Clinical observations on adoptive immunotherapy with vaccine-primed T-lymphocytes secondarily sensitized to tumor in vitro

The adoptive immunotherapy of human malignancy requires reliable methods to sensitize and expand patients' T-cells reactive to autologous tumors. In animal studies, we have generated therapeutic effector cells against a poorly immunogenic tumor by a two-step procedure: vaccination of the host followed by the secondary stimulation of vaccine-primed lymph node (LN) cells by in vitro sensitization (IVS) with tumor in the presence of interleukin 2 (IL-2). Based on these observations, we performed a clinical trial in patients with advanced cancer to evaluate the antitumor efficacy of vaccine-primed LN cells which were similarly activated in vitro. Patients were vaccinated with irradiated autologous tumor admixed with Bacillus Calmette-Guérin and had draining LN excised 10 days later for IVS culture. During IVS culture, LN cells expanded up to 14-fold (average of 8.4-fold). A mean of 6.7 x 10(9) cells was infused in ten patients (seven melanoma, three renal cell cancer) along with the concomitant i.v. administration of IL-2 (180,000 IU/kg every 8 h for 5 days). Phenotype analysis of IVS-LN cells revealed 78 +/- 4% CD3+ T-cells which were predominantly CD4+ (67 +/- 5%) with expression of HLA-DR and IL-2 receptor. IVS-LN cells displayed relative specificity of autologous tumor lysis in four of ten cases compared to zero of seven IVS-peripheral blood lymphocytes derived from the same patients as measured by the 51Cr release assay. One mo after therapy, seven of nine patients treated with IVS-LN cells and IL-2 developed delayed-type hypersensitivity reactivity to autologous tumor compared to zero of nine patients treated with tumor vaccination and IL-2 only (P < 0.002). These observations suggest that antitumor reactivity was passively transferred with the IVS-LN cells. Major toxic side effects including fever, hepatic dysfunction, and weight gain associated with the capillary leak syndrome were associated with exogenous IL-2 administration. Tumor vaccination and cell transfer were well tolerated without significant complications. Of the ten patients treated with IVS-LN cells and IL-2, there were one partial and one minor response, and one patient has had stable disease for 27+ mo. There was no evidence of tumor response in ten patients treated with tumor vaccination and IL-2 only. Further clinical studies evaluating the antitumor reactivity of vaccine-primed LN cells are warranted.     

Adoptive transfer of anti-CD3-activated CD4+ T cells plus cyclophosphamide and liposome-encapsulated interleukin-2 cure murine MC-38 and 3LL tumors and establish tumor-specific immunity

The infusion of anti-CD3-activated murine T cells plus interleukin-2 (IL-2) exerts antitumor effects against several tumors in murine immunotherapy models. This study compares the therapeutic efficacy of anti-CD3-activated CD4+ or CD8+ T-cell subsets, when given with cyclophosphamide (Cy) and liposome-encapsulated IL-2 (L-IL2) in a murine model. C57BL/6 mice bearing subcutaneous (S.C.) MC-38 colon adenocarcinoma, 3LL Lewis lung carcinoma, or 38C13 lymphoma for 7 to 14 days were pretreated with low-dose intraperitoneal (I.P.) Cy before intravenous (I.V.) injection of anti-CD3-activated T cells or T-cell subsets. Cell administration was followed by I.P. administration of L-IL2 for 5 days. Mice receiving activated CD4+ T cells showed significantly reduced tumor growth or complete remissions with prolonged disease-free survival in MC-38, 3LL, and 38C13. The timing of Cy doses in relation to adoptive transfer was critical in obtaining the optimal antitumor effect by CD4+ cells. Injecting Cy 4 days before the infusion of CD4+ cells greatly enhanced the antitumor effect of the CD4+ cells and improved survival of the mice compared with other Cy regimens. C57BL/6 mice cured of MC-38 after treatment with CD4+ T cells developed tumor-type immunologic memory as demonstrated by their ability to reject rechallenges with MC-38, but not 3LL. Similarly, mice cured of 3LL tumors rejected rechallenges of 3LL, but not MC-38. The immunologic memory could be transferred with an I.V. injection of splenocytes from mice cured of MC-38 or 3LL. No cytotoxic T-lymphocyte activity was detected in T cells or T-cell subsets from mice cured of MC-38 or 3LL. Increased IL-2 and interferon-gamma (IFN-gamma) production was observed from CD4+ subsets in cured animals when stimulated in vitro with the original tumor, but not with an unrelated syngeneic tumor. These results suggest that tumor-specific immunity can be achieved in vivo with anti-CD3-stimulated CD4+ T cells in this cellular therapy model.     

