European Journal of Immunology

doi: 10.1002/eji.1830181101pmid: N/A

Merkenschlager, Matthias; Terry, Linda; Edwards, Robert; Beverley, Peter C. L.

doi: 10.1002/eji.1830181102pmid: 2974420

Using limiting dilution analysis, we investigated proliferative responses of UCHL1+ and UCHL1− T cell populations to compare the precursor cell frequencies following recall and alloantigen stimulation, and the complexity of cellular interactions within UCHL1+ and UCHL1− populations. We find high frequencies of recall antigen responses among UCHL l+, but not UCHL1− T cells. In contrast, both populations contain similar frequencies of alloantigen responsive cells. Our results are consistent with single‐hit kinetics in recall as well as alloantigen responses and show no complex cellular interactions within the responding populations. In conclusion, the difference between the recall antigen response of UCHL 1+ and UCHL1− cells observed in conventional proliferation assays is most probably due to a high frequency of recall antigen‐responsive UCHL1+ cells and not to suppressive phenomena in the UCHL1−population. These data suggest that memory T cells are largely of the UCHL 1+ phenotype. The relation of post‐thymic T cell maturation and differential CD45 expression is briefly discussed.

Kisaki, Tomonari; Leung, Donald Y. M.; Jardieu, Paula; Geha, Raif S.; Ishizaka, Kimishige

doi: 10.1002/eji.1830181103pmid: 3264529

We have previously established human T cell hybridomas which produce IgE‐binding factors. Incubation of one of the T cell hybridomas, 166A2, with human IgE dimer in the presence of 1 μg/ml bradykinin resulted in the formation of IgE‐binding factors having affinity for lentil lectin. The factors selectively enhanced both IgE‐forming cell responses of rat mesenteric lymph node (MLN) cells and spontaneous IgE synthesis by human peripheral blood B cells of atopic patients, without affecting the IgG response. The same factors that enhanced IgE synthesis of B cells from atopic patients also enhanced IgE synthesis induced under bystander conditions by activated alloreactive T cells. Fractionation of the affinity‐purified IgE‐binding factors by gel filtration revealed three molecular mass species, i.e., 60 kDa, 30 kDa and 15 kDa. The 60‐kDa and 15‐kDa IgE‐binding factors selectively enhanced both the spontaneous IgE synthesis by B cells of atopic patients and IgE response of rat MLN cells. In contrast, the 30‐kDa IgE‐binding factors had only marginal enhancing effects on the IgE synthesis by both human B cells and rat MLN cells. When the 166142 hybridoma cells were incubated with IgE dimer in the presence of glycosylation‐inhibiting factor (GIF), essentially all IgE‐binding factors formed by the cells had affinity for peanut agglutinin (PNA) but for neither lentil lectin nor concanavalin A. All of the 60‐kDa, 30‐kDa and 15‐kDa species, having affinity for PNA, selectively suppressed the potentiating factor‐enhanced IgE response of rat MLN cells. The factors also suppressed the IgE synthesis of human B cells from atopic patients when the synthesis was enhanced by IgE‐potentiating factor. The results indicate that human IgE‐binding factors regulate IgE synthesis by both human and rat lymphocytes.

Jitsukawa, Setsuko; Triebel, Frédéric; Faure, Florence; And, Christine Miossec; Hercend, Thierry

doi: 10.1002/eji.1830181104pmid: 2849548

We have assessed the organization of T cell γ rearranging genes (TRG) in circulating TcRγ/δ+ lymphocytes which do not express Vγ9‐encoded TiγA+ γ chain. Following purification of the minor TcRγ/δ+ TiγA− fraction, cloned cell lines were developed from peripheral blood of 5 individuals. Out of the 26 clones studied, only 3 TcRγ/δ+ TiγA‐ cells were found to express a disulfide‐linked C1‐encoded γ chain. The remaining 23 TiγA− clones with a C2‐encoded nondisulfide‐linked receptor were found to display rearrangements of various V genes to 52 segments on both chromosomes; there was no predominance of a unique rearrangement even though the TRG‐V3 and ‐V4 genes belonging to subgroup I were frequently employed. Together, these findings further strengthen the hypothesis that lymphocytes with a Cγ1 encoded chain are produced earlier in T cell ontogeny than the Cγ2 counterparts. The “non‐major histocompatibility complex (MHC) requiring” (i.e., “natural killer‐like”) cytotoxicity mediated by many TcRγ/δ+ TiγA− cells appeared to be very low as compared to that of Ti γA+ clones. Yet, treatment by the OKT3 monoclonal antibody revealed a strong lytic potential in the TiγA− lymphocytes with little, if any, natural killer‐like activity. Thus, with respect to the latter function, a substantial heterogeneity is found in cells expressing distinct γ chains. In an attempt to characterize undefined specificities of TiγA− lymphocytes, they were screened against a panel of Epstein‐Barr virus‐transformed B cell lines homozygous for HLA‐DR1 to DR10 determinants; one of the clones was found to recognize DR7. In light of reports from other groups describing class I‐related specificities, it is apparent that TcRγ/δ+ lymphocytes are able, like the TcRα/β+, to recognize and kill target cells through either an MHC‐dependent (with involvement of either class I or class II gene products) or a non‐MHC‐requiring pathway.

