HIV-1包膜V3区在病毒侵入靶细胞中的增强作用

目的　明确Ⅰ型人免疫缺陷病毒 (humanimmunodeficiencyvirustype 1,HIV 1)包膜糖蛋白gp12 0第三可变区 (variableregion ,V3)在病毒进入靶细胞的早期阶段中的作用。方法　合成了 3个环状V3多肽 ,包括V3 BH10、V3 ADA和V3 89.6以及直链和短链多肽V3 BH10 /CA、V3 NNT2 4和V3 IRI12。构建了 3株分别带HIVHXB2株、ADA株和 89.6株包膜的伪病毒并观察了多肽对不同嗜性HIV侵入靶细胞能力的影响。结果　 (1)HIV 1V3区多肽以毒株特异性模式增强HXB2、ADA和 89.6进入靶细胞 ;(2 )直链V3多肽与其环状多肽具有相似作用 ;带有C末端的短肽V3 NNT2 4也有与长链V3 BH10 /CA相近的作用 ,只有中央保守区的短链V3 IRI12没有类似功能。结论　本研究首次发现HIV 1V3区在病毒进入靶细胞早期阶段中具有毒株特异性增强病毒侵入的作用。该发现有助于进一步明确HIV 1进入靶细胞的机制     

A broad range of mutations in HIV-1 neutralizing human monoclonal antibodies specific for V2, V3, and the CD4 binding site

The HIV vaccine-induced neutralizing antibodies (Abs) display low rates of mutation in their variable regions. To determine the range of neutralization mediated by similar human monoclonal Abs (mAbs) but derived from unselected chronically HIV-1 infected subjects, we tested a panel of 66 mAbs specific to V3, CD4 binding site (CD4bs) and V2 regions. The mAbs were tested against 41 pseudoviruses, including 15 tier 1 and 26 tier 2, 3 viruses, showing that the neutralization potency and breadth of anti-V3 mAbs were significantly higher than those of the anti-CD4bs and anti-V2 mAbs, and only anti-V3 mAbs were able to neutralize some tier 2, 3 viruses. The percentage of mutations in the variable regions of the heavy (VH) and light (VL) chains varied broadly in a range from 2% to 18% and correlated moderately with the neutralization breadth of tier 2, 3 viruses. There was no correlation with neutralization of tier 1 viruses as some mAbs with low and high percentages of mutations neutralized the same number of viruses. The electrostatic interactions between anti-V3 mAbs and the charged V3 region may contribute to their neutralization because the isoelectric points of the VH CDR3 of 48 anti-V3 mAbs were inversely correlated with the neutralization breadth of tier 2, 3 viruses. The results demonstrate that infection-induced antibodies to CD4bs, V3 and V2 regions can mediate cross-clade neutralization despite low levels of mutations which can be achieved by HIV-1 vaccine-induced antibodies.     

Complement-dependent cytotoxic antibodies to human T lymphotropic virus type I (HTLV-I)-infected cells in the sera of HTLV-I-infected individuals

To investigate whether HTLV-I induces the development of complement-dependent cytotoxic antibodies in humans, sera of asymptomatic HTLV-I carriers and of patients suffering from tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) or adult T cell leukaemia (ATL) were used in a cytotoxicity assay against a panel of target cells. This panel included uninfected cell lines (CEM, Jurkat, Molt and H9), cell lines chronically infected with HTLV-I (MT2, MT4, C91PL and HUT102), as well as lines H36 (H9 infected with HTLV-I), H9-IIIB (H9 infected with HIV_IIIB) and H9-MN (H9 infected with HIV_MN). HTLV-I⁺ sera induced lysis of H36 and of lines expressing HTLV-I antigens in the presence of rabbit complement, but did not lyse cells in presence of human complement. The HTLV-I⁺ sera also failed to lyse the HTLV-I⁻ lines and H9 cells, suggesting that lysis was specific for HTLV-I. H36 cell lysis was prevented by IgG depletion of the sera and by dialysis of rabbit complement against EGTA or EDTA. Rabbit complement-dependent cytotoxic antibodies were present in the sera of 14/14 HTLV-I-infected individuals; the highest titres were predominantly found in the sera of the TSP/HAM patients. Such antibodies were also detected in 5/5 individuals coinfected with HIV-1 and HTLV-I, although no cytotoxic antibody could be found against HIV-infected cells. Vice versa, sera of HIV-1-infected individuals did not exert a lytic effect in the presence of complement (of human or rabbit origin) against HIV-1- or HTLV-I-infected cells. Incubation of the sera of four HTLV-I-infected patients with HTLV-I env-specific synthetic peptides demonstrated that some of the complement-dependent cytotoxic antibodies recognized epitopes located on gp46 between amino acids 190 and 209. There is no correlation of rabbit complement-dependent cytotoxic HTLV-I antibodies with the development of disease.     

Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop

The crystal structure of the human immunodeficiency virus type 1 (HIV-1) neutralizing, murine Fab 83.1 in complex with an HIV-1 gp120 V3 peptide has been determined to 2.57 A resolution. The conformation of the V3 loop peptide in complex with Fab 83.1 is very similar to V3 conformations seen previously with two other neutralizing Fabs, 50.1 and 59.1. The repeated identification of this same V3 conformation in complex with three very different, neutralizing antibodies indicates that it is a highly preferred structure for V3 loops on some strains of the HIV-1 virus.     

Permissive residues within the minimal epitopes of neutralizing monoclonal antibodies to the V3 loop of HIV-1

Neutralizing monoclonal antibodies (mAb) specific for the third variable (V3) domain of gp120, the HIV-1 surface envelope protein, are mainly isolate specific. We have studied the composition and the permissivity of the minimal epitopes interacting with two of these, the 110-A and 19.26.4 mAb, which are strictly LAI isolate specific. Screening a hexapeptide phage library displayed on the surface of filamentous phage with the 110-A mAb has allowed selection of 49 phage sequences, permitting the definition of a consensus sequence. Based on this sequence, substituted synthetic peptides were prepared and used in binding assays. Our results show that both mAb interact with the same narrow region (316–320) of the V3 domain. The minimal epitope of the 110-A mAb was identified as a five amino acid sequence, Hy x R G p, where Hy represents any non-aromatic hydrophobic amino acid. By contrast, the minimal epitope of the 19.26.4 mAb was identified as x Q Pos G P, where Pos is any positively charged amino acid. Core residues of the epitope, critical for the binding to the mAb (written in uppercase letters), were set apart from permissive amino acid positions that tolerate substitutions (written in lowercase letters). Interestingly, the identified core residues Q_2/317 (19.26.4 mAb) and R_3/318 (110-A mAb) do not tolerate substitution and correspond to the QR insertion in the V3 domain, characteristic of the LAI isolate as compared to other isolates. This result may explain the strict isolate specificity of most anti-V3 LAI mAb. The two epitopes have totally different patterns of permissivity; thus, the effect of substitutions will differ depending on the mAb involved in the interaction. This suggests that the diversity of the antibody response is high enough to delay the emergence of HIV-1 variants resistant to neutralization by V3-specific antibodies.     

Focusing the immune response on the V3 loop, a neutralizing epitope of the HIV-1 gp120 envelope

Rabbits were immunized with a novel regimen designed to focus the immune response on a single neutralizing epitope of HIV-1 gp120 and thereby preferentially induce neutralizing antibodies (Abs). Animals were primed with gp120 DNA from a clade A Env bearing the GPGR V3 motif and/or a clade C Env bearing the GPGQ V3 motif, and boosted with one or more fusion proteins containing V3 sequences from clades A, B and/or C. Immune sera neutralized three of four Tier 1 primary isolates, including strains heterologous to the immunizing strains, and potent cross-clade-neutralizing activity was demonstrated against V3 chimeric pseudoviruses carrying in a Tier 1 Env, the consensus V3 sequences from clades A1, AG, B, AE, or F. The broadest and most potent neutralizing responses were elicited with the clade C gp120 DNA and a combination of V3-fusion proteins from clades A, B and C. Neutralizing activity was primarily due to V3-specific Abs. The results demonstrate that the immune response can be focused on a neutralizing epitope and show that the anti-V3 Abs induced recognize a diverse set of V3 loops.     