Paxillin phosphorylation and association with Lck and Pyk2 in anti-CD3- or anti-CD45-stimulated T cells

Antibodies to either CD3 or CD45 have been shown to induce dramatic changes in cell morphology, increased tyrosine phosphorylation of cellular proteins, and the association of a subset of these proteins with the tyrosine kinase Lck. The current study was initiated to determine the identity of the tyrosine-phosphorylated 70-80 kDa protein that becomes Lck-associated after stimulation with anti-CD45 or anti-CD3. We demonstrate that the cytoskeletal protein paxillin becomes tyrosine-phosphorylated when cells are plated on immobilized antibodies specific for CD45 or CD3. Only tyrosine-phosphorylated paxillin is associated with Lck, suggesting that the association is through the SH2 domain of Lck. Consistent with this we demonstrate that the SH2 domain of Lck binds tyrosine-phosphorylated paxillin. In contrast, the association of paxillin with the FAK-related kinase Pyk2 was found to be constitutive and not altered by the phosphorylation of either protein. Finally, we establish that the phosphorylation of paxillin is dependent on the expression of Lck. Taken together, these results demonstrate that paxillin is physically associated with kinases from two different families in T cells and suggest that paxillin may function as an adaptor protein linking cellular signals with cytoskeletal changes during T cell activation.     

Phase I trial of anti-CD3-stimulated CD4+ T cells, infusional interleukin-2, and cyclophosphamide in patients with advanced cancer

PURPOSE: We performed a phase I trial to determine whether in vivo expansion of activated CD4+ T cells was possible in cancer patients. 111Indium labeling was used to observe trafficking patterns of the infused stimulated CD4+ T cells. The influence of cyclophosphamide (CTX) dosing on immunologic outcome was also examined. PATIENTS AND METHODS: Patients with advanced solid tumors or non-Hodgkin's lymphoma received CTX at 300 or 1,000 mg/m2 intravenously (i.v.). Leukapheresis was performed to harvest peripheral-blood mononuclear cells (PBMCs) either just before the CTX dose, or when the patient was either entering or recovering from the leukocyte nadir induced by CTX. An enriched population of CD4+ T cells was obtained by negative selection. The CD4+ T cells were activated ex vivo with anti-CD3, cultured with interleukin-2 (IL-2) for 4 days, and adoptively transferred. After adoptive transfer, patients received IL-2 (9.0 x 10(6) IU/m2/d) by continuous infusion for 7 days. RESULTS: The absolute number of CD4+, CD4+/DR+, and CD4+/CD45RO+ T cells increased in a statistically significant fashion in all cohorts after the first course of therapy. The degree of CD4 expansion was much greater than CD8 expansion, which resulted in a CD4:CD8 ratio that increased in 26 of 31 patients. The greatest in vivo CD4 expansion occurred when cells were harvested as patients entered the CTX-induced nadir. One complete response (CR), two partial responses (PRs), and eight minor responses were observed. Trafficking of 111Indium-labeled CD4 cells to subcutaneous melanoma deposits was also documented. CONCLUSION: CD4+ T cells can be expanded in vivo in cancer patients, which results in increased CD4:CD8 ratios. The timing of pheresis in relation to CTX administration influences the degree of CD4 expansion. Tumor responses with this regimen were observed in a variety of tumors, including melanoma and non-Hodgkin's lymphoma; a high percentage of patients had at least some tumor regression from the regimen that produced the greatest CD4+ T-cell expansion.     

