Changes in peripheral lymphocyte subsets in patients after partial microwave ablation of the spleen for secondary splenomegaly and hypersplenism: a preliminary study.

PURPOSE: Microwave ablation therapy for secondary splenomegaly and hypersplenism has been shown to be effective from pre-clinical animal models and clinical investigations. This study was performed to determine its effects on the status of peripheral lymphocyte subsets in patients receiving microwave ablation of the spleen. MATERIALS AND METHODS: Ten patients with secondary splenomegaly and hypersplenism received microwave ablation of the spleen during laparoscopy or percutaneously under ultrasound guidance. The percentage peripheral blood T cells, B lymphocytes and NK cells were measured using flow cytometry before and on days 1, 3 and 7 after therapy, as well as 1 and 3 months afterwards. RESULTS: Percentages of CD3(+) and CD4(+) cells increased rapidly 1 month after therapy. There was no significant change in CD8(+), CD4(+)/CD8(+) or NK cells of the pre- and post-therapy levels and B lymphocytes increased significantly after therapy. In patients with an ablation volume (AV) less than 20% (group A), T cells increased 1 month after ablation but decreased 3 months after ablation. B lymphocytes increased significantly after surgery. Levels of NK cells were lower than that before therapy on each testing. In patients with 20-40% AV (group B), levels of T cells, B lymphocytes and NK cells showed an increase. Levels of CD4(+) cells were significantly higher in group B than in group A, 3 months after therapy. CONCLUSIONS: Microwave ablation therapy for splenomegaly and hypersplenism appears to have a favourable effect on peripheral lymphocyte subsets. A relationship may exist between the ablation volume and the level of peripheral lymphocyte subsets.     

Silent NK/T cell reactions to dasatinib during sustained deep molecular response before cessation are associated with longer treatment‐free remission

This study presents the final report of the multicenter, prospective tyrosine kinase inhibitor discontinuation study, D‐STOP, after a 3‐year follow‐up of 54 patients with chronic CML who discontinued dasatinib after a sustained deep molecular response (DMR) for ≥2 years with dasatinib treatment. Estimated treatment‐free remission (TFR) rates at 12 and 36 months were 63.0% [95% confidence interval (CI): 48.7‐74.3] and 59.3% (95% CI: 45.0‐71.0), respectively. CD3^?CD56⁺ NK, CD16⁺CD56⁺ NK, and CD57⁺CD56⁺ NK large granular lymphocyte (NK‐LGL), CD8⁺CD4^– cytotoxic T cell, and CD57⁺CD3⁺ T‐LGL cell numbers were relatively elevated throughout the 24‐month consolidation only in failed patients who molecularly relapsed within 12 months. In successful patients, these subsets elevated transiently after 12 months, but returned to basal levels after 24‐month consolidation. Therefore, smaller changes in NK/T, particularly the NK subset throughout consolidation, reflected higher TFR rates. TFR rates of those patients exhibiting elevation in CD3^?CD56⁺ NK >376 cells/μL, CD16⁺CD56⁺ NK > 241 cells/μL, or CD57⁺CD56⁺ NK‐LGL >242 cells/μL during consolidation compared with others were 26.7% (8.3%‐49.6%) vs 78.3% (55.4%‐90.3%), HR 0.032 (0.0027‐0.38; P = .0064), 31.2% (11.4%‐53.6%) vs 85.0% (60.4%‐94.9%), HR 0.039 (0.0031‐0.48; P = .011), or 36.8% (16.5%‐57.5%) vs 77.3% (53.7%‐89.8%), HR 0.21 (0.065‐0.69; P = .010), respectively. Therefore, silent responses of T/NK subsets to dasatinib throughout consolidation were significant for longer TFR. Elevated NK/T, particularly NK lymphocytes responsive to dasatinib, may be immunologically insufficient to maintain TFR. Their decline, subsequently replaced by altered lymphocyte population with less response to dasatinib during sustained DMR, might be immunologically significant. (D‐STOP, NCT01627132).     

