Cytotoxic T Lymphocytes In Patients Research Articles

Spontaneous high clonal expansion of Wilms’ tumor gene 1-specific cytotoxic T-lymphocytes in patients with Wilms’ tumor gene 1-expressing solid tumor

Wilms’ tumor protein 1 (WT1)-targeted immunotherapy has been used in patients with leukemia and solid tumors. However, the spontaneous WT1-specific immune response before WT1 peptide vaccination in patients with WT1-expressing tumors (PTs) remains unclear. Therefore, we investigated whether WT1-specific cytotoxic CD8+ T-lymphocytes (CTLs) are clonally expanded in the peripheral blood outside of tumor sites. Clonal expansion of WT1126 peptide (a.a.126–134)-specific CTLs (WT1126-CTLs) was compared between seven PTs and five healthy volunteers (HVs), and their T-cell receptors (TCRs) were analyzed at the single-cell level. Overall, 433 and 351 TCR β-chains of WT1126-CTLs were detected from PTs and HVs, respectively, and complementarity-determining region 3 was sequenced for clonality analysis. The frequencies of WT1126-CTLs were higher in human leukocyte antigen (HLA)-A*02:01+ PTs than in HLA-A*02:01+ HVs, although the difference was not statistically significant. WT1126-CTLs of differentiated types, including memory and effector, were higher in PTs than in HVs; whereas, those of the naïve type were higher in HVs than in PTs. WT1126-CTL clonality was significantly higher in PTs than in HVs. Furthermore, the frequency of effector WT1126-CTLs positively correlated with WT1126-CTL clonality in PTs; whereas, the frequency of naïve phenotype WT1126-CTLs tended to be negatively correlated with clonality. In conclusion, these results suggest that the WT1 protein in tumor cells is highly immunogenic, thereby stimulating endogenous naïve-type WT1126-CTLs and enabling them to clonally expand and differentiate into effector-type WT1126-CTLs.

An observational study on the relationship between tyrosine kinase inhibitor treatment-free remission and cytotoxic T-lymphocytes in patients with chronic phase chronic myeloid leukemia.

171 Background: Since the introduction of tyrosine kinase inhibitors (TKIs), the effectiveness of treating chronic phase chronic myeloid leukemia has seen remarkable improvement. A current challenge in TKI therapy is achieving treatment-free remission (TFR) safely. Numerous TKI-stop trials and translational studies have investigated factors influencing TFR, including TKI treatment duration, duration of deep molecular response (DMR), cellular immunity activation, and bcr::abl1 transcript type. However, identifying reliable markers for achieving TFR safely remains uncertain. In this multicenter study, we focused on examining the association between TFR and cytotoxic T-lymphocytes (CTLs). Methods: Between June 2020 and March 2022, a total of 45 patients were enrolled: 38 patients with DMR for at least 1 year on TKIs (on TKI group) and 7 patients with DMR for at least 1 year after discontinuing TKIs (off TKI group). The study included 30 males, with a median age of 59 years (range: 29–87). Median TKI treatment duration was 7.7 years (range: 1.5–18.3), and median TKI cessation duration in the off TKI group was 4.9 years (range: 1.7–7.1). Molecular response and cellular immunity were monitored over 12 months post-consent for patients in the off TKI group and after TKI discontinuation for patients in the on TKI group. Results: During the observation period, 25 patients (65.8%) in the on TKI group maintained DMR, while 13 patients (34.2%) lost DMR. Conversely, all patients in the off TKI group sustained DMR. The median TKI treatment duration in the group maintaining DMR (9.45 years, range: 3.3–18.3, N = 32) significantly exceeded that in the group losing DMR (4.9 years, range: 1.5–11.9, N = 13) (P = 0.0048). Patients taking TKIs for over 7 years with a higher percentage of total memory CTLs than total effector CTLs maintained DMR. Conversely, patients losing DMR despite over 10 years of TKI treatment had a higher percentage of total effector CTLs than total memory CTLs. Among the 32 patients maintaining DMR, 19 exhibited a higher percentage of total effector CTLs than total memory CTLs, with 15 (78.9%) maintaining MR5 throughout the study. Furthermore, of the six patients maintaining DMR with relatively short TKI administration (median 5.5 years, range: 3.3–6.4), three displayed a higher percentage of total memory CTLs than total effector CTLs. Among the remaining three patients, two showed strong CTL activation, with one patient having an effector CTL clone over 30% and the other having a memory CTL clone over 30%. Conclusions: This study indicates that a sufficiently prolonged TKI treatment duration (>7 years) and maintaining deeper DMR (≥ MR4.5, preferably MR5) are conducive to safely achieving TFR. Furthermore, a high percentage of total memory CTLs and intense CTL activation may serve as meaningful markers for TFR.

Potential biomarkers for treatment response in advanced non-pancreatic neuroendocrine tumors.