Heterogeneity of dendritic cells in human superficial lymph node: in vitro maturation of immature dendritic cells into mature or activated interdigitating reticulum cells

A target identification paradigm was used to study cross-modal spatial cuing effects on auditory and visual target identification. Each trial consisted of an auditory or visual spatial cue followed by an auditory or visual target. The cue and target could be either of the same modality (within-modality conditions) or of different modalities (between-modalities conditions). In 3 experiments, a larger cue validity effect was apparent on within-modality trials than on between-modalities trials. In addition, the likelihood of identifying a significant cross-modal cuing effect was observed to depend on the predictability of the cue-target relation. These effects are interpreted as evidence (a) of separate auditory and visual spatial attention mechanisms and (b) that target identification may be influenced by spatial cues of another modality but that this effect is primarily dependent on the engagement of endogenous attentional mechanisms.     

Signaling via CD28 costimulates lymphokine production, but does not reverse unresponsiveness to interleukin-2 in anti-CD3 triggered Th1 cells

Previously, it has been described that the ability of murine Th1 cells to proliferate in response to exogenous interleukin (IL)-2 is blocked when these cells are exposed to immobilized anti-CD3 antibodies. In the present study we examined whether simultaneous triggering of the T cell antigen CD28 can prevent the induction of unresponsiveness to IL-2 in Th1 cells. We report that costimulation of Th1 cells with anti-CD28 monoclonal antibodies (mAb) did not overcome unresponsiveness to IL-2 induced by various amounts of immobilized anti-CD3 antibodies. However, stimulation with anti-CD28 mAb strongly augmented IL-2 and interferon-gamma production in anti-CD3-exposed Th1 cells. Thus, despite the fact that anti-CD28 mAb is a potent costimulus for lymphokine production, signaling through CD28 does not seem to be sufficient to trigger proliferation in Th1 cells activated via the T cell receptor. These data suggest the existence of at least three signals to trigger Th1 cell activation. The first is mediated by ligation of the T cell receptor. One cosignal, delivered by the CD28 molecule, leads to IL-2 production. A third, still undefined, signal is required for proliferation in response to IL-2.     

Lymph node eosinophilic granuloma. Apropos of 2 cases of Langerhans-cell histiocytosis with isolated lymph node involvement

The authors report two cases of isolated lymph node involvement by Langerhans' cell histiocytosis which affected two young children. The histologic aspect reveals that lymph nodes have been modified by a proliferation of large histiocyte-like cells, associated with eosinophils. An immunohistochemical study on paraffin sections and for one case on frozen sections, reveals the usual phenotype of Langerhans' cells: these cells stain positively with S 100 protein and CD1 and are negative for both lysozyme and al antichymotrypsine. After a period of two years for one child and four years for the other, these children are in total remission, one spontaneously, the other after chemotherapy.     

Adhesion molecule expression by epidermal Langerhans cells and lymph node dendritic cells: a comparison

BACKGROUND: Nasal gliomas are uncommon neurogenic malformations, which derive from the prenasal space. They may appear as intranasal masses, frontonasal masses, or deformities of the nose, brow, or lower central forehead. Almost all of these tumors were diagnosed shortly after birth. The clinical findings of meningoencephaloceles, nasal fistulas, dermoides and epidermoid cysts are presented additionally for differential diagnosis. PATIENTS: Following some interesting case reports, the management of these types of benign tumors is discussed. RESULTS: Complete radiologic evaluation with computed tomography and magnetic resonance imaging should be performed, because a possible intracranial connection must be considered. The preferred surgical treatment of glioma is an endoscopically controlled procedure. In most cases craniotomy is not required. Open rhinoplasty can be helpful for removal of ectodermic malformations. CONCLUSIONS: The surgeon should be familiar with the diagnosis and management of the rare congenital tumors of the nose to ensure proper therapy and to provide the requisite information for patients, parents, and colleagues.     