Mimicking the humoral immune response in vitro results in antigen-specific isotype switching supported by specific autologous T helper cells: generation of human HIV-1-neutralizing IgG monoclonal antibodies from naive donors

Molecular and cellular requirements for antigen-specific isotype switch of human B cells have been investigated by mimicking signaling occurring in germinal centers. Peripheral blood mononuclear cells from healthy seronegative blood donors were first primary immunized in vitro, using a synthetic immunogen containing both a T and B cell epitope, which generated specific IgM-secreting B cells. We used the apex of the V3 loop of gp120 as B cell epitope linked to a promiscuous T helper epitope from tetanus toxin. In parallel, CD4⁺ T helper cell clones specific for the T epitope of the immunogen were established. In a secondary in vitro stimulation period, we co-cultured the antigen-specific T and B cells on CD32-transfected fibroblasts, together with an anti-CD40 monoclonal antibody. This resulted in isotype switching and human antigen-specific, IgG-secreting B cells were detected. This response was strictly dependent upon the presence of autologous T helper cells and the immunogen. Antigen-specific human B cells derived from this primary and secondary in vitro immunization were subsequently subjected to electrofield-induced somatic cell hybridization and hybridomas secreting human anti-V3 IgG monoclonal antibodies were isolated. One human antibody was further characterized and shown to be specific for the immunizing antigen with an affinity constant of 24 nM. This antibody also effectively neutralized different isolates of HIV-1, achieving a 50% neutralization at 0.46 μg/ml.     

广西HIV-1 CRF01-AE重组毒株env基因V3环序列变异及其与生物表型的关系

目的研究广西HIV-1 CRF01-AE重组毒株env基因V3环序列变异及其与生物表型间的关系。方法从广西主要流行区收集来的50份HIV-1感染者血液样本中提取前病毒DNA，使用巢式聚合酶链反应（nested-PCR）扩增HIV-1 env基因片段并进行亚型鉴定，选择38份CRF01-AE重组型HIV-1毒株env，基因V3环及邻近区域的序列进行系统树和氨基酸变异分析。结果38份CBF01-AE重组毒株中36份与分离于广西地区的CRF01-AE.97CNGX2f和泰国代表毒株THCM240接近，另外2份与中非共和国代表株90CF402聚成一簇；CRF01-AE重组毒株V3环顶端四肽存在着4种类型：CPCQ、GPGR、GPGH和GPGA；根据V3环关键氨基酸推测辅助受体使用情况，结果显示：71．05％的CRF01-AE重组毒株可能使用CCR5作为辅助受体，28．95％不能对其辅助受体的使用情况做出预测。结论广西HIV-1 CRF01-AE重组毒株V3顶端四肽变异较大，而且大部分毒株可能为NSI型。这可为广西该毒株的防治和诊断试剂的更新提供参考。     

Antibody-mediated activation of T cell clones as a method for screening hybridomas producing antibodies to the T cell receptor

In this study, supernatants (SN) of hybridomas established by fusing P3X63.Ag8.653 to spleen cells from C57L mice (Vß8 family of T cell receptor (TcR) gene negative) immunized with the H-Y specific Vß8 allotype positive helper T cell (HTL) clone OI6 were screened for the capacity to activate cloned T cells in the absence of interacting stimulator cells. In the first assay, SNs were mixed with Vß8⁺ H-Y specific CTL OH2 and ⁵¹Cr-labelled non-specific B lymphoma (L10.A). In this system, antibodies (Ab) which can bind to L10.A by Fc-Fc receptor interaction and recognize TcR can facilitate lysis of L10.A target cells by OH2 CTL. In the second assay, OI6 clone cells were cultured in microtiter well, previously coated with hybridoma supernatants (SN). In this assay, Ab recognizing OI6 TcR complex and bound to plastic plates can stimulate OI6 cells to proliferate in the absence of stimulator cells. Using these two screening methods, nine hybridomas were established. Analysis of these hybridoma SN using surface staining, inhibition of T cell function and immunoprecipitation of radiolabelled surface molecules followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) showed that five Ab were directed to the allotypic determinant (Vß8) of TcR and four Ab were specific to the clonotypic determinant of OI6 TcR. These results suggest that this Ab-mediated activation of T cell clone can be used for the screening of hybridomas secreting anti-TcR Ab and the immunogenicity of OI6 clonotypic determinants is similar to that of the Vß8 allotypic determinant even in strains which do not express the Vß8 TcR allotype.     