Adoptive transfer of ex vivo-activated memory T-cell subsets with cyclophosphamide provides effective tumor-specific chemoimmunotherapy of advanced metastatic murine melanoma and carcinoma

Autolymphocyte therapy (ALT) is adoptive cellular therapy of neoplastic disease using ex vivo activation of autologous (human) or syngeneic (murine) lymphocytes from tumor-bearing hosts (TBH) by low doses of anti-CD3 monoclonal antibody (MAb) and a mixture of previously prepared autologous cytokines (T3CS). Ex vivo activation by T3CS without tumor antigen results in expansion of CD44⁺ (memory) T cells. These memory T cells (ALT cells) mediate in vivo anti-tumor specificity and with cyclophosphamide (CY) are capable of curing metastatic disease in murine TBH. To determine whether CY could enhance the effectiveness of CD4⁺ or CD8⁺ subsets of ALT cells, C57BL/6J TBH with B16 melanoma or Lewis lung (3LL) carcinoma were treated with adoptive chemoimmunotherapy (ACIT) using CD4-depleted or CD8-depleted ALT cells and CY. ALT cells were derived from splenocytes of B16 or 3LL-TBH and activated ex vivo with T3CS. Depletion of CD4⁺ or CD8⁺ T cells was performed before or after activation with T3CS. B16-TBH or 3LL-TBH that received ACIT using CY with B16-derived or 3LL-derived CD8-depleted ALT cells, respectively, demonstrated cure of metastatic disease regardless of whether CD8⁺ T cells were depleted before or after T3CS activation. B16 or 3LL-TBH that received ACIT using CY with B16 or 3LL-derived CD4-depleted ALT cells also cured metastatic disease but only if CD4⁺ T cells were depleted after T3CS activation. Interleukin (IL)-2 added to pre-T3CS CD4-depleted ALT cells cultured with T3CS restored anti-tumor activity when combined with CY. TBH cured by ACIT using CY and ALT-cell subsets derived from syngeneic TBH with the identical tumor displayed tumor-specific immunity in rejecting a lethal challenge of identical but not reciprocal tumor. TBH given ACIT using CY and ALT-cell subsets derived from splenocytes of syngeneic TBH with reciprocal tumors rejected lethal challenges of both tumors. Tumor specificity measured by interferon (IFN)-γ and ⁵¹Cr-release assays was demonstrated in pre- or post-T3CS/CD8-depleted, post-T3CS/CD4-depleted and pre-T3CS + IL-2/CD4-depleted ALT-cell subsets. Our data demonstrate that ACIT using CY combined with ex vivo T3CS-activated CD44⁺ memory T-cell subsets conveys long-term tumor-specific immunity. © 1995 Wiley-Liss, Inc.     

Immune reconstitution of B‐cell lymphoma patients receiving CHOP‐based chemotherapy containing rituximab

Sixty‐six B‐cell lymphoma patients were treated with a CHOP‐based chemotherapy containing rituximab (R‐CHOP‐like regimen). Immune reconstitution was assessed by measuring lymphocyte subsets and immunoglobulins. Fifty‐five patients (83.3%) underwent six cycles of the R‐CHOP‐like regimen. CD19⁺ and CD20⁺ cells were completely eliminated from the peripheral blood after one cycle of the R‐CHOP‐like regimen; they were detected again 6 months after the therapy. One year after the therapy, B‐cell numbers recovered to their level at diagnosis and almost doubled 2 years after the therapy. The lowest numbers of CD3⁺, CD8⁺ and CD56⁺ cells were seen after three cycles of the regimen, but continued to increase until 1 year after the therapy, remaining stable thereafter. CD4⁺ T‐cells were at their lowest after six cycles of the regimen and recovered slowly for 2 years after the therapy; however, their numbers were still lower than that at diagnosis. Immunoglobulins decreased over six cycles of the R‐CHOP‐like regimen and then recovered gradually for 2 years after the therapy. The percentage of IgG, IgA and IgM 2 years after the therapy compared with those at diagnosis was 93.9%, 90.1%, 76.3%, respectively. Twelve infectious complications occurred which consisted of three febrile neutropenia, three Herpes Zoster, two tympanitis, two Pneumocystis jiroveci pneumonia and two hepatitis B virus reactivation. B‐cells required 1 year and CD4⁺ T‐cells and immunoglobulins needed 2 years to recover after the therapy. Copyright © 2010 John Wiley & Sons, Ltd.     

