Cell Immunity In Patients Research Articles

Distribution of the T cell element of the immune system in post-COVID patients with first identified hyperglycemia

Post-COVID syndrome combines many adverse outcomes with often new-onset conditions, including type 2 diabetes mellitus (DM). The relevance of the study is the insufficient data on T cell immunity in patients with hyperglycemia after COVID-19. The purpose of this study was to identify the characteristics of T cell immunity in individuals with post-COVID syndrome and newly diagnosed carbohydrate metabolism disorders (CDM) in the post-COVID period. The study included 100 patients (32 men and 68 women) aged from 38 to 70 years (mean age (M±m) was 59.40±8.80 years) with a verified post-COVID-19 condition. All patients were divided into three groups: group 1 – patients with concomitant newly diagnosed impaired glucose tolerance (IGT) in the post-COVID period (prior to SARS-CoV-2 infection, no increase in glycemia was detected according to primary medical documentation) (n = 30), group 2 – patients with concomitant newly diagnosed type 2 diabetes mellitus (n = 30), and group 3 – patients with the development of post-COVID syndrome without hyperglycemia (n = 40). During the study, the following parameters were determined: CD45+, CD3+ (T lymphocytes), CD45+, CD3+, CD4+ (helper inducers), CD45+, CD3+, CD8+ (cytotoxic T lymphocytes), CD45+, CD3+, CD4+, CD25+, CD127- (T regulatory cells), CD45+, CD3+, CD4+, CD25+ (T lymphocytes – early activation), CD45+, CD3+, HLA-DR+ (T lymphocytes – late activation). Infection with SARS-CoV-2 with the subsequent development of post-COVID syndrome and persistent hyperglycemia (IGT, type 2 diabetes) in the post-COVID period was accompanied by a higher level of the total number of T lymphocytes. A study of the T lymphocyte population revealed changes in the subpopulation composition. In the group of patients with CDM, a significant increase in T cytotoxic and T regulatory cells was noted. More pronounced changes in the subpopulation composition were identified in the group of patients with newly diagnosed type 2 diabetes in the post-COVID period: an increase in the relative and absolute numbers of T helper cells and T cells of late activation of lymphocytes. Disorders of carbohydrate metabolism associated with COVID-19 are accompanied by changes in the subpopulation composition of T lymphocytes. In individuals with newly diagnosed type 2 diabetes in the post-COVID period, hyperactivation of the T cell component of the immune system was revealed. Heterogeneous data were obtained in individuals with newly diagnosed impaired glucose tolerance, which requires further study of this group of patients.

Neoadjuvant chemotherapy enhances tumor-specific T cell immunity in patients with HPV-associated oropharyngeal cancer.

Treatment of patients with newly diagnosed HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) with neoadjuvant chemotherapy (NAC) results in a high rate of 5-year recurrence free survival with few patients requiring adjuvant treatment. We hypothesized that NAC enhances primary tumor HPV-specific T cell responses. HPV-specific responses in tumor infiltrating lymphocytes (TILs) before and after NAC were determined using autologous co-culture assays. Greater HPV16-specific TIL responses, sometimes polyclonal, were observed after NAC compared to before in 8 of 10 patients (80%) with PCR-verified HPV16-positive tumors. A significant association was observed between net-negative change in HPV-specific TIL response and disease relapse (p = 0.04, Mann-Whitney test), whereas pathologic complete response at time of surgery did not correlate with recurrence. NAC induces HPV-specific tumor T cell responses in patients with newly diagnosed HPV-associated OPSCC; whereas lack of an increase following NAC may associate with risk of relapse.

Characteristics of T-cell immunity and level of the natural killer cells content in COVID-19 convalescents with carbohydrate metabolism disorders

