SC3 monoclonal antibody defines a novel specific human B-cell surface antigen differentially expressed on B-cell leukaemias and lymphomas and involved in the proliferation of normal and malignant B lymphocytes

The monoclonal antibody SC3 was raised against the NK leukaemia cell line YTindi. It detected a 98-kDa surface antigen with weak expression on a restricted number of leukaemia cell lines under reducing conditions. SC3 mAb labelled 5-10% of normal peripheral blood lymphocytes corresponding almost exclusively to B lymphocytes, and 60-70% of tonsillar B cells. It did not react with erythrocytes, platelets or monocytes whereas it stained granulocytes. The aim of the present study was to examine the expression and functional effects of SC3 mAb reactive epitope on normal and malignant B cells. Most SC3+ B cells from healthy donors were CD23+, some co-expressed CD5 and CD27 and a few were CD38+. SC3 epitope was expressed exclusively by B-lineage malignant proliferations, including B-lineage ALL. Practically, all B-CLL studied expressed SC3 mAb reactive epitope although with variable intensity, while MCL and PLL were negative. Other low grade and high grade B-NHL were variably stained. SC3 mAb alone triggered the proliferation of CD2-depleted PBL and significantly increased the proliferation induced by suboptimal concentrations of LPS. This effect was much weaker with B-CLL cells but was increased after cross-linking with an anti-IgM antibody. The restricted expression pattern combined with molecular weight and functional data indicate that SC3 mAb may detect a novel B-cell antigen mostly expressed by early and naive B cells. Although its expression in B-cell malignancies was not limited to a single differentiation stage, it might confer specific functional characteristics to the positive malignant cells.     

Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR⁺ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19⁺ tumor targets. This clone can be used to detect CD19-specific CAR⁺ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR⁺ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.     

CD20-specific chimeric antigen receptor-expressing T cells as salvage therapy in rituximab-refractory/relapsed B-cell non-Hodgkin lymphoma

Background aimsThe infusion of chimeric antigen receptor (CAR) T cells that target specific tumor-associated antigens is a promising strategy that has exhibited encouraging results in clinical trials. However, few studies have focused on the effectiveness and safety of CD20 CAR T cells in rituximab-refractory/relapsed (R/R) B-cell non-Hodgkin lymphoma (B-NHL) patients, particularly those treated with rituximab for a short time. This prospective study aimed to assess the effectiveness and toxicity of CD20 CAR T cells in R/R B-NHL patients previously treated with rituximab.MethodsThe authors conducted a prospective, single-center phase I study on the effectiveness and toxicity of CD20 CAR T cells in rituximab-treated R/R B-NHL patients (no. ChiCTR2000036350). A total of 15 patients with R/R B-NHL were enrolled between November 21, 2017, and December 1, 2021.ResultsAn overall response rate of 100% was shown in enrolled patients, with 12 (80%) achieving complete remission and three (20%) achieving partial remission for the best response. The median follow-up time was 12.4 months. Progression-free survival and overall survival were not yet reached by the data cutoff day. No patient developed grade 4 cytokine release syndrome, and only one patient had immune effector cell-associated neurotoxicity syndrome.ConclusionsAll enrolled B-NHL patients who were previously R/R to rituximab achieved different degrees of clinical response with tolerable toxicities. Notably, patients who had received rituximab within 3 months had a poorer prognosis.     

Suppression in murine experimental autoimmune thyroiditis: in vivo inhibition of CD4+ T cell-mediated resistance by a nondepleting rat CD4 monoclonal antibody.

Genetically susceptible mice become resistant to experimental autoimmune thyroiditis (EAT) induction with mouse thyroglobulin (MTg) and lipopolysaccharide after pretreatment with deaggregated MTg (dMTg). Recent work showed this suppression to be mediated by CD4+ suppressor T cells (Ts). To study Ts action in vivo, we used a rat IgG2a monoclonal antibody (mAb), YTS 177.9, which modulates CD4 antigen in vivo without depleting CD4+ cells. Initial studies showed that after two 1-mg doses of mAb 7 days apart, extensive CD4 antigen modulation of peripheral blood leukocytes occurred within 4 days. Mice given CD4 mAb 24 hr before dMTg (2 doses, 7 days apart) were resistant to EAT induction when immunized with MTg and LPS 20 days later. Also, anti-rat IgG2a titers were reduced following challenge with heat-aggregated rat IgG2a compared to controls. Subsequent analysis of serum in CD4 mAb-treated animals revealed that mAb was present in the circulation for 14 days. Moreover, mice given CD4 mAb and dMTg, then challenged after only 10 days, when CD4 mAb was still circulating, developed a significantly higher incidence of thyroid damage than controls. These findings suggest that modulation of CD4 antigen does not interfere with Ts activation, but the presence of CD4 mAb, at the time of autoantigenic challenge, can interfere with tolerance to EAT induction. Thus, the direct relationship between the presence of CD4 mAb and inhibition of EAT suppression implicates a role for CD4 molecules in the mediation of suppression.     