Dasatinib cessation after deep molecular response exceeding 2 years and natural killer cell transition during dasatinib consolidation

Tyrosine kinase inhibitors (TKI) improve the prognosis of patients with chronic myelogenous leukemia (CML) by inducing substantial deep molecular responses (DMR); some patients have successfully discontinued TKI therapy after maintaining DMR for ≥1 year. In this cessation study, we investigated the optimal conditions for dasatinib discontinuation in patients who maintained DMR for ≥2 years. This study included 54 patients with CML who were enrolled in a D‐STOP multicenter prospective trial, had achieved DMR, and had discontinued dasatinib after 2‐year consolidation. Peripheral lymphocyte profiles were analyzed by flow cytometry. The estimated 12‐month treatment‐free survival (TFS) was 62.9% (95% confidence interval: 48.5%‐74.2%). During dasatinib consolidation, the percentage of total lymphocytes and numbers of CD3⁻ CD56⁺ natural killer (NK) cells, CD16⁺ CD56⁺ NK cells and CD56⁺ CD57⁺ NK‐large granular lymphocytes (LGL) were significantly higher in patients with molecular relapse after discontinuation but remained unchanged in patients without molecular relapse for >7 months. At the end of consolidation, patients whose total lymphocytes comprised <41% CD3⁻ CD56⁺ NK cells, <35% CD16⁺ CD56⁺ NK cells, or <27% CD56⁺ CD57⁺ NK‐LGL cells had higher TFS relative to other patients (77% vs 18%; P < .0008; 76% vs 10%; P < .0001; 84% vs 46%; P = .0059, respectively). The increase in the number of these NK cells occurred only during dasatinib consolidation. In patients with DMR, dasatinib discontinuation after 2‐year consolidation can lead to high TFS. This outcome depends significantly on a smaller increase in NK cells during dasatinib consolidation.     

NK- and T-cell subsets in malignant mesothelioma patients: Baseline pattern and changes in the context of anti-CTLA-4 therapy

Malignant mesothelioma (MM) is a highly aggressive form of cancer with limited treatment options. Although the role of NK cells has been studied in many solid tumors, the pattern of NK-cell subsets and their recognition of mesothelioma cells remain to be explored. We used RNA expression data of MM biopsies derived from the cancer genome atlas to evaluate the immune cell infiltrates. We characterized the phenotype of circulating NK and T cells of 27 MM patients before and after treatment with an anti-CTLA-4 antibody (tremelimumab). These immune cell profiles were compared to healthy controls. The RNA expression data of the MM biopsies indicated the presence of NK cells in a subgroup of patients. We demonstrated that NK cells recognize MM cell lines and that IL-15 stimulation improved NK cell-mediated lysis in vitro. Using multivariate projection models, we found that MM patients had a perturbed ratio of CD56^bright and CD56^dim NK subsets and increased serum concentrations of the cytokines IL-10, IL-8 and TNF-α. After tremelimumab treatment, the ratio between the CD56^bright and CD56^dim subsets shifted back towards physiological levels. Furthermore, the improved overall survival was correlated with low TIM-3⁺CD8⁺ T-cell frequency, high DNAM-1⁺CD56^dim NK-cell frequency and high expression levels of NKp46 on the CD56^dim NK cells before and after immune checkpoint blockade. Together, our observations suggest that NK cells infiltrate MM and that they can recognize and kill mesothelioma cells. The disease is associated with distinct lymphocytes patterns, some of which correlate with prognosis or are affected by treatment with tremelimumab.     

Differences in kinetics of donor lymphoid cells in response to G-CSF administration may affect the incidence and severity of acute GvHD in respective HLA-identical sibling recipients

Granulocyte-colony stimulating factor (G-CSF), in addition to myeloid and stem cells, mobilizes a large number of lymphoid cells. We examined which lymphoid populations were mobilized in 21 consecutive donors of peripheral blood stem cells (PBSC) and whether the differences in mobilization could affect the incidence of acute and chronic GvHD in respective HLA-identical recipients. G-CSF administration induced significant increases of donor B (CD3⁻CD19⁺) lymphocytes and slight increases of T (CD3⁺) and cytotoxic (CD16⁺CD56⁺) NK cells. The number of extrathymic cells (CD3⁺ cells with NK markers, or CD7⁺) remained unchanged except for an increase of CD3⁺CD57⁺CD8⁺ cells. Donors of patients without subsequent grade II–IV acute GvHD compared to donors of patients who developed significant acute GvHD were found to have in peripheral blood stable numbers of CD3⁺CD4⁺ cells producing IL2, with a concomitant increased number of CD3⁺CD4^low+CD25⁺ T regulatory cells and decreased NK-mediated cytotoxicity, together with a higher number of suppressive extrathymic CD57⁺CD3⁺ cells in the blood and G-PBMC grafts. Increasing numbers of activated T and NK cells in the blood were associated with the development of chronic GvHD. We suggest that differences in steady-state levels and kinetics of G-CSF induced mobilization of donor lymphoid cells may in addition to other well-known factors affect the incidence of GvHD in HLA-identical recipients. However, owing to the small number of donor-recipient pairs studied, our results must be verified in a larger group of patients. Both authors contributed equally to this study.     