599 Background: Well-differentiated NETs are highly vascular and hence, inhibition of angiogenesis is of interest. VEGF, a key molecule in promoting angiogenesis, has also been shown to promote an immunosuppressive tumor microenvironment. Nintedanib, an oral inhibitor of FGFR1–3, VEGFR1–3 and PDGFRA, has previously been evaluated in a phase II clinical trial in well-differentiated, non-pancreatic NETs, where 83% of the evaluable patients were progression-free at 16 weeks and median PFS was 11 months. In preclinical studies, nintedanib has been shown to increase PD-L1 expression in tumor cells, which then results in improved outcomes with immune checkpoint inhibition. There are multiple indicators of T-cell exhaustion, including PD-L1 and LAG-3. LAG-3+ T-cells have been found to correlate with poor response to treatment in neuroendocrine neoplasms. Given these findings, this study evaluates serum VEGF and LAG-3 expression to determine potential biomarkers of response in patients treated with nintedanib. Methods: 27 sets of paired samples collected at baseline and 8-weeks post treatment with nintedanib, from clinical trial NCT02399215 were used for this study. With IRB approval, we tested for serum VEGFa and serum LAG-3 using Luminex test kits: HCKP1-11K-01 (LAG-3) and HCYTA-60K-01 (VEGFa). We compared the baseline, 8-week, and percentage change in serum VEGF and serum LAG-3 levels with Rd Cd8+ pAkt+ T-cells (activated cytotoxic T-cells) as a marker of immune activation, Rd CD25+ pAkt+ T-cells (regulatory T-cells) as a marker of immune suppression, and PFS. Results: The median VEGF levels at baseline and 8-weeks were 10.50 pg/mL and 11.90 pg/mL respectively. The median LAG-3 level at baseline and 8-weeks were 59000 pg/mL and 62900 pg/mL respectively. Higher VEGFa levels in the post-treatment samples were associated with poorer progression free survival (HR= 1.009, 95% HR CI: [0.999, 1.019], with P= 0.0760). Higher levels of VEGF at baseline were also associated with lower numbers of Rd CD8+ pAkt + cells (Pearson coefficient = -0.42, p-value 0.04) at baseline. Increased baseline CD25+ pAkt+ levels were positively correlated with percent change in serum LAG-3 levels between baseline and post-treatment samples. (Pearson Co-efficient: 0.45; p=value: 0.02). Conclusions: Elevated serum VEGFa levels at 8-weeks post treatment correlated with lower PFS. Serum VEGFa level is a potential indicator of effector T-cell suppression, as evidenced by low number of activated cytotoxic T-cells in patients with higher VEGFa levels at baseline. Higher baseline regulatory T-cells were associated with a greater increase in serum LAG-3 levels, suggesting that serum LAG-3 may be an indicator of an immunosuppressive tumor microenvironment.

Randomized phase I/II study of vascular endothelial growth factor receptor peptide vaccines for patients with hepatocellular carcinoma.

We evaluated the safety and efficacy of vascular endothelial growth factor receptor (VEGFR)-targeted peptide vaccines for the immunization of patients with unresectable hepatocellular carcinoma (HCC) who had responded to transarterial chemoembolization. Twenty-two patients were randomized 1:1 to receive VEGFR-targeted peptides or placebo. The primary end-point was the safety assessment of the immunization. The secondary end-points were evaluation of immunological responses and clinical outcomes. No severe adverse events were induced by the study agents. Among the 12 patients in the vaccine group, a VEGFR1-specific cytotoxic T lymphocyte (CTL) response was induced in eight (66.7%) patients and a VEGFR2-specific CTL response was induced in 10 (83.3%). The median progression-free survival (PFS) and overall survival (OS) rates were 4.8 and 52.0months, respectively, in the vaccine group, and 2.7 and 21.8months, respectively, in the placebo group. No statistically significant differences were found between the two groups (PFS p=0.925, OS p=0.190). When divided into two groups according to immunoreactivity, the median PFS of patients with and without a strong immune response to VEGFR1 were 7.4 and 2.7months, and that to VEGFR2 were 10.6 and 2.7months, respectively; there were significant differences according to the immune response. Immunotherapy with peptide vaccines targeting VEGFR1 and VEGFR2 was well tolerated with no serious adverse events. It also effectively induced peptide-specific CTLs in patients with unresectable HCC.

Circulating antigen-primed cytotoxic T-cells in patients with renal tumors treated with surgery.

Systemic immunotherapy has changed the paradigm of treatment of advanced renal cell carcinoma, but nephrectomy continues to benefit selected patients. While we continue to identify mechanisms behind drug resistance, the effect of surgery on natural anti-tumor immunity is poorly understood. Specifically, peripheral blood mononuclear cell (PBMC) profile and tumor reactive cytotoxic T lymphocytes changes secondary to tumor resection have not been extensively characterized. Hence, we aimed to evaluate the effect of nephrectomy on PMBC profile and circulating antigen-primed CD8+ T-cells for patients undergoing solid renal mass resection. Patients with localized or metastatic solid renal masses who underwent nephrectomy from 2016 to 2018 were enrolled. Blood samples were collected at 3 timepoints for PBMCs analysis (pre-op, 1 day, and 3 months post-op). Flow cytometry was used to identify CD11ahigh CD8+ T lymphocytes that were then further characterized according to the expression of CX3CR1/GZMB, Ki67, Bim, and PD-1. Changes in circulating CD8+ T-cells from pre-op to 1 day and 3 months post-op were evaluated using Wilcoxon signed rank tests. Antigen-primed CX3CR1+GZMB+ T-cells significantly increased by 3 months after surgery among patients with RCC (0.8 × 109 cells; P = 0.01). In contrast, there was a decrease in absolute numbers of Bim+ T-cells at 3 months (-1.9 × 109 cells; P = 0.02). There were no significant absolute changes in PD-1+ (-1.4 × 109; P = 0.7) and CD11ahigh CD8+ T lymphocytes (1.3 × 109; P = 0.9). Ki67+ T-cells decreased by 3 months (-0.8 × 109; P < 0.001). Nephrectomy is associated with an increase in cytolytic antigen-primed CD8+ T-cells and specific PBMC profile changes. Further studies are warranted to ascertain the role surgery may have in the restoration of anti-tumor immunity.

