抗人黑素瘤双特异性抗体增强LAK细胞毒效应的实验研究

抗肿瘤单克隆抗体的高度特异性与杀伤细胞对肿瘤的细胞毒效应相互结合起来，是增强ＬＡＫ细胞抗肿瘤作用的重要途径。本研究用化学连接法制备了既可识别淋巴细胞表面ＣＤ３抗原又可识别ＬｉＢｒ黑素瘤细胞表面肿瘤相关抗原的双特异性抗体ＣＤ３－ＨＢ８７５９。ＦＡＣＳ分析证实，ＣＤ３－ＨＢ８７５９具有结合两种抗原的活性。４ｈ５１Ｃｒ释放细胞毒实验结果表明，ＣＤ３－ＨＢ８７５９在诱导相可显著增强ＬＡＫ对ＬｉＢｒ细胞的细胞毒效应，也能使非ＬＡＫ细胞获得细胞毒性；在杀伤相加入ＣＤ３－ＨＢ８７５９对ＬＡＫ细胞的细胞毒作用也具有促进作用。上述结果表明，ＣＤ３－ＨＢ８７５９是一种抗人黑素瘤的双特异性抗体，为体内应用提供了良好的实验基础。     

Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies

We have previously described two human cold agglutinin MoAbs 216 and A6(H4C5), that are derived from the VH4-34 (VH4.21) gene that bind specifically to a cell surface ligand on human B lymphocytes. In this study, we report that binding of 216 and A6(H4C5) leads to rapid killing of target B cells. This complement-independent cytotoxicity was measured by three independent assays, cell viability dye uptake on FACS, ³H-thymidine uptake, and the 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was specific for CD20⁺ mononuclear cells in human spleen and peripheral blood. The MoAbs were also cytotoxic to human B cell lines Nalm-6, OCI-LY8, Arent and SUP-B8, but not to T cell lines HuT 78 and PEER. As observed by scanning electron microscopy, membrane pores were formed within 15 min of exposure to the MoAbs. Cytotoxic activity was dependent on MoAb concentration and temperature of exposure. Killing was greater at 4°C than 37°C. Sodium azide and EDTA did not block the cytotoxic activity. No DNA fragmentation typical of apoptosis was observed. This rapid cytotoxic activity, independent of physiologic cellular processes and independent of complement, suggests a novel mechanism of cell death via membrane perturbations.     

A 55,000 Mr surface antigen on activated human T lymphocytes defined by a monoclonal antibody

A T cell growth factor-dependent alloreactive human T cell line has been used to generate a monoclonal antibody B1.49.9 that reacts with an antigen present on most if not all mitogen or alloantigen activated T cells but not on resting T cells. The T lymphoblastoid cell line HUT-102 is also strongly reactive with B1.49.9 but all other T and non-T leukemia-lymphoma cell lines tested were negative. The B1.49.9 antigen is a glycoprotein of 55,000 Mr on mitogen or alloantigen activated T cells and 50,000 Mr on the cell line HUT-102. Pulse labeling experiments showed that a 40,000 Mr precursor (at approximately 0.7 h) which does not bind to ricin lectin precedes the appearance of the ricin-binding 55,000 Mr form. Comparisons of the monoclonal antibody anti-Tac, which recognizes the IL-2 receptor, to B1.49.9 suggest that B1.49.9 also recognizes a structure similar or identical to the IL-2 receptor.     

Preparation of anti-T idiotype monoclonal antibody reacting with human T leukaemic cell lines and with a small percentage of peripheral T lymphocytes.

Monoclonal antibody (MoAb) KT38 raised against a human T leukaemic cell line TALL-1, reacted with another T leukaemic cell line Jurkat, but not with any other cell lines tested. The co-modulation of CD3 and KT38 antigen was observed with stimulations of either MoAb T3 or KT38 on both TALL-1 and Jurkat. Upon radioimmunoprecipitation and SDS-PAGE analysis, MoAb KT38 precipitated the heterodimer of 40-60 kD from Jurkat and TALL-1 under reducing conditions. Thus, MoAb KT38 is considered to be an anti-T idiotype (Ti) antibody to TALL-1 and Jurkat cells. MoAb KT38 was also shown to react with a minor population of peripheral blood lymphocytes (PBL) and with very few cells (0.5-2.0%) in the paracortical area of the lymph node. When PBL were stimulated in a KT38-coated culture flask for 5 days, the percentage of KT38-positive PBL was markedly increased. The CD3 antigen on these cultured PBL in the flask was modulated by the stimulation with MoAb KT38. Thus, it is suggested that a common idiotope exists on the T cell receptor of Jurkat, TALL-1 and a small percentage (1.9-6.1%) of PBL.     