Mackay, Charles R.; Hein, Wayne R.; Brown, Marion H.; Matzinger, Polly

doi: 10.1002/eji.1830181105pmid: 2462499

The CD2 adhesion/activation molecule on the surface of mammalian T lymphocytes binds to a ubiquitous receptor, LFA‐3. We show that CD2 in sheep differs significantly in its expression from CD2 in humans, and this most likely relates to the high level of expression of the sheep LFA‐3 molecule. In sheep, in contrast to man, CD2 was weakly expressed on peripheral T cells and thymocytes. Moreover, a large subset of T cells identified by the monoclonal antibody T19 and considered to be γ δ receptor‐bearing T cells completely lacked the CD2 molecule. T19+ cells constituted up to 50% of peripheral blood T cells in lambs, and 20–30% of T cells in older sheep, whereas the CD4+ and CD8+ subsets, which are both CD2+, constituted relatively small subsets in peripheral blood. Only those T cells which did express CD2 adhered as “rosettes” to dendritic cells, and the localization of CD2 to the membrane junction indicated that CD2 was critical for this adhesion. However, CD2 adhesion was not necessary for CTL‐mediated killing of allogeneic target cells, since T19+ cells generated in bulk mixed lymphocyte culture were extremely efficient at killing appropriate target cells. Some of the behavioral differences between T19+ and CD4+/CD8+ subsets might be explained by the presence or absence of CD2. The results also indicate that the expression of CD2 (and LFA‐3) may differ markedly between species.

Ulaeto, David; Waace, Lesley; Morgan, Andrew; Morein, Bror; Rickinson, Alan B.

doi: 10.1002/eji.1830181106pmid: 2904885

Specific T cell proliferation was observed in short‐term blood mononuclear cell cultures set up from Epstein‐Barr virus (EBV)‐immune individuals and challenged either with UV‐irradiated EB virions or with a candidate subunit vaccine preparation, the purified envelope glycoprotein gp340 incorporated into immune stimulating complexes (gp340 iscoms). Limiting dilution culture of the activated T lymphoblasts in interleukin 2‐containing medium generated stable CD3+CD4+CD8‐ T cell clones. Particular clones showing virus‐specific proliferation in preliminary screening assays were selected for more detailed study. Three gp340 iscoms‐induced clones from EBV‐immune donor CG responded specifically to restimulation either with UV‐EBV or with purified gp340 iscoms in the presence of autologous antigen‐presenting cells (APC). Both T cell‐depleted blood mononuclear cells and the EBV‐transformed B cell line (treated with Acyclovir to block endogenous gp340 production) could be used for presentation, the latter being the more efficient when gp340 iscoms was the source of antigen. Blocking studies with monoclonal antibodies to HLA class II antigens and experiments using HLA‐typed allogeneic APC indicated that all three gp340‐specific CG clones were restricted through the HLA‐DR2 antigen. One gp340 iscoms‐induced clone from another EBV‐immune donor, MR, likewise showed gp340‐specific proliferation, in this case restricted through a HLA‐DR4 antigen. Using HLA‐DR‐homozygous B cell lines representing the five known DR4 subtypes, efficient presentation of gp340 to this T cell clone was observed with both DR4Dw4 and DR4Dw14 antigens. Parallel experiments on one UV‐EBV‐induced T cell clone from donor MR gave a different pattern of results; these cells appeared to be specific for a virus structural component other than gp340 and to be restricted through an HLA‐DP determinant.

Yang, Xiao‐Dong; De Weck, Alain L.; Stadler, Beda M.

doi: 10.1002/eji.1830181107pmid: 3144452

Recombinant human interleukin 4 (IL4) alone enhanced the spontaneous IgE synthesis in cultures of peripheral blood leukocytes (PBL) from atopic patients as well as from nonatopic individuals, suggesting the existence of preactivated PBL sensitive for IL4. Preactivated cells were also obtained by stimulation with Staphyloccus aureus strain Cowan I (SAC). However, co‐stimulation of PBL by IL4 with SAC or anti‐IgM antibody and pokeweed mitogen did not result in an enhanced IgE synthesis. Optimal IL4 concentrations for the induction of IgE synthesis coincided with optimal proliferative responses in PBL. The effect of IL4 was not isotype specific, and in terms of protein even more IgG and IgM antibodies were formed. The effect of IL4 on IgE synthesis was counteracted by very low concentrations of interferon‐γ (IFN‐γ), suggesting that both IL 4 and IFN‐γ might be decisive cytokines for the human in vitro IgE synthesis.