Transfection of the CD20 cell surface molecule into ectopic cell types generates a Ca2+ conductance found constitutively in B lymphocytes

CD20 is a plasma membrane phosphoprotein expressed exclusively by B lymphocytes. mAb binding to CD20 alters cell cycle progression and differentiation, indicating that CD20 plays an essential role in B lymphocyte function. Whole-cell patch clamp and fluorescence microscopy measurements of plasma membrane ionic conductance and cytosolic-free Ca2+ activity, respectively, were used to directly examine CD20 function. Transfection of human T and mouse pre-B lymphoblastoid cell lines with CD20 cDNA and subsequent stable expression of CD20 specifically increased transmembrane Ca2+ conductance. Transfection of CD20 cDNA and subsequent expression of CD20 in nonlymphoid cells (human K562 erythroleukemia cells and mouse NIH-3T3 fibroblasts) also induced the expression of an identical transmembrane Ca2+ conductance. The binding of a CD20-specific mAb to CD20+ lymphoblastoid cells also enhanced the transmembrane Ca2+ conductance. The mAb-enhanced Ca2+ currents had the same conductance characteristics as the CD20- associated Ca2+ currents in CD20 cDNA-transfected cells. C20 is structurally similar to several ion channels; each CD20 monomer possesses four membrane spanning domains, and both the amino and carboxy termini reside within the cytoplasm. Biochemical cross-linking of cell-surface molecules with subsequent immunoprecipitation analysis of CD20 suggests that CD20 may be present as a multimeric oligomer within the membrane, as occurs with several known membrane channels. Taken together, these findings indicate that CD20 directly regulates transmembrane Ca2+ conductance in B lymphocytes, and suggest that multimeric complexes of CD20 may form Ca2+ conductive ion channels in the plasma membrane of B lymphoid cells.     

甘草酸二铵对于HCV相关性B-NHLCD25-和CD25+T细胞增殖影响研究

目的:本研究初步探究甘草酸二铵(Diammonium Glycyrrihizinate,DG)对HCV相关性B细胞非霍奇金淋巴瘤(B-cellnon-Hodgkin's lymphoma,B-NHL)CD25-T细胞、CD25+T细胞的免疫调控作用。方法:应用流式细胞分析仪检测HCV相关性B-NHL CD4+CD25+T细胞占CD4+T细胞的比例、CD25+细胞占总PBMC比例,并与单纯HCV感染患者、健康人检测结果相对照。应用免疫磁珠分离法(MACS)分选获得CD25-和CD25+细胞,将两者和未分选的外周血单个核细胞(peripheral blood mononuclearcell,PBMC)以CFSE标染孵育72小时后,应用流式细胞分析仪将APC CD3阳性细胞设定为门检测CD25-T细胞、未分选T细胞、CD25+T细胞的增殖情况,及甘草酸二铵干预后的CD25-T细胞、CD25+T细胞增殖情况。结果:应用流式细胞分析仪检测健康人、单纯HCV感染者和HCV相关性B-NHL患者在CD4+CD25+占总CD4+细胞比例和CD25+占总细胞比例上均呈现逐步递增的关系(分别为33.94%±2.18%,57.95%...     

Identification and characterization of a hamster monoclonal antibody anti-2.28 directed against a 70-kilodalton activation antigen on human monocytes

Hamster mAb against activated human monocytes were examined for their reactivities against monocyte activation Ag. One mAb, anti-2.28, stained only monocytes activated with LPS plus IFN-gamma, but not unactivated peripheral blood monocytes, polymorphonuclear leukocytes, lymphocytes, RBC, and platelets. However, it stained peripheral blood T cells activated with PMA plus anti-CD3 and peripheral blood and tonsillar B cells activated with PMA plus anti-mu. Of the 35 cell lines of diverse origin examined for immunofluorescence staining by anti-2.28, only EBV-transformed cell lines showed strong staining by this mAb. One pre-B cell line, Nalm-12, could be induced by PMA to exhibit intermediate staining. Immunoprecipitation studies identified the 2.28 Ag as a 70- to 85-kDa monomer. Immunofluorescence staining, immunoprecipitation, and peptide mapping studies indicated that 2.28 was different from a number of monocyte and lymphocyte surface Ag including Mo3e, B-4 (CD19), B-5, CD39, and the G28-8 Ag Bgp 95. These studies suggest that 2.28 may be a novel hemopoietic non-lineage-specific activation Ag.     

