Surface expression of alternative forms of the TCR/CD3 complex.

T-cell antigen receptor (TCR) heterodimers of both the alpha beta and gamma delta types are expressed at the surface of T cells only in association with a complex of invariant chains called CD3. The requirement for individual CD3 components to achieve TCR surface expression was examined by cotransfection of a non-T-cell line with TCR alpha and beta, as well as CD3 delta, epsilon, gamma, and zeta, cDNAs. Both transient and stable transfectants expressing TCR and CD3 epitopes at the cell surface were generated. By transfection of TCR and CD3 components in different combinations, the TCR chains, as well as the CD3 epsilon and zeta chains, were each shown to be essential for reconstituting surface expression. On the other hand, CD3 delta and gamma chains could be used alternatively, providing evidence for two different types of TCR/CD3 complexes.     

Synergism in the activation of human CD8 T cells by cross-linking the T-cell receptor complex with the CD8 differentiation antigen.

Resting human T cells can be activated and induced to proliferate by cross-linking the T-cell receptor complex (Ti/CD3) with anti-CD3 (T3) antibodies, such as OKT3, together with interleukin 2. Here we describe functional properties of another monoclonal anti-CD3 antibody (BMA 030) that, cross-linked in various ways, only weakly stimulates accessory-cell-depleted T-cell cultures. However, when cross-linked to anti-CD4 or anti-CD8 antibodies a markedly enhanced proliferation of the corresponding subpopulation is observed. We have concentrated on the analysis of CD8 cells and have found that BMA 030, when cross-linked together with anti-CD8 (T811), induced proliferation more than 100-fold greater than BMA 030 alone, whereas cross-linking with antibodies to other T-cell membrane antigens (HLA-A, B, or CD5) provided no or marginal synergistic signals. There was no synergistic effect when only one of the two antibodies, BMA 030 or T811, was cross-linked and the other was applied in soluble form. In contrast, each of the two antibodies alone, when applied in soluble form, inhibited activation induced by the cross-linked antibodies. The T-cell differentiation antigen CD8 has been implicated in the major histocompatibility complex (MHC) class I restricted specificity of CD8 T cells. In previous work from other laboratories only the negative influences of soluble anti-CD8 antibodies have been noted. In contrast, our results suggest that cross-linking between Ti/CD3 and CD8 may be a critical event in the activation of mature CD8 cells. We hypothesize that, in antigen-induced T-cell activation, CD8 and Ti/CD3 become cross-linked by their simultaneous binding to class I-associated structures. Such a mechanism, if required for proliferation in early T-cell ontogeny, could generate a selective pressure for CD8 cells to recognize class I-associated antigens.     

Circulating T cells in patients with untreated acute myelogenous leukemia are heterogeneous and can be activated through the CD3/TCR complex

T lymphocyte defects may contribute to the immune insufficiency seen in acute myelogenous leukemia (AML). We therefore characterized the T cell system for untreated AML patients. T lymphocyte subsets were analyzed by flow cytometry for 45 AML patients. The in vitro interferon-gamma (IFNgamma) release in response to stimulation with anti-CD3 plus anti-CD28 in the presence of autologous AML cells was examined for 31 patients. The majority of circulating lymphocytes were CD3(+)T cells, and CD19(+)B cells usually constituted < 10% of the lymphocytes. Most T cells expressed the alphabeta T cell receptor (TCRalphabeta(+)), and only a minority of the cells was TCRgammadelta(+). Both CD4(+) and CD8(+)T cells were detected, the CD4:CD8 ratio showed a wide variation but was generally >1.0. The majority of CD4(+) and CD8(+)T cells were CD45RA(+) cells. The T cells could be stimulated to release IFNgamma in response to anti-CD3 plus anti-CD28 ligation even in the presence of excess autologous AML blasts, and for a subset of patients (6 of 27) these IFNgamma levels could be further increased by the novel protein kinase C (PKC) agonist PEP005. In conclusion, circulating T cells in patients with untreated AML are mainly CD4(+) or CD8(+) TCRalphabeta(+); both CD45RA(+) and CD45R0(+) can be detected, and these cells can be activated through the CD3/TCR complex even in the presence of excess AML cells. For a subset of patients T cell responsiveness can be further increased by targeting PKC and these data therefore suggest that T cell function is not inhibited in AML patients.     

