Memory T-cell Responses Research Articles

Loss of Preexisting Immunological Memory Among Human Immunodeficiency Virus-Infected Women Despite Immune Reconstitution With Antiretroviral Therapy.

It is unclear whether human immunodeficiency virus (HIV) infection results in permanent loss of T-cell memory or if it affects preexisting antibodies to childhood vaccinations or infections. We conducted a matched cohort study involving 50 pairs of HIV-infected and HIV-uninfected women. Total memory T-cell responses were measured after anti-CD3 or vaccinia virus (VV) stimulation to measure T cells elicited after childhood smallpox vaccination. VV-specific antibodies were measured by means of enzyme-linked immunosorbent assay (ELISA). There was no difference between HIV-infected and HIV-uninfected study participants in terms of CD4+ T-cell responses after anti-CD3 stimulation (P = .19) although HIV-infected participants had significantly higher CD8+ T-cell responses (P = .03). In contrast, there was a significant loss in VV-specific CD4+ T-cell memory among HIV-infected participants (P = .04) whereas antiviral CD8+ T-cell memory remained intact (P > .99). VV-specific antibodies were maintained indefinitely among HIV-uninfected participants (half-life, infinity; 95% confidence interval, 309 years to infinity) but declined rapidly among HIV-infected participants (half-life; 39 years; 24-108 years; P = .001). Despite antiretroviral therapy-associated improvement in CD4+ T-cell counts (nadir, <200/μL; >350/μL after antiretroviral therapy), antigen-specific CD4+ T-cell memory to vaccinations or infections that occurred before HIV infection did not recover after immune reconstitution, and a previously unrealized decline in preexisting antibody responses was observed.

Human Immunodeficiency Virus Exposure but Not Early Cytomegalovirus Infection Is Associated With Increased Hospitalization and Decreased Memory T-Cell Responses to Tetanus Vaccine.

Human immunodeficiency virus (HIV)-exposed, uninfected (HEU) infants experience high rates of infectious morbidity. We hypothesized that early cytomegalovirus (CMV) infection was associated with increased hospitalization rates and decreased vaccine responses in HEU compared with HIV-unexposed (HUU) infants. Among infants enrolled in the Tshipidi study in Botswana, we determined CMV infection status by 6 months of age and compared hospitalization rates and responses to tetanus and Bacille Calmette-Guérin vaccines among HEU and HUU vaccinees. Fifteen of 226 (6.6%) HEU infants and 17 (19.3%) of 88 HUU infants were CMV-infected by 6 months. The HEU infants were approximately 3 times as likely to be hospitalized compared with HUU infants (P = .02). The HEU peripheral blood cells produced less interleukin (IL)-2 (P = .004), but similar amounts of interferon-γ, after stimulation with tetanus toxoid. Antitetanus immunoglobulin G titers were similar between groups. Cellular responses to purified protein derivative stimulation did not differ between groups. Maternal receipt of 3-drug antiretroviral therapy compared with zidovudine was associated with increased IL-2 expression after tetanus toxoid stimulation. The infants' CMV infection status was not associated with clinical or vaccine response outcomes. We observed that increased rates of hospitalization and decreased memory T-cell responses to tetanus vaccine were associated with HIV exposure and incomplete treatment of maternal HIV infection, but not early CMV infection.

Role and plasticity of Th1 and Th17 responses in immunity to Staphylococcus aureus

ABSTRACT The human commensal Staphylococcus aureus (SA) is a leading cause of skin/soft tissue and surgical-site infections, and bacteremia. Functional antibodies and T-cell-mediated immunity, particularly Th1/Th17 responses, are thought to mediate protection. Vaccine development may be hindered by modulation of vaccine-induced T cells by pathogen-activated immunoregulatory responses, e.g., via IL-10. We screened SA proteins for CD4+ T-cell-activating and IL-10/IL-17-inducing capacities using healthy donor-derived PBMCs. Responses were characterized (Th1/Th17/Th22/immunosuppressive IL-10-producing cells) using intracellular cytokine staining and flow cytometry. Phenotypic plasticity of Th1/Th17 cells was evaluated under pro- or anti-inflammatory conditions using modulatory cytokines. The impact of vaccination on SA-specific memory responses was assessed using samples from a clinical trial evaluating AS03-adjuvanted and non-adjuvanted multicomponent (CPS5/CPS8/α-toxin/ClfA) vaccines (NCT01160172). The donors exhibited SA-specific memory T-cell responses, indicative of pre-existing immunity to SA. We identified effective activators of Th1 responses (EbhA/IsaA/SdrE/MntC/Aaa/α-toxin), and Th17 and Th1/Th17 responses (EbhA/IsaA/SdrE and, to a lesser extent, α-toxin), but not of Th22 responses or IL-10 production. MRPII, IsdA, and ClfA were inefficient CD4+ T-cell activators in our assays. IL-10, likely produced by innate immune cells, influenced mainly Th1 cells by suppressing IFN-γ production. The memory CD4+ T-cells observed after long-term stimulation with α-toxin and ClfA indicated that vaccination with these proteins had induced expansion of pre-existing Th1 but not Th17 responses, without apparent adjuvant effect, confirming the trial data. The Th1/Th17-driving proteins (EbhA/IsaA/SdrE) shared low IL-10-promoting abilities and restricted phenotypic plasticity under pro- and anti-inflammatory conditions. Given the complex immunopathology and multiple virulence factors, identification of Th1/Th17-driving antigens, adjuvants and administration routes, and delineation of the role of memory responses, may advance vaccine development.