A Suboptimal Response in CML Is Associated with Expression of the Immune Checkpoint VISTA

Introduction: Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukemia (CML) leading to molecular remission in most patients. The next challenges are decreasing resistance and improving the odds of treatment-free remission. Understanding determinants of disease persistence and suboptimal responses is critical for improving these probabilities. The role of the immune system in the control or eradication of CML has been demonstrated in multiple settings including successful graft-vs-leukemia effect and the requirement of active T-cells and natural killer (NK) cells for sustained remission prior to therapy discontinuation. Herein, we aim to demonstrate a comparison of immune determinants of response in CML in those who achieve a deep molecular response (MR4.5) compared to suboptimal responders. Methods: We prospectively collected longitudinal blood and bone marrow samples from patients receiving treatment for CML and characterized them using cytometry by time of flight (CyTOF). 20 patients that achieved MR4.5 at any time during their treatment were compared to a cohort of 32 patients with suboptimal response to therapy according to the European LeukemiaNet (ELN) response milestones (Table 1). We custom-designed this CyTOF panel (36-plex) to include markers of leukemia/stem progenitor cells, lineage defining, intracellular signaling, immune exhaustion and activation markers. Samples were barcoded, pooled and processed simultaneously to overcome technical variations and batch effects. CD45hi cells (leukocytes) were grouped using PhenoGraph clustering, based on expression of these markers in the earliest samples available, collected at various timepoints during treatment. Results: Analysis of differential expression patterns revealed 25 distinct clusters. Patients with suboptimal response to therapy had a higher proportion of CD45hiCD3+ cells (T-cells) compared to those with an optimal response (median of 59% vs 53%, P=0.04). Notably, this included a higher proportion of exhausted CD8+ cytotoxic T-cells in patients with suboptimal response characterized by high Eomes and low Tbet expression (median of 8.0% vs 1.9%, P=0.04). In contrast, CD56+ NK-cells were more prevalent in the optimal response group (median of 5.5% vs 3.2%, P=0.04), particularly an interferon gamma releasing (CD56hiCD16loTbet+) subpopulation (median of 4.1% vs 2.5%, P=0.04). While CD68+ cells (macrophages) were detected at similar rates in both the optimal and suboptimal response groups (median of 16.5% vs 15.5%, P=0.07), the phagocytic subpopulation (CD16-CD14+CD68+HLA-DRlo) was more prevalent in those who achieved an optimal response (median of 15.1% vs 13.2%, P=0.02). Patients with suboptimal response had a higher proportion of CD68+HLA-DRloVISTAhi cells (median of 5.6% vs 1.8%, P=0.004), referring to macrophages expressing the immune regulatory ligand V-domain immunoglobulin suppressor of T cell activation (VISTA). Like PD-L1 and other B7 family ligands, VISTA attenuates T-cells response to antigens and VISTA overexpression has been associated with resistance to immune checkpoint inhibitors in solid tumors. Furthermore, cells characterized by CD66b+CXCR4+VISTAlo were increased in patients with optimal response (median of 20.6% vs 0.6%, P=0.01), indicating a higher proportion of mature granulocytes with low expression of VISTA in the optimal response group (Figure 1). Conclusion: We found that higher expression of the immune checkpoint VISTA, in addition to markers of T-cells exhaustion is associated with suboptimal response to therapy in CML. These findings highlight the importance of the immune microenvironment in response to targeted therapies such as TKIs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CTLs From Patients With Atherosclerosis Show Elevated Adhesiveness and Distinct Integrin Expression Patterns on 2D Substrates.

Atherosclerosis is the major cause of cardiovascular disease that is characterized by plaque formation in the blood vessel wall. Atherosclerotic plaques represent sites of chronic inflammation with diverse cell content that is shifted toward the prevalence of cytotoxic T-lymphocytes (CTLs) upon plaque progression. The studies of CTL recruitment to atherosclerotic plaques require adequate in vitro models accounting for CTL interactions with chemokine-ligands and extracellular matrix fibers via surface chemokine receptors and integrins. Here we applied such a model by investigating CTL adhesion and migration on six types of coated surfaces. We assessed adhesion and motility metrics, the expression of chemokine receptors, and integrins in CTLs of patients with atherosclerosis and healthy donors. Using fibronectin, platelet-poor plasma from patients with atherosclerosis, and conditioned medium from atherosclerotic plaques we revealed the role of substrate in CTL adhesiveness: fibronectin alone and fibronectin combined with platelet-poor plasma and conditioned medium elevated the CTL adhesiveness – in patients the elevation was significantly higher than in healthy donors (p = 0.02, mixed 2-way ANOVA model). This was in line with our finding that the expression levels of integrin-coding mRNAs were elevated in the presence of fibronectin (p < 0.05) and ITGB1, ITGA1, and ITGA4 were specifically upregulated in patients compared to healthy donors (p < 0.01). Our experimental model did not affect the expression levels of mRNAs CCR4, CCR5, and CX3CR1 coding the chemokine receptors that drive T-lymphocyte migration to plaques. Thus, we demonstrated the substrate-dependence of integrin expression and discriminated CTLs from patients and healthy donors by adhesion parameters and integrin expression levels.