Chimeric antigen receptor‐engineered cytokine‐induced killer cells overcome treatment resistance of pre‐B‐cell acute lymphoblastic leukemia and enhance survival

Pre‐emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine‐induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft‐versus‐host‐disease. CIK cells are a heterogeneous effector cell population including T cells (CD3⁺ CD56^?), natural killer (NK) cells (CD3^?CD56⁺) and natural killer T (T‐NK) cells (CD3⁺ CD56⁺) that exhibit non‐major histocompatibility complex (MHC)‐restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)‐γ, anti‐CD3 antibody, interleukin‐2 (IL‐2) and interleukin‐15 (IL‐15). To facilitate selective target‐cell recognition and enhance specific cytotoxicity against B‐cell acute lymphoblastic leukemia (B‐ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28‐CD3ζ domain for signaling and a CD19‐specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19‐targeted CIK/63.28.z cells against otherwise CIK‐resistant cancer cell lines and primary B‐ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre‐B‐ALL. Our results demonstrate potent antileukemic activity of CAR‐engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre‐B‐ALL.     

宫颈癌患者外周血T淋巴细胞亚群和NK细胞数量与临床分期的相关性分析

目的 探讨宫颈癌患者外周血T淋巴细胞亚群和NK细胞数量的变化及其与宫颈癌分期的相关性.方法 收集2010年6月-2011年6月期间在沈阳医学院奉天医院妇科治疗的146例宫颈癌患者及同期在我院进行体检的50例健康女性(对照组),宫颈癌患者根据临床分期分为0期、Ⅰ期、Ⅱ期、Ⅲ期、Ⅳ期5组.采用免疫荧光法和流式细胞仪测定各组患者外周血T淋巴细胞亚群和NK细胞数量.结果 宫颈癌患者CD3+,CD3+ CD4+,NK细胞数量及CD3+ CD4+/CD3+ CD8+比值均低于对照组,且随分期增加而逐渐降低;而CD3+ CD8+细胞数量高于对照组,且随分期增加而逐渐增高.CD3+、CD3+CD4+、NK细胞数量及CD3+ CD4+/CD3+ CD8+细胞比值与宫颈癌临床分期呈负相关(P＜0.01);CD3+ CD8+细胞数量与宫颈癌分期呈正相关(P＜0.01).结论 宫颈癌患者细胞免疫功能低下,且临床分期越晚,免疫功能越低,检测T淋巴细胞亚群、NK细胞可用于宫颈癌患者的免疫监测.     

Transgenic expression of IL-33 activates CD8 T cells and NK cells and inhibits tumor growth and metastasis in mice

IL-33 is a multifunctional cytokine in immune regulation that activates Th1 cells, Th2 cells, CD8⁺ T cells and NK cells. Our study showed that transgenic expression of IL-33 attenuated tumor metastasis in the B16 melanoma and Lewis lung carcinoma (LLC) metastatic models. The percentages and cytotoxicity of CD8⁺ T cells and NK cells and their infiltration into the tumor tissues were significantly increased by the transgenic expression of IL-33 in tumor-bearing mice. Treatment with recombinant IL-33 could also increase the cytotoxicity of CD8⁺ T cells and NK cells in vitro. In addition, depletion of CD8⁺ T cells and NK cells using anti-CD8 or anti-asialo GM1 antibody abolished the pulmonary metastasis inhibition mediated by IL-33. Furthermore, IL-33 stimulated the activation of NF-κB and increased CD69 expression, which is a marker of the activated form of the two cell subsets, in CD8⁺ T cells and NK cells. Our results suggest that IL-33 stimulated NF-κB signaling and promoted the proliferation, activation and infiltration of CD8⁺ T cells and NK cells, which resulted in the inhibition of pulmonary metastasis in B16 melanoma and LLC mice models.     