Mycobacterium indicus pranii Induced Memory T-Cells in Lung Airways Are Sentinels for Improved Protection Against M.tb Infection.

The lungs are the most vulnerable site for air-borne infections. Immunologic compartmentalization of the lungs into airway lumen and interstitium has paved the way to determine the immune status of the site of pathogen entry, which is crucial for the outcome of any air-borne infections. Vaccination via the nasal route with Mycobacterium indicus pranii (MIP), a prospective candidate vaccine against tuberculosis (TB), has been reported to confer superior protection as compared to the subcutaneous (s.c.) route in small-animal models of TB. However, the immune mechanism remains only partly understood. Here, we showed that intranasal (i.n.) immunization of mice with MIP resulted in a significant recruitment of CD4+ and CD8+ T-cells expressing activation markers in the lung airway lumen. A strong memory T-cell response was observed in the lung airway lumen after i.n. MIP vaccination, compared with s.c. vaccination. The recruitment of these T-cells was regulated primarily by CXCR3–CXCL11 axis in “MIP i.n.” group. MIP-primed T-cells in the lung airway lumen effectively transferred protective immunity into naïve mice against Mycobacterium tuberculosis (M.tb) infection and helped reducing the pulmonary bacterial burden. These signatures of protective immune response were virtually absent or very low in unimmunized and subcutaneously immunized mice, respectively, before and after M.tb challenge. Our study provides mechanistic insights for MIP-elicited protective response against M.tb infection.

540P - A phase Ib study of E7046 (AN0025) in combination with radiotherapy/chemoradiotherapy (RT/CRT) in preoperative treatment of rectal cancer

BackgroundAN0025 (previously E7046) is a selective inhibitor of the EP4 receptor and targets macrophages and immunosuppressive cells of myeloid lineage in tumor microenvironment. Preclinical studies have shown potent antitumor activity with AN0025 combining with RT and animal model data suggested antitumor memory T-cell response development by the combination. Neoadjuvant treatment of high-risk rectal cancer provides the platform to test such novel agents to clarify safety and efficacy and provide biopsy and surgical specimens for biomarker analysis. MethodsThis is a multicenter, open-label, Phase 1b study in patients (pts) with poor prognosis locally advanced rectal cancer as defined by standard MRI. This ongoing study enrolled pts into two groups, AN0025 in combination with Long Course Radio Chemotherapy (LCRT), or Short Course Radiotherapy (SCRT) followed by chemotherapy. The dose escalation design comprised 2 dose levels, 250mg and 500mg QD for both SCRT and LCRT. Treatment duration was 10 weeks followed by surgery at week 14-16. Pre-surgery MRI was done at week 11-13. Primary objective was safety and tolerability of AN0025 + CRT. ResultsAs of 23 Apr 2019, 27 pts were enrolled. 14 pts were treated in 250mg cohort with 7 pts in LCRT and in SCRT. No DLT was observed among 13 evaluable pts in this dose level. 500mg cohort is ongoing, 13 pts enrolled. Overall, 18 (66.7%) pts had treatment-related AEs (TRAE), most commonly fatigue (25.9%), diarrhea (14.8%), nausea (11.1%), decreased appetite (11.1%), headache (11.1%), and paraesthesia (11.1%). 2 pts experienced grade 3 TRAEs (diarrhea and fatigue) and 1pt had serious TRAEs (abdominal pain, vomiting, and fatigue). Median age was 59, 74% were male, 41% had ECOG 1, 56% with T stage T3c-T4b, and 63% were EMVI+. For 250mg cohort, pre-surgery MRI showed 6/13 pts had downstaging in T stage (3 each in LCRT and SCRT) and mrTRG 1-2 rate was 46% (43% and 50% in LCRT and SCRT respectively). Clinical responses led to 5/13 pts (38%) managed by a watch-and-wait approach. ConclusionsAN0025 was well tolerated in combination with chemoradiation and preliminary efficacy results are encouraging. Additional safety and efficacy data will be presented. Clinical trial identificationNCT03152370. Legal entity responsible for the studyAdlai Nortye USA. FundingAdlai Nortye USA. DisclosureL. Wyrwicz: Honoraria (self), Honoraria (institution): EISAI; Honoraria (self): Adlai Nortye USA. M.P. Saunders: Honoraria (self): Servier, Merck, Amgen, Sanofi. M. Hall: Advisory / Consultancy, Travel / Accommodation / Expenses: Roche, Tesaro, Clovis Oncology; Advisory / Consultancy: AstraZeneca, Amgen. V. Bhagawati Prasad: Full / Part-time employment: Eisai. N. Lautermilch: Full / Part-time employment: Adlai Nortye. M.M. Rashford: Full / Part-time employment: Adlai Nortye. J. Jin: Full / Part-time employment: Adlai Nortye. S. Formenti: Research grant / Funding (self): Bristol-Myers Squibb, Varian, Janssen, Regeneron, Eisai, Merck, Celldex; Honoraria (self): Bristol-Myers Squibb, Varian, Elekta, Janssen, Regeneron, GlaxoSmithKline, Eisai, AstraZeneca, Merck, Viewray, Bayer. R. Glynne-Jones: Advisory / Consultancy: Eisai, Sanofi, Servier, Amgen. All other authors have declared no conflicts of interest.