Monoclonal anti-CD44 antibody acts in synergy with anti-CD2 but inhibits anti-CD3 or T cell receptor-mediated signaling in murine T cell hybridomas

We investigated the effects of anti-murine CD44 monoclonal antibodies on the activation of antigen-specific T cell hybridomas. Anti-murine CD44 antibodies by themselves did not induce the production of IL-2 by antigen-specific T cell hybridomas. However, anti-murine CD44 monoclonal antibodies were able to either inhibit or enhance the production of IL-2, depending on the other monoclonal antibodies used as comitogenic stimuli. When a T cell hybridoma was activated by antigen and antigen-presenting cells or anti-CD3 antibodies, addition of anti-CD44 antibodies inhibited IL-2 production. In contrast, monoclonal anti-CD44 antibodies acted in synergy with anti-human CD2 antibodies in stimulating a murine T cell hybridoma stably transfected with the human CD2 gene to produce IL-2. Therefore, cross-linking of surface CD44 is able to deliver either a positive or a negative signal in murine antigen-specific T cell hybridomas. One of the ligands for CD44 is hyaluronic acid. Hyaluronic acid in vitro significantly increased the activation of murine T cell hybridomas. Hyaluronic acid itself was mitogenic for T cell hybridomas. Therefore, in addition to being an adhesion molecule, CD44 functions as a signal-transducing molecule on murine T cell hybridomas.     

p-Stat3、VEGF-C和MMP-2在非小细胞肺癌中的表达及其与淋巴结转移的关系

目的:检测非小细胞肺癌组织中磷酸化信号转导和转录激活子-3(p-Stat3)、血管内皮生长因子C(VEGF-C)和基质金属蛋白酶2(MMP-2)的表达情况,探讨p-Stat3与VEGF-C、MMP-2之间,以及这三者与临床病理指标之间的相关性.方法:采用免疫组织化学SP法,检测53例非小细胞肺癌组织和10例正常肺组织中p-Star3、VEGF-C和MMP-2的表达情况,并对p-Star3、VEGF-C和MMP-2的表达情况进行相关性分析.结果:在53例非小细胞肺癌病例中,P-Stat3、VEGF-C和MMP-2的阳性表达率分别为45.2%(24/53)、77.3%(41/53)和58.4%(31/53).其中,p-Stat3的表达在腺癌中最显著,阳性表达率为75%(9/12),且在低分化肺癌组织中的表达明显高于高分化肺癌组织(P<0.01),在有淋巴结转移的肺癌组织中高于无淋巴结转移的肺癌组织(P<0.05),而阳性表达率与患者的年龄、性别和肿瘤的大小无明显相关(P>0.05).VEGF-C和MMP-2在有淋巴结转移的非小细胞肺癌组织中的阳性表达率高于无淋巴结转移的肺癌组织(P<0.05),且VEGF-C、MMP-2的表达与p-Stat3的表达呈正相关(依次为r=0.427和0.345,P均<0.05).结论:在非小细胞肺癌组织中,p-Stat3、VEGF-C和MMP-2表达均上调,高表达的p-Stat3与肺癌的发生、发展及淋巴结转移密切相关,其机制可能与VEGF-C和MMP-2有关.     

Antigen-specific IgA response of NALT and cervical lymph node cells in antigen-primed rats

Three models to estimate energy requirement as a function of growth curve pattern were applied to controlled experimental data of male vs female of broilers and turkeys. The share of maintenance out of total feed requirement was 55% for the average of the three models with major divergence due to age. Comparison of the ratio between actual and estimated feed consumption reveals that the relative energy requirement was always lower in females than in males in the range of 5 to 10% for the three models, with an average of 7.7%. It appears, therefore, that in estimating the energy requirement for use in practical feeding, specific models should be assigned for males and females in both broilers and turkeys.     

