell transplants, recipients of single or double umbilical cord blood transplants (UCBT) receive lower numbers of donor T cells that have not previously been primed to CMV and are at increased risk for early and recurrent CMV infections. At our institution, the rate of CMV reactivation in CMV seropositive patients undergoing CBT is close to 100% with standard dose Acyclovir as prophylaxis [Delaney unpublished data]. Here, we systematically analyzed the kinetics of recovery, durability, and specificity of CMV-specific CD8+ and CD4+ T cell responses in UCBT recipients. CD8 T cell responses to CMV were analyzed by interferon γ (IFN-γ) intracellular cytokine staining after stimulating recipient peripheral blood mononuclear cells (PBMC) obtained at various time points after CBT with autologous patient fibroblasts infected with the RV798 virus, which is a mutant CMV strain that lacks the viral US genes that downregulate class I MHC and can present all potentially immunogenic epitopes of the virus. The mean absolute CD8 T cell counts were 59, 93 and 213 cells/μl and the mean CD4 T cell counts were 154, 223 and 397 cells/μl in PBMC at day 56, 180 and 365 respectively. Direct assays of PBMC after a 4–6 hour stimulation with RV798-infected fibroblasts did not detect a significant frequency of IFN-γ+ CD8+ T cells in CBT recipients, in contrast to normal CMV+ donors that exhibited frequencies of CD8+ T cells of 2–10%. However, IFN-γ+ CMV specific CD8 T cells were readily detectable in PBMC obtained as early as day 42 after UCBT from 8 out of 8 CMV positive CBT recipients after a 10 day stimulation with RV798 infected fibroblasts. These responses were sustained at multiple time points through day 365 post transplant. This result was not a consequence of in vitro priming of CD8 T cells by prolonged stimulation with RV798 since we did not detect a CMV-specific T cell response in 3 out of 3 CMV seronegative recipients at any time through day 365 with the same assay. To assess CD4+ T cell responses, we performed lymphoproliferative assays (LPA) by stimulating patient PBMC obtained at the same time points with whole CMV antigen. The proportion of patients with a positive response at day 56, 80, 180 and 365 was 0.38, 0.50, 0.88, and 1.0 respectively. All of the CMV positive CBT recipients in our study had multiple occurrences of CMV reactivation throughout the first year post CBT requiring antiviral drug therapy. The CMV-specific CD8 T cell response in normal CMV+ individuals recognizes a large number of distinct dominant and subdominant antigens and a potential explanation for the failure to control CMV after CBT is that the T cell response may not be sufficiently diverse. We analyzed the specificity of CMV specific CD8+ T cells that developed after CBT in 4 recipients by assessing recognition of COS cells transfected with the class I HLA restricting alleles and with a CMV plasmid library consisting of 142 ORFs, subdivided into pools. A response was seen in 3 out of 4 patients to at least 3 different CMV antigens by day 80 post CBT, including previously defined dominant epitopes in pp65 and this diversity was maintained through 6–12 months post transplant. One patient had a less diverse response early post CBT and the response changed over time to include recognition of new epitopes. Collectively, our results demonstrate that CD8+ and CD4+ T cells are primed to CMV antigens very early after CBT despite the infusion of limited numbers of naïve T cells and the administration of post transplant immunosuppression. The inability to control CMV infection may be due to a quantitative deficiency of CMV-specific T cells resulting from the inability of CMV-specific T cells to expand in vivo to numbers sufficient to eliminate virus replication. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3199-3207 ◽  
Author(s):  
Gennadiy Zelinskyy ◽  
Kirsten K. Dietze ◽  
Yvonne P. Hüsecken ◽  
Simone Schimmer ◽  
Savita Nair ◽  
...  

AbstractCytotoxic CD8+ T cells control acute viremia in many viral infections. However, most viruses that establish chronic infections evade destruction by CD8+ T cells, and regulatory T cells (Treg) are thought to be involved in this immune evasion. We have infected transgenic mice, in which Treg can be selectively depleted, with Friend retrovirus (FV) to investigate the influence of Treg on pathogen-specific CD8+ T-cell responses in vivo. We observed that Treg expansion during acute infection was locally defined to organs with high viral loads and massive activation of virus-specific effector CD8+ T cells. Experimental ablation of Treg resulted in a significant increase of peak cytotoxic CD8+ T-cell responses against FV. In addition, it prevented the development of functional exhaustion of CD8+ T cells and significantly reduced FV loads in lymphatic organs. Surprisingly, despite the massive virus-specific CD8+ T-cell response after temporary Treg depletion, no evidence of immunopathology was found. These results demonstrate the important role of Treg in controlling acute retrovirus-specific CD8+ T-cell responses, and suggest that temporary manipulation of Treg might be a possible therapeutic approach in chronic infectious diseases.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 606-606 ◽  
Author(s):  
Louis J. Picker ◽  
Andrew W. Sylwester ◽  
Bridget L. Mitchell ◽  
Cara Taormina ◽  
Christian Pelte ◽  
...  