Long-term HPV-specific immune response after one versus two and three doses of bivalent HPV vaccination in Dutch girls

BackgroundIn view of further reduction of HPV vaccination schedules, gaining more insight into humoral and cellular immune responses after a single HPV vaccine is of great interest. Therefore, these responses were evaluated after different doses of the bivalent (2v) HPV-vaccine in girls. MethodsBlood was collected yearly up to seven years post-vaccination with one-, two- or three-doses of the 2vHPV vaccine (N = 890). HPV-type-specific IgG and IgA-antibody levels, IgG-isotypes and avidity indexes were measured by a virus-like-particle-based multiplex-immuno-assay for two vaccine and five non-vaccine HPV types. HPV-type-specific memory B-cell numbers- and T-cell cytokine responses were determined in a subpopulation. ResultsHPV-type-specific antibody concentrations were significantly lower in one- than in two- and three-dose vaccinated girls but remained stable over seven years. The lower antibody response coincided with reduced HPV-type-specific B- and T-cell responses. There were no differences in both the IgG subtypes and the avidity of the HPV16-specific antibodies between the groups. ConclusionsOne-dose of the 2vHPV vaccine is immunogenic, but results in less B- and T-cell memory and considerable lower antibody responses when compared with more doses. Therefore, at least of some of girls receiving the one-dose of the vaccination might be at higher risk for waning immunity to HPV in the long-term.

Single-cell transcriptomics reveal polyclonal memory T-cell responses in skin with positive abacavir patch test results

Capsule Summary. Single-cell responses in HLA-B*57:01 abacavir patch test positive skin remote to the acute hypersensitivity reaction demonstrate polyclonal T-cell activation and proliferation characterized by a transcriptional and cellular response consistent with memory responses to altered peptides.

Immune responses against enterovirus A71 infection: Implications for vaccine success.

Enterovirus A71 (EV-A71) from the Picornaviridae family is an important emerging pathogen causing hand, foot, and mouth disease (HFMD) outbreaks worldwide. EV-A71 also caused fatal neurological complications in young children especially in Asia. On the basis of seroepidemiological studies from many Asian countries, EV-A71 infection is very common. Children of very young age are particularly vulnerable. Large-scale epidemics that occur every 3 to 4 years are associated with accumulation of an immunologically naive younger population. Capsid proteins especially VP1 with the presence of major B- and T-cell epitopes are the most antigenic proteins. The nonstructural proteins mainly contribute to T-cell epitopes that induce cross-reactive immune responses against other enteroviruses. Dominant epitopes and their neutralization magnitudes differ in mice, rabbits, and humans. Neutralizing antibody is sufficient for immune protection, but poorer cellular immunity may lead to severe neurological complications and deaths. Some chemokines/cytokines are consistently found in severely ill patients, for example, IL-6, IL-10, IL-17A, MCP-1, IL-8, MIG, IP-10, IFN-γ, and G-CSF. An increase in white cell counts is a risk factor for severe HFMD. Recent clinical trials on EV-A71 inactivated vaccine showed >90% efficacy and a robust neutralization response that was protective, indicating neutralizing antibody correlates for protection. No protection against other enteroviruses was observed. A comprehensive understanding of the immune responses to EV-A71 infection will benefit the development of diagnostic tools, potential therapeutics, and subunit vaccine candidates. Future development of a multivalent enterovirus vaccine will require knowledge of correlates of protection, understanding of cross-protection and memory T-cell responses among enteroviruses.