CD19-CD21 complex regulates an intrinsic Src family kinase amplification loop that links innate immunity with B-lymphocyte intracellular calcium responses

CD19 is a B-lymphocyte cell surface molecule that functions as a general response regulator or rheostat, which defines intrinsic and B-cell antigen receptor-induced signalling thresholds that are critical for humoral immunity and expansion of the peripheral B-cell pool. In addition, B-cell responses are influenced by signals transduced through a CD19-CD21 cell surface receptor complex, where the binding of complement C3d to CD21 links humoral immune responses with the innate immune system. This review outlines recent biochemical and genetic studies that characterize the signal transduction pathways utilized by this receptor complex to regulate B-cell intracellular calcium responses.     

B-cell subpopulations in humans and their differential susceptibility to depletionwith anti-CD20 monoclonal antibodies

In humans, different B-cell subpopulations can be distinguished in peripheral bloodand other tissues on the basis of differential expression of various surface markers.These different subsets correspond to different stages of maturation, activation anddifferentiation. B-cell depletion therapy based on rituximab, an anti-CD20 mAb, iswidely used in the treatment of various malignant and autoimmune diseases. Rituximabinduces a very significant depletion of B-cell subpopulations in the peripheral bloodusually for a period of 6 to 9 months after one cycle of therapy. Cells detectedcirculating during depletion are mainly CD20 negative plasmablasts. Data on depletionof CD20-expressing B cells in solid tissues are limited but show that depletion issignificant but not complete, with bone marrow and spleen being more easily depletedthan lymph nodes. Factors influencing depletion are thought to include not only thetotal drug dose administered and distribution into various tissues, but also B-cellintrinsic and microenvironment factors influencing recruitment of effector mechanismsand antigen and effector modulation. Available studies show that the degree ofdepletion varies between individuals, even if treated with the same dose, but that ittends to be consistent in the same individual. This suggests that individual factorsare important in determining the final extent of depletion.     

Morphological and immunological changes of hairy cell leukemia during alpha-2-interferon therapy

We describe a patient who presented with the clinical picture of hairy cell leukemia (HCL). Bone marrow and peripheral blood lymphoma cells showed morphological and immunological features of HCL. Under recombinant alpha-2-interferon (alpha-2-IF) therapy the characteristic morphology changed from HCL to prolymphocytic leukemia (PLL). At diagnosis the lymphoma cells expressed CD24 and FMC7 surface antigen, but stained negative for surface immunoglobulins, light chains and anti-CD5. During alpha-2-IF treatment surface antigen expression changed to CD24, CD5 and FMC7. Surface IgD and lambda light chains became strongly positive. Southern Blot analysis of peripheral blood mononuclear cells showed two rearranged immunoglobulin bands at diagnosis but only one upon alpha-2-IF therapy. These data suggest, that this patient suffered from a biclonal lymphoma, HCL and PLL. While undergoing alpha-2-IF treatment the HCL came into remission, whereas the PLL clone proved to be poorly sensitive to alpha-2-IF therapy.     

Isolation and characterization of a monoclonal antibody that inhibits HIV-1 infection

To identify a cell surface molecule other than CD4 involved in infection of cultured cells with human immunodeficiency virus type 1 (HIV-1), mice were immunized with the CD4-negative Raji human B-cell line in order to isolate a monoclonal antibody (mAb). We isolated mAb 33A, which inhibited the infection of CD4-positive T cells, B cells, human peripheral blood lymphocytes (PBL), and brain-derived cells with HIV-1. Formation of viral DNA was also blocked when CD4-positive Raji cells were treated with 33A after adsorption of HIV-1, but not before its adsorption. mAb 33A had little effect on syncytium formation induced by cocultivation with HIV-1-producing cells. Flow cytometry revealed that 33A reacted with HTLV-I-positive T-cell lines, Burkitt's lymphoma cell lines, phytohemagglutinin (PHA) -stimulated PBL, brain-derived fibroblast-like cells, and some adherent cell lines, but hardly at all with immature T-cell lines. Immunoblotting experiments showed that 33A recognized an antigen with an apparent molecular mass of 32 kDa, but did not recognize chemokine receptors such as CXCR4, CCR5, or CCR3. The distribution characteristic of the antigen recognized by 33A on various cells and its molecular weight suggest that mAb 33A recognizes a new cellular antigen that is necessary for HIV-1 entry.     