The pandemic of the new COVID-19 coronavirus infection has created a public health emergency in the Russian Federation in 2020-2022. COVID-19 causes various consequences, often manifested by the endocrine system dysfunction. The rationale for our study is insufficient data on T and NK cell immunity in patients with hyperglycemia after COVID-19. The study was aimed at the features of T cell immunity in individuals with post-COVID syndrome and disorders of carbohydrate metabolism, depending on the NK cells count. Materials and methods: Sixty-four post-COVID patients with carbohydrate metabolism disorders were divided into three groups: with reduced, normal, or elevated NK cell counts. Carbohydrate metabolism disorders included impaired glucose tolerance (n = 36) and type 2 diabetes mellitus (n = 28). The comparison group comprised 60 post-COVID persons with no history of carbohydrate metabolism disorders. The assessment of the lymphocytic link of immunity included the definition of: CD45+CD3+ (T lymphocytes), CD45+CD3+CD4+ (T helpers), CD45+CD3+CD8+ (T cytotoxic), CD45+CD3+CD16+CD56+ (T-NK cells), CD45+CD3-CD16+CD56+ (natural killers), CD45+CD3+CD4+CD25+ (T lymphocytes – early activation), CD45+CD3+HLA-DR+ (T lymphocytes – late activation). Results and Discussion. As the study showed, a decrease in the number of natural killers was accompanied by a higher level of T helpers in the group with carbohydrate metabolism disorders, the latter is probably associated with a compensatory increase in T lymphocytes and dysregulation of the T cell link of the immune system. It can also be concluded that in patients with long-COVID and CMD, with normal NK cell counts, an altered subpopulation composition remains, namely, a significant increase in total T lymphocytes. Many authors associate the obtained data on a significant decrease in T-NK lymphocytes with a decrease in the antiviral activity of the immune system, which can lead to a poor response to new viral agents or contribute to the activation of chronic viral infections. Dysregulation of the T cell link in individuals with long-COVID and disorders of carbohydrate metabolism requires a more detailed study, including an assessment of the cytokine profile in this category of patients.

SAT-376 - Osteopontin serves as a potential regulator of T cell immunity in patients with hepatocellular carcinoma

SAT-376 - Osteopontin serves as a potential regulator of T cell immunity in patients with hepatocellular carcinoma

Circulating eosinophils associated with responsiveness to COVID-19 vaccine and the disease severity in patients with SARS-CoV-2 omicron variant infection

ObjectiveThis study aimed to investigate the longitudinal circulating eosinophil (EOS) data impacted by the COVID-19 vaccine, the predictive role of circulating EOS in the disease severity, and its association with T cell immunity in patients with SARS-CoV-2 Omicron BA.2 variant infection in Shanghai, China.MethodsWe collected a cohort of 1,157 patients infected with SARS-CoV-2 Omicron/BA.2 variant in Shanghai, China. These patients were diagnosed or admitted between Feb 20, 2022, and May 10, 2022, and were classified as asymptomatic (n = 705), mild (n = 286) and severe (n = 166) groups. We compiled and analyzed data of patients’ clinical demographic characteristics, laboratory findings, and clinical outcomes.ResultsCOVID-19 vaccine reduced the incidence of severe cases. Severe patients were shown to have declined peripheral blood EOS. Both the 2 doses and 3 doses of inactivated COVID-19 vaccines promoted the circulating EOS levels. In particular, the 3rd booster shot of inactivated COVID-19 vaccine was shown to have a sustained promoting effect on circulating EOS. Univariate analysis showed that there was a significant difference in age, underlying comorbidities, EOS, lymphocytes, CRP, CD4, and CD8 T cell counts between the mild and the severe patients. Multivariate logistic regression analysis and ROC curve analysis indicate that circulating EOS (AUC = 0.828, p = 0.025), the combination of EOS and CD4 T cell (AUC = 0.920, p = 0.017) can predict the risk of disease severity in patients with SARS-CoV-2 Omicron BA.2 variant infection.ConclusionsCOVID-19 vaccine promotes circulating EOS and reduces the risk of severe illness, and particularly the 3rd booster dose of COVID-19 vaccine sustainedly promotes EOS. Circulating EOS, along with T cell immunity, may have a predictive value for the disease severity in SARS-CoV-2 Omicron infected patients.

Intradermal vaccination of HPV-16 E6 synthetic peptides conjugated to an optimized Toll-like receptor 2 ligand shows safety and potent T cell immunogenicity in patients with HPV-16 positive (pre-)malignant lesions

BackgroundAmplivant is a molecularly optimized Toll-like receptor 2 ligand that can be covalently conjugated to tumor peptide antigens. In preclinical models, amplivant-adjuvanted synthetic long peptides (SLPs) strongly enhanced antigen presentation...

Long-Lasting Virus-Specific T Cell Response with Divergent Features in Self-Resolved and Chronic Hepatitis C Virus Patients 35 Years Postinfection.