Characterization of antibody and memory T-cell response in H7N9 survivors: a cross-sectional analysis

ObjectivesDespite the importance of immunological memory for protective immunity against viral infection, whether H7N9-specific antibodies and memory T-cell responses remain detectable years after the original infection is unknown. MethodsA cross-sectional study was conducted to investigate the immune memory responses of H7N9 patients who contracted the disease and survived during the 2013–2016 epidemics in China. Sustainability of antibodies and T-cell memory to H7N9 virus were examined. Healthy individuals receiving routine medical examinations in a physical examination centre were recruited as control. ResultsA total of 75 survivors were enrolled and classified into four groups based on the time elapsed from illness onset to specimen collection: 3 months (n = 14), 14 months (n = 14), 26 months (n = 28) and 36 months (n = 19). Approximately 36 months after infection, the geometric mean titres of virus-specific antibodies were significantly lower than titres in patients 3 months after infection, but 16 of 19 (84.2%) survivors in the 36-month interval had microneutralization (MN) titres ≥40. Despite the overall declining trend, the percentages of virus-specific cytokine-secreting memory CD4+ and CD8+ T cells remained higher in survivors at nearly all time-points in comparison with control individuals. Linear regression analysis showed that severe disease (mean titre ratio 2.77, 95% CI 1.17–6.49) was associated with higher haemagglutination inhibition (HI) titre and female sex for both HI (1.92, 1.02–3.57) and MN (3.33, 1.26–9.09) antibody, whereas female sex (mean percentage ratio 1.69, 95% CI 1.08–2.63), underlying medical conditions (1.94, 95% CI 1.09–3.46) and lack of antiviral therapy (2.08, 95% CI 1.04–4.17) were predictors for higher T-cell responses. ConclusionsSurvivors of H7N9 virus infection produced long-term antibodies and memory T-cell responses. Our findings warrant further serological investigation in general and high-risk populations and have important implications for vaccine design and development.

IRF4-dependent dendritic cells regulate CD8+ T-cell differentiation and memory responses in influenza infection.

Acute respiratory disease caused by influenza viruses is imperfectly mitigated by annual vaccination to select strains. Development of vaccines that elicit lung-resident memory CD8+ T cells (TRM) would offer more universal protection to seasonal and emerging pandemic viruses. Understanding how lung-resident dendritic cells (DCs) regulate TRM differentiation would be an important step in this process. Here, we used CD11c-cre-Irf4f/f (KO) mice, which lack lung-resident IRF4-dependent CD11b+CD24hi DCs and show IRF4 deficiency in other lung cDC subsets, to determine if IRF4-expressing DCs regulate CD8+ memory precursor cells and TRM during influenza A virus (IAV) infection. KO mice showed defective CD8+ T cell memory, stemming from a deficit of T regulatory cells and memory precursor cells with decreased Foxo1 expression. Transfer of wild-type CD11b+CD24hi DCs into KO mice restored CD8+ memory precursor cell numbers to wild-type levels. KO mice recovered from a primary infection harbored reduced numbers of CD8+ TRM and showed deficient expansion of IFNγ+CD8+ T cells and increased lung pathology upon challenge with heterosubtypic IAV. Thus, vaccination strategies that harness the function of IRF4-dependent DCs could promote the differentiation of CD8+ TRM during IAV infection.

Neo-antigen specific memory T-cell responses in healthy individuals

ABSTRACTThe driver mutations in exon 9 of the calreticulin protein have only been identified in patients with myeloid cancers. We recently demonstrated that healthy individuals display strong and frequent T-cell responses towards this mutation. This memory T-cell response is likely evidence of the elimination of mutated cells in healthy individuals.