系统性红斑狼疮患者T细胞TCR/CD3复合物介导的信号传导研究

目的：证实系统性红斑狼疮（SLE）患者T细胞功能异常是否与其生物化学信号传导异常有关。方法：用CD3单抗与羊抗鼠二抗IgG相交联刺激T细胞并用Thapsigargin和依地酸（EGTA）干预后，分别用粘附细胞仪连续观察10min T细胞[Ca^2 ]i的变化，并评价[Ca^2 ]i反应与CD3分子和三磷酸肌醇（InsP3）生成量的相关性。结果：正常人和SLE患者T细胞[Ca^2 ]i反应的基准值相似（P＝0．105）；SLE患者高峰者，平台值T细胞的[Ca^2 ]i反应明显高于正常对照（P＜0．001，P＜0．001），加入Thapsigargin后二者[Ca^2 ]i反应差异无显著性，而加入EGTA后二者[Ca^2 ]i反应差异有显著性，二者的T细胞CD3阳性率和InsP3生成量差异无显著性（P＝0．665，P=0．537）。结论：SLE患者T细胞TCR／CD介导的信号传导途径存在异常，SLE患T细胞功能异常可能是因细胞内生物化学信号传导途径异常所致。     

Dysfunctional T regulatory cells in multiple myeloma

Multiple myeloma (MM) is characterized by the production of monoclonal immunoglobulin and is associated with suppressed uninvolved immunoglobulins and dysfunctional T-cell responses. The biologic basis of this dysfunction remains ill defined. Because T regulatory (T(reg)) cells play an important role in suppressing normal immune responses, we evaluated the potential role of T(reg) cells in immune dysfunction in MM. We observed a significant increase in CD4+ CD25+ T cells in patients with monoclonal gammopathy of undetermined significance (MGUS) and in patients with MM compared with healthy donors (25% and 26%, respectively, vs 14%); however, T(reg) cells as measured by FOXP3 expression are significantly decreased in patients with MGUS and MM compared with healthy donors. Moreover, even when they are added in higher proportions, T(reg) cells in patients with MM and MGUS are unable to suppress anti-CD3-mediated T-cell proliferation. This decreased number and function of T(reg) cells in MGUS and in MM may account, at least in part, for the nonspecific increase in CD4+ CD25+ T cells, thereby contributing to dysfunctional T-cell responses.     

HIV感染者/AIDS患者外周血T细胞抗原受体γδT细胞的检测及其临床意义

目的　探讨外周血T细胞抗原受体 (TCR)γδT细胞在艾滋病病毒 (HIV)感染者 /艾滋病 (AIDS)患者疾病进程中的作用。方法　应用流式细胞仪采用三色荧光抗体染色技术分别检测 18例HIV感染者、2 9例AIDS患者、2 5例伴有机会性感染的AIDS患者、2 0例高效抗逆转录病毒联合疗法 (HAART)治疗后的AIDS患者及 2 0例正常对照者外周血的TCRγδT细胞与TCRαβT细胞。 结果　HIV感染者组和AIDS患者组的外周血TCRγδT细胞的百分率比正常对照组明显地增高 (P <0 0 1) ,而TCRαβT细胞的百分率明显地低于正常对照组 (P <0 0 1) ,AIDS患者组的CD+ 3 细胞的百分率明显地低于HIV感染者组和正常对照组 (P <0 0 1)。AIDS患者伴机会性感染组的外周血TCRγδT细胞的百分率明显地高于HAART治疗后的AIDS患者组 (P <0 0 1) ,该组的TCRαβT细胞的百分率及CD+ 3 T细胞的百分率则明显地低于经HAART治疗后的AIDS患者组 (P <0 0 1)。结论　TCRγδT细胞数量增多是机体受到病毒感染时的一种早期非特异性免疫反应 ,在AIDS的疾病进程中及机会性感染时起一定的免疫防御作用。     

Future of CAR T cells in multiple myeloma

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G-banding analysis of complex aneuploidy in multiple myeloma bone marrow cells.

Chromosome studies with the banding technique have been performed in a considerable number of cases of myeloproliferative diseases, but technical difficulties have so far prevented detailed studies of chromosomal abnormalities in multiple myeloma. The karyotypes of bone marrow cells from two patients with multiple myeloma have been analyzed by a trypsin-Giemsa banding technique. Evidence is given for clonal evolution which in one patient has probably occurred by cell fusion and subsequent chromosome loss. Eight different marker chromosomes are characterized. Nonrandom chromosomal participation in the translocations and the existence of specific vulnerable points on chromosomes 1, 3, and 16 are suggested.     