Evaluation of the peripheral blood T and B cell subsets and IRF-7 variants in adult patients with severe influenza virus infection.

Background and AimsInfluenza virus is one of the leading infections causing death among human being. Despite known risks, primary immune deficiency due to Interferon Regulatory Factor‐7 (IRF7) gene defect was reported as a possible cause of the risk factors for complicated influenza. We aimed to investigate the changes in peripheral T and B cell subsets in adult patients with severe seasonal influenza virus infection and the investigation of variants of IRF7 gene.MethodsIn this study, 32 patients, hospitalized due to influenza infection‐related acute respiratory failure were included.ResultsThe median age of the patients was 76 years (26‐96), and 13/32 (40.6%) were in the intensive care unit. Central memory Th, effector memory Th, TEMRA Th, cytotoxic T lymphocytes (CTL), central memory CTL of the patients were found to be increased, naive CTL were decreased. There was a significant increase in the percentage of effector memory Th, and a decrease in the percentage of naive CTL in patients ≥65 years‐old compared to patients <65 years old (P = .039, and P = .017, respectively). IRF7 gene analysis revealed two different nucleotide changes in three patients; c.535 A > G; p.Lys179Glu ( K179E ) and c584A > T; p.His195Leu ( H195L ), located in the fourth exon of the IRF7 gene.DiscussionThe increases in central and effector memory Th, central memory CTL and decrease of naive CTLs may be secondary to the virus infection. K179E (rs1061502) and H195L (rs139709725) variants were not reported to be related with susceptibility to an infection yet. It is conceivable to investigate for novel variants in other genes related to antiviral immunity.

Phenotypic characterization of lymphocytes and the immunopathogenetic significance of cytotoxic T-lymphocytes in patients with ulcerative colitis severe exacerbation

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First-line Therapy With Donor-derived Human Cytomegalovirus (HCMV)-specific T Cells Reduces Persistent HCMV Infection by Promoting Antiviral Immunity After Allogenic Stem Cell Transplantation.

Human cytomegalovirus (HCMV) infection, especially persistent HCMV infection, is an important cause of morbidity and mortality after allogenic stem cell transplantation (allo-SCT). Antiviral agents remain the first-line therapy but are limited by side effects and acquired resistance. We evaluated the safety and efficacy of donor-derived HCMV-specific cytotoxic T cells (CTLs) as a first-line therapy for HCMV infection after allo-SCT and investigated the underlying mechanisms. In humanized HCMV-infected mice, first-line therapy with CTLs effectively combated systemic HCMV infection by promoting the restoration of graft-derived endogenous HCMV-specific immunity in vivo. In a clinical trial, compared with the pair-matched, high-risk control cohort, first-line therapy with CTLs significantly reduced the rate of persistent (2.9% vs 20.0%, P = .018) and late (5.7% vs 20.0%, P = .01) HCMV infection and cumulative incidence of persistent HCMV infection (hazard ratio [HR], 0.13; 95% confidence interval [CI], 0.10-0.82; P = .02), lowered 1-year treatment-related mortality (HR, 0.15. 95% CI, 0.11-0.90. P = .03), and improved 1-year overall survival (HR, 6.35; 95% CI, 1.05-9.00; P = .04). Moreover, first-line therapy with CTLs promoted the quantitative and functional recovery of CTLs in patients, which was associated with HCMV clearance. We provide robust support for the benefits of CTLs combined with antiviral drugs as a first-line therapy for treating HCMV infection and suggest that adoptively infused CTLs may stimulate the recovery of endogenous HCMV-specific immunity. NCT02985775.

Relationship between effect of lamivudine in the treatment of cirrhotic patients with uncompensated hepatitis B with HBV genotypes and cytotoxic T lymphocyte

To explore relationship between effect of Lamivudine in the treatment of cirrhotic patients with uncompensated hepatitis B with hepatitis B virus (HBV)genotypes and HBV specific cytotoxic T lymphocytes (CTL). 80 cases of uncompensated cirrhotic hepatitis B (40 cases with genotype B and 40 with genotype C), HBV DNA positive, HBeAg positive and human leukocyte antigen (HLA)-A2 positive,were treated with Lamivudine 100 mg/d, one year later, its effect and relationship with HBV genotypes and HBV specific CTL were observed. HBV DNA turned negative:40 cases with genotype B turned negative (100%). In the 9th and 10th month of treatment, there was one case with genotype C had YMDD variation respectively and Adefovir dipivoxil was used for treatment, of the rest 38 cases, HBV DNA of 26 cases (68.42%) turned negative,HBV DNA negative rate of patients with genotype is lower than that of patients with genotype B, chi2 = 14.91, P < 0.01. HBeAg turned negative: 18 cases with genotype B (45%) turned negative, more than that of patients with genotype C (7 cases, 18.42%), chi2 = 6.32, P < 0.05. Peripheral blood HBV specific CTL level: before treatment, it was (0.33 +/- 0.03)% of patients with genotype B,higher than that of patients with genotype C [(0.11 +/- 0.02)%], t = 8.12, P < 0.001. 1 year after treatment: it was (0.44 +/- 0.04)% of patients with genotype B, higher than that before treatment, t = 4.01, P < 0.001, it was also higher than that of patients with genotype C 1 year after treatment [(0.23 +/- 0.03)%], t = 5.63, P < 0.01, alanine amino-transferase (ALT) returned to normal: 38 cases with genotype B (95%) returned to normal, more than that of patients with genotype C (28 cases, 73.68%), X2 = 6.79, P < 0.01. Effect of Lamivudinein the treatment of cirrhotic patients with uncompensated hepatitis B is better in patients with genotype B than patients with genotype C, its mechanism may be related to lower level of HBV specific CTL in patients with genotype C than patients with genotype B.