Eosinophils and serum eosinophilic cationic proteins in interleukin-2-based immunotherapy for cancer

BACKGROUND: Extramedullary hematopoiesis (EMH) occurs in many disorders, including thalassemias and other hemoglobinopathies, and commonly presents in the spleen and liver. We present a case of spinal cord compression in a patient with beta-thalassemia intermedia, and review the literature and available treatment options. PATIENT AND METHODS: A 35-year-old black female with beta-thalassemia intermedia presented with a 3-week history of back pain and lower extremity weakness. Neurologic examination was consistent with spinal cord compression, and gadolinium enhanced magnetic resonance imaging (MRI) confirmed this diagnosis. She was given intravenous steroids and radiotherapy was begun in 200 cGy fractions to a total dose of 2000 cGy. RESULTS: At the completion of radiotherapy the patient was ambulatory with mild residual weakness. MRI scans 16 months later showed smaller, but persistent masses, and she remains asymptomatic 5 years from her diagnosis. CONCLUSION: Recognition of spinal cord EMH requires prompt physical examination and MRI for accurate diagnosis. EMH can be managed with radiation, surgery, transfusions, or a combination of these therapies. Radiation in conservative doses of (750-3500 cGy) is non-invasive, avoids the surgical risks of potentially severe hemorrhage and incomplete resection, and has a high complete remission rate in the majority of patients. Relapse rates are moderate (37.5%), but retreatment provides excellent chance for second remission.     

Autologous peripheral blood stem cell transplantation and adoptive immunotherapy with activated natural killer cells in the immediate posttransplant period

Relapse after high-dose chemotherapy supported by peripheral blood stem cell transplantation (HDC-PBSCT) is the main cause of therapeutic failure in patients with lymphoma and breast cancer. Adoptive immunotherapy with activated natural killer (A-NK) cells and interleukin 2 might eliminate surviving residual tumor without adding to toxicity. Eleven patients with relapsed lymphoma and one with metastatic breast cancer were entered on a pilot clinical trial of HDC-PBSCT followed on day 2 after transplant by infusion of cultured autologous A-NK cells. Simultaneously, recombinant human interleukin 2 (rhIL-2) was initiated as a 4-day continuous i.v. infusion at 2 x 10(6) IU/m2/day, referred to as high-dose rhIL-2. Therapy with high-dose rhIL-2 was followed by a 90-day continuous i. v. infusion at 3 x 10(5) IU/m2/day, referred to as low-dose rhIL-2. All patients engrafted and nine completed treatment. Posttransplant days to a neutrophil count of 500/microliter and to a platelet count of 50,000/microliter were similar to comparable patients treated with HDC-PBSCT alone. Generation of A-NK cells for therapy was feasible in all patients except the three patients with Hodgkin's disease, whose cells did not proliferate in culture. Overall toxicity associated with early posttransplant transfer of A-NK cells and interleukin 2 did not differ from that observed with peripheral blood stem cell transplantation alone in comparable patients. There was early amplification of natural killer cell activity in the peripheral blood of four patients that appeared to result from the transfused A-NK cells. Adoptive transfer of A-NK cells and rhIL-2 during the pancytopenic phase after HDC-PBSCT was feasible and well tolerated, did not adversely affect engraftment, and resulted in amplified natural killer activity in the peripheral blood during the immediate posttransplantation period.     