In vivo peripheral expansion of naive CD4+CD25high FoxP3+ regulatory T cells in patients with multiple myeloma

In solid tumors, leukemias, and lymphomas, increased frequencies of functional CD4+CD25(high) regulatory T cells (T(reg) cells) have been previously demonstrated. In healthy individuals, T(reg) cells consist not only of memory but also of naive T cells, which can undergo peripheral expansion and are characterized by a relative enrichment for autoreactive T-cell receptors. Here, we demonstrate in patients with premalignant monoclonal gammopathy of undetermined significance and patients with multiple myeloma that functional FoxP3(+) T(reg) cells of naive, central, and effector memory phenotype as determined by CCR7 and CD45RA expression are significantly expanded. Low frequencies of T-cell receptor excision circles in naive T(reg) cells in both healthy controls and multiple myeloma patients point to peripheral expansion as the prominent mechanism of increased frequencies of naive T(reg) cells in these cancer patients. These findings strongly suggest that the increase of functional T(reg) cells in cancer patients is a response to the process of malignant transformation.     

Tumor-specific recognition of human myeloma cells by idiotype-induced CD8(+) T cells

Immunoglobulin secreted by myeloma cells contains a unique antigenic determinant (idiotype [Id]) that may serve as a tumor-specific antigen. Although Id-protein-specific T-cell responses have been reported in patients with myeloma, it is not known whether primary myeloma tumor cells can present naturally processed Id peptides on their surface as a target. We immunized 2 healthy human stem-cell donors with Id proteins from their recipients. T cells from the immunized donors released high levels of T-helper 1-type cytokines in response to stimulation with myeloma cells from their recipients. The T-cell-mediated cytokine response to tumor cells was blocked by a major histocompatibility complex (MHC) class I monoclonal antibody, whereas the response to soluble Id protein was dependent on MHC class II. To investigate whether Id-specific CD8(+) T cells can recognize and kill autologous myeloma cells, we generated T cells from peripheral blood mononuclear cells from a third patient with myeloma by means of in vitro stimulation with autologous dendritic cells pulsed with Id protein. Tumor-specific lysis of myeloma cells was demonstrated by the lack of killing of autologous nonmalignant B cells or natural killer-sensitive K562 cells. Lysis of autologous myeloma targets was restricted by MHC class I molecules. These data represent the first report of class I-restricted T-cell recognition of fresh autologous myeloma targets and formally demonstrate that human myeloma cells can serve as targets of an Id-specific T-cell response. (Blood. 2000;96:2828-2833)     

Clonal populations of CD4+ and CD8+ T cells in patients with multiple myeloma and paraproteinemia

Patients with paraproteinemia have abnormalities in their T-cell subsets including inversion of the CD4:CD8 ratio and increased expression of activation markers. Recently, distortions in T-cell receptor (TCR) TCRAV and TCRBV gene segment expression have been reported, although the significance of these observations is unclear given the finding of clonal populations of CD8+ T cells in healthy elderly individuals. We have used an extensive range of TCR V-region- specific monoclonal antibodies to assess TCRAV and TCRBV expression in patients with myeloma and paraproteinemia. TCR sequence analysis was used to assess the clonality of expansions and 3-color fluorescence- activated cell sorting analysis determined the phenotype of the expanded populations. The patients show novel oligoclonal expansions within the CD4+ subset and show an increased frequency of CD8+ expansions. Oligoclonal CD4+ T cells belong to the rare CD4+CD28- T- cell subset, a phenotype associated with granular morphology. CD45RA and CD11b are expressed on many of the CD8 T-cell expansions. Comparison of T-cell receptor sequences from two T-cell clones in one patient suggests a possible role for a common peptide antigen in the generation of the expansions. Further work is needed to identify the relevance of such T cells to the B-cell proliferation.     

Pseudo-Gaucher cells in multiple myeloma.

A bone biopsy specimen from a patient with multiple myeloma showed numerous Gaucher-like cells scattered throughout a homogeneous background of plasma cells. Further studies using histochemical stains, immunofluorescence, and light and electron microscopy were carried out to further define these cells. Light microscopy of Wright-stained and hematoxylin and eosin-stained marrow preparations showed large, round cells with fibrillar appearing cytoplasm and eccentric, pyknotic nuclei. These cells were periodic acid-Schiff positive, resistant to diastase digestion. Electron microscopy demonstrated plasma cells containing crystals in membrane-bound vesicles. Also, large macrophages among these plasma cells contained similar crystals surrounded by a single limiting membrane. Immunofluorescence staining of thin sections of marrow with fluorescein-labelled specific antiserums showed fluorescence of these large cells. Strong immunofluorescence was seen with polyvalent kappa and gamma antiserums but not with anti-albumin or serums with anti-lambda, mu or alpha specificity. It appears that these large cells have the light microscopic and histochemical characteristics of true Gaucher cells but, when studied with immunofluorescence and electron microscopy, it appears that the pseudo-Gaucher cells of multiple myeloma are bone marrow macrophages engorged with immunoglobulin.     