Phase I clinical trial of a peptide vaccine combined with tegafur-uracil plus leucovorin for treatment of advanced or recurrent colorectal cancer

Recently, analysis of tumor antigens using micro-arrays has revealed upregulation of cancer-testis antigens RNF43 and TOMM34 and vascular endothelial growth factor receptors VEGFR1 and VEGFR2 in colorectal cancer. A phaseI clinical trial of peptide vaccine therapy together with oral anticancer drugs was conducted to treat advanced colorectal cancer using synthesized peptides of these tumor antigens in order to confirm the safety, immunogenicity and activity of this treatment. The subjects were patients with a human leukocyte antigen (HLA) type of A2402 who had inoperable colorectal cancer but had failed to respond to or were unable to undergo standard chemotherapy. Four peptides (RNF43, TOMM34, VEGFR1 and VEGFR2) were emulsified with incomplete Freund's adjuvant (Montanide), and the resulting solution was administered subcutaneously once a week. Patients received the oral anticancer drug tegafur-uracil plus leucovorin for four weeks continuously as part of one course followed by one week of rest. The primary endpoint of the trial was observation of adverse events as determined by the NCI-CTCAE criteria, and the secondary endpoints were the size of the tumor and the number of cytotoxic Tlymphocytes (CTLs) in the peripheral blood after treatment. Vaccine therapy was administered 148times to 10patients from July2008 to December2009. The adverse events were grade 1 redness and induration, a grade2 skin ulcer at the vaccination site and grade 1 pyrexia. All patients tolerated treatment. Tumor imaging revealed that after 1 course of treatment 1 patient had partial response (PR), 7had stable disease (SD) and 2had progressive disease. A CTL assay of 10 patients revealed an increase in peptide-specific CTLs in patients with PR and SD, and the clinical responses of those patients were observed. Kaplan‑Meier analysis indicated that patients who had a strong CTL reaction had a tendency to have longer progression‑free survival and overall survival.

Detailed Investigation On Characteristics of Japanese Patients with Chronic Phase CML Who Achieved a Durable CMR After Discontinuation of Imatinib – an Updated Result of the Keio STIM Study.

AbstractAbstract 2788 Background and Purpose:Tyrosine kinase inhibitor (TKI) therapy has become the standard treatment for patients with chronic myelogenous leukemia (CML). It can induce durable hematologic, cytogenetic and molecular response, leading to a marked improvement of progression-free survival (PFS). On the other hand, long-term discontinuation of TKIs has recently been investigated by many groups. Our study was designed to confirm whether TKI could be safely discontinued in Japanese patients who have maintained complete molecular response (CMR) for at least 2 years, and to identify possible factors associated with prolonged drug-free survival (DFS), including immunologic profile. The effect of imatinib discontinuation in terms of quality of life (QOL) was also assessed. Method:Adult patients with CML who have sustained CMR (defined as negative quantitative and qualitative PCR of bcr-abl in the bone marrow) for more than 2 years were enrolled in the study. Treatment with imatinib or one of the other TKIs was initiated if the peripheral blood quantitative PCR (TMA method) value exceeded 100 copies. Lymphocyte subset analysis was performed before discontinuation of the drug, and at 6 months after discontinuation or re-induction of the drug in case of relapse. In 6 patients, WT-1 specific cytotoxic T lymphocyte (CTL) frequency was also assessed before, 3 and 6 months after drug discontinuation. QOL analysis was performed using SF-36 questionnaire before, 2 months and 1 year after discontinuation of imatinib. Patients:41 patients were enrolled in the study, among which 40 patients were analyzed. The median age of the patients was 54 (range 28 – 83) years old. The Sokal risk score was low in 24 (60%), intermediate in 10 (25%) and high in 3 (7.5%) patients. The median time on imatinib treatment was 98 (range 24–126) months and the median duration of CMR was 49.5 months (range 24–106). Results:The median follow-up of the patients at the time of this analysis was 15.5 months (range 2–18). Treatment was restarted in 18 patients (45%), and the estimated DFS rate at 12 months was 55.4% (Fig 1). In 5 patients, imatinib was commenced again, whereas 13 patients were re-treated with dasatinib. All but one patient restored CMR after commencing TKIs.Among various factors including age, previous interferon treatment, duration of imatinib treatment, duration of CMR, time until CMR, sex, cytomegalovirus serology and Sokal risk score, duration of CMR was identified as a significant factor associated with prolonged DFS on univariate analysis (p=0.027), the difference which was also significant upon multivariate analysis (p=0.014).Regarding lymphocyte subsets in the peripheral blood, no significant changes were observed in CD4, 19, 56, ab TCR, gd TCR, CD4/CD25 positive cell population, but, there was a significant increase in the proportion of CD8 positive T cells among those who relapsed and those who did not (2.4% vs −2.4%, p=0.04). There was a trend for increased proportion of WT-1 specific CTL in patients who were restarted on TKI therapy.QOL scores of both physical and mental domains did not differ significantly with the discontinuation of imatinib or re-initiation of treatment, although symptoms such as facial puffiness or muscle cramping were markedly decreased with discontinuation. There was also no difference in the patients' QOL according to the choice of drug used for re-treatment.Altogether 6 patients had fluctuating PCR copy number during follow-up, of which 2 were restarted on treatment. Others have maintained low copy number or have returned to negative during follow-up. Due to the small number of patients, no specific clinical factors or immunophenotypes associated with sustained low count PCR were identified. Conclusion:Sustained CMR was achieved in a substantial proportion of patients who had been in CMR for over 2 years. All patients restarted on TKI treatment remained sensitive to treatment. Longer time in CMR was identified as a significant factor related to sustained CMR in our patient population. Increase in CTL may also correlate with the necessity to restart treatment. Longer observation period and increased number of patients is necessary to draw a concrete conclusion, and to identify the role of immunologic profiles relative to persistence of CMR. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Higher Bone Marrow Lymphocyte Percentage 3 to 6 Month After Therapy with Hypercvad + Dasatinib Than That with Hypercvad + Imatinib or Hypercvad Alone In Patients with Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