Differentially regulated expression and function of CD22 in activated B-1 and B-2 lymphocytes

CD22 is a B cell-restricted transmembrane protein that apparently controls signal transduction thresholds initiated through the B cell Ag receptor (BCR) in response to Ag. However, it is still poorly understood how the expression of CD22 is regulated in B cells after their activation. Here we show that the expression levels of CD22 in conventional B-2 cells are markedly down-regulated after cross-linking of BCR with anti-IgM mAb but are up-regulated after stimulation with LPS, anti-CD40 mAb, or IL-4. In contrast, treatment with anti-IgM mAb barely modulated the expression levels of CD22 in CD5(+) B-1 cells, consistent with a weak Ca(2+) response in anti-IgM-treated CD5(+) B-1 cells. Moreover, in CD22-deficient mice, anti-IgM treatment did not trigger enhanced Ca(2+) influx in CD5(+) B-1 cells, unlike CD22-deficient splenic B-2 cells, suggesting a relatively limited role of CD22 in BCR signaling in B-1 cells. In contrast, CD22 levels were markedly down-regulated on wild-type B-1 cells in response to LPS or unmethylated CpG-containing oligodeoxynucleotides. These data indicate that the expression and function of CD22 are differentially regulated in B-1 and conventional B-2 cells, which are apparently implicated in innate and adaptive immunity, respectively.     

HCV-associated lymphomas

Epidemiologic and molecular observations have recently suggested that hepatitis C virus (HCV) may be the causative agent of some B-cell non-Hodgkin lymphomas (B-NHL). Epidemiologic data suggest that in Italy about 5% of B-NHL are caused by HCV. Molecular data indicate a close relationship between HCV-associated B-NHL and type II mixed cryoglobulinemia. The latter disorder appears to reflect the benign monoclonal proliferation of B cells expressing a specific cross-reactive idiotype, known as WA, that may recognize an antigen of HCV, perhaps the E2 protein. Genetic abnormalities occurring during this phase of antigen-induced clonal expansion may drive the neoplastic transformation into low- or high-grade lymphoma. The recent demonstration that splenic B cell lymphomas associated with HCV-infection may regress after successful antiviral therapy confirms a role for this virus in B-cell lymphomagenesis.     

甘草酸二铵对于HCV相关性B—NHLCD25-和CD25＋T细胞增殖影响研究

目的：本研究初步探究甘草酸二铵（Diammonium Glycyrrihizinate,DG）对HCV相关性B细胞非霍奇金淋巴瘤（B-cellnon-Hodgkin-Slymphoma,B-NHL）CD25-T细胞、CD25＋T细胞的免疫调控作用。方法：应用流式细胞分析仪检测HCV相关性B-NHLCD4＋CD25＋T细胞占CD4＋1r细胞的比例、CD25＋细胞占总PBMC比例,并与单纯HcV感染患者、健康人检测结果相对照。应用免疫磁珠分离法（MACS）分选获得CD25-和CD25＋细胞,将两者和未分选的外周血单个核细胞（peripheralblood mononuclear cell。PBMC）以CFSE标染孵育72小时后,应用流式细胞分析仪将APCCD3阳性细胞设定为门检测CD25T细胞、未分选T细胞、CD25＋T细胞的增殖情况,及甘草酸二铵干预后的CD25-T细胞、CD25＋T细胞增殖情况。结果：应用流式细胞分析仪检测健康人、单纯HCV感染者和HCV相关性B—NHL患者在CD4＋CD25＋占总CD4＋细胞比例和CD25＋占总细胞比例上均呈现逐步递增的关系（分别为33．94％±2．18％,57．95％±1．77％,70．24％±12．75％,P〈0．05;18．16％±2．23％,33．45％±1132％,54.69％±8．66％,P〈0．05）。CFSE标染后孵育72小时检测（MI＋M2）分裂相百分比呈现CD25-T细胞最高、未分选T细胞其次、CD2YT细胞最低的表现（分别为74．4％±1．2％,63．1％±5．4％,47．2％±5．9％,P〈0．05）。甘草酸二铵干预后CD25-T细胞较未干预CD25T细胞M1分裂相百分比增加（分别为57．7％±4．2％,46．5％±5．6％,P〈0．05）。甘草酸二铵干预后CD25＋T细胞较未干预cD25＋1r细胞M1分裂相百分比减少（分别为14．7％±1．3％,22．1％±4．1％,P〈0．05）。结论：HCV相关性B-NHLCIM＋CD25＋、CD25＋细胞百分比较单纯HCV感染者、健康人明显增加,提示存在T细胞免疫抑制,且抑制程度重于单纯HCV感染。去除CD25＋细胞后CD25T细胞增殖较未去除CD25＋的T细胞增殖活跃,说明HCV相关性B-NHL患者的CD25＋细胞能够抑制CD25-T细胞增殖。而DG干预后HCV相关性B-NHLCD25＋T细胞增殖M1分裂相减少,而CD25T细胞增殖M1分裂相增加,表明DG对HCV相关性B-NHL具有抑制CD25＋T细胞增殖而促进CD25-T细胞增殖的积极免疫调节作用。     