The bi-specific CD3 x NCAM antibody: a model to preactivate T cells prior to tumour cell lysis

To target the neural cell adhesion molecule (NCAM, CD56) on neuroblastoma by T cell-based immunotherapy we have generated a bi-specific CD3 x NCAM antibody (OE-1). This antibody can be used to redirect T cells to NCAM+ cells. Expectedly, the antibody binds specifically to NCAM+ neuroblastoma cells and CD3+ T cells. OE-1 induces T cell activation, expansion and effector function in peripheral blood mononuclear cell (PBMC)-derived CD4+ and CD8+ T cells. T cell activation was shown to depend on the presence of normal natural killer (NK) cells in the culture. Interestingly, while PBMC- derived T cells were activated by OE-1, NK cells were almost completely depleted, suggesting that T cells activated by OE-1 deleted the NK cells. Activated CD4+ and CD8+ T cells differentiate into a larger CCR7+ central memory and a smaller CCR7- effector memory cell population. Most importantly, preactivated T cells were highly cytotoxic for neuroblastoma cells. In eight of 11 experiments tumour-directed cytotoxicity was enhanced when NK cells were present during preactivation with OE-1. These data strongly support a bi-phasic therapeutic concept of primarily stimulating T cells with the bi-specific antibody in the presence of normal NCAM+ cells to induce T cell activation, migratory capacity and finally tumour cell lysis.     

抗人CD28激发性单抗对T细胞淋巴瘤的抑制作用研究

目的:研究鼠抗人CD28单克隆抗体(克隆号:2F5)对T淋巴细胞瘤的抗肿瘤效应,为抗体介导肿瘤靶向治疗提供新的方法和思路。方法:流式细胞术检测鼠抗人CD28单克隆抗体2F5对人T淋巴瘤细胞株Jurkat细胞表面膜型CD28分子的识别;MTT法检测2F5对Jurkat细胞体外增殖的影响;将Jurkat细胞株接种于裸鼠腋下,建立Jurkat细胞荷瘤裸鼠模型;经瘤内多点注射2F5(注射剂量1μg/mm3),逐日观察肿瘤的大小和裸鼠的生存状态。结果:2F5能够识别Jurkat细胞表面CD28分子,阳性结合率可达93.37%;2F5与Jurkat细胞共培养后,显微镜下观察可见胞质中出现黑色颗粒,胞膜破裂,细胞的折光性和立体感减弱;MTT法分析结果显示,抗CD28单抗对Jurkat细胞的体外增殖具有明显的抑制作用;将Jurkat细胞接种35只裸鼠(成瘤率达70%);经瘤内多点注射2F5,35天后荷瘤小鼠体内肿瘤的生长速率明显降低;而生理盐水对照组肿瘤生长无明显改变。结论:鼠抗人CD28单克隆抗体2F5通过与Jurkat细胞表面膜型CD28分子结合对其增殖具有明显的抑制作用,提示CD28分子可作为治疗T淋巴瘤的一个潜在靶点,抗体对治疗CD28分子阳性的肿瘤具有潜在临床应用价值。     

致敏树突状细胞活化细胞毒性T细胞抗肿瘤作用的研究

目的：研究树突状细胞（DCs）激活的细胞毒性T细胞的抗肿瘤及预防肿瘤发生的作用。 方法： 细胞因子诱生人PBMC未成熟DCs，加入肿瘤细胞抗原提取物致敏DCs产生成熟DCs；通过细胞形态、表面标记鉴定成熟DCs，MTT法测成熟DCs活化的细胞毒性T细胞(CTL)的体外杀伤活性；裸鼠体内注射活化CTL观察其抑制移植瘤生长及发生的作用。 结果： 经过7 d培养，获得大量形态典型、具有强烈刺激增殖能力、高表达CD80(63.5%)、CD83（67.6%）和CD3/ HLA-DR(83.2%)的DCs。其活化的CTL在20∶1效靶比时对抗原来源细胞株自身的杀伤率达75%以上，对同系细胞株的杀伤活性为35%-45%，对其它种系肿瘤细胞仅有微弱杀伤力（P<0.01）。CTL对裸鼠结肠癌HT-29移植瘤有特异性的生长抑制和预防生成作用（P<0.05）。CTL治疗组肿瘤组织中PCNA表达水平显著低于对照组（P<0.05）。 结论： 肿瘤细胞抗原活化的DC诱导CTL对肿瘤有特异性的杀伤作用，体内应用可特异性抑制移植结肠癌的生长或预防小鼠结肠癌移植瘤的发生。     

