Chronic lymphocytic leukemia-reactive T cells during disease progression and after autologous tumor cell vaccines.

PURPOSE: Tumor-reactive T cells were measured in patients with chronic lymphocytic leukemia (CLL) because vaccines that increase the activity of these cells might lead to better disease control. EXPERIMENTAL DESIGN: Proliferation and ELISPOT assays (for T cells producing IFN-gamma after stimulation by CD40-activated CLL cells) were used to determine the prevalence of tumor-reactive T cells in 25 CLL patients at various stages of disease progression. The effects of vaccines, composed of autologous-oxidized tumor cells, on both the clinical course and tumor-reactive T-cell numbers were then determined in 2 patients. RESULTS: CLL-reactive T cells were found at frequencies of > or =10(-3) in 6 of 11 patients. Significant proliferation was found in 15 of 25 patients and correlated with clinical stage. The inability to measure CLL-reactive T cells in the remaining patients was not uniformly a result of generalized T-cell dysfunction or defective antigen presentation by CD40-activated CLL cells. CLL-reactive T-cell frequencies increased in response to vaccination with oxidized autologous tumor cells in a patient with preexisting CLL-reactive T cells but not in a patient where tumor-reactive T cells were undetectable in the ELISPOT assay. CONCLUSIONS: Tumor-reactive T cells exist in some CLL patients (mainly during earlier stages of disease) and may potentially mediate therapeutic responses if their numbers and activation states can be sufficiently increased by tumor vaccines.     

Evidence for involvement of clonally expanded CD8+ T cells in anticancer immune responses in CLL patients following nonmyeloablative conditioning and hematopoietic cell transplantation.

We have analyzed the clonotype composition of CD8+ T cells following nonmyeloablative (NMA) conditioning and hematopoietic cell transplantation (HCT), of patients with chronic lymphocytic leukemia (CLL). Consecutive analyses of blood samples taken up to 2 years following HCT, demonstrated that CD8+ T-cell clonality was highly dynamic in the early phases after HCT, but became more stable after 4-5 months. Moreover, donor lymphocyte infusion (DLI) given for disease progression in one of the patients led to establishment of recurrent as well as new T-cell clonotypes. This coincided with disease remission, strongly suggesting that these T cells were engaged with anti-CLL cytotoxicity. To examine the functional capacity of stable clonally expanded T cells after HCT, CD8+ T cells isolated post-transplant from the recipients were stimulated ex vivo with CLL cells and subsequently analyzed by FACS for surface expression of the marker for cytotoxic activity, CD107a. Stimulation with CLL cells indeed led to surface expression of CD107a, and clonotype analyses of sorted cells demonstrated that CD107a positive T cells were stably expanded following HCT. Our data suggest that clonally expanded CD8+ T-cell clones participate in the ongoing T-cell response against CLL cells following HCT with NMA conditioning.     

Expansion of effector T cells associated with decreased PD-1 expression in patients with indolent B cell lymphomas and chronic lymphocytic leukemia

In patients with chronic lymphocytic leukemia (CLL), numbers of CD8 + CD45RA +/- CD27- effector T cells are expanded. We investigated whether this expansion is also present in other B cell malignancies and the possible mechanism underlying these changes. Whereas an increase in total CD4+and CD8+ T cell numbers was found only in CLL, numbers of CD4+ and CD8+ effector T cells were significantly increased in both CLL and indolent lymphoma, but not aggressive lymphoma and myeloma. Interestingly, PD-1 expression was decreased on effector T cells and inversely correlated with effector T cell numbers, suggesting a functional role for PD-1 in regulating T cell homeostasis. In vitro experiments revealed impaired up-regulation of PD-1 upon T cell activation in the presence of malignant but also healthy B cells. Our data suggest that in CLL and indolent lymphoma, the malignant B cells affect PD-1 expression on effector T cells, resulting in an expansion of these subsets.     

Autoreactive, cytotoxic T lymphocytes specific for peptides derived from normal B-cell differentiation antigens in healthy individuals and patients with B-cell malignancies.