The role of protein kinase C in transmembrane signaling by the T cell antigen receptor complex. Effects of stimulation with soluble or immobilized CD3 antibodies

Phorbol esters, such as phorbol myristate acetate (PMA), are known to be potent co-stimulants with calcium ionophores for activation of T lymphocytes. The most extensively studied intracellular effect of PMA is its ability to activate the cytoplasmic enzyme protein kinase C (pkC). Herein, we examined the role of pkC activation during T cell activation. During physiologic activation, this enzyme is activated by diacylglycerol which is generated through the hydrolysis of polyphosphoinositides. Therefore, we studied the activation of T lymphocytes induced by a synthetic diacylglycerol, dioctanoylglycerol. In contrast to PMA, this compound can be metabolized in T cells and presumably more closely mimics physiologic activation of pkC. Dioctanoylglycerol together with reagents that induce increases in intracellular free Ca2+ concentration, Ca2+ ionophores, or anti-cluster designation (CD)3 monoclonal antibodies (mAb) were able to induce interleukin 2 receptor expression and proliferation of T lymphocytes. Previous studies have demonstrated that the stimulation of T cells via the CD3/T cell antigen receptor complex by mAb against CD3 leads to an increase in cytoplasmic free Ca2+ and to an activation of pkC. Paradoxically, however, soluble CD3 antibodies do not cause proliferation of resting purified T cells. Inasmuch as immobilization of CD3 mAb has been shown to influence the agonist properties of such antibodies, we compared the ability of soluble and immobilized CD3 mAb to activate pkC. We demonstrated herein that soluble CD3 mAb cause only a very transient activation of pkC in the T cell leukemic line Jurkat. This pkC activation is markedly prolonged when Jurkat cells are stimulated with immobilized rather than soluble CD3 antibodies. These studies suggest that activation of pkC plays a major role in T cell activation and that the activation of pkC is influenced by the form in which CD3 mAb is presented to T cells.     

Normal development but differentially altered proliferative responses of lymphocytes in mice lacking CD81.

CD81 (TAPA-1) is a member of the transmembrane 4 superfamily (TM4SF) which is expressed on the cell surface of most cells of the body throughout their cellular differentiation. It has been recognized in several cell surface complexes of lymphocytes, suggesting that it may have diverse roles in lymphocyte development and activation regulation. Mice with a CD81 null mutation revealed normal T- and conventional B-cell development, although CD19 expression on B cells was dull and B-1 cells were reduced in number. However, both T and B cells of mutant mice exhibited strikingly enhanced proliferation in response to various types of stimuli. Interestingly, while proliferative responses of T cells following T-cell antigen receptor (TCR) engagement was enhanced in the absence of CD81, B-cell proliferation in response to B-cell antigen-receptor (BCR) cross-linking was severely impaired. Despite these altered proliferative responses, both tyrosine phosphorylation and intracellular calcium flux in response to cross-linking of cell surface antigen receptors were normal in mutant mice, reflecting apparently normal initial signaling of antigen receptors. In conclusion, though CD81 is not essential for normal T- and conventional B-cell development, it plays key roles in controlling lymphocyte homeostasis by regulating lymphocyte proliferation in distinct manners, dependent on the context of stimulation.     