Activation of thymic T cells by MHC alloantigen requires syngeneic, activated CD4+ T cells and B cells as APC

We examine here the in vitro requirements to activate immunocompetent T cells, present among thymocytes, to give rise to CTL, CD4+ T cells producing IL-2 and CD8+ T cells producing IFN-gamma. These thymocytes are naive in not having received antigen-dependent signals characteristic of the periphery. Their activation, upon stimulation with allogeneic spleen cells depleted of T cells, referred to here as allogeneic antigen-presenting cells (APCs), to produce allo-MHC-specific effector T cells, requires activated (radiation resistant) CD4+ T cells, syngeneic with the responding thymocytes. We refer here to these T cells as 'help'. Furthermore, optimal T cell activation requires an Ig+ B220+ cell in the allogeneic APC population, most probably a B cell. The allogeneic APCs cannot be replaced by conventional bone marrow (BM)-derived dendritic cells (DCs) activated by CD40 ligation or exposure to LPS. The requirements for both help and allogeneic B cells in the activation of thymocytes contrast with the requirements to generate substantial responses from splenic T cell populations. Activated, BM-derived DCs stimulate substantial splenic responses without help. These different requirements for activation could reflect the fact that thymocytes have not received an exit-thymus signal and/or that splenic T cells are heterogeneous, containing naive, memory and partially-activated T cells.     

Identification of two distinct CD5- B cell subsets from human tonsils with different responses to CD40 monoclonal antibody

This study investigated the response of different CD5^? B cell subsets to CD40 monoclonal antibody (mAb) in various combinations with interleukin (IL)-4 or rabbit anti-human μ chain antibody (a-μ-Ab). The different CD5 B cell subsets were isolated from tonsillar B cell suspensions depleted of CD5⁺ B cells and subsequently fractionated on Percoll density gradients. While resting CD5⁺ B cells proliferated and produced IgM molecules in response to a-μ-Ab, IL-4 and CD40 mAb as well as to Staphylococcus aureus Cowan strain I (SAC) and IL-2, resting CD5^? B cells, which were co-purified in the same 60% Percoll fractions, consistently failed to respond. These cells were, however, activated by the stimuli employed, as demonstrated by their capacity to express the surface activation markers CD69, CD25 and CD71. Resting CD5⁺ B cells had the typical phenotype of mantle zone B cells (IgM⁺ IgD⁺ CD39+ CD38^? CD10^? CDw75^dim), whereas resting CD5^? B cells were CD38^? CD39^? CD 10^? CDw75 intermediate and expressed surface IgM but relatively little surface IgD and could not be classified as mantle zone or germinal center cells. The finding that purified germinal center cells (CD38⁺ CD10⁺ CD39^? CDw75^bright, IgG⁺) responded to CD40 mAb and IL-4 and also to SAC plus IL-2 further underlined the differences to resting CD5^? B cells. However, some of the data collected suggest possible relationships between CD5^? B cells and germinal center cells. The CD5^? B cells isolated from the 50 % Percoll fraction proliferated in response to a-μ-Ab, CD40 mAb and IL-4 as well as to SAC and IL-2. These cells had the same mantle zone B cell phenotype as the CD5⁺ B cells, but their capacity to respond to the stimuli in vitro was unrelated to a possible contamination with CD5⁺ B cells, as documented by the appropriate controls. Furthermore, upon exposure to SAC or phorbol esters, the large majority of CD5^? B cells from the 50 % Percoll fraction did not express surface CD5 and there was very little if any accumulation of CD5 mRNA. Finally, most of the cycling cells in the stimulated CD5^? B cells did not express CD5. The CD5^? B cells from the 50 % Percoll fraction were comprised of a consistent proportion of cells that expressed surface activation markers. The removal of these cells abrogated the capacity of the suspensions to respond to the stimuli in vitro, possibly suggesting that these cells additional activation signals in vivo which were essential to acquire the capacity to respond and that could not be reproduced in vitro. The present study underlines the phenotypic and functional heterogeneity of CD5^? B cells and contributes to the identification of two subsets of these cells which differ in phenotype, tissue distribution and in vitro responses to different stimuli.     