Although recognized as a curable disease, the persistence of hepatitis C virus (HCV) in chronically infected patients remains a great burden for public health. T cell immune responses serve a key role in anti-HCV infection; however, the features of T cell immunity in patients after a long-term infection are not well explored. We recruited a special cohort of patients with similar genetic background and natural developing progression of disease who were infected with HCV through blood donation 35 y ago. We found that self-resolved individuals had higher levels of cytokine-secreting T cells than individuals with chronic infections, indicating HCV-specific T cell immunity could be sustained for >35 y. Meanwhile, virus-specific CD8+ T cells in chronic patients were characterized by programmed cell death-1high, TIM-3high expression, which was related to liver injury characterized by aspartate transaminase/alanine aminotransferase levels and morphopathological changes. Unexpectedly, the expression of Lymphocyte-activation gene 3 on CD8+ T cells was lower in chronic patients and negatively correlated with alanine aminotransferase/aspartate transaminase. Our findings provided new insights into HCV-specific T cell responses and may shed light on a way to figure out novel effector targets and explore a way to reverse chronic infections.

Humoral and cellular immunogenicity two months after SARS-CoV-2 messenger RNA vaccines in patients with cancer

SummaryLittle is known on the long-lasting humoral response and the T cell activation induced by SARS-CoV-2 mRNA vaccines in patients with cancer. The study assessed the efficacy of the SARS-CoV-2 mRNA vaccines through measuring the seroconversion rate at pre-specified time points and the effect on the T cell immunity in patients with cancers. The study included 131 adult patients with solid or hematological cancer, who received SARS-CoV-2 mRNA vaccines. 96.2% of them exhibited adequate antibody response to the SARS-CoV-2 mRNA vaccines 2 months after the booster dose. SARS-CoV-2 mRNA vaccines could induce T cell activation; however, this is more likely in patients who have a positive seroconversion (94%) compared with the patients who did not (50%). Further research into the clinical relevance of low antibodies titers and lack of T cell activity is required to set up an effective vaccination strategy within this group of patients.

Ibrutinib protects T cells in patients with CLL from proliferation-induced senescence

BackgroundThe development of Bruton’s tyrosine kinase inhibitors (BTKi) for the treatment of chronic lymphocytic leukaemia (CLL) has provided a highly effective and relatively non-toxic alternative to conventional chemotherapy. Some studies have shown that BTKi can also lead to improvements in T cell immunity in patients despite in vitro analyses suggesting an immunosuppressive effect of BTKi on T cell function.MethodsIn this study, we examined both the in vitro effect and long-term in vivo effect of two clinically available BTKi, ibrutinib and zanubrutinib. Additional in vitro assessments were undertaken for a third BTKi, acalabrutinib. Immune subset phenotyping, cytokine secretion, T cell degranulation and proliferation assays were performed on peripheral blood mononuclear cells isolated from untreated CLL patients, and CLL patients on long-term (> 12 months) BTKi treatment.ResultsSimilar to prior studies we observed that long-term BTKi treatment normalises lymphocyte subset frequency and reduces PD-1 expression on T cells. We also observed that T cells from patients taken prior to BTKi therapy showed an abnormal hyper-proliferation pattern typical of senescent T cells, which was normalised by long-term BTKi treatment. Furthermore, BTKi therapy resulted in reduced expression of the T cell exhaustion markers PD-1, TIM3 and LAG3 in late generations of T cells undergoing proliferation.ConclusionsCollectively, these findings indicate that there are critical differences between the in vitro effects of BTKi on T cell function and the effects derived from long-term BTKi exposure in vivo. Overall long-term exposure to BTKi, and particularly ibrutinib, resulted in improved T cell fitness in part due to suppressing the abnormal hyper-proliferation of CLL T cells and the associated development of T cell senescence.

Age peculiarities of CD4+ T cell immunity in patients with atherosclerosis.

Background and Aims: Age-related changes in the immune system can contribute to “inflammaging” accompanying the development of chronic inflammatory diseases. There are no unambiguous data on the peculiarities of T cell immunity parameters in the elderly. The aim of this study was to analyze the content of CD4+ T cell subsets in patients with atherosclerosis depending on age.

Immunotherapy with adoptive cytomegalovirus-specific T cells transfer: Summarizing latest gene engineering techniques.