AbstractAbstract 4336 Background:Recent reports have suggested that aside from the tyrosine kinase inhibitory effect, dasatinib also promotes a clonal expansion of cytotoxic T-cells in patients with Philadelphia-chromosome positive (Ph+) leukemias. We sought to determine whether there is an absolute lymphocytosis in patients with Ph+ Acute Lymphocytic Leukemia (ALL) treated at our institution on 3 consecutive protocols. Method:We collected data on 122 patients with Ph+ ALL treated with 3 different regimens (hyperCVAD, hyperCVAD + imatinib, and hyperCVAD + dasatinib) from 1994 to 2010, who achieved a complete hematological response (CHR). We examined potential differences between the absolute numbers lymphocytes in peripheral blood (PB) and percentage lymphocytes in bone marrow (BM) at 3 stages: pretreatment, at CHR, and 3–6 months after CHR. Result:Thirty four (28%) patients received hyperCVAD, 43 (35%) hyperCVAD + imatinib, and 45 (37%) hyperCVAD + dasatinib. Twenty five (74%) of patients treated with hyperCVAD, 14 (33%) of patients treated with hyperCVAD + imatinib, and 5 (11%) of patients treated with hyperCVAD + dasatinib have relapsed on these sequential studies. The median PB absolute lymphocyte count for the 3 groups are as follow: Pretreatment: hyperCVAD: 3570.5/uL, hyperCVAD + imatinib: 2262/uL, hyperCVAD + dasatinib: 2376/uL (p = 0.1); at CR: hyperCVAD: 291.5/uL, hyperCVAD + imatinib: 168/uL, hyperCVAD + dasatinib: 239/uL (p = 0.3); 3 to 6 months after CR: hyperCVAD: 485/uL, hyperCVAD + imatinib: 450/uL, hyperCVAD + dasatinib: 632.5/uL (p = 0.26). The median BM lymphocyte percentages are as follow: Pretreatment: hyperCVAD: 3%, hyperCVAD + imatinib: 6%, hyperCVAD + dasatinib: 5% (p = 0.5). CHR: hyperCVAD: 6%, hyperCVAD + imatinib: 4%, hyperCVAD + dasatinib: 4% (p = 0.4). Three to 6 months after CHR: hyperCVAD: 5%, hyperCVAD + imatinib: 3%, hyperCVAD + dasatinib: 7% (p = 0.005). We then divided all patients by their 3 to 6 month BM lymphocyte percentages to 3 groups (< 3%, 3–6%, >6%). There was no difference in remission-free survival for these 3 groups (p=0.76)(Figure 1) Conclusion:Patients who received hyperCVAD + dasatinib had a statistically significant higher BM lymphocyte percentages, 3 to 6 month after achieving CHR than patients treated with the imatinib-containing regimen or chemotherapy alone. Whether this is a clonal population of effector T-cells and whether it plays an important role on the activity of dasatinib compared to imatinib or chemotherapy alone will need to be further investigated. Disclosures:Ravandi:Novartis: Honoraria, Speakers Bureau; Bristol- Myere- Squibb: Honoraria, Research Funding. Kantarjian:BMS, Pfizer and Novartis: Research Funding; Novartis: Consultancy. Cortes:Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Pfizer: Consultancy, Research Funding. O'Brien:Novartis: Research Funding; BMS: Research Funding. Thomas:Novartis: Honoraria; Bristol-Meyer-Squibb: Honoraria; Amgen: Research Funding. [Display omitted]

Interstitial Foxp3‐positive T cells may predict renal survival in patients with myeroperoxidase anti‐neutrophil cytoplasmic antibody‐associated glomerulonephritis