Blocking-ELISA for detection of specific antibodies to the glycoproteins of the human immunodeficiency virus type 1 (HIV-1)

A blocking ELISA was developed to confirm the specificity of screening tests for anti-HIV-1 antibodies. A murine monoclonal antibody (McAb) raised against recombinant gp160 was used in combination with a commercial technique (ELA-VIA-1). After determining the optimal experimental conditions, the assay was applied to 92 samples presenting different reactivities by Western blot (WB) analysis. All the sera containing antibodies to gp160/gp120 (53) were positive in our assay. The six patients who sero converted showed a low positivity by ELAVIA-1 (optical density near the cutoff value) reacted by blocking-ELAVIA-1 with an McAb binding inhibition greater than 85%. By contrast, negative samples (29) and specimens that exhibited reactivity only against gag-proteins (10) were not detected (McAb binding inhibition smaller than 15%). This sensitive and specific blocking-ELAVIA-1 represents a convenient alternative to WB as a confirmatory test. The technique is time-saving and inexpensive and can easily be integrated with a screening test for diagnostic or epidemiologic studies on HIV-1 infection.     

HIV-1 determinants of thrombocytopenia at the stage of CD34+ progenitor cell differentiation in vivo lie in the viral envelope gp120 V3 loop region

HIV-1 V3 loop clones of virus isolates derived from patients suffering from thrombocytopenia were used for infection of the human thymus/liver conjoint hematopoietic organ that developed in the severe combined immunodeficient mouse (SCID-hu Thy/Liv). The V3 loop clones showed a significantly greater degree of inhibition of megakaryopoiesis than myelopoiesis and erythropoiesis of the human CD34+ progenitor cells, in vivo. Inhibition of megakaryopoiesis occurs through reduction in c-Mpl expression and consequent decrease in STAT5 activation. Therefore HIV-1 V3 loop sequences of thrombocytopenic patients exhibit preferential inhibition of megakaryocyte lineage-specific differentiation of CD34+ progenitor cells, thus reflecting the patients' clinical condition.     

Activation of human T lymphocytes II. Involvement of the T3 antigen in polyclonal T cell activation by mitogenic lectins and oxidation

The effect of monoclonal antibodies against the T cell surface antigens T3 and T4 on accessory cell-dependent mitogen-induced proliferation of human antigen-specific T4⁺ T lymphocyte clones and purified peripheral blood T cells was studied. To avoid the Fc receptor-dependent mitogenic effects of the OKT3 antibodies, monocytes were replaced by Epstein-Barr virus-transformed B lymphoblastoid cells as accessory cells. Monoclonal antibody OKT3 but not OKT4 inhibited the response of both T lymphocyte clones and purified T cells to mitogenic lectins and oxidation. The inhibition was not due to nonspecific effects of the monoclonal antibodies since it affected only the initial triggering but not the proliferation of activated T cells and it could be overcome by higher concentrations of lectin indicating a competition between OKT3 antibody and lectin. Furthermore, OKT3 antibody at the same concentration that was inhibitory in this system was mitogenic in the presence of Fc receptor-bearing monocytes. Surface modulation of T3 but not of T4 antigens led to unresponsiveness to a mitogen pulse given directly after modulation. These findings suggest that antigen-specific and mitogen-induced T cell triggering are due to interaction with the same receptors of the T lymphocyte.     