Human double-negative (CD4-CD8-) T cells bearing alpha beta T cell receptor possess both helper and cytotoxic activities.

Expression of CD4 or CD8 on the cell surface is an important guide for discriminating the immunological functions of T cells. However, a minor T cell subset, which lacks both CD4 and CD8 molecules but bears the usual form of T cell receptor (TCR) alpha beta (CD4-CD8-TCR alpha beta+ T cells), has recently been found not only in mice but also in humans, and its role in immune response is now of considerable interest. In order to clarify the characteristics of this newly defined T cell subpopulation, we established five IL-2-dependent CD4-CD8-TCR alpha beta+ T cell clones from the peripheral blood of a healthy individual, and examined their various biological functions. It was found that all clones not only helped B cells in immunoglobulin production, but also exerted major histocompatibility complex-unrestricted cytotoxicity. Although their CD3/TCR complexes were functionally competent, the cytotoxicity seemed to be mediated via unknown molecules other than the CD3/TCR complex, as evidenced by the failure of CD3 MoAb to inhibit the cytotoxic activity. Our present findings showed that CD4-CD8-TCR alpha beta+ T cells possess potential bifunction, i.e. helper and cytotoxic activities. Their roles in the pathogenesis of immunodeficiency are discussed.     

CD45 expression by murine B cells and T cells: Alteration of CD45 isoforms in subpopulations of activated B cells

The CD45 family of high molecular weight cell surface glycoproteins is abundantly expressed by virtually all hematopoietic cells. CD45 molecules exist as multiple isoforms whose extracellular portions vary in protein structure and carbohydrate content but whose intracellular portions are highly conserved and possess tyrosine phosphatase activity. In this review we summarize current studies describing CD45 isoform expression on peripheral and thymic lymphocytes. Further, we analyze changes in CD45 isoform expression by selective populations of activated B cells.     

The expression analysis of ICOS-L on activated T cells and immature dendritic cells as well as malignant B cells and Grave's-disease-derived thyroid tissues by two novel mAbs against human ICOS-L

ICOS-L, a newly identified member of B7 superfamily, plays an important role in immune responses. In this article, we report on two novel mouse anti-human ICOS-L monoclonal antibodies (mAbs) named as 11C4 and 12B11, whose specificities were verified by methods of flow cytometry, western blotting, and epitope competition assay. The two mAbs bound to distinct ICOS-L epitopes on B cells. Interestingly, mAb 11C4 could well recognize ICOS-L molecule on activated T cells and Jurkat cell lines, which is different from commercial anti-ICOS-L mAb (clone number MIH12) and the other mAb 12B11. In addition, we found that the expression of ICOS-L molecule was only detected on the surface of immature monocyte-derived dendritic cells (Mo-DCs) and was sharply decreased after induction of mature Mo-DCs activated by tumor necrosis factor-alpha or CD40. Furthermore, we showed that 11C4 could effectively suppress the maturation of Mo-DCs in vitro as evidenced by the low expression of CD80, CD86, CD83, and human leukocyte antigen-DR, which suggested that ICOS-L may be involved in the maturation of Mo-DCs. Using immunohistochemistry staining with mAb 11C4, the expression of ICOS-L was found in B lymphoma tissues and thyroid tissues from the Grave's disease but not in thyroid adenoma and normal thyroid tissues.     

Characterization of two monoclonal antibodies (UCL4D12 and UCL3D3) that discriminate between human mantle zone and marginal zone B cells.