Plasmacytoid dendritic cells respond to Epstein-Barr virus infection with a distinct type I interferon subtype profile

AbstractInfectious mononucleosis, caused by infection with the human gamma-herpesvirus Epstein-Barr virus (EBV), manifests with one of the strongest CD8+ T-cell responses described in humans. The resulting T-cell memory response controls EBV infection asymptomatically in the vast majority of persistently infected individuals. Whether and how dendritic cells (DCs) contribute to the priming of this near-perfect immune control remains unclear. Here we show that of all the human DC subsets, plasmacytoid DCs (pDCs) play a central role in the detection of EBV infection in vitro and in mice with reconstituted human immune system components. pDCs respond to EBV by producing the interferon (IFN) subtypes α1, α2, α5, α7, α14, and α17. However, the virus curtails this type I IFN production with its latent EBV gene products EBNA3A and EBNA3C. The induced type I IFNs inhibit EBV entry and the proliferation of latently EBV-transformed B cells but do not influence lytic reactivation of the virus in vitro. In vivo, exogenous IFN-α14 and IFN-α17, as well as pDC expansion, delay EBV infection and the resulting CD8+ T-cell expansion, but pDC depletion does not significantly influence EBV infection. Thus, consistent with the observation that primary immunodeficiencies compromising type I IFN responses affect only alpha- and beta-herpesvirus infections, we found that EBV elicits pDC responses that transiently suppress viral replication and attenuate CD8+ T-cell expansion but are not required to control primary infection.

Neoantigen identification strategies enable personalized immunotherapy in refractory solid tumors.

Recent genomic and bioinformatic technological advances have made it possible to dissect the immune response to personalized neoantigens encoded by tumor-specific mutations. However, timely and efficient identification of neoantigens is still one of the major obstacles to using personalized neoantigen-based cancer immunotherapy. Two different pipelines of neoantigens identification were established in this study: (1) Clinical grade targeted sequencing was performed in patients with refractory solid tumor, and mutant peptides with high variant allele frequency and predicted high HLA-binding affinity were de novo synthesized. (2) An inventory-shared neoantigen peptide library of common solid tumors was constructed, and patients' hotspot mutations were matched to the neoantigen peptide library. The candidate neoepitopes were identified by recalling memory T-cell responses in vitro. Subsequently, neoantigen-loaded dendritic cell vaccines and neoantigen-reactive T cells were generated for personalized immunotherapy in six patients. Immunogenic neo-epitopes were recognized by autologous T cells in 3 of 4 patients who utilized the de novo synthesis mode and in 6 of 13 patients who performed shared neoantigen peptide library, respectively. A metastatic thymoma patient achieved a complete and durable response beyond 29 months after treatment. Immune-related partial response was observed in another patient with metastatic pancreatic cancer. The remaining four patients achieved the prolonged stabilization of disease with a median PFS of 8.6 months. The current study provided feasible pipelines for neoantigen identification. Implementing these strategies to individually tailor neoantigens could facilitate the neoantigen-based translational immunotherapy research.TRIAL REGSITRATION. ChiCTR.org ChiCTR-OIC-16010092, ChiCTR-OIC-17011275, ChiCTR-OIC-17011913; ClinicalTrials.gov NCT03171220. This work was funded by grants from the National Key Research and Development Program of China (Grant No. 2017YFC1308900), the National Major Projects for "Major New Drugs Innovation and Development" (Grant No.2018ZX09301048-003), the National Natural Science Foundation of China (Grant No. 81672367, 81572329, 81572601), and the Key Research and Development Program of Jiangsu Province (No. BE2017607).

Analysis of exotic squirrel trade and detection of human infections with variegated squirrel bornavirus 1, Germany, 2005 to 2018.

Following the discovery in 2015 of the variegated squirrel bornavirus 1 (VSBV-1) in fatal encephalitis cases among exotic squirrel breeders and a zoo animal caretaker in Germany, a case definition was developed. It was employed during trace-back animal trade investigations and sero-epidemiological studies among breeders and zoo animal caretakers of holdings with VSBV-1 infected squirrels. During the investigation, two possible human cases who had died of encephalitis were identified retrospectively among the squirrel breeders. Moreover, one probable human case was detected among the breeders who had a positive memory T-cell response to VSBV-1 antigen and antibodies against VSBV-1. The low rate of seropositivity found among living persons in risk groups that handle exotic squirrels privately or at zoos may reflect rareness of exposure to VSBV-1 during animal contact, a high lethality of infection or a combination of these factors. As a precaution against human exposure, testing of exotic squirrels for VSBV-1 infection and/or avoiding direct contact with exotic squirrels in zoos and private holdings is strongly advised.