PURPOSE: To investigate potential immunotherapeutic strategies in B lymphocytic malignancies we looked for CTLs recognizing CD19 and CD20 epitopes. EXPERIMENTAL DESIGN: Three CD19 and CD20 peptides binding to HLA-A*0201 were identified and used to detect peptide specific CTLs by a quantitative real-time PCR to measure IFN-gamma mRNA expression in 23 healthy individuals and 28 patients (18 chronic lymphocytic leukemia (CLL), 7 follicular lymphoma, 2 acute lymphocytic leukemia, and 1 large cell lymphoma). Peptide-specific CTLs were expanded in culture with CD40-activated B cells to test lytic activity in three patients. RESULTS: In healthy individuals, CD8+ T-cell responses were detected in one to CD19(74-82), in three to CD20(127-135), and three to CD20(188-196). Seven of 27 patients (6 with CLL) had CD8+ T cells recognizing CD19(74-82). Seven patients responded to CD20(127-135) and three to CD20(188-196). All were CLL patients. CD19(74-82)-specific CTLs from three patients were expanded over 4 weeks. These cells were HLA-A*0201 specific and lytic for peptide-loaded antigen-presenting cells but not to malignant or unpulsed B cells. CONCLUSIONS: CTLs that recognize CD19 and CD20 epitopes exist in healthy individuals and may be increased in CLL patients. They are of low avidity and require high doses of peptide for activation. Strategies to increase T-cell avidity would be necessary for T-cell immunotherapeutic approaches using the peptides studied.     

Expression levels of CD38 on leukemic B cells but not on non-leukemic T cells are comparably stable over time and predict the course of disease in patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by a number of T-cell abnormalities, which may play a causative role in disease progression and immune dysfunction. Recently, expression levels of CD38 in T cells have been suggested as a novel adverse prognostic factor in male CLL patients. In the current study, CD38 expression on CLL T cells was examined by flow cytometry in 126 patients with B-CLL and correlated with clinical parameters and established molecular risk factors. In line with previous results we observed a positive correlation of CD38 expression on leukemic B and non-leukemic T cells with clinical stage. CD38 expression on B-CLL cells, cytogenetic aberrations and mutations of IgVH genes were found to significantly influence treatment-free survival. By contrast, CD38 expression on T cells was not significantly associated with an adverse clinical outcome.     

Imatinib impairs the proliferation and function of CD4+CD25+ regulatory T cells in a dose-dependent manner

The tyrosine kinase inhibitor imatinib has been reported to inhibit CD8+ T lymphocytes. Little is known about its effects on CD4+CD25+ regulatory T cells (T(reg) cells) which might regulate the graft-vs.-leukemia (GVL) reaction after allogeneic stem cell transplantation (allo-SCT) and donor lymphocyte infusion (DLI). This is of particular interest in patients with relapse of chronic myeloid leukemia (CML) after allo-SCT, as the two therapeutical options DLI and imatinib might interact reversely. Here, we demonstrate that the proliferation of CD4+CD25+ T(reg) cells and their production of IL-10, TGF-beta1 and granzyme B as markers of activation were significantly down-regulated by imatinib in a dose-dependent manner. In addition, the expression of surface CD69, both surface and intracellular GITR, FoxP3, CD152 (CTLA) of activated CD4+CD25+ T(reg) cells were inhibited by imatinib in a dose-dependent manner. In light of these findings, clinical administration of imatinib might not result in a reduction of the GVL effect on CML patients receiving imatinib after allo-SCT and/or DLI or other CD8+ T lymphocyte based immunotherapies as the function of CD8+ cytotoxic T lymphocytes and CD4+CD25(hi) Treg cells is hampered in a similar way by imatinib.     

Free circulating soluble CD52 as a tumor marker in chronic lymphocytic leukemia and its implication in therapy with anti-CD52 antibodies