初诊肺癌患者多次化疗外周血免疫相关指标动态变化的研究

  目的  探索免疫治疗与化疗相结合的有利切入点,为临床免疫治疗介入提供试验依据。  方法  收集2015年11月至2016年12月新乡医学院第一附属医院初诊的23例肺癌患者,全部患者均完成连续5个周期的化疗,采用流式细胞术方法检测化疗过程中患者外周血免疫相关指标如CD4+T细胞、CD8+T细胞、CD19+B细胞和CD16+、CD56+、NK细胞比率,T细胞表面共信号分子PD-1、PD-L1、CD137、CTLA-4、CCR-4、LAG-3以及细胞因子表达情况。分析多周期过程中,上述指标动态变化趋势。  结果  在肺癌患者多个周期的化疗过程中,机体CD8+T淋巴细胞、CD19+B淋巴细胞和CD16+、CD56+、NK细胞水平下调,CD4+T淋巴细胞数量增加,差异具有统计学意义（P＜0.05）;T细胞表面共抑制分子PD-1、CTLA-4和CCR-4随治疗进行表达下调,差异具有统计学意义（P＜0.05）。  结论  肺癌患者在多个周期的化疗中,实施淋巴细胞亚群数量及T细胞表面共抑制分子监测具有重要意义,化疗中后期针对免疫检查点高表达患者,联合应用PD-1抗体、PD-L1抗体、CTLA-4抗体或CCR-4抗体可能具有更优的治疗效果。      

Clinical significance of regulatory T cells and CD8+ effector populations in patients with human endometrial carcinoma

BACKGROUND:

A study was carried out to determine the functional attributes of CD4⁺ CD25⁺ regulatory T cells in cancer progression by suppressing antitumor immunity.

METHODS:

Triple‐color flow cytometry was used to study the phenotype expression of CD4⁺ CD25⁺ regulatory T cells and CD8⁺ T cells in the peripheral blood lymphocytes (PBLs) and tumor‐infiltrating lymphocytes (TILs) of 57 cases of stage I to IV endometrial carcinoma. The expression of T cell subsets was correlated with clinical prognostic parameters.

RESULTS:

The prevalence of CD4⁺ CD25⁺ T cells was significantly higher in the TILs than PBLs. The expression of CD4⁺ CD25⁺ regulatory T cells in cancer milieu correlated with the tumor grade, stage, and myometrium invasion. The expression of FOXP3 and GITR in CD4⁺ CD25⁺ regulatory T cells was lower in PBLs than TILs. Most tumor‐infiltrating CD8⁺ T cells were CD28^? CD45RA^? CD45RO⁺ CCR7^?, suggesting good terminal differentiation. Most of them had an activated role with CD69⁺ CD103⁺ CD152⁺. Functionally, both granzyme B and perforin were scarcely expressed in peripheral regulatory T cells but were highly expressed in peripheral regulatory T cells in the tumor microenvironment. In contrast, CD8⁺ cytotoxic T cells derived from PBLs expressed both granzyme B and perforin, and at significantly higher levels than in TILs. Further functional assays demonstrated that Th1 cytokines and cytotoxic molecules can be synchronously up‐regulated in CD8⁺ cytotoxic T cells.

CONCLUSIONS:

Regulatory T cells in the tumor microenvironment may abrogate CD8⁺ T cell cytotoxicity in a granzyme B‐ and perforin‐dependent conduit. Decreases in both Th1 cytokines and cytotoxic enzymes are relevant for regulatory T cell‐mediated restraint of tumor clearance in vivo. Of clinical significance, the expression of regulatory T cells in TILs may mediate T cell immune repression within cancer milieu and thus greatly correlate with cancer progression. Cancer 2010. © 2010 American Cancer Society.     