Cytomegalovirus (CMV) infection remains a major complication following allogeneic hematopoietic stem cell transplantation (HSCT). T cell response plays a critical role in inducing long‐term immunity against CMV infection/reactivation that impairs during HSCT. Adoptive T cell therapy (ACT) via transferring CMV‐specific T cells from a seropositive donor to the recipient can accelerate virus‐specific immune reconstitution. ACT, as an alternative approach, can restore protective antiviral T cell immunity in patients. Different manufacturing protocols have been introduced to isolate and expand specific T cells for the ACT clinical setting. Nevertheless, HLA restriction, long‐term manufacturing process, risk of alloreactivity, and CMV seropositive donor availability have limited ACT broad applicability. Genetic engineering has developed new strategies to produce TCR‐modified T cells for diagnosis, prevention, and treatment of infectious disease. In this review, we presented current strategies required for ACT in posttransplant CMV infection. We also introduced novel gene‐modified T cell discoveries in the context of ACT for CMV infection. It seems that these innovations are enabling to improvement and development of ACT utilization to combat posttransplant CMV infection.

A Missing Link: Engagements of Dendritic Cells in the Pathogenesis of SARS-CoV-2 Infections.

Dendritic cells (DC) connect the innate and adaptive arms of the immune system and carry out numerous roles that are significant in the context of viral disease. Their functions include the control of inflammatory responses, the promotion of tolerance, cross-presentation, immune cell recruitment and the production of antiviral cytokines. Based primarily on the available literature that characterizes the behaviour of many DC subsets during Severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19), we speculated possible mechanisms through which DC could contribute to COVID-19 immune responses, such as dissemination of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to lymph nodes, mounting dysfunctional inteferon responses and T cell immunity in patients. We highlighted gaps of knowledge in our understanding of DC in COVID-19 pathogenesis and discussed current pre-clinical development of therapies for COVID-19.

A blood transcriptome-based analysis of disease progression, immune regulation, and symptoms in coronavirus-infected patients

COVID-19 patients show heterogeneity in clinical presentation and outcomes that makes pandemic control and strategy difficult; optimizing management requires a systems biology approach of understanding the disease. Here we sought to potentially understand and infer complex disease progression, immune regulation, and symptoms in patients infected with coronaviruses (35 SARS-CoV and 3 SARS-CoV-2 patients and 57 samples) at two different disease progression stages. Further, we compared coronavirus data with healthy individuals (n = 16) and patients with other infections (n = 144; all publicly available data). We applied inferential statistics (the COVID-engine platform) to RNA profiles (from limited number of samples) derived from peripheral blood mononuclear cells (PBMCs). Compared to healthy individuals, a subset of integrated blood-based gene profiles (signatures) distinguished acute-like (mimicking coronavirus-infected patients with prolonged hospitalization) from recovering-like patients. These signatures also hierarchically represented multiple (at the system level) parameters associated with PBMC including dysregulated cytokines, genes, pathways, networks of pathways/concepts, immune status, and cell types. Proof-of-principle observations included PBMC-based increases in cytokine storm-associated IL6, enhanced innate immunity (macrophages and neutrophils), and lower adaptive T and B cell immunity in patients with acute-like disease compared to those with recovery-like disease. Patients in the recovery-like stage showed significantly enhanced TNF, IFN-γ, anti-viral, HLA-DQA1, and HLA-F gene expression and cytolytic activity, and reduced pro-viral gene expression compared to those in the acute-like stage in PBMC. Besides, our analysis revealed overlapping genes associated with potential comorbidities (associated diabetes) and disease-like conditions (associated with thromboembolism, pneumonia, lung disease, and septicemia). Overall, our COVID-engine inferential statistics platform and study involving PBMC-based RNA profiling may help understand complex and variable system-wide responses displayed by coronavirus-infected patients with further validation.