BACKGROUND: The CD52 antigen is a glycoprotein anchored on the cell membrane of mature B and T lymphocytes, monocytes, and eosinophils. Alemtuzumab (CAMPATH-1H; anti-CD52) is currently approved for the treatment of patients with refractory chronic lymphocytic leukemia (CLL). The authors investigated the possibility that CD52 may be shed from cells and, once soluble, may bind to injected alemtuzumab, forming immune complexes. METHODS: The authors used Western blot analysis, immunoprecipitation, and enzyme-linked immunoadsorbent assay to investigate the presence of soluble CD52 (sCD52) in the plasma specimens of 117 patients with CLL. They also used in vitro mixing experiments to examine the ability of sCD52 to compete with cells and sequester therapeutic alemtuzumab. RESULTS: The authors detected high levels of sCD52 in the plasma specimens of patients with CLL. sCD52 can compete with cells in vitro for binding to alemtuzumab, and can form complexes in patients receiving alemtuzumab. Plasma levels of sCD52 were found to be correlated (r)with Rai stage (P = 0.0001), beta-2-microglobulin (beta-2M) levels (P = 0.00002), soluble CD23 levels (r = 0.42, P < 0.001), and immunoglobulin mutation status (P = 0.003). In the multivariate analysis adjusted for beta-2M level, patients with sCD52 levels > 2336 nM/L had a nearly 4-fold increase in risk of death. Higher levels of plasma alemtuzumab were achieved when levels of sCD52 were lower. CONCLUSIONS: These data not only demonstrated that sCD52 was detectable and useful in the staging and monitoring of patients with CLL, but also showed that sCD52 formed immune complexes with alemtuzumab and may influence the efficacy and toxicity of alemtuzumab therapy.     

CD+4CD+25Foxp+3调节性T细胞在T细胞非霍奇金淋巴瘤中的研究进展

 CD+4 CD+25 调节性T细胞（CD+4 CD+25 Treg）是一个具有独特免疫调节功能的T细胞亚群, 天然的CD+4 CD+25 Treg起源于胸腺,获得性CD+4 CD+25 Treg可在外周由CD+4 CD-25 T细胞诱导产生,其分子表面表达特异性的转录抑制因子Foxp3,又可表达CD4、CD25、CTLA-4 （CD152） 、GITR等膜分子,主要功能具有免疫抑制性和免疫无能性。近年来研究发现,其在非霍奇金淋巴瘤（NHL）中存在表达异常,某些NHL外周血中或瘤内均存在CD+4 CD+25 Treg表达升高,且有研究表明其表达量随肿瘤增长和分期而增加。增加的CD+4 CD+25 Treg可加速肿瘤生长及再发,且可抑制自身效应性T细胞（CD+4 T／CD+8 T）功能,在肿瘤免疫逃逸机制中发挥一定作用。文章就CD+4 CD+25 Foxp3+调节性T细胞在T细胞非霍奇金淋巴瘤（T-NHL）（主要为皮肤T细胞淋巴瘤及成年人T细胞淋巴瘤）中的研究进展进行综述。     

IL-2 induces and altered CD4/CD8 ratio of splenic T lymphocytes from transgenic mice overexpressing the glucocorticoid-induced protein GILZ

We used transgenic mice to investigate the effect of IL-2 stimulation on T lymphocyte functions of GILZ-overexpressing splenic T cells. When compared to their controls, T cells from transgenic mice underwent normal activation after stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies, as evaluated by CD25 expression, CD2 up-regulation and proliferation. IL-10, IL-13 and IFN-gamma increased more consistently in CD3/CD28-triggered TG compared to WT splenic CD4(+)cells. Analysis of the CD4(+)and CD8(+)T cells demonstrated a decreased CD4(+)/CD8(+)T-cell ratio (1:1 instead of 1:2) in response to IL-2 stimulation, possibly due to an unresponsiveness of IL-2 receptor beta and/or gamma chains. Finally, the total number of T cells was significantly increased in aged mice and this was due to the augmentation of CD4(+)T cells. These results support the hypothesis that GILZ regulates, at least in part, peripheral T-cell functions by influencing their responsiveness to IL-2.     

Ctla-4 blockade confers lymphocyte resistance to regulatory T-cells in advanced melanoma: surrogate marker of efficacy of tremelimumab?