Obesity-induced changes in T-cell metabolism are associated with impaired memory T-cell response to influenza and are not reversed with weight loss

Obesity-induced changes in T-cell metabolism are associated with impaired memory T-cell response to influenza and are not reversed with weight loss

Obesity-Induced Changes in T-Cell Metabolism Are Associated With Impaired Memory T-Cell Response to Influenza and Are Not Reversed With Weight Loss.

Obesity is an independent risk factor for increased influenza mortality and is associated with impaired memory T-cell response, resulting in increased risk of infection. In this study, we investigated if weight loss would restore memory T-cell response to influenza. Male C57BL/6J mice were fed either low-fat or high-fat diet to induce obesity. Once obesity was established, all mice received primary infection with influenza X-31. Following a recovery period, we switched half of the obese group to a low-fat diet to induce weight loss. Fifteen weeks after diet switch, all mice were given a secondary infection with influenza PR8, and memory T-cell function and T-cell metabolism were measured. Following secondary influenza infection, memory T-cell subsets in the lungs of obese mice were decreased compared to lean mice. At the same time, T cells from obese mice were found to have altered cellular metabolism, largely characterized by an increase in oxygen consumption. Neither impaired memory T-cell response nor altered T-cell metabolism was reversed with weight loss. Obesity-associated changes in T-cell metabolism are associated with impaired T-cell response to influenza, and are not reversed with weight loss.

Potent Antitumor T-Cell Memory Is Generated by Curative Viral Oncolytic Immunotherapy But Not Curative Chemotherapy.

Late developing breast cancer metastases are common and lethal despite treatment with adjuvant chemotherapy at the time of primary tumor excision. Stimulation of an antitumor immune response is an alternative strategy for preventing this devastating development. A mouse model of the human epidermal growth factor receptor 2 (HER2/neu)-positive mammary cancer was used to compare the antitumor memory T-cell response following tumor cure by viral oncolytic immunotherapy, chemotherapy, surgical excision, or surgical excision plus virus infection. Memory T-cell response was assessed by functional in vivo assays. Antitumor T-cell memory was generated most powerfully by curative viral oncolytic immunotherapy and poorly by curative chemotherapy. Cure by surgical excision generated an immune antitumor response which was increased by neo-adjuvant virus infection. CD4 memory T-cells were most potent. Virus infection of tumor generates an antitumor memory immune response and chemotherapy suppresses this response. Clinical trials testing adjuvant immune stimulation instead of chemotherapy may be worth exploring because memory antitumor T-cells have the unique potential to find and eliminate small nests of metastatic cancer cells in sanctuary sites and prevent emergence of tumors from dormant cancer cells.

The HLA Ligandome Landscape of Chronic Myeloid Leukemia Delineates Novel T-Cell Epitopes for Immunotherapy