Abstract 5587: Characterization of shared immunogenic frameshift mutations in microsatellite unstable cancers to guide the design of off-the-shelf immunotherapies

Abstract Microsatellite instability-high (MSI-H) cancers are an important model for evaluating neoantigen-based immunotherapies, given their high loads of tumor-specific antigens, evidence of T cell infiltration and exceptional response rates to checkpoint blockade. Importantly, MSI-H tumors are enriched in short nucleotide insertion/deletions in microsatellite regions (“hot spots”) within open reading frames, potentially leading to frameshift mutations shared across patients with MSI-H tumors and Lynch Syndrome, a hereditary cancer predisposition syndrome with MSI-H phenotype. We hypothesized that MSI-H tumors are enriched in recurrent frameshift mutations encoding immunogenic neoantigens and pre-selection of an optimal set of recurrent frameshift mutations, based on the multiplicity of encoded neoepitopes, diversity of interaction with common major histocompatibility complex (MHC) alleles and immunogenicity, will inform the design of off-the-shelf immune therapies for patients with MSI-H phenotype. Utilizing the whole-exome sequencing information available at The Cancer Genome Atlas, we evaluated the neoantigen profiles of patients with colorectal (n=461), stomach (n=443) and endometrial carcinoma (n=560) (COAD, STAD and UCEC, respectively) according to the aforementioned criteria and identified 37, 23 and 9 frameshift mutations encoding potentially immunogenic neoantigens, which were shared across the MSI-H population in COAD, STAD and UCEC, respectively. In an array of immunological assays utilizing peripheral blood mononuclear cells (PBMCs) from healthy donors, we characterized the immunogenicity of the neoantigens derived from the 9 frameshift peptides shared in MSI-H UCEC to assess the extent and specificity of the engendered T cell immunity. We found that each frameshift peptide could elicit antigen-specific CD8+ T cell responses in a subset of subjects tested, which suggests that MSI-H UCEC patients have an increased frequency of high-quality T cell epitopes derived from shared frameshift peptides, binding to a broad spectrum of MHC alleles, capable of inducing CD8+ T responses. Currently, utilizing blood and tissue specimens from patients with MSI-H COAD, STAD and UCEC, we are interrogating (1) the expression levels and frequencies of the selected shared neoantigens, (2) whether these neoantigens are presented in the context of MHC and (3) can elicit endogenous T cell responses. Findings from this research will guide the selection of shared neoantigens eliciting antitumor T cell immunity in patients with MSI-H tumors and will ultimately lay the foundation for common neoantigen-based, off-the-shelf cancer immunotherapy designs, which can be utilized in therapeutic and preventative settings in MSI-H cancers and Lynch Syndrome, respectively. Citation Format: Cansu Cimen Bozkus, Vladimir Roudko, Theofani Orfanelli, Stephanie Blank, Benjamin Greenbaum, Nina Bhardwaj. Characterization of shared immunogenic frameshift mutations in microsatellite unstable cancers to guide the design of off-the-shelf immunotherapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5587.

An RNA vaccine for advanced melanoma.

An RNA vaccine targeting tumour-associated antigens promotes T cell immunity in patients with advanced melanoma.

Next Generation Sequencing-Based Characterization of T Cell Receptor Repertoire of Patients with Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by increased platelet destruction and impaired platelet production. Abnormal T cell immunity plays a critical role in the pathogenesis of ITP. Analysis of T cell receptor (TCR) repertoire can identify specific T cell population proliferations in response to antigenic stimulation. No systematic next-generation sequencing (NGS)-based TCR repertoire characterization of pediatric ITP patients has been reported to date. We herein report our comprehensive analysis of TCR-β repertoires of peripheral blood samples from a cohort of pediatric patients with ITP. Method: Pediatric patients from Lucile Packard Children's Hospital (Stanford Children's Health) with newly diagnosed ITP (n=11), chronic ITP (n=8), resolved from ITP (n=3), and healthy individuals (n=4) were consented to be included in the study. Total RNA was extracted from peripheral blood samples of the study subjects and reverse transcribed into cDNA. Completely rearranged TRB gene locus was amplified and used for library preparation, which were subsequently sequenced on the Illumina MiSeq platform. The sequencing reads were analyzed with IMGT (ImMunoGeneTics)/HighV-QUEST tools. Further analysis was performed to evaluate TRB repertoire diversity, V-J gene segment usage, clonotype sharing among patient samples, clonality pattern, predominant clonotype frequency changes in different disease state, and sequence similarity among expanded clonotypes. Result: The normalized clonotype counts and the calculated TRB repertoire diversity indices (Shannon and Simpson indices) did not reveal significant difference among newly diagnosed ITP, chronic ITP and healthy control groups. In terms of the V-J gene segment usage among the top 200 clonotypes (as defined by CDR3 amino acid sequences), newly diagnosed ITP group used TRBV12-3 significantly more frequently, and TRBV6-4 significantly less frequently, compared to the healthy control group. The means of overlap coefficient, reflective of TRB clonotype sharing, for newly diagnosed ITP, chronic ITP and healthy control groups are 0.0045 (range 0.005-0.008), 0.0057 (range 0-0.016), 0.0041(range 0.004-0.005), respectively. Chronic ITP patients had significantly more overlap in TRB clonotypes than healthy donors. No common or "public" clonotypes shared within each group were identified. The nucleotide sequence and CDR3 amino acid sequence of dominant TRB clonotypes in ITP patients were compared, and the percent similarity ranged from 30.18% to 88.26% for the former, and from 16.9% to 36.04% for the latter. Conclusion: We performed comprehensive analysis of TCR repertoire of peripheral blood samples from pediatric ITP patients by next-generation sequencing. TCR repertoire diversity at different disease states was analyzed. Differences in V-J gene segment usage and clonotype sharing among patients were observed. These findings provide unique insights into the potential pathophysiology of T cell immunity in patients with ITP. Disclosures No relevant conflicts of interest to declare.