CD20生物学功能的研究进展

B细胞抗原受体（BCR）信号传导起始于持续的钙离子向细胞内流动,这种钙离子的内流对于B细胞的生长、分化、活化是必需的。CD20是B细胞膜上特有的4次跨膜蛋白,参与了BCR活化的钙离子流入。最近的研究提供了直接的证据,证明CD20形成的同源寡聚体是四聚体。CD20单抗诱导的钙信号也得到研究,研究表明只有Ⅰ型CD20单抗能引起钙离子内流。CD20还通过钙池调控钙离子进入（SOCE）参与了细胞信号传导。我们就CD20形成同源寡聚体、与BCR的相互作用、参与调节B淋巴细胞钙离子的流动等进行简要综述。     

The CD2 ligand LFA-3 activates T cells but depends on the expression and function of the antigen receptor

The T cell Ag receptor (CD3/Ti) and the sheep E receptor (CD2) expressed on the surface of human T cells are both capable of initiating intracellular signals necessary for T cell activation. CD3/Ti interacts with Ag to initiate cellular immune responses. Although the exact function of CD2 is unknown, lymphocyte function-associated Ag 3 (LFA-3), a 55- to 70-kDa receptor expressed on a broad spectrum of hemopoietic and nonhemopoietic cells, has recently been shown to be its natural ligand. We show here that although purified multimeric LFA-3 is not capable of initiating transmembrane signaling events on its own, the combination of LFA-3 and the anti-CD2 mAb CD2.1 induces intracellular calcium increases, phosphatidylinositol second messenger generation and lymphokine secretion in the T cell leukemic line Jurkat. In order to study the signaling requirements of CD2, we compared the ability of CD2 mAb and LFA-3 to initiate activation signals in Jurkat and in three Jurkat-derived mutants. A CD3-CD2+ mutant failed to increase calcium or exhibit phosphatidylinositol hydrolysis to either the combination of agonist CD2 mAb 9-1 and 9.6 or LFA-3 and CD2.1. Reconstitution of the Ag receptor by transfection of the Ti-beta-chain restored the expression of the CD3/Ti complex and the ability to respond to either combination of CD2 ligands. However, no response to CD2 ligands was detected in a CD3+CD2+ mutant selected for signaling defects to CD3/Ti ligands. Complementation of the CD3/Ti signaling defect by cell fusion also restored competency to respond to CD2 agonists. These results demonstrate that LFA-3 under appropriate conditions can activate T cells via the CD2 complex and that this activation requires not only the cell surface expression of the CD3/Ti complex but also a functional Ag receptor pathway.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Association of human lymph node homing receptor (Leu 8) with the TCR/CD3 complex.

Cross-linking of the human homologue of the murine MEL-14 lymph node homing receptor (Selectin-1, LECAM-1, Leu 8) on both T and B cells results in modification of cell function. To investigate this phenomenon, we performed studies to determine if the Leu 8 molecule influences T cell activation via the TCR/CD3 complex. In initial studies, we treated T cells with immobilized anti-CD3 (OKT3 mAb) in the presence or absence of immobilized Leu 8 mAb. We found that although Leu 8 mAb alone had no effect on T cell proliferation, this antibody markedly augmented immobilized OKT3 mAb-induced proliferation. In further studies, we immunoprecipitated surface radioiodinated T cell lysates with OKT3 and Leu 8 mAb to determine if molecules in the TCR/CD3 complex associate with Leu 8 molecules. Although Leu 8 mAb immunoprecipitated only a single protein of approximately 80 kDa from T cell lysates treated with Nonidet P-40 under reducing condition, it coimmunoprecipitated additional proteins of 48, 42, 28, 24, and 22 kDa from T cell lysates treated with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. These additional proteins were identified as the alpha-, beta-, gamma-, delta-, and epsilon-chains of the TCR/CD3 complex by one-dimensional and two-dimensional diagonal SDS-PAGE. Densitometric scanning showed that, on average, 18% of the TCR/CD3 complex associates with Leu 8. In a final study, we showed by immunoblotting analysis using anti-zeta peptide antibody that Leu 8 mAb coimmunoprecipitates the zeta-chain of CD3. These results indicate that the human lymph node homing receptor homologue (Leu 8) participates in the activation of T cells, probably via its association with the TCR/CD3 complex.