Lymphokine-activated killer cell activity of peripheral blood,spleen, regional lymph node,and tumor infiltrating lymphocytes in gastric cancer patients

Lymphokine-activated killer (LAK) cell activity of peripheral blood mono-nuclear cells (PBM), spleen cells (SPC), regional lymph node cells (LNC), and tumor-infiltrating lymphocytes (TIL), induced by activation with in-terleukin 2 (IL 2) for 4 days, was evaluated in patients with gastric carcinoma. TIL exhibited the lowest LAK activity and the cytotoxicity of LNC was significantly lower than that of either PBM or SPC. There was no difference between PBM and SPC. Then, there were significant correlations of LAK activity among PBM, SPC, and LNC, whereas poor correlations were observed in the cytotoxicity between TIL and PBM, SPC, or LNC. Phenotypic analysis of each cell population was performed before and after activation with IL 2. Before culture, the cells mediating natural killer (NK) activity such as CD16⁺, CD56⁺, and CD57⁺ cells were few in LNC and TIL. However, CD56⁺ and CD57⁺ cells in TIL were increased after culture. Then, CD4⁺ Leu8⁺ and CD8⁺ CD11⁺ cells, which identify suppressor cell function, were not elevated in LNC or TIL, as compared to that in PBM or SPC. Further, the proportions of OKIa1⁺ and CD25⁺ cells expressing T-cell activation and IL 2 receptor were uniformly increased in all cell populations after culture. These results indicate the differential reactivity of each lymphocyte population to IL 2 and fundamental dysfunction of LNC and, especially TIL, suggesting the specific influence of the local tumor environment on the lymphocyte function in the area in patients with gastric carcinoma. © Wiley-Liss, Inc.     

CD80 expression in an HLA-A2-positive human non-small cell lung cancer cell line enhances tumor-specific cytotoxicity of HLA-A2-positive T cells derived from a normal donor and a patient with non-small cell lung cancer

To generate non-small cell lung cancer (NSCLC)–reactive lymphocytes, we transfected an HLA-A2-expressing human NSCLC line (1355) with the cDNA encoding the lymphocyte co-stimulatory molecule CD80. Following selection in G418, 1355.7 demonstrated stable cell-surface expression of CD80. Allogeneic mixed lymphocyte tumor cell cultures (MLTCs) were established in 600 IU/ml IL-2 using HLA-A2⁺ normal donor peripheral blood mononuclear cells (PBMCs) stimulated with 1355-P (parental), 1355.7 or IL-2 alone. In 7 of 9 MLTCs, those stimulated with 1355.7 demonstrated enhanced growth after 30 to 45 days of culture. The predominant lymphocyte to grow in all MLTCs was a CD3⁺αβ⁺CD4⁺ T cell. In one case, lymphocytes stimulated with 1355.7 (MLTC 2389.7) exhibited preferential lysis of 1355. MLTC 2389.7 was cloned by limiting dilution, and 2 resultant cloids were shown to be NSCLC-reactive and dependent on both MHC class I and CD3 in their recognition of tumor cells. Additionally, allogeneic MLTCs were established using three HLA-A2⁺ NSCLC patients' PBMC. The predominant lymphocyte to grow in these MLTCs was a CD3⁺ αβ⁺CD8⁺ T cell. In cytotoxicity studies, MLTC-UKY25.7 demonstrated preferential lysis of 1355-P, 1355.7 and an HLA-A2⁺ NSCLC cell line, 1650. Lymphocytes from this MLTC did not lyse K562, Daudi or an HLA-A2^– NSCLC cell line, 647. Our data suggest that CD80-expressing NSCLC tumor cells may enhance the generation of specific CTLs in vitro. These CTLs could be important reagents for use in cellular immunotherapy and/or in isolating tumor antigens for potential tumor vaccine development. Int. J. Cancer 78:685–694, 1998. © 1998 Wiley-Liss, Inc.     

Immune recovery of lymphocyte subsets 6 years after autologous peripheral blood stem cell transplantation (PBSCT) for lymphoproliferative diseases. A comparison between NHL, HD and MM in group of 149 patients