T Cell Immunity Toward Viral- and Tumor-Antigens Is Preserved in MDS Patients

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by bone marrow failure and a propensity to progress to acute myeloid leukemia (AML). A core component of the underlying pathogenesis in MDS is deregulation of inflammatory cytokines, such as tumor growth factor-β (TGFβ), which impact the function of immune cells and hence their capacity to mount anti-infective or anti-tumor responses. However, little is known about antigen-specific T cell function in patients with MDS. We hypothesized that virus-specific T cell (VST) function might be preserved in patients with MDS, and that the functional capacity of T cells reactive against tumor-associated antigens aberrantly overexpressed by clonal MDS cells such as Cyclin A1 (CCNA1) and Proteinase (PR3) might also be preserved and exploited for immunotherapeutic purposes. Following informed consent, we collected peripheral blood samples from 10 patients (pts) with MDS and 17 healthy donors. Most pts (9 out of 10) were transfusion dependent and 3 subsequently underwent an allogeneic HSCT. Table 1 summarizes the other clinical characteristics, karyotypic and mutational profile at the time of blood collection. Compared with T cells isolated from healthy donors, MDS patient-derived T cells had a similar CD4 to CD8 ratio (1.5-2.5:1 for healthy donors and 3:1 for MDS pts), but displayed a more exhausted profile at baseline (CD3+TIM3+: 1% in healthy donors and 5% in MDS pts) and produced higher levels of inflammatory cytokines [IFNγ (18±3pg/ml vs 36±16pg/ml, healthy donor vs MDS; p=0.12), and IL-8 (56±32 vs 704±446 pg/ml, p=0.01)]. Next, to assess the capacity of MDS pts to mount ex vivo functional virus-directed responses, we stimulated patient-derived PBMCs (n=5) with overlapping peptide libraries (pepmixes) spanning immunogenic AdV, CMV, EBV, BK and HHV-6 antigens. Similar to healthy donor-derived T cell lines (n=5, 3 specific for 4 viruses and 2 for 5 viruses), all 5 MDS patient-derived lines demonstrated specificity for one or more of the target viruses (1 for 5 viruses, 1 for 4, 2 for 3 and 1 for 1 virus) as observed by IFNγ ELISpot assay with comparable magnitude (range Adv: 43-730 vs 384-941 in healthy donors, CMV: 0-1599 vs 0-3002, EBV: 0-1486 vs 0-541, BK: 0-839 vs 38-275 and HHV6: 0-794 vs 5-407 SFU/2x105 cells, respectively). We next examined the feasibility of expanding autologous MDS-antigen directed T cell products (n=10) to determine whether an adoptive immunotherapeutic approach might be applicable for MDS treatment. Thus, we exposed patient-derived PBMCs to autologous dendritic cells (DC) loaded with pepmixes spanning 6 MDS-associated antigens (CCNA1, survivin, WT1, PRAME, PR3 and NYESO1). After 3 rounds of stimulation, the products obtained were predominantly CD3+ T cells (mean 88±1.3%) that were polyclonal (CD4: 46±5% and CD8: 41±4%) containing predominantly memory T cells (TEM: 36±6% TCM: 37±5% and Tnaïve =13±3%). Six lines (60%) showed specific recognition to at least one of the target antigens: 4 lines specific for PRAME, 1 for CCNA1, 1 for WT1 and 1 for NYESO1 (range 0-40, 0-184, 0-1386 and 0-179 SFU/2x105 cells, respectively by IFNγ ELIspot). T cell lines were capable of specifically secreting multiple effector cytokines in response to targets (TNFα: 12% and IFNγ: 16% in response to PRAME in a representative patient-derived T cell line). Furthermore, this line was capable of killing PRAME+ targets in a 4hr 51Cr release assay [60% specific lysis, E:T 20:1]. In conclusion, functional virus-directed T cell immunity in patients with MDS is preserved, potentially explaining the lower rates of viral reactivation seen in these patients compared with other infections. Moreover, T cells specific for MDS-expressed tumor antigens can also be successfully expanded ex vivo from patients. Taken together this raises the possibility of applying an adoptive immunotherapeutic approach for the treatment of MDS. Disclosures Ramos: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding. Leen:Allovir: Consultancy, Other: Cofounder, Ownership Interest; Marker Therapeutics: Consultancy, Other: Cofounder, Ownership Interest.