Chronic myeloid leukemia (CML) is characterized by the translocation t(9;22), which leads to the formation of the BCR-ABL fusion transcript. Several approved tyrosine kinase inhibitors (TKIs) target the resulting fusion protein, leading to an improved prognosis of CML patients. Currently, the main treatment goal is the achievement of a deep molecular response (MR), in which TKI therapy can be terminated. Several studies provide evidence that immunological control plays a major role for the course of CML and contributes to the achievement of deep MR in CML patients under TKI treatment (CMLTKI). This implies that reinforcing these immune responses might sustain long-term TKI-free survival or even cure for CML patients. Besides unspecific immunotherapy, such as interferon or immune checkpoint blocking antibodies, a more specific and minor side effect targeting of CML cells might be achieved by antigen-specific immunotherapy approaches. The prerequisite for such strategies is the identification of T-cell targets represented by tumor-associated human leukocyte antigen (HLA)-presented peptides on malignant cells.In this study, we used a mass spectrometry-based approach to identify naturally presented, CML-associated peptides in primary CML samples (HLA class I, n=21, 11,945 peptides, 5,478 source proteins; class II, n=20, 5,991 peptides, 1,302 proteins). Comparative HLA peptidome profiling using a comprehensive dataset of various benign tissues (e.g. blood, bone marrow, spleen, and lung) revealed frequently presented and strictly CML-associated antigens. In detail, the benign tissue dataset comprises hematological benign samples (class I, n=108, 51,233 peptides, 11,437 proteins; class II, n=88, 42,753 peptides, 4,877 proteins) and non-hematological benign tissues (28 tissues, n=166; class I, 128,590 peptides, 16,405 proteins; class II, 143,652 peptides, 13,410 proteins). We identified 50 CML-associated, HLA class I-restricted peptides with HLA allotype adjusted representation frequencies of ≥38% presented on HLA-A*02, -A*03, -A*11, and -B*07. HLA class II comparative profiling delineated 36 peptides exclusively and frequently presented in the HLA peptidome of ≥20% analyzed CML patients.For immunological characterization, we selected 8 HLA class I- and 6 class II-restricted highly CML-associated antigens. These peptides were analyzed in IFNγ ELISPOT assays using PBMCs from CMLTKI patients and healthy volunteers (HVs). Peptide-specific immune recognition was detected for 1/8 (13%) HLA class I peptides in 2/17 (12%) of CMLTKI patients. We hypothesized that this weak immune response might be due to an impaired CD8+ T cell function that reportedly is caused by TKI treatment. Thus, we compared T-cell responses against viral epitopes in IFNγ ELISPOT assays of CMLTKI patients with that of HVs and chronic lymphocytic leukemia (CLL) patients: in line with our hypothesis, we observed significantly reduced IFNγ release of T cells from CMLTKI patients compared to HVs and CLL patients (p<0.001), whereas CD8+ T-cell counts were not reduced. In contrast, no reduced IFNγ production was observed for HLA class II-restricted viral epitopes. These results were confirmed by memory T-cell responses detected for 6/6 (100%) HLA class II CML-associated peptides with frequencies up to 24% (4/17) of analyzed CMLTKI patients.To assess the immunogenicity of all HLA class I peptides, we performed in vitro artificial antigen presenting cell-based priming experiments using CD8+ T cells of HVs and CML patients. Effective priming of T cells was observed for 8/8 CML-associated peptides in ≥70% of analyzed HVs with frequencies of 0.1-33.9% (mean 2.2%) of CD8+ peptide-specific T cells. Notably, peptide-specific CD8+ T cells with frequencies of 0.1-2.2% (mean 0.4%) could also be induced in samples of CMLTKI patients that had not displayed preexisting immune responses. For 6/8 peptides, we observed multifunctionality of peptide-specific T cells by IFNγ and TNF production as well as upregulation of the degranulation marker CD107a. Cytotoxicity assays with polyclonal peptide-specific effector T cells confirmed the capacity to induce antigen-specific lysis for 3/4 analyzed peptides.Taken together, we here identified novel, naturally presented, CML-associated antigens and validated them as promising targets for tailored T cell-based immunotherapeutic approaches for CML patients. DisclosuresSalih:Several patent applications: Patents & Royalties: e.g. EP3064507A1. Kowalewski:Immatics Biotechnologies GmbH: Employment. Schuster:Immatics Biotechnologies GmbH: Employment. Brümmendorf:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy. Niederwieser:Miltenyi: Speakers Bureau; Novartis: Research Funding.

Somatic PRAME Deletions Are Associated with Decreased Immunogenicity, Apoptosis Resistance and Poor Outcomes in Diffuse Large B-Cell Lymphoma