To evaluate the normalization of lymphocyte subsets several years after autologous peripheral blood stem cell transplantation (aPBSCT) and to detect any differences based on the underlying lymphoproliferative diseases, we analyzed the immunological recovery of 149 patients with Non Hodgkin's Lymphoma (NHL), Hodgkin's Disease (HD), Multiple Myeloma (MM). Lymphocyte recovery was assessed before the transplant, on days 15, 30, 60, 90, 120 and on years 1, 2, 4, 6. Analysis of a total of 709 lymphocytes, including total lymphocyte count, CD3 + , CD4 + , CD8 + , CD4 + /CD8 + ratio, CD19 + , CD3 + HLA-DR + , CD16 + 56 + , was performed. The normalization of total lymphocyte counts was achieved between days 14 to 22 following PBSCT. CD3 + cells count showed a normalization after 2 years in the HD and NHL groups and after 4 years in MM group. CD4 + subset achieved normalization during the sixth year in the 3 groups. The CD8 + and CD19 + lymphocytes subsets achieved normal values in the 3 groups at day 60 and at day 120 respectively. CD16 + 56 + and CD3 + /HLA-DR + lymphocytes showed median values above the normal range starting from day 30. Immunological recovery was similar in all 3 groups. Moreover, the recovery of all subsets evaluated was similarly demonstrated within 6 years after aPBSCT.     

Numbers and cytotoxicities of CD3+CD56+ T lymphocytes in peripheral blood of patients with acute myeloid leukemia and acute lymphocytic leukemia

Recent reports have highlighted the role of cellular immunity in anti-tumor defenses. T lymphocytes are known to play important part in anti-cancer immunity. The number and function of T lymphocytes are altered in chronic leukemia patients. CD3⁺CD56⁺ T lymphocytes have also been found to be abnormal in cancer patients. We therefore investigated changes in the number and cytotoxicity of CD3⁺CD56⁺ T lymphocytes in the peripheral blood of acute leukemia (AL) patients (excluding acute promyelocytic leukemia), to improve our understanding of the role of this T lymphocyte subset. We analyzed CD3⁺CD56⁺ T lymphocyte numbers and cytotoxicities in healthy controls, AL patients, and AL patients with complete remission. Lymphocyte counts were performed in peripheral blood and flow cytometry was used to determine cell numbers and cytotoxicities. The absolute number of CD3⁺CD56⁺ T lymphocytes was increased in AL patients (including acute myeloid [AML] and acute lymphocytic leukemia [ALL]) compared with healthy controls (P < 0.05), but their functioning was significantly reduced (P < 0.05). The number of CD3⁺CD56⁺ T lymphocytes in AML and ALL patients who achieved remission following chemotherapy was close to healthy controls (P > 0.05), but their functioning was still significantly reduced (P < 0.05). In addition, the number of CD3⁺CD56⁺ T lymphocytes increased significantly in AML patients with increased peripheral blood white blood cell (WBC) counts, and in ALL patients without increased WBCs. These results suggest that cellular immunity may respond to AML and ALL, but that lymphocyte cytotoxicity remains impaired. Dysfunction of CD3⁺CD56⁺ T lymphocytes in AML and ALL patients may contribute to the failure of the host immune response against leukemic blasts.     

Immune correlates of clinical benefit in a phase I study of hyperthermia with adoptive T cell immunotherapy in patients with solid tumors

Abstract

Purpose: To characterize the T cell receptor (TCR) repertoire, serum cytokine levels, peripheral blood T lymphocyte populations, safety, and clinical efficacy of hyperthermia (HT) combined with autologous adoptive cell therapy (ACT) and either salvage chemotherapy (CT) or anti-PD-1 antibody in patients with previously treated advanced solid tumors.

Materials and methods: Thirty-three (33) patients with ovarian, pancreatic, gastric, colorectal, cervical, or endometrial cancer were recruited into the following therapeutic groups: HT?+?ACT (n?=?10), HT?+?ACT?+?anti-PD-1 inhibitor (pembrolizumab) (n?=?11) and HT?+?ACT?+?CT (n?=?12). Peripheral blood was collected to analyze TCR repertoire, measurements of cytokines levels and lymphocyte sub-populations before and after treatment.

Results: The objective response rate (ORR) was 30% (10/33), including three complete responses (CR) (9.1%) and seven partial responses (PR) (21.2%) and a disease control rate (DCR?=?CR?+?PR?+?SD) of 66.7% (22 of 33). The most common adverse reactions, blistering, subcutaneous fat induration, local heat-related pain, vomiting and sinus tachycardia, were observed in association with HT. IL-2, IL-4, TNF-α, and IFN-γ levels in peripheral blood were significantly increased among the clinical responders (p?<?0.05) while IL-6 and IL-10 were elevated among those with progressive disease (p?<?0.05). Peripheral blood CD8⁺/CD28⁺ T cells increased (p?=?0.002), while the CD4⁺/CD25⁺/CD127⁺Treg cells decreased after therapy (p?=?0.012). TCR diversity was substantially increased among the clinical responders.