Changing sensitivity to broadly neutralizing antibodies b12, 2G12, 2F5, and 4E10 of primary subtype B human immunodeficiency virus type 1 variants in the natural course of infection

The conserved nature of the epitopes of the four broadly neutralizing antibodies (BNAbs), b12, 2G12, 2F5, and 4E10, may imply that the sensitivity of HIV-1 for these BNAbs remains fairly constant over the course of infection. Here, we demonstrate that viruses isolated early during the course of infection were mostly sensitive to HIVIg and antibody neutralization, although variation was observed in neutralization sensitivity of coexisting viruses to the different antibodies as well as between viruses from different patients. HIV-1 resistance to HIVIg developed relatively early during follow-up in three out of five patients, while early, b12 sensitive viruses in three out of five patients were replaced by b12 resistant variants relatively late in infection. In contrast, viruses generally remained sensitive to 2F5 and 4E10 neutralization over the course of infection, although 2F5 and/or 4E10 resistant variants did emerge later in infection in four out of five patients. In most patients, HIV-1 resistance to 2F5 or 4E10 did not correlate with mutations at critical amino acid positions in their defined epitopes. Viruses resistant to 2G12-mediated neutralization were present throughout the course of infection. As viral resistance against BNAb-mediated neutralization generally developed when autologous serum neutralizing activity had faded, it seems unlikely that these changes are driven by escape from autologous humoral immunity.     

Restriction molecules involved in the interaction of human T cell clones with antigen-presenting cells

Two of three distinct human Ia molecules detected by murine monoclonal antibodies (MoAb) have been suggested to be involved in antigen presentation for T cell responses to purified protein derivatives (PPD) and herpes simplex virus (HSV). This observation was first suggested from studies on the inhibition of proliferative responses of whole T cell populations with MoAb against human Ia molecules. To determine whether a single T cell recognizes the antigen in the context of both Ia molecules or in the context of each one of two Ia molecules, we isolated and propagated PPD-reactive T cell clones from an HLA-DR heterozygous individual. They showed four different restriction patterns: type I and type II clones each appeared to be restricted to one of two HLA-DR antigens, type III clone gave anomalous patterns of response and seemed to be restricted to non-DR antigens, and type IV clone recognized antigen when both DR antigens were presented on the same antigen-presenting cells (APC) surface. Blocking study with monoclonal anti-Ia antibodies suggested that type I, II and IV clones are restricted to DR molecules and type III clones are restricted to 1B4 molecules distinct from DR or MB1 molecules. These data imply that human T cell clones recognizing PPD in the context of each one of two Ia molecules are clonally distributed.     

Restricted T cell receptor expression by human T cell clones specific for mycobacterial 65-kDa heat-shock protein: Selective in vivo expansion of T cells bearing defined receptors

We have examined the T cell receptor (TcR) expression of clones specific for epitopes of mycobacterial 65-kDa heat-shock protein (hsp65) in the context of two different HLA molecules, and used this system as a model to assess the selection of T cells responsive to this antigen in vivo. DR3-restricted clones were raised from both the synovial fluid (SF) and peripheral blood (PB) of a patient with reactive arthritis in three separate cloning events. Five of five SF-derived clones tested expressed either Vβ5.2 or a closely related β chain, Vβ5.6.The α chains expressed by Vβ5.2⁺ and Vβ5.6⁺ clones were from different families, Vα2.4 and Vα23.2, respectively. Nine of ten clones derived from two cloning procedures on PB taken 3 years later also expressed either Vβ5.2 or Vβ5.6. This suggests that the TcR repertoire for recognizing this major histocompatibility complex/peptide complex is relatively restricted and favors the use of Vβ5. Conservation of the β chain third complementarity-determining region (CDR3) sequence was not evident, however. Sequencing α and β chains of representative Vβ5.2⁺ and Vβ5.6⁺ PB-derived clones revealed TcR which were identical to those utilized by the SF-derived clones, showing that the repertoire for recognition of this antigen is stable over time. Similar studies of TcR expression were carried out on hsp65-specific, DP4-restricted clones derived from the SF of a patient with rheumatoid arthritis by two independent cloning procedures. There was conservation of a chain usage, since all clones expressed a member of the Vαl family, but again CDR3 sequence conservation was not apparent, β chain usage was not restricted since different clones expressed Vβ6.7, Vβ22.3 and Vβ12. Subtle differences in epitope specificity were detected for two clones with differing TcR. Once more, T cell clones with identical α and β TcR chains were obtained from the separate cloning procedures, suggesting oligoclonalty of T cells with this defined specificity in the patient's SF.     