Rapid generation of antiplasma cell activity in the bone marrow of myeloma patients by CD3-activated T cells

We have recently shown that peripheral blood T cells of multiple myeloma (MM) patients are very susceptible to stimulation of the T-cell receptor/CD3 complex with anti-CD3 monoclonal antibodies (MoAbs). CD3 stimulation is currently under clinical investigation as a nonspecific approach to boost antitumor effector mechanisms. The aim of this study was to determine whether the hyperreactivity of MM T cells to CD3 stimulation could be exploited to generate antitumor activity. Bone marrow mononuclear cells (BMMCs) from 65 MM patients were stimulated with the anti-CD3 MoAb OKT3 and the effect of this stimulation on autologous T cells and plasma cells was evaluated. The number of CD3+ CD25+ cells on day 6 was significantly higher in MM than the controls (30 normal individuals) (P = .001). Kinetic studies showed that 3H- thymidine incorporation peaked on day 3 and that the T-cell expansion peaked on days 5 and 6. In MM, T-cell activation markedly affected the survival of autologous plasma cells; their number in OKT3-treated cultures was significantly lower than in unstimulated cultures (P < .0001). T-cell activation and plasma cell decrease were not observed when T cells were removed from BMMC preparations. MM produced significantly higher levels of interferon-gamma (P = .005) and tumor necrosis factor-beta (P = .001), but lower levels of tumor necrosis factor-alpha (P < .001) than normal individuals. Interferon-gamma only was partially involved in CD3-induced plasma cell killing. Transwell cultures showed that the main mechanism by which CD3+ CD25+ cells affected plasma cells was direct cell-to-cell contact rather than cytokines. In conclusion, T cells in MM BMMCs possess distinct features in terms of susceptibility to CD3 stimulation and cytokine production compared with normal bone marrow T cells that can be exploited to generate antiplasma cell activity.     

Tc-99m methoxyisobutylisonitrile bone marrow imaging for predicting the levels of myeloma cells in bone marrow in multiple myeloma: correlation with CD38/CD138 expressing myeloma cells

The percentage of myeloma cells in bone marrow is subsequently an important index of disease in patients with multiple myeloma (MM). Bone marrow myeloma cells can be detected by strong CD38/CD138 positivity and light scatter characteristics using flow cytometry. The aim of the study was to evaluate the relationship between the degree of Tc-99m methoxyisobutylisonitrile (MIBI) uptake and the percentage of CD38/CD138 expressing myeloma cells in the bone marrow of patients with MM. A total of 15 patients with MM (mean age: 61.7+/-2.4 years; 7 F and 8 M) were included in the study. Tc-99m MIBI imaging was obtained 20 min after injection of 740 MBq Tc-99m MIBI. Planar spot images of the pelvis and thorax were acquired. The uptake of Tc-99m MIBI in the bone marrow was evaluated using a qualitative and also a semiquantitative scoring system for the bone marrow in areas that included the proximal femurs, anterior iliac crest, and sternum. In all patients, flow cytometry was performed for assessing the percentage of CD38/CD138 expressing myeloma cells in the bone marrow samples. There was a statistically significant positive correlation between the percentage of CD38/CD138 expressing plasma cells in bone marrow and both mean qualitative (r=0.689, p=0.005) and semiquantitative (r=0.669, p=0.006) results of Tc-99m MIBI uptake. In conclusion, our results indicate that increased Tc-99m MIBI uptake of bone marrow is related to the percentage of plasma cell infiltration of bone marrow. Tc-99m MIBI bone marrow imaging may be a useful tool for predicting the levels of myeloma cells in bone marrow of patients with MM.     

Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells.