Conclusions: Combining HT with ACT plus either CT or anti-PD-1 antibody was safe, generated clinical responses in previously treated advanced cancers, and promoted TCR repertoire diversity and favorable changes in serum IL-2, IL-4, TNF-α, and IFN-γ levels in clinical responders.     

Alterations of T‐cell surface markers in older women with persistent human papillomavirus infection

We previously reported decreased lymphocyte proliferative responses among older women with persistent human papillomavirus (HPV) infection. To characterize the phenotype of peripheral lymphocytes associated with persistent HPV infection, we evaluated the expression of different cell surface markers in peripheral blood mononuclear cells (PBMCs) from a case–control study within a 10,049 woman population‐based cohort study in Guanacaste, Costa Rica. Women in the cohort aged 46–74 and with HPV results at their 5th year anniversary visit were considered, and all women (n = 87) with persistent HPV infections, all women (n = 196) with transient HPV infections and a random sample of HPV DNA‐negative women (n = 261) frequency‐matched to cases on age were selected for this study. A median of 3 years after the case–control matching visit, cervical cells were collected for liquid‐based cytology and repeat HPV DNA genotyping. Blood was obtained from which PBMCs were extracted and cryopreserved for immunological phenotyping via flow cytometry. Significant increases in risk of HPV persistence were observed for 3 marker subsets indicative of immune cell activation/differentiation. Relative risk estimates were 5.4 (95% CI = 2.2–13.3) for CD69⁺CD4⁺, 2.6 (95% CI = 1.2–5.9) for HLADR⁺CD3⁺CD4⁺ and 2.3 (95% CI = 1.1–4.7) for CD45RO⁺CD27^?CD8⁺. A significant decrease in HPV persistence was observed for a subset marker indicative of an immature, undifferentiated memory state CD45RO⁺CD27⁺CD4⁺ (OR = 0.36; 95% CI = 0.17–0.76). Adjustment for these markers only partially explained the previously reported association between decreased lymphoproliferative responses and persistent HPV infection. Whether phenotypic alterations observed predispose to HPV persistence or result from it should be the focus of future studies.     

Analysis of T-cell immune response in renal cell carcinoma: Polarization to type 1-like differentiation pattern,clonal T-cell expansion and tumor-specific cytotoxicity

We assessed the naturally occurring T-cell immune response in primary renal cell carcinoma (RCC) tumors from 12 unselected patients. A predominance of CD3⁺ T-cell receptor (TCR)α/β⁺ T cells was observed in tumor-infiltrating lymphocytes (TILs), in contrast with peripheral blood lymphopenia found in some patients. Activation antigen expression on TILs revealed an imbalance in the activation status, with a significant percentage of CD69⁺ and HLA-DR⁺ and a low percentage of CD25⁺ and CD71⁺ TILs. The lymphocyte activation gene-3 (LAG-3) was detected in some TIL subpopulations and especially in one patient in whom TILs were predominantly TCRα/β⁺CD8⁺DR⁺LAG-3⁺. In addition, we found that RCC TILs are polarized to a global type 1-like (Th1/Tc1) differentiation pattern (strong secretion of interferon-γ and interleukin-2 (IL-2) following CD3/TCR cross-linking) but are under the influence of the down-modulatory cytokines IL-6 (secreted by tumor cells) and IL-10, within the tumor microenvironment. In 3 of 5 patients, clonal T-cell expansion at the tumor site was found for several Vβ specificities, suggesting that in situ stimulation of specific clonotypes in response to potential tumor antigens is a frequent event in RCC. Furthermore, in one patient, selective intratumor amplification of a Vβ1 subpopulation (5% of TCR α/β⁺ cells) corresponding to 2 distinct Vβ1-Jβ1.6 and Vβ1-Jβ2.3 tumor-specific MHC class I-restricted cytotoxic T lymphocytes supports the view that discrete T-cell subsets contribute readily to in situ immunosurveillance. Int. J. Cancer 72:431–440, 1997. © 1997 Wiley-Liss, Inc.