The differential effects of hydrocortisone on activation and tolerance induction in human T lymphocyte clones

The in vitro effects of hydrocortisone on T cell activation and tolerance induction were investigated using human influenza virus immune T cell lines and clones. Hydrocortisone at 10⁻⁹ to 10⁻⁶ molar concentrations was able to inhibit the antigen induced but not the T cell growth factor (TCGF) mediated proliferative response of both the lines and clones. However, hydrocortisone was able to inhibit TCGF production by cloned T cells. The proliferative response of cloned T cells to intact influenza virus A/Texas/1/77 was more markedly inhibited by equivalent concentrations of hydrocortisone than was the response of that clone to a 24 amino acid sequence (p20) of the haemagglutinin molecule implying that hydrocortisone may also act at the level of antigen processing. Furthermore hydrocortisone was able neither to induce T cell tolerance alone nor to inhibit antigen specific tolerance induction. However, hydrocortisone did lower the antigen threshold for tolerance induction. The possible mechanisms of hydrocortisone activity in the modulation of T cell regulation in autoimmune disease are discussed.     

T cell activation by monoclonal antibodies directed to different epitopes on the human T cell receptor/CD3 complex: evidence for two different modes of activation

The mouse monoclonal antibody (mAb) BMA031 (IgG2b) has recently been described to be directed against a monomorphic part of the human T cell receptor (TcR) alpha/beta. In vitro analysis of its stimulatory potential for mononuclear cells revealed two patterns of responsiveness. Out of 35 tested individuals only 2 generated a proliferative response to low antibody concentrations (15 ng/ml; "high responders"), the others ("low responders") responded only to high antibody concentrations (1.5 micrograms/ml); the anti-CD3 mAb UCHT1 (IgG2b) stimulated only the two high responders. This response pattern to BMA031 was determined by the accessory cell compartment in the culture. Stimulation by BMA031 in low responders demonstrated some unusual features: (a) high antibody concentrations were required, (b) addition of autologous serum had no inhibitory effect and (c) vigorous depletion of macrophages reduced but did not abolish the proliferative response. These characteristics were shared by two other mAb, BMA032 and BW239/347, presumably directed against the TcR alpha/beta but not by several other antibodies to the TcR/CD3 complex. Thus, the results demonstrate unusual stimulatory properties of three anti-TcR alpha/beta mAb, inducing a proliferative response without antibody cross-linking. This suggests that the stimulatory effect of anti-TcR/CD3 complex mAb is not only determined by their isotype, but also strongly depends on their epitope specificity.     

HIV envelope-directed signaling aberrancies and cell death of CD4+ T cells in the absence of TCR co-stimulation

HIV-1 infection in CD4₊ T cells initiates a viral cytopathiceffect (CPE) that is dependent on the activation of intracellularprotein tyrosine kinases (PTK). PTK in T cells are also activatedduring the course of TCR or CD4 receptor engagement and themanner of receptor engagement may generate signals leading eitherto cell proliferation, tolerance induction (anergy) or programmedcell death (PCD). We have identified PTK triggered during theinteraction of cells stably expressing surface HIV envelope(gp120/gp41; HIVenv) and CD4⁺ T cells, which leads to extensiveand rapid individual cell death. We have found that this killingis accompanied by tyrosine phosphorylation and activation ofthe CD4-associated p56^ick kinase, and by activation of a secondmember of the src family of PTK, p59^fyn kinase, normally associatedwith T cell stimulation through the TCR. Interestingly, in contrastwith normal T cell signaling, the subunit of the TCR failsto become tyrosine-phosphorylated during signaling accompanyingHIV-directed cell killing. Downstream activation of the ZAP-70PTK also does not occur. Unlike T cell apoptosis triggered bysoluble HIVenv glycoproteins, which requires co-stimulationof CD4 and the antigen-specific TCR, T cell killing by membrane-associatedHIVenv does not require TCR co-stimulation, because aberrantsignaling and cell death are triggered by CD4+ but TCR^–cell lines. These results are the first report where dual activationof the Lck and Fyn PTK does not result in normal downstreamsignaling through the ZAP PTK. We suggest by analogy to SCIDresulting from ZAP-70 mutations, that the dissociation of upstreamPTK activation from ZAP-70 signaling contributes to T cell depletionby HIV and to the development of AIDS.     