Two new monoclonal antibodies (MoAbs), UCL3D3 and UCL4D12 were obtained following immunization with follicular lymphoma (UCL3D3) or low-grade primary B cell gastric lymphoma cells (UCL4D12). In normal splenic white pulp, tonsil and small intestinal Peyer's patches, UCL4D12 recognizes marginal zone B cells and a subpopulation of follicle centre cells, whereas mantle zone B cells are UCL4D12 negative. In contrast, UCL3D3 recognizes mantle zone B cells and follicular dendritic cells, but not marginal zone B cells or follicle centre B cells. Double-immunofluorescence studies showed that in the splenic white pulp, these antibodies stain reciprocally. The majority of UCL3D3+ cells are sIgM+ and sIgD+ whereas a higher proportion of UCL4D12+ cells express surface IgM (sIgM) but not surface IgD (sIgD). Less than 10% of splenic B cells express both 3D3 and 4D12 antigens. None of the cell lines tested expressed either antigen. Functional studies showed that both antigens play a role in B cell activation as the MoAbs increase the mitogenic effect of Staphylococcus aureus Cowan I on tonsil B cells. This effect was maximal at 72 h in culture. TPA activation was reduced, and no effect was observed with anti-immunoglobulin (anti mu) or CDw40 (G28.5). UCL3D3 and UCL4D12 did not show any stimulatory effect on their own. Biochemical studies show that both MoAbs recognize proteins of 80-90 kD under reducing conditions. These two MoAbs appear to recognize new B cell surface antigens which may be useful for identifying subpopulations of B cells.     

T cell lymphoblastic leukaemia/lymphoma associated with a microenvironment of thymic asteroid B cells in the bone marrow

Naresh K N, May P C, Reid A G, Marks A J, Macdonald D & Kanfer E
(2010) Histopathology 57, 549–554
T cell lymphoblastic leukaemia/lymphoma associated with a microenvironment of thymic asteroid B cells in the bone marrow Aims: Asteroid B cells are a component of normal thymus. It is currently unclear whether these cells are identifiable in T cell lymphoblastic leukaemia/lymphoma (T‐ALL/LBL) of the thymus. The aim of this study was to identify asteroid B cells both in thymic and extrathymic tissue involved by T‐ALL/LBL. Methods and results: Thymic, lymph node (LN) and bone marrow trephine biopsy (BMTB) samples from eight patients with T‐ALL/LBL were reviewed. All had been investigated by immunohistochemistry and one by fluorescent in situ hybridization (FISH). The BMTB samples of two of eight T‐ALL/LBLs and LN sample in one of them showed the presence of asteroid‐shaped B cells with dendritic cytoplasmic processes. These B cells also expressed CD23 and the features were akin to the unique thymic asteroid B cells. Both patients had aggressive/resistant disease. Cytogenetic analysis in one showed a complex translocation involving the T cell receptor beta (TCRB) gene at 7q35 and a distal region of 9q known to harbour the NOTCH1 gene. Conclusion: This is the first report of T‐ALL/LBL documenting the presence of an asteroid B cell‐rich microenvironment at bone marrow and LN sites. In this small subset, T‐ALL/LBL cells are possibly dependent upon asteroid B cells, and whether targeting of asteroid B cells with anti‐CD20 monoclonal antibody in such cases will result in clinical benefit remains to be determined.     

Specific activation of resting T cells against tumour cells by bispecific antibodies and CD28-mediated costimulation is accompanied by Th1 differentiation and recruitment of MHC-independent cytotoxicity

Specific activation of resting lymphocytes for tumour targeting can be achieved by bispecific monoclonal antibodies (bi-MoAbs) with specificity for tumour antigens and T cell-activating antigens in combination with a costimulatory anti-CD28 antibody. In this study we focus on the immunomodulatory function of an anti-CD3/CA19-9 bi-MoAb in combination with a costimulatory anti-CD28 antibody which may result not only in antigen-specific, T cell-mediated tumour cell lysis but also in recruitment of other cellular effector functions. In combination with costimulatory anti-CD28 antibodies, resting peripheral lymphocytes could be activated specifically to secrete high amounts of Th1 cytokines (IL-2, interferon-gamma (IFN-γ)) characterizing a cellular immune response. In contrast, no IL-4 and only low amounts of IL-10 could be detected. Furthermore, bi-MoAb-mediated CA19-9-specific activation of T cells was accompanied by recruitment of MHC- and CA19-9-independent cytotoxicity, as was determined by lysis of different CA19-9⁻cell lines. This MHC-independent cytotxicity was mediated at least in part by activated natural killer (NK) cells, as depletion of CD16⁺ NK cells resulted in substantial decrease of cytotoxicity against CA19-9⁻ targets. Our results indicate that specific activation of resting T cells with CD3-associated bi-MoAbs in combination with an anti-CD28 antibody leads to a Th1 differentiation pathway and is accompanied by recruitment of MHC-independent lymphokine-activated killer (LAK) cell cytotoxicity which can possibly be directed against a heterogeneous tumour.     