1. Regulatory T cells (T(reg)) and cytotoxic T cells (CTL) are involved in various immune diseases. However, the prognostic impact of T(reg) and CTL in patients with myeroperoxidase anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (MPO-ANCA-GN) is not well known. Therefore, in the present study, we examined the relationship between expression of forkhead box P3 (Foxp3) and T cell intracytoplasmic antigen (TIA)-1, T(reg) and CTL markers and renal survival in patients with MPO-ANCA-GN. 2. Forty patients with MPO-ANCA-GN and 10 patients with minimal change nephrotic syndrome (MCNS) underwent physical examination, determination of blood chemistry and renal biopsy. Immunohistochemical staining for Foxp3 and TIA-1 was performed on paraffin-embedded renal sections. 3. Although almost all patients received standard immunosuppressive treatment for 6 months, seven MPO-ANCA-GN patients needed maintenance haemodialysis (HD), whereas 33 patients did not (non-HD). Both Foxp3- and TIA-1-positive cells were detected in the interstitium and glomeruli of MPO-ANCA-GN patients, whereas they were rarely detected in patients with MCNS. The total crescent rate was significantly higher in the HD group than in the non-HD group (35.9 +/- 3.5 vs 65.8 +/- 7.4, respectively). In the interstitium, the age-adjusted Foxp3/TIA-1 ratio was significantly higher in the non-HD group than in the HD group (0.016 +/- 0.016 vs 0.004 +/- 0.008, respectively; P < 0.05). The Foxp3/TIA-1 ratio, but not the Foxp3/CD3 ratio, remained significantly higher in the non-HD group than in the HD group even after adjustment for crescent rate. Age- and total crescent rate-adjusted renal survival rates were higher in patients with a Foxp3/TIA-1 ratio > or = 0.06 than in patients with a Foxp3/TIA-1 ratio < 0.06 (P = 0.02). 4. The results of the present study suggest that T(reg) could play a protective role against MPO-ANCA-GN and that a decreased Foxp3/TIA-1 ratio in interstitial areas may predict future renal failure in patients with MPO-ANCA-GN.

Tumor Burden Influences Cytotoxic T Cell Development in Metastatic Breast Cancer Patients—A Phase I/II Study

The influence of tumor burden on the generation of tumor antigen-specific cytotoxic T-lymphocytes (CTL) was investigated in a phase I/II clinical adoptive immunotherapy trial. Four previously treated metastatic breast cancer patients, two with macroscopic disease and two with no evidence of disease, in complete remission (CR), were enrolled. Each apheretic peripheral blood mononuclear cell (PBMC) sample was stimulated twice with MUC-1 before infusion back into the patients. CTL responses against MCF-7 cell line and cytokine production were measured before infusion. Patients received two monthly CTL infusions and were monitored for toxicity, tumor response as well as tumor marker levels. The CTL generated from patients with high tumor burdens had less cytokine production and lower cytotoxicity of MCF-7 than the CTL of patients in CR. The differences between the two groups were observed after the two MUC1 in vitro stimulations of the cells obtained in first apheresis. This difference increased after the two MUC1 stimulations of the cells obtained in the second apheresis. The cytotoxicity function was sustained from the first infusion to the second apheresis only for the patients in CR. This suggests that tumor burden had an inverse effect on the function of the generated CTL.

Dynamic CD8 T-cell responses to tumor-associated Epstein-Barr virus (EBV) antigens in patients with EBV-negative Hodgkin's disease

8573 Background: Multiple translational efforts in HD are actively investigating augmentation of the anti-tumor immune response by stimulating cytotoxic T lymphocytes (CTL) against tumor-associated EBV antigens. It has previously been believed that this therapeutic strategy and presence of EBV-specific CTLs are limited to EBV-positive HD. Here, we challenge this belief by characterizing EBV-specific CTL responses in EBV-negative HD. Methods: Among 52 consecutive patients with EBV-negative HD, CTL responses to latent antigens (LMP2, LMP2a) and lytic antigens (BMLF, BRLF) were serially assessed at diagnosis, during chemotherapy, and throughout followup for 2 years by IFN-γ Elispot and flow cytometric tetramer analysis. Results: We detected weak EBV-specific responses to both lytic and latent antigens by IFN-γ Elispot among patients with EBV-negative HD. Response to lytic antigen, BMLF1, was more robust in function and size among healthy donors compared to patients as determined by IFN-γ Elispot and flow cytometry of BMLF1-tetramer positive, CD8 T cells. Chemoradiotherapy was associated temporally with an initial decrease in LMP2A- and BMLF1-specific responses during the first 5–15 weeks of treatment, which subsequently became more robust 20–50 weeks after diagnosis, 2 to 4-fold greater compared to response at diagnosis. At final follow-up (24 months), increases in both lytic (2.6-fold) and latent (2.5-fold) CTL responses were observed with robustness of lytic-specific response equivalent to healthy controls. No significant change in control peptide response was observed. Conclusions: We confirm evidence of EBV-specific CTLs in patients with EBV-negative HD and provide the first report of dynamic variance in this population during treatment, challenging prior belief that patients with HD remain immunodeficient following therapy and arguing that the clinical significance of EBV-specific immune responses in EBV-negative HD should be further investigated. [Table: see text] No significant financial relationships to disclose.