Histamine increases anti-CD3 induced IL-5 production of TH2-type T cells via histamine H2-receptors

Besides its proinflammatory functions histamine released from basophils and mast cells during immediate-type hypersensitivity reactions is known to inhibit several lymphocyte functions like IL-2 and gamma-IFN production. Recently, it has been shown that T helper cells of type 2 phenotype (TH2) represent the T cell fraction which may play a pivotal role in the promotion of the allergic inflammatory eosinophilic late-phase reaction by secretion of cytokines, especially IL-4 and IL-5. We have investigated the effect of histamine on anti-CD3 induced IL-4 and IL-5 production by TH2 cells. Histamine in concentrations between 10(-7) and 10(-5) mol/l concentration-dependently increased anti-CD3 induced IL-5 production up to 120%, whereas IL-4 production was not affected. The activity of histamine in increasing IL-5 production was mimicked by the H2-receptor agonist dimaprit. Histamine induced increase in IL-5 production was inhibited by histamine H2-receptor antagonists, but remained unaffected by H1- or H3-receptor antagonists. Administration of forskolin which directly stimulates the production of cAMP, the second messenger of the H2-receptor, also resulted in an increase in anti-CD3 induced IL-5 production. These results indicate that the histamine-mediated increase in anti-CD3 induced IL-5 production is mediated via H2-receptors. Consequently, histamine released from mast cells and basophils during the early-phase allergic reaction may act as an important stimulatory signal for the initiation of the allergic inflammatory late-phase reaction by increasing local IL-5 production of allergen triggered TH2 cells.     

S-phase fraction and proportion of cells with high DNA content as prognostic factors of lymph node negative breast cancer

Breast cancer is the most frequent female cancer in Western Europe. Roughly half of the patients have no axillary lymph node involvement at time of operation. In this group of patients adjuvant treatment should only be undertaken in the subgroup with a high risk of recurrence. Suitable significant prognostic parameters are needed to identify patients at risk. S-phase fraction and the percentage of cells with a high DNA content are two prognostic factors that are determined by measuring total nuclear DNA and cell cycle analysis. Both were analyzed in large studies and have shown their impact on prognosis. However, divergent results underline the necessity for larger studies with a longer follow-up.     

Cytokine production by lymph node cells from mice infested with Ixodes ricinus ticks and the effect of tick salivary gland extracts on IL-2 production

In BALB/c mice repeatedly infested with nymphal Ixodes ricinus ticks, lymphocytes from axillary and brachial lymph nodes which drain the tick attachment site produced significant levels of IL-2, TNF-alpha and GM-CSF when stimulated in vitro with Con A or anti-CD3 antibodies. Cytokine production by cells from lymph nodes of the opposite flank was equivalent to that of cells from uninfested mice. Nine days after the first infestation and IL-2, GM-CSF were produced primarily by the CD4+ T cells, while some other cell types contributed also to the TNF-alpha production. In mice repeatedly infested, a gradual increase of lymph node cell production of IL-2 was observed. The IL-2 levels regularly increased from the first to the third infestation compared to TNF-alpha levels which gradually decreased. The in vitro production of GM-CSF was not affected by successive infestations. Spleen lymphocytes from naive mice produced higher levels of IL-2 than lymphocytes from axillary and brachial lymph nodes. Both tick salivary gland extracts and D-mannose inhibited IL-2 production by these lymphocytes.     

A phase-I/II study on the local hyperthermia of cervical N2/N3 lymph node metastases

A solid complex of C60 with gamma-cyclodextrin (gamma-CyD) was examined with NMR spectroscopic methods in order to understand the dynamics of C60, and the interaction between C60 and gamma-CyD. A 13C solid-state cross-polarization magic angle spinning (CP/MAS) NMR spectra shows C60 resonance at 142.6 ppm. This provides the evidence of interaction between 13C spins in C60 and 1H spins in the gamma-CyD host. Ambient temperature experiments on the 13C CP/MAS NMR, with varying contact time, shows that the water associated with gamma-CyDs plays an important role in the nuclear relaxation processes. The dynamics of C60 in gamma-CyD was investigated using temperature and field-dependent 13C spin-lattice relaxation time measurements. The influence of water on the dynamics of C60 was less significant below 250 K.