Evidence for quantitative and qualitative differences in functional activation of MIs-reactive T cell clones and hybridomas by antigen or TcR/CD3 antibodies

In this study, we demonstrated that some Vp6⁺, CD4⁺, Mls-l^a-specific T cell clones had cytolytic activity when stimulated with anti-T cell receptor(TcR)/CD3 monoclonal antibodies (mAb), but not with targets expressing Mls-1^a, although they produced lymphokines (interleukin 2 and interferon-y) in response to both types of stimuli. To examine the possibility that lack of cytolysis resulted from expression of the Mls-l^a antigen on merely a fraction of splenic B blasts, we (a) used the B cell lymphoma LBB.3.4.16 and (b) measured esterase secretion which is generally concurrent with cytotoxic T lymphocyte (CTL) activity. The B cell lymphoma maximally stimulated the T cell clone for interferon-y production when responding and stimulating cells were incubated at a 1:1 ratio, but it was never killed by the Mls-1^a-specific T cell clone unless TcR/CD3-specific mAb were added. Furthermore, a fivefold excess of the Mls-1^a B cell lymphoma did not induce any secretion of esterase, which was observed only in the presence of the TcR/CD3-specific mAb. Comparison of the reactivity of two Mls-1^a-specific T cell hybridomas expressing the same TcR at similar surface density, revealed both quantitative and qualitative differences between CD3-specific mAb and Mls stimulation of the hybridomas. A small quantitative difference in the sensitivity of hybridoma FJ22.5 to stimulation with V_β6 or CD3-specific mAb resulted in a marked decrease in efficiency of stimulation by Mls-1^a for interleukin 2 production and to inability to detect growth inhibition by Mls-expressing cells. A qualitative difference was observed when analyses of inositol phosphate production were performed under optimal conditions of stimulation of the highly responsive T cell hybridoma (FJ8.1): only stimulation with CD3-specific mAb, but not Mls-expressing cells, could induce detectable inositol phosphate production. Lack of cytolysis of Mls-1^a class II-expressing B cells may have evolutionary significance in view of the recent mapping of Mls to mouse mammary tumor virus genes.     

Human allospecific T cell proliferative clones: Different T cell clones expressing an apparently identical HLA class II specificity are heterogeneous in regard to their proliferative inhibition by a panel of monoclonal antibodies against human Ia-like antigens

Four independent PLT clones displaying an apparently identical class II specificity (i.e., Dw/DR3) were found to give a heterogeneous pattern of inhibition in relation to 15 anti-class II mAb and an anti-ß2m mAb used as a control. Some clones were inhibited by all anti-class II mAbs, irrespective of the cluster of molecular subsets with which they reacted. Such clones were also inhibited by the control anti-ß2m mAb. Other clones were inhibited by only a few of the mAb tested. Within this group of T clones following the addition of a limiting amount of conditioned medium the inhibitory data of all independent clones with an identical specificity were inhibited by the same mAb; under these conditions, it was possible to relate the mAb inhibition patterns with the specificity of the T cell clones and these T cell specificities with the epitopic cluster/molecular subsets defined by these mAbs. A new level of T cell subset heterogeneity within T cell clones with apparent identical proliferative specificities is demonstrated, in relation to the T cell clone “susceptibility” or “resistance” to the effects of anti-class II mAbs directed towards their own Ia-like antigens.