B7/BB1, the ligand for CD28, is expressed on repeatedly activated human T cells in vitro

The activation of T cells is now thought to require at least two distinct signals. One signal is delivered through the interaction of the antigen-specific T cell receptor with major histocompatibility complex (MHC) molecules and peptide, while the other is received from interactions with less precisely defined accessory or costimulatory molecules. In the absence of this second costimulatory signal, some T cells subsequently become unresponsive to antigenic stimulation. One of the major candidates for providing such a second signal to T cells is the molecule B7 interacting with the T cell glycoprotein CD28. In the present study we have investigated whether B7 is expressed on human T cell lines and clones, since these cells have the capacity to present antigen to each other by expressing MHC class II molecules. Our results demonstrate that B7 can be detected on T cell clones and on repeatedly activated but not freshly isolated peripheral blood T cells. The expression of B7 is dependent on the state of activation of the cells, being maximally expressed shortly after restimulation and becoming undetectable as the cells quiesce. Together, these results suggest that B7 expression may be of importance to T cells, perhaps in the avoidance of anergy.     

Expansion of CD4+ T cells with a cytotoxic phenotype in patients with B-chronic lymphocytic leukaemia (B-CLL).

Abnormal CD4/CD8 ratios and T-cell function have previously been shown in patients with B-chronic lymphocytic leukaemia (B-CLL). We have demonstrated that CD4+ T cells containing both serine esterase and perforin (PF) are increased in the blood of these patients. Using flow cytometry, we have shown that the CD4+ PF+ cells were CD57+ but lacked expression of CD28, suggesting a mature population. The same phenotype in CD8+ T cells is characteristic of mature cytotoxic T cells. However, in contrast to the CD8+ T cells, the CD4+ T cells were more frequently CD45RO positive than CD45RA positive, indicating prior antigen experience. In contrast, this population lacked expression of either CD69 or HLA-DR, arguing that they were not activated or that they are an abnormal population of T cells. Their constitutive cytokine levels showed them mainly to contain IL4 and not IFNgamma, suggesting a Th2 phenotype. The role of the CD4+ PF+ T-cell population is at present uncertain. However, this potentially cytotoxic T-cell population could contribute both to enhancing survival of the B-CLL tumour cells through production of IL4, and to the immunodeficient state frequently seen in patients with this tumour, independent of drug treatment.     

Differential staining of human alpha beta and gamma delta T cells by the fluorescein conjugate of an anti-CD3 monoclonal antibody.

The enumeration of total T cells, an important function of the clinical immunology laboratory, utilizes antibodies to CD3, the macromolecular complex associated with the antigen-specific receptors of T cells. We compared the ability of some commonly employed commercial anti-CD3 reagents to stain human peripheral blood lymphocytes. Surprisingly, the fluorescein isothiocyanate (FITC) conjugate of Coulter clone T3 (FITC-T3) stained most T cells brightly, but selectively stained gamma delta T cells very dimly or not at all. In contrast, the other anti-CD3 reagents studied (FITC-Leu 4, PE-T3, PE-Leu 4, and indirectly labelled T3 and Leu 4) stained all T cells equivalently. Dual-colour flow cytometric analysis with FITC-T3 and PE-Leu 4 readily demonstrated a FITC-T3-/PE-Leu 4+ population of T cells. This unique population stained dimly or not at all with a combination of anti-CD4 and anti-CD8 monoclonal antibodies and positively with the pan-gamma delta T cell antibody TCR delta 1. Moreover, an excellent correlation was found between the number of FITC-T3-/PE-Leu 4+ cells and the number of TCR delta 1+ cells in 32 normal individuals. Thus, the FITC-T3-/PE-Leu 4+ phenotype accurately marks all gamma delta T cells. In contrast to FITC-T3, both PE-conjugated and unconjugated T3 stained gamma delta T cells brightly. Therefore, T3 binds to an epitope present on all T cells, but fluoresceinylation specifically attenuates this antibody's ability to bind to gamma delta T cells. These findings indicate that the use of FITC-T3 can result in a significant and variable underestimation of peripheral blood T cell number and demonstrate further that the CD3 complexes of human alpha beta and gamma delta T cells are significantly different.