Abstract Human Cytomegalovirus (HCMV) is among the largest and most complex of known viruses with 150–200nm virions enclosing a double stranded 230kb DNA genome capable of coding for >200 proteins. HCMV infection is life-long, and for the vast majority of immune competent individuals clinically benign. Disease occurs almost exclusively in the setting of immune deficiency, suggesting that the stable host-parasite relationship that characterizes these infections is the result of an evolutionarily “negotiated” balance between viral mechanisms of pathogenesis and the host immune response. In keeping with, and perhaps because of this balance, the human CD4+ T cell response to whole HCMV viral lysates is enormous, with median peripheral blood frequencies of HCMV-specific cells ~5–10 fold higher than for analogous preparations of other common viruses. Although certain HCMV ORFs have been identified as targets of either the CD4+ or CD8+ T cell response, the specificities comprising the CD4+ T cell response, and both the total frequencies and component parts of the CD8+ T cell response are unknown. Here, we used cytokine flow cytometry and ~14,000 overlapping 15mer peptides comprising all 213 HCMV ORFs encoding proteins >100 amino acids in length to precisely define the total CD4+ and CD8+ HCMV-specific T cell responses and the HCMV ORFs responsible for these responses in 33 HCMV-seropositive, HLA-disparate donors. An additional 9 HCMV seronegative donors were similarly examined to define the extent to which non-HCMV responses cross-react with HCMV-encoded epitopes. We found that when totaled, the median frequencies of HCMV-specific CD4+ and CD8+ T cells in the peripheral blood of the seropositive subjects were 4.0% and 4.5% for the total CD4+ or CD8+ T cell populations, respectively (which corresponds to 9.1% and 10.5% of the memory populations, respectively). The HCMV-specific CD4+ and CD8+ T cell responses included a median 12 and 7 different ORFs, respectively, and all told, 73 HCMV ORFs were identified as targets for both CD4+ and CD8+ T cells, 26 ORFs as targets for CD8+ T cells alone, and 43 ORFS as targets for CD4+ T cells alone. UL55, UL83, UL86, UL99, and UL122 were the HCMV ORFs most commonly recognized by CD4+ T cells; UL123, UL83, UL48, UL122 and UL28 were the HCMV ORFs most commonly recognized by CD8+ T cells. The relationship between immunogenicity and 1) HLA haplotype and 2) ORF expression and function will be discussed. HCMV-seronegative individuals were non-reactive with the vast majority of HCMV peptides. Only 7 potentially cross-reactive responses were identified (all by CD8+ T cells) to 3 ORFs (US32, US29 and UL116) out of a total of almost 4,000 potential responses, suggesting fortuitous cross-reactivity with HCMV epitopes is uncommon. These data provide the first glimpse of the total human T cell response to a complex infectious agent, and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2910-2910
Author(s):  
Katayoun Rezvani ◽  
Agnes S. M. Yong ◽  
Abdul Tawab ◽  
Behnam Jafarpour ◽  
Rhoda Eniafe ◽  
...  

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.


2006 ◽  
Vol 81 (4) ◽  
pp. 2002-2011 ◽  
Author(s):  
David Masopust ◽  
Kaja Murali-Krishna ◽  
Rafi Ahmed

ABSTRACT Measuring the magnitudes and specificities of antiviral CD8 T-cell responses is critical for understanding the dynamics and regulation of adaptive immunity. Despite many excellent studies, the accurate measurement of the total CD8 T-cell response directed against a particular infection has been hampered by an incomplete knowledge of all CD8 T-cell epitopes and also by potential contributions of bystander expansion among CD8 T cells of irrelevant specificities. Here, we use several techniques to provide a more complete accounting of the CD8 T-cell response generated upon infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV). Eight days following infection, we found that 85 to 95% of CD8 T cells exhibit an effector phenotype as indicated by granzyme B, 1B11, CD62L, CD11a, and CD127 expression. We demonstrate that CD8 T-cell expansion is due to cells that divide >7 times, whereas heterologous viral infections only elicited <3 divisions among bystander memory CD8 T cells. Furthermore, we found that approximately 80% of CD8 T cells in spleen were specific for ten different LCMV-derived epitopes at the peak of primary infection. These data suggest that following a single LCMV infection, effector CD8 T cells divide ≥15 times and account for at least 80%, and possibly as much as 95%, of the CD8 T-cell pool. Moreover, the response targeted a very broad array of peptide major histocompatibility complexes (MHCs), even though we examined epitopes derived from only two of the four proteins encoded by the LCMV genome and C57BL/6 mice only have two MHC class I alleles. These data illustrate the potential enormity, specificity, and breadth of CD8 T-cell responses to viral infection and demonstrate that bystander activation does not contribute to CD8 T-cell expansion.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 552-552 ◽  
Author(s):  
Geoffrey L. Rogers ◽  
Roland W Herzog

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.


Sign in / Sign up

Export Citation Format

Share Document