Renard, Dominique; Petit‐Koskas, Elisabeth; Gé, Elisabeth; Dugas, Bernard; Poggioli, Josiane; Kolb, Jean‐Perre

doi: 10.1002/eji.1830181108pmid: 2849549

The possible role of phosphatidylinositol breakdown in the induction of proliferation of human activated B cells by low molecular weight B cell growth factor (LMW‐BCGF) was examined. LMW‐BCGF was found to induce a rapid rise in the concentration of inositol trisphosphate (InsP3) in (3H)inositol‐loaded B cell blasts, obtained by prior anti‐μ antibody activation. A concomitant decrease in the concentration of phosphatidylinositol 4,5‐bisphosphate could be detected at the same time. Maximum generation of InsP, occurred within 15–30 s after the addition of the LMW‐BCGF ligand to the activated B cells, then was followed by a slow decrease and return to control values. The amount of InsP3 generated by phosphatidylinositol hydrolysis was dependent on the concentration of LMW‐BCGF. This effect was only detected in B cells already preactivated by a first signal such as anti‐μ antibody and not in resting unstimulated B cells. In contrast, under similar conditions, interleukin 2, another B cell growth‐promoting lymphokine, did not alter the rate of formation of the various phosphatidylinositol breakdown products. An augmentation of the (Ca2+), concentration was also detected in activated B cells upon addition of LMW‐BCGF and this increase could be blocked by TMB‐8, a specific inhibitor of endoplasmic reticulum calcium release. Hydrolysis of phosphoinositides thus represents an essential component in the mechanism of transduction of the signal provided by LMW‐BCGF.

Smorodinsky, Nechama Ina; Ghendler, Yoseph; Bakimer, Ronit; Chaitchuk, Samario; Keydar, Iafa; Shoenfeld, Yehuda

doi: 10.1002/eji.1830181109pmid: 2849550

Previously we have reported on the production of two sets of human monoclonal antibodies reacting with mouse mammary tumor virus (MMTV) and human mammary tumor virus (HuMTV) cross‐reacting antigens. B11 is a monoclonal IgG generated by the human‐human hybridoma technique using axillary lymph node of breast cancer patients. 4.616 is a monoclonal IgG established by the mouse‐human hybridoma procedure using peripheral blood lymphocytes of a healthy investigator working with the breast cancer cell line T47D which secretes HuMTV antigens. Two anti‐idiotypic (Id) antibodies were produced by immunizing rabbits with B11 or 4.616. Following exhaustive adsorption on unrelated human‐Ig, fetal calf serum and purification on B11 or 4.616 affinity columns, the anti‐Id antibodies were shown to react specifically with their respective Id. The binding of these anti‐Id antibodies to the respective Id was specifically inhibited by prior incubation of the Id with MMTV and/ or HuMTV antigens Rabbit anti‐B11 and rabbit anti‐4.6/6 anti‐Id antibodies were employed to immunize female C3Heb mice. Following immunizations, humoral and cellular immune responses to MMTV‐related antigens could be demonstrated. The sera of the mice contained anti‐MMTV antibodies and delayed‐type hypersensitivity was specifically expressed when irradiated cells of the Mm5MT line (which carry surface MMTV antigens) were injected Our results support the notion that anti‐Id antibodies harboring the internal image can immunize animals against tumor cells bearing on their surface viral‐associated antigens.

Pessara, Ulrich; Momburg, Frank; Koch, Norbert

doi: 10.1002/eji.1830181110pmid: 3144453

The regulation of the invariant chain (Ii) expression was studied in the human colon carcinoma cell line HT‐29 that constitutively expressed neither Ii nor class II antigens. Upon stimulation of HT‐29 cells with a combination of human recombinant tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), expression of mRNA and protein of the invariant chain were induced. In contrast, administration of TNF‐α or IFN‐γ alone had no effect. A delayed induction of Ii mRNA, which was first detected 10–12 h after stimulation, was observed; this suggests an indirect regulatory mechanism. Stimulation with both IFN‐γ and TNF‐α led to the co‐expression of class II antigens with the invariant chain. In order to study the genetic basis for this stimulation the murine invariant chain gene (800 bp 5′ flanking sequences and the structural gene) was transfected into HT‐29 cells and transfected cells were tested for the ability to respond to IFN‐γ and TNF‐α. Simultaneous application of both cytokines had a strong effect on the induction of the murine invariant chain. IFN‐γ alone had no effect and TNF‐α only marginally stimulates murine invariant chain expression. The transfection experiment indicates that the murine invariant chain gene construct contains the structural elements which are responsible for regulation with IFN‐γ and TNF‐α. We determined whether the cooperative effect of TNF‐α and IFNγ is also found in vivo. Stimulations of mice were performed with TNF‐α, IFN‐γ and a combination of both. The immunohistological analysis of kidney tissue sections revealed that TNF‐α had no effect on Ii and Ia expression. Upon IFN‐Y treatment a minor subset of renal tubules showed staining for Ii, and less prominently also for Ia. However, simultaneous application of both cytokines led a strong induction of both Ii and Ia antigens in renal epithelial cells, thus suggesting that this synergistic effect potentially occurs under physiological conditions.