bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C‐activating phorbol esters augment expression of T cell receptor genes
Authors: Noonan Daniel J. , Isakov Noah , Theofilopoulos Argyrios N. , Dixon Frank J. , Altman Amnon
Published: March 2007

Tumor‐promoting phorbol esters (PE) can modulate cellular functions and cell surface determinant expression in a variety of cell types, including T lymphocytes, presumably by activating the enzyme, protein kinase C (PKC). To examine whether PKC might be involved in regulating the expression of genes encoding the antigen‐specific T cell receptor (TCR), we cultured the murine thymoma line, EL4, in the presence of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and analyzed the expression of TCR α or β‐chain genes by Northern blots. TPA stimulation of an interleukin 2 (IL 2)‐producing variant, EL4+, induced a 3–4‐fold increase in TCR β, but not α, chain mRNA. Maximal increase was obtained with 3 ng/ml TPA and 12 h of stimulation. This effect appeared related to PKC activation because other tumor‐promoting PE known to be PKC activators, but not inactive PE, induced the same increase. TPA stimulation of EL4+ cells also induced de novo expression of the IL 2 gene and subsequent secretion of this lymphokine. However, the increased expression of the TCR β‐chain gene and the induction of the IL 2 gene were not linked since (a) expression of TCR β‐chain mRNA was increased to a similar degree in EL4+ and IL 2‐nonproducing EL4− sublines, and (b) cyclosporin A selectively blocked TPA‐induced IL 2‐gene expression in EL4+ cells without affecting the increase in TCR β‐chain mRNA. These findings suggest that PKC activation, an event that supposedly occurs after antigen‐mediated triggering of the TCR, can regulate the expression of at least some of the genes encoding this receptor.
Proliferation of Ly‐1 B cells in autoimmune NZB and (NZB × NZW)F1 mice
Authors: Wofsy David , Chiang Nancy Y.
Published: March 2007

Spontaneous autoimmune disease in NZB and (NZB × NZW)F1 (B/W) mice is associated with a spectrum of lymphoproliferative abnormalities, but the relationship between autoimmunity and lymphoproliferation is poorly understood. Lymphomas occur commonly in NZB mice, but they appear to be rare in B/W mice, perhaps because B/W mice die of murine lupus before the lymphomas are evident. We recently reported that autoimmune disease in B/W mice could be reversed by weekly treatment with monoclonal antibodies to the L3T4 antigen on “helper/inducer” T cells. This has enabled us to examine the evolution of lymphoproliferation in B/W mice that survive beyond the usual life span, both in long‐term survivors of treatment with anti‐L3T4 and in the occasional B/W mouse that spontaneously survives beyond 1 year of age. We find that all of these mice develop marked proliferation of a distinct subpopulation of B cells that express the Ly‐1 antigen in low density. These Ly‐1+ B cells account for a 2‐10‐fold increase in the number of splenic, lymph node and peripheral blood lymphocytes. The Ly‐1 B cells in individual mice are restricted in their expression of immunoglobulin light chains, suggesting a clonal origin. NZB mice develop similar proliferation of Ly‐1 B cells, suggesting that this is due to underlying genetic and/or viral factors in NZB and B/W mice, and that it is not the result of treatment with anti‐L3T4. Although recent studies have implicated Ly‐1 B cells in the production of autoantibodies, proliferation of Ly‐1 B cells in B/W mice was not associated with production of anti‐DNA antibodies or with any paraprotein.
Activation of B lymphocytes by Epstein‐Barr virus/CR2 receptor interaction
Authors: Masucci Maria G. , Szigeti Robert , Ernberg Ingemar , Hu Cheng‐Po , Torsteinsdottir Sigurbjörg , Frade Raymond , Klein Eva
Published: March 2007

Epstein‐Barr virus (EBV) infects and transforms human B lymphocytes. The virus receptor was shown to be identical to the complement receptor CR2. The consequences of EBV/CR2 receptor interaction on B lymphocyte activation were analyzed by infection of B cells with the transforming (B95‐8) and the nontransforming (P3HR1) virus strains and with UV‐inactivated B95‐8 virus.

Similar to mitogens and antibodies against surface IgM and CR2 receptor, transforming and nontransforming EBV induced the release of leukocyte migration inhibitory factor (LIF). LIF production occurred early after infection and was not affected by irradiation of the B95‐8 virus with doses of UV‐light which prevented the expression of viral functions. B lymphocytes infected with UV‐inactivated virus responded to T cell‐derived B cell growth factors.