Regulatory T Cell Adoptive Immunotherapy Promotes B Cell Immunity after Haploidentical Transplantation

Background: Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Tregs) effectively prevents conventional T cell (Tcons) mediated Graft versus Host Disease (GvHD) while it does not impair graft-versus-leukemia effect in haploidentical hematopoietic cell transplantation (haplo-HCT). Moreover Treg immunotherapy promotes fast donor T cell recovery after transplant. Recent studies showed that mouse bone marrow (BM) Tregs localize in the hematopoietic stem cell (HSC) niche, where they contribute to HSCs maintenance and promote donor engraftment and B cell lymphopoiesis. We are investigating if human Tregs promote B cell reconstitution and immunity in preclinical models and in haplo-HCT patients. Methods: Human sample analysis: B cell reconstitution was analysed monthly by FACS in BM and peripheral blood (PB) samples from 66 patients who underwent either Treg/Tcon haplo-HCT (45 patients), or T-cell depleted haplo-HCT (8 patients) or haplo-HCT with post-transplant cyclophosphamide (PTCy, 13 patients). Diagnosis was acute leukemia in 52 patients, lymphoma in 11 and multiple myeloma in 3. PB total immunoglobulin (Ig), anti-Cytomegalovirus (CMV) IgM and CMV viremia were monitored. Humanized mouse model: donor derived human Tregs and purified CD34+ HSCs were co-infused in sublethally irradiated (2 Gy) immune-deficient NSG mice. Donor engraftment and B cell reconstitution were analysed by FACS and histology. Results: B cell reconstitution was faster after Treg/Tcon haplo-HCT when compared to other haplo-HCT protocols. B cell counts were higher in PB of patients that received Treg/Tcon haplo-HCT (p<.05) and were comparable to those of healthy subjects by 4 months after transplant (117±148 cells/mm, Fig.1A). We could detect early frequencies of CD34+CD38+CD10+CD127+ common lymphoid progenitors, CD45+CD10+CD38+CD19- Pre/Pro-B, CD45+CD10+CD38+CD19+ Pre-B, and Pro-B cells in the BM of these patients, that resulted in an increased production of CD38+CD19+CD5-IgM+ immature B cells, CD38+CD19+CD5+IgM+ transitional B cells and CD19+CD20+ mature B cells. We used a mouse model of xenotransplantation to understand whether donor B cell reconstitution in Treg/Tcon haplo-HCT is boosted by a Treg-mediated effect on donor human HSCs. We found that infusion of human Tregs facilitated donor HSC engraftment. BM and PB human chimerism was increased (p<.05) in mice that received Treg infusion. BM histology revealed the presence of in vivo expanded human CXCR4+ Tregs in the femurs of Treg receiving mice 1 month after transplant. In the same samples we observed a lower number of CD34+ HSCs, suggesting BM Tregs facilitate donor HSC differentiation. Moreover, Treg treatment allowed human HSCs to localize preferentially in the epiphyseal areas of the femurs, where donor cells started to engraft (p<.04). When looking at donor B cell reconstitution, we found HSC-derived mature B cells rapidly abundant and easily detectable starting 30 days after HSC infusion in PB of Treg-treated animals (p<.05). To further assess whether HSC-derived B cells were functional in patients that received Treg/Tcon haplo-HCT, we analysed total Ig production and specific responses to CMV reactivation. Post-transplant hypogammaglobulinemia was rapidly corrected in Treg/Tcon haplo-HCT patients. Total IgM were higher compared to other haplo-HCT protocols and reached normal levels by 3 months after transplant (96±155 mg/dL, Fig.1B). CMV reactivation rate was lower (47% vs 79%) and it occurred later after Treg/Tcon immunotherapy (51±19 vs 40±48 days). New production of anti-CMV specific IgM was documented in 43% of CMV seropositive patients 98±48 days after Treg/Tcon haplo-HCT, while anti-CMV specific IgM were almost undetectable after other haplo-HCT protocols within the first 6 months after transplant (Fig.1C). Such potent B cell responses coupled with T cell reconstitution resulted in a reduction of CMV second reactivations (Fig.1D p<.05) with no patient that died with CMV disease. Conclusions: Adoptive transfer of human Tregs boosts donor HSC engraftment and facilitates the reconstitution of functional HSC-derived donor B cells. Such results suggest that Treg/Tcon haplo-HCT patients could be early vaccinated after transplant. Treg/Tcon immunotherapy promotes control of infections and B cell immunity in patients undergoing haplo-HCT. Disclosures No relevant conflicts of interest to declare.