Dynamic CD8 T-Cell Responses to Tumor-Associated Epstein-Barr Virus Antigens in Patients With Epstein-Barr Virus-Negative Hodgkin's Disease

In almost half of patients diagnosed with Hodgkin's disease (HD), the malignant Reed-Sternberg (RS) cells express Epstein-Barr virus (EBV) antigens. Multiple translational efforts are actively investigating antitumor immune strategies by stimulating cytotoxic T lymphocytes (CTL) against tumor-associated EBV antigens. It has previously been believed that this therapeutic strategy and presence of EBV-specific CTLs are limited to EBV-positive HD. In an effort to explore the EBV-specific immune response, here we characterize EBV-specific CTL responses to lytic and latent EBV antigens in 12 consecutive EBV carriers with EBV-negative HD. Compared to healthy donors, we detected weak, baseline EBV-specific responses to both lytic and latent antigens by IFN-gamma ELISPOT in patients with EBV-negative HD at diagnosis. Chemoradiotherapy was associated temporally with a decrease EBV-specific responses. At final follow-up (24 months), recovery of EBV-specific CTL responses was observed with robustness of lytic-specific response equivalent to healthy controls. We confirm evidence of EBV-specific CTLs in patients with EBV-negative HD and provide the first report of dynamic variance in this population during treatment. Our observation challenges prior belief that patients with HD remain immunodeficient following therapy and argues that the clinical significance of the EBV-specific immune response in EBV-negative HD should be further investigated.

The Role of Proteinase Inhibitor 9 (PI-9) in Granzyme B/Perforin-Mediated Cytotoxicity Induced by Cytotoxic T Cells in Allogeneic Hematopoietic Stem Cell Transfusion.

The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)/perforin-induced apoptosis and cytotoxicity, which is one of the important mechanisms in immunotherapy against tumors. However, the role of PI-9, which specifically inhibits GrB, in tumor immune escape is still under debate. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least partly through apoptotic molecules GrB and perforin produced by the donor-derived cytotoxic T cells (CTLs) and natural killer cells. However, the role of PI-9 in GVHD and GVL is unknown. In this study we analyzed the role of PI-9 in GVHD and GVL after HSCT through expression of PI-9 relative to GrB and perforin.First, we analyzed the expressions of PI-9 and three apoptosis-related molecules GrB, perforin and Fas ligand by quantitative RT-PCR in leukocytes of five patients with chronic GVHD and six patients without obvious GVHD, from whom a written informed consent was obtained. The level of GrB expression was significantly increased in three of the patients with GVHD and two of them exhibited elevation both in GrB/perforin and FasL. One of the cases with GVHD showed an isolated high expression of FasL. However, significantly, the expression of PI-9 was prominently decreased in all of the cases with GVHD relative to those without GVHD. None of the patients with GVHD who were positive for any of the apoptosis-related molecules relapsed. Two of the six patients without GVHD showed an elevated expression of GrB and perforin. The one of these two patients suffered from relapsed leukemic infiltration into various tissues and the other from herpes zoster reactivation. Thus, the expression of these molecules was thought to reflect the recipients' protective reactions against the intrinsic diseases. We observed a tendency that the ratio of GrB and PI-9 expressions were higher in patients with GVHD.Next, human CTL and natural killer cell (YT-N10) lines were analyzed to check the time course of PI-9 and GrB expressions in vitro. The CTL line had been established by immortalization of normal T cells with herpesvirus saimiri. The stimulation of these cells with phorbol 12-myristate 13 acetate (PMA) resulted in the prior peak of expression of PI-9 within 3 hours followed by the late expression of GrB peaked after 8 hours. The co-stimulation of these cells with IL-2 and IL-12 showed similar results.These results have indicated that the earlier synthesis of PI-9 might be important for escaping autolysis of immunocompetent cells and that the expressions of PI-9 and the apoptosis-related molecules may be controlled in a different manner. The low expression of PI-9 relative to GrB/perforin in patients who received HSCT may indicate that the balance between the expressions of PI-9 and GrB/perforin could monitor the activity of CTLs in patients with post-transplantation GVHD (and possibly with various other immune disorders) and predict the prognosis of these diseases. It could also be correlated with the GVL activity and hence predict the probability of relapses.

Identification of α‐fetoprotein‐derived peptides recognized by cytotoxic T lymphocytes in HLA‐A24+ patients with hepatocellular carcinoma

Alpha-fetoprotein (AFP) has been proposed as a potential target forT-cell-based immunotherapy for hepatocellular carcinoma (HCC), but the number of its epitopes that have been identified is limited and the status of AFP-specific immunological responses in HCC patients has not been well-characterized. To address the issue, we examined the possibility of inducing AFP-specific cytotoxic T cells (CTLs) using novel HLA-A*2402-restricted T-cell epitopes (HLA, human leukocyte antigen) derived from AFP and then analyzed the relationship between its frequency of occurrence and clinical features associated with patients having HCC. Five AFP-derived peptides containing HLA-A*2402 binding motifs and showing high binding affinity to HLA-A*2402 induced CTLs to produce IFN-gamma and kill an AFP-producing hepatoma cell line. The frequency of AFP-specific CTLs was 30-190 per 1 x 10(6) peripheral blood mononuclear cells, which was the same as that of other immunogenic cancer associated antigen-derived epitopes. Analyses of the relationships between AFP-specific CTL responses and clinical features of patients with HCC revealed that AFP epitopes were more frequently recognized by CTLs in patients with advanced HCC correlating to tumor factors or the stage of TNM classification. The analyses of CTL responses before and after HCC treatments showed that the treatments changed the frequency of AFP-specific CTLs. In conclusion, we identified five HLA-A*2402-restricted T-cell epitopes derived from AFP. The newly identified AFP epitopes could be a valuable component of HCC immunotherapy and for analyzing host immune responses to HCC.