Background: The current standard of care in diffuse large B-cell lymphoma (DLBCL) consists of chemotherapy and therapeutic monoclonal antibodies that have significantly improved patient outcomes over the past 15 years. However, a large proportion of patients suffer from refractory or relapsed disease. Therefore, the development of new therapeutic strategies for this subgroup of patients, who are threatened by a high chance of disease-related death, represents an important unmet clinical need.Methods: We enrolled into our study 347 de novo DLBCL patients uniformly treated with R-CHOP from the BC Cancer population-based cohort between September 2000 and January 2012. RNAseq and high-resolution copy number analysis were performed and correlated with clinical outcome data and tumor microenvironment composition. We also performed functional studies to investigate PRAME-mediated memory T-cell responses and gene expression changes.Results: We discovered novel, highly focal deletions of 22q11.22, including the PRAME gene in 13% (44/338) of the cases. The deletions cluster in a narrow chromosomal region that includes a very small number of genes (VpreB1, ZNF280A/B, PRAME, GGTLC2, miR-650). Of clinical importance, 22q11.22 deletions were found significantly more frequently in germinal centre B-cell-like (GCB) type DLBCL (17% (31/180) vs. activated B-cell-like (ABC) type: 8% (8/98), P < 0.01), and were also significantly associated with worse outcome, which was specifically observed in GCB-DLBCL (5-year disease specific survival, non-PRAME-deleted: 84.5% vs. PRAME-deleted: 67.2%, P = 0.026). Homozygous deletions were more strongly associated with poor outcome than heterozygous deletions. Interestingly, 90% of PRAME-deleted cases were Ig-lambda restricted (P < 0.001).PRAME is a prominent member of the cancer testis antigen (CTA) family of proteins that are expressed in various types of cancers, but not in normal tissues, including normal mature B-cells, apart from male germinal cells. Due to the cancer-specific expression of CTAs, these molecules are considered promising targets for cancer immunotherapy using cytotoxic T-cells and tumor vaccination approaches. To determine the association with tumor microenvironment composition, we analyzed CD4/CD8 flow cytometry data from DLBCL patient samples. The numbers of CD4 and CD8-positive T cells were significantly lower in PRAME-deleted cases compared to wild type (CD4: P < 0.001, CD8: P = 0.013). Notably, RNAseq analysis revealed that the HLA-A*0201 genotype was seen significantly more often in PRAME deleted cases (PRAME wt: 2.5% vs. PRAME deleted: 10.8%, P = 0.005). In order to functionally characterize its interaction with the immune microenvironment, we utilized enzyme-linked immunoSpot (ELISPOT) assays to investigate memory T-cell reactions of patient-derived T cells to PRAME antigens using patient-derived peripheral blood mononuclear cells (PBMC) and measured IFN-g production (7 control healthy donors, 4 PRAME-deleted and 4-wild type patients). While T cells from PRAME-replete patients had no reaction to PRAME antigens, PRAME-deleted patient-derived T-cells had significant reactions to 4 independent PRAME peptides. These data suggest that PRAME-deleted tumor cells can escape from cytotoxic T-cell attack to gain growth advantage.Next, we performed PRAME knock-out (KO) experiments using CRISPR/Cas9 genome editing to clarify the cell autonomous effects of PRAME deletions. Using 2 different cell lines (Karpas422 and SUDHL-4), we found TNFSF10 (TRAIL) expression was significantly down-regulated in homozygous PRAME-KO cell lines compared to wild type. The soluble form of TRAIL (sTRAIL) was also reduced, as measured with enzyme-linked immunosorbent assays. These results suggest that PRAME downregulated cells may contribute to cell survival via TRAIL and sTRAIL reduction.Conclusion: We identified recurrent PRAME deletions and characterized their clinical and functional role in DLBCL. Our findings contribute to the understanding of cell-autonomous and extrinsic roles of PRAME deletions in lymphomagenesis and may lead to the discovery of new therapeutic avenues to simultaneously treat the tumor and the host. DisclosuresGascoyne:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Janssen: Research Funding; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Tioma: Research Funding; Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

638. CMV-Specific T-Cell Immune Responses in Older vs. Younger Kidney Transplant Recipients

BackgroundCompared with younger patients on similar immunosuppression regimens, older solid-organ transplant recipients experience increased rates of infection and death, but decreased rates of rejection. The mechanism behind these differences has yet to be defined, but may be related to ÒinflammagingÓ driven by CMV infection. The objective of this study was to evaluate older vs. younger solid-organ transplant recipients for CMV-specific T-cell immune responses.MethodsPeripheral blood mononuclear cells were isolated from 20 older (3 age 60) and 25 matched younger (ages 30–59) kidney transplant recipients at 3 months after transplantation. Eight recipients were high risk by CMV serology (D+/R−) and 37 were intermediate risk (D−/R+). Overlapping CMV peptide pools were used for stimulation. Intracellular staining to determine cytokine stimulation was performed by multiparameter flow cytometry. Statistical analysis was performed using Jmp Pro 11 software.ResultsThere was no association between patient age and CMV risk status (P = 0.728). There was no difference between older and younger kidney transplant recipients in release of IFNγ, TNFα, or IL-2 from CD4+ or CD8+ T cells in response to CMV antigen stimulation. However, Older recipients had similar frequencies of CD8+ naive cells but decreased frequency of CD8+ terminally differentiated effector memory CD45RA+ (TEMRA) T cells releasing both IFNg and TNFa (P = 0.037) (figure). Interestingly, development of CMV viremia was associated with a weaker CMV-specific immune response: Patients who had a history of CMV viremia had a decreased frequency of CD8+ TEMRA cells releasing both IFNγ and TNFα (P = 0.041).ConclusionOlder kidney transplant recipients demonstrated a decreased frequency of CMV-specific polyfunctional CD8+ TEMRA T cells. This impaired memory T-cell response to CMV suggests a possible mechanism for the increased vulnerability of older recipients to CMV infection or reactivation, which may in turn worsen age-related immune dysfunction. Furthermore, patients with subsequent CMV viremia had a decreased frequency of CMV-specific polyfunctional CD8+ TEMRA T cells. This finding may explain patient vulnerability to CMV viremia despite modern protocols for antiviral prophylaxis. Disclosures All authors: No reported disclosures.