The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investigate the mechanism by which CD4 cross-linking induces cell death. We have found that CD4 cross-linking results in a small but rapid increase in levels of cell surface Fas, a member of the tumor necrosis factor receptor family implicated in apoptotic death and maintenance of immune homeostasis. Importantly, CD4 cross-linking triggered the ability of Fas to function as a death molecule. Subsequent to CD4 cross-linking, CD4+ splenocytes cultured overnight became sensitive to Fas-mediated death. Death was Fas-dependent, as demonstrated by cell survival in the absence of plate-bound anti-Fas antibody, and by the lack of CD4-induced death in cells from Fas-defective lymphoproliferative (lpr) mice. We demonstrate here that CD4 regulates the ability of Fas to induce cell death in Cd4+ T cells.     

Clinical experience of CAR T cells for multiple myeloma

Important advances in the treatment landscape of multiple myeloma (MM) had been seen over the past two decades leading to improved overall survival but despite the progress multiple myeloma is still considered incurable and the prognosis of the pentarefractory patients have been poor. The development of immunotherapy and in particular adoptive cell therapy with chimeric antigen receptor (CAR) T cells have dramatically improved the outcomes of heavily pretreated relapsed/refractory MM patients. The bulk of CAR T-cell constructs currently in clinical development target the B-cell maturation antigen (BCMA) and to date only idecabtagene vicleucel (ide-cel) is approved by the Food and Drug Administration (FDA) for commercial use in adult patients with relapsed or refractory MM based on the promising clinical responses and positive safety record shown in the pivotal KarMMa study. This review focus on the development of CAR T-cell therapy for multiple myeloma as well as a brief review of the mechanisms of resistance, toxicity and new approaches under development.     

Detection and quantitation of malignant cells in the peripheral blood of multiple myeloma patients.

One of the distinguishing features of multiple myeloma (MM) is the proliferation of plasma cells that home to the bone marrow (BM). However, there still remains some uncertainty concerning the presence of related malignant cells in the peripheral blood of myeloma patients. Using consensus oligonucleotide primers, we amplified the third complementary determining region (CDR3) of rearranged immunoglobulin heavy chain alleles from MM marrow samples by polymerase chain reaction (PCR). From the sequences of the products, we derived allele-specific oligonucleotides (ASO), and these were used in subsequent amplification reactions to detect malignant clones in the peripheral blood of myeloma patients. This method is highly specific and sensitive to 1 malignant cell in the background of 10(5) normal cells. Using this method we detected circulating malignant cells in 13 of 14 previously untreated MM patients. Furthermore, by applying ASO-PCR to artificial titrations of initial BM DNA sample into normal peripheral blood lymphocyte (PBL) DNA we were able to generate standard curves and quantitate the amount of tumor in the patient PBL. We observed a wide variation in the amount of circulating tumor between patients. In addition, we found that the incidence of circulating tumor cells was independent of BM tumor burden and stage of disease. The detection and quantitation of circulating tumor cells in the PBL of MM patients may offer an alternative assessment of the disease and may be an important consideration in the use of peripheral stem cells in bone marrow transplantation.     

CD5 positive immunoregulatory B cells in spleen populations from multiple myeloma patients.

CD19+CD5+ lymphocytes constitute a minority of peripheral blood B cells. In view of the importance of these cells in the pathogenesis of the immunoregulation of myeloma, their incidence in another lymphoid organ was determined. CD5+ B cells were studied in 9 spleens from patients with multiple myeloma and in 10 spleens from normal individuals removed secondary to trauma. The total number of CD19+ B cells were increased in myeloma spleens (44.4% +/- 12.6%) as compared to normal spleens (20.4% +/- 7.4%). Likewise, the percentage of CD19 cells which co-expressed CD5 were increased in myeloma (25.3% +/- 12.4%) versus normal (4.4% +/- 2.3%) spleen. CD5+ B cells isolated from myeloma spleens, but not normal spleens, inhibit production of immunoglobulin in a pokeweed mitogen driven assay. Thus the spleen appears to be an important source of immunoregulatory B cells in multiple myeloma.     

Expansion of CD8+ T cells that express low levels of the B cell-specific molecule CD20 in patients with multiple myeloma

An expanded cytotoxic/memory T-cell subpopulation expressing low levels of the B-cell-specific CD20 molecule was found in peripheral blood and bone marrow of patients with multiple myeloma at the time of diagnosis, but returned to normal levels following treatment. CD3+CD20dim cells were also increased in monoclonal gammopathy of unknown significance albeit at lower levels. Lower CD3+CD20dim cell numbers at baseline may be associated with lack of response to treatment and a poor outcome. Because expansion of these T cells may be related to disease status, further studies should investigate their potentially unique function in plasma cell dyscrasias.