PURPOSE: Anti-CTL antigen-4 (CTLA-4) monoclonal antibody (mAb) has led to encouraging antitumor activity associated with immune-related adverse events in patients with heavily pretreated melanoma. However, mechanisms of action and surrogate immunologic markers of efficacy have not been reported thus far. EXPERIMENTAL DESIGN: We monitored the immune responses of 10 melanoma patients included in a phase II clinical trial, which evaluated the efficacy of a second line of therapy of tremelimumab anti-CTLA-4 mAb in patients with metastatic melanoma. The frequency of blood leukocyte populations in association with T cell and regulatory T cell (Treg) functions were evaluated. RESULTS: Prior to therapy, patients with advanced melanoma presented with a severe CD4+ and CD8+ T cell lymphopenia associated with blunted T-cell proliferative capacities that could be assigned to Treg. Tremelimumab rapidly restored the effector and memory CD4+ and CD8+ T-cell pool and TCR-dependent T-cell proliferation that became entirely resistant to Treg-mediated suppression. Progression-free survival and overall survival was directly correlated with the acquisition of a biological response defined as the resistance of peripheral lymphocytes to Treg-inhibitory effects (obtained in 7 of 10 patients). CONCLUSION: CTLA-4 blockade seems to be a valuable strategy to revive reactive memory T cells anergized in the context of stage IV melanoma, and our work suggests that memory T-cell resistance to Treg resulting from anti-CTLA-4 treatment might be a biological activity marker for tremelimumab in patients with melanoma.     

The effect of peripheral blood lymphocyte stimulation on zeta chain expression and IL-2 production in Hodgkin's disease

It has been reported that peripheral blood T cells and NK cells express reduced levels of the T-cell receptor signal-transducing zeta chain in Hodgkin's disease (HD). The zeta chain has emerged as a key subunit of the T-cell antigen receptor, which plays a central role in the signal-transducing events leading to T and NK-cell activation. We were interested in determining whether the low zeta chain expression in HD could be corrected by anti-CD3, anti-CD3-rIL-2 ex vivo stimulation. Zeta chain expression was analysed by dual immunofluorescence on permeabilized cells before and after 72 hours of culture. The IL-2 concentration in the culture supernatants was measured by ELISA. Zeta chain was significantly reduced on unstimulated CD4+, CD8+ and CD56+ cells from patients in active disease compared with normal subjects. In patients in complete remission, the values were normal except for CD8+ cells, on which zeta expression remained significantly reduced. Stimulation with anti-CD3 did not change zeta expression. Co-stimulation with rIL-2 increased but did not normalize the proportions of CD4(+)/zeta(+), CD8(+)/zeta(+)and CD56(+)/zeta(+)cells and IL-2 production in active disease. Stimulation of cells from patients in clinical remission with anti-CD3(+)rIL-2 increased the proportion of CD8(+)zeta(+)cells and normalized IL-2 production levels. Considering the pivotal role of CD3-zeta in immune response, our data suggest that successful immunotherapy approaches in active HD should consider inclusion of other potent cytokines, as well as genetically engineered tumour vaccines.     

Disruption of T cell suppression in chronic lymphocytic leukemia by CD200 blockade

CD200 plays a key role in regulating the immune system and has been shown to be upregulated on the surface of different tumors including chronic lymphocytic leukemia. In this study we addressed the effects of CD200 over-expression in CLL cells on autologous T cells in a mixed lymphocyte reaction system. We used native and CD40 ligand (CD40L)-stimulated CLL cells as antigen-presenting cells (APCs) to expand autologous T cells of 14 patients. T cell proliferation over 3 weeks of in vitro culture was significantly enhanced compared to control cells when using CD40L-stimulated APCs and the anti-CD200 antibody 1B9 (p=0.0004). CD200 blockade was further shown to stimulate antigen-specific T cell responses towards the CLL-associated antigen fibromodulin (p=0.04). Finally, the number of CD4+/CD25high/FOXP3+ T cells (T(reg)) was significantly decreased adding anti-CD200 antibody (p=0.04). In summary, CD200 blockade may provide therapeutic benefits in CLL by augmenting an antigen-specific T cell response with suppression of regulatory T cells.     

CC chemokine ligand 25 enhances resistance to apoptosis in CD4+ T cells from patients with T-cell lineage acute and chronic lymphocytic leukemia by means of livin activation