Preclinical Development of a Lentiviral Vector for Gene Therapy of X-Linked Severe Combined Immunodeficiency

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the interleukin-2 receptor γ chain gene (IL2RG), and it is characterized by profound defects in T, B, and natural killer (NK) cell functions. Transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with early murine leukemia retrovirus (MLV)-derived gammaretroviral vectors showed restoration of T cell immunity in patients, but it resulted in vector-induced insertional oncogenesis. We developed a self-inactivating (SIN) lentiviral vector carrying a codon-optimized human IL2RG cDNA driven by the EF1α short promoter (EFS-IL2RG), and we tested its efficacy and safety in vivo by transplanting transduced Il2rg-deficient Lin− HSPCs in an Il2rg−/−/Rag2−/− mouse model. The study showed restoration of T, B, and NK cell counts in bone marrow and peripheral blood and normalization of thymus and spleen cellularity and architecture. High-definition insertion site analysis defined the EFS-IL2RG genomic integration profile, and it showed no sign of vector-induced clonal selection or skewing in primarily and secondarily transplanted animals. The study enables a phase I/II clinical trial aimed at restoring both T and B cell immunity in SCID-X1 children upon non-myeloablative conditioning.

Hemacytotoxicity and natural killer lytic index: New parameters to evaluate natural killer cell immunity for clinical use in cancer.

Cytotoxicity assays with patient peripheral blood mononuclear cell (PBMC)-derived natural killer (NK) cells are useful in evaluating the innate immunity of patients with cancer. However, the size of the NK cell population in PBMC preparations may have significant effects on the assay outcome. Therefore, the present study examined the effect of NK cell frequency in a cytotoxicity system to investigate NK cell immunity in post-surgical colorectal cancer patients. For this, hemacytotoxicity was assessed using PBMC preparations, and lymphocyte subset populations were analyzed in samples obtained from 47 patients and 45 healthy volunteers. In addition, a new theoretical parameter, the 'NK lytic index', was termed to represent the per-cell cytotoxicity and compensate for the NK cell frequency effect during PBMC preparations. Notably, the patterns of hemacytotoxicity and NK lytic index did not coincide in follow-up studies with consecutive patients following surgical intervention. In addition, it was determined that NK cell NKG2D expression influences NK lytic index, but not hemacytotoxicity. Transforming growth factor (TGF)-β-bound lymphocytes influenced hemacytotoxicity and NK lytic index. These findings indicate that total cell activity (hemacytotoxicity) is not a sum of per-cell activities (NK lytic indexes), suggesting that clinicians should employ NK lytic index in addition to hemacytotoxicity in order to precisely determine how to enhance NK cell immunity in patients with cancer, either focusing on recovering the number of NK cells or boosting NK cell activity in single cell levels, or both.