We investigated CD4 and CD8 double-positive thymocytes, CD4(+) T cells from typical patients with T-cell lineage acute lymphocytic leukemia (T-ALL) and T cell lineage chronic lymphocytic leukemia (T-CLL), and MOLT4 T cells in terms of CC chemokine ligand 25 (CCL25) functions of induction of resistance to tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. We found that CCL25 selectively enhanced resistance to TNF-alpha-mediated apoptosis in T-ALL and T-CLL CD4(+) T cells as well as in MOLT4 T cells, but CD4 and CD8 double-positive thymocytes did not. One member protein of the inhibitor of apoptosis protein (IAP) family, Livin, was selectively expressed in the malignant cells at higher levels, particularly in T-ALL CD4(+) T cells, in comparison with the expression in CD4 and CD8 double-positive thymocytes. After stimulation with CCL25 and apoptotic induction with TNF-alpha, the expression levels of Livin in these malignant cells were significantly increased. CCL25/thymus-expressed chemokine (TECK), by means of CC chemokine receptor 9 (CCR9) ligation, selectively activated Livin to enhance resistance to TNF-alpha-mediated apoptosis in c-jun-NH(2)-kinase 1 (JNK1) kinase-dependent manner. These findings suggested differential functions of CCR9/CCL25 in distinct types of cells. CD4 and CD8 double-positive thymocytes used CCR9/CCL25 for migration, homing, development, maturation, selection, cell homeostasis, whereas malignant cells, particularly T-ALL CD4(+) T cells, used CCR9/CCL25 for infiltration, resistance to apoptosis, and inappropriate proliferation.     

Imatinib mesylate suppresses cytokine synthesis by activated CD4 T cells of patients with chronic myelogenous leukemia.

Although imatinib mesylate (IM) is highly effective at inducing complete cytogenetic remission in patients with chronic myelogenous leukemia (CML), it is known to suppress T-cell proliferation in vitro. As cytokines are required for T-cell proliferation, we investigated the effects of IM on cytokine synthesis by T cells of CML patients by assessing cytokine synthesis by activated CD4+ and CD8+ T cells in vitro. The activation of T cells in the whole blood of IM-treated patients (CML-IM) with Staphylococcus enterotoxin B resulted in significantly lower percentages of CD4+ T cells that synthesized interleukin 2 (P = 0.017), interferon-gamma (P = 0.010), and tumor necrosis factor-alpha (P = 0.009) than did the activated T cells of control subjects. The addition of exogenous IM to the cultures of peripheral blood mononuclear cells of CML-IM patients reduced Th1 cytokine synthesis by the CD4+ T cells. Furthermore, IM therapy at clinical doses suppressed the tyrosine phosphorylation of ZAP70. These findings suggest that inhibition of ZAP70 signaling pathway and suppression of Th1 cytokine synthesis by CD4+ T cells required the presence of IM at the time of T-cell activation through the T-cell receptor.     

Poorly expressed CD2 antigen on the leukemic cells of adult T-cell leukemia and chronic lymphocytic leukemia of T-cell lineage

Adult T-cell leukemia (ATL) and T-cell chronic lymphocytic leukemia (T-CLL) are hematologic neoplasms in differentiated stages of peripheral mature T cells. This is suggested by the presence of CD2 (E rosette receptor) and mature and/or pan-T cell membrane surface antigens on their leukemic cells. We recently encountered one patient with ATL and another with T-CLL; their leukemic cells poorly expressed CD2 antigens, but clinical presentation, morphology of the leukemic cells and other marker studies were characteristic of either ATL or CLL. The clinical significance of the poor expression of CD2 remains to be further studied. The two patients reported here died of severe complications 4 and 9 weeks after diagnosis. The poor expression of CD2 in the peripheral T cells in these neoplasms is likely to indicate an aggressive nature of the disease.     

Reduced cell turnover in lymphocytic monkeys infected by human T-lymphotropic virus type 1

Understanding cell dynamics in animal models have implications for therapeutic strategies elaborated against leukemia in human. Quantification of the cell turnover in closely related primate systems is particularly important for rare and aggressive forms of human cancers, such as adult T-cell leukemia. For this purpose, we have measured the death and proliferation rates of the CD4+ T lymphocyte population in squirrel monkeys (Saimiri sciureus) infected by human T-lymphotropic virus type 1 (HTLV-1). The kinetics of in vivo bromodeoxyuridine labeling revealed no modulation of the cell turnover in HTLV-1-infected monkeys with normal CD4 cell counts. In contrast, a substantial decrease in the proliferation rate of the CD4+ T population was observed in lymphocytic monkeys (e.g. characterized by excessive proportions of CD4+ T lymphocytes and by the presence of abnormal flower-like cells). Unexpectedly, onset of HTLV-associated leukemia thus occurs in the absence of increased CD4+ T-cell proliferation. This dynamics significantly differs from the generalized activation of the T-cell turnover induced by other primate lymphotropic viruses like HIV and SIV.