The results demonstrate that binding of EBV to the CR2 receptor activates the B cells. Progression of the infected cells in the activation pathway required helper cell‐derived factors or signals provided by the intact viral genome. The nontransforming P3HR1 virus induced a low level of RNA synthesis and increase of cell size and major histocompatibility complex class I antigen expression. Only the transforming virus induced expression of EBV nuclear antigens and cellular DNA synthesis.
A functional idiotypic network of T helper cells and antibodies, limited to the compartment of “naturally” activated lymphocytes in normal mice
Authors: Coutinho António , Marquez Carlos , Araujo Paulo M. F. , Pereira Pablo , Toribio Maria Luisa , Marcos Miguel Angel R. , Martinez‐A Carlos
Published: March 2007

As shown previously, idiotype (Id) sharing between anti‐2,4,6‐trinitrophenyl T helper (Th) cells and antibodies in BALB/c mice results from immunoglobulin (Ig)‐dependent selection of the T cell repertoire. In contrast, a clonotype defined by the same F6(51) anti‐Id antibody is expressed by C57BL/6 anti‐(4‐hydroxy‐3‐nitrophenyl)acetyl Th cells independently of Ig influences. We have now used these systems to test the hypothesis that Ig‐dependent Th cell repertoire selection occurs in the compartment of “naturally” activated lymphocytes.

“Naturally” activated or resting splenic L3T4+ cells were separated from normal BALB/c and C57BL/6 mice and tested, either directly or after in vitro priming, in hapten‐specific helper assays for expression of the clonotope defined by the F6(51) anti‐Id antibody. The results show the selective expression of the antibody‐dependent T cell Id in the “naturally” activated helper cell compartment. In contrast, when the T cell Id is expressed in the absence of Ig‐dependent selection, it is only detected in the resting helper cell repertoire. Furthermore, BALB/c “natural” IgM antibodies with anti‐Id specificities similar to F6(51) show functionally relevant interactions with syngeneic “naturally” activated Th cells. These are also characterized by high paratopic/Id degeneracy, as compared to helper cells obtained by conventional immunization. These results demonstrate repertoire differences between the set of (resting) lymphocytes participating in immune responses, vs. those “internally” activated in normal individuals. They also suggest the importance of Id network interactions in the compartment of “naturally” activated T and B cells.
Soluble factors secreted by naturally occurring suppressor cells that interfere with in vivo graft‐vs.‐host disease and with T cell responsiveness in vitro
Authors: Knaan‐Shanzer Shoshan , Van Bekkum Dirk W.
Published: March 2007

A potent immunosuppressive factor (SUF) is found in the supernatant of short‐term cultures of unstimulated thymocytes or spleen cells of neonatal mice and rats and in culture medium of hybridoma cell lines established by fusing neonatal mouse spleen cells with T lymphoma cells (the BW 5147 line). In vitro incubation of spleen cells with SUF suppresses the acute in vivo graft‐vs.‐host disease, normally induced by allogeneic spleen cells in lethally irradiated mice. Incubation of bone marrow cells with SUF does not affect the hemopoietic stem cells. The addition of SUF to mixed lymphocyte reaction cultures strongly suppresses lymphocyte proliferation. The non‐species‐restricted inhibition of cell proliferation induced by SUF is shown not to be due to toxicity or nonspecific interference with DNA synthesis.

Molecular size fractionation of crude SUF revealed two active moieties: a large moiety of molecular mass > 100 kDa and a small moiety of < 3 kDa. The high kDa moiety mediates T cell unresponsiveness both in vivo and in vitro. In vitro studies revealed that this moiety primarily affects an early event in the proliferative response to alloantigen and mitogen, that prevents interleukin 2 (IL 2) receptor expression and, consequently, blastogenesis and DNA duplication. It does not affect, however, the synthesis of IL 2. The suppressive activity of the low kDa moiety can be demonstrated only in in vitro systems. Pre‐treatment of donor lymphocytes with this fraction cannot prevent graft‐vs.‐host disease mortality. The inhibition of cell proliferation induced by this fraction in vitro is most likely due to interference with the utilization of IL 2, as suggested by its suppressive effect on the proliferation of CTLL‐2 cells (an IL 2‐dependent cell line).
Expression of the Fcγ receptor on Ly‐1+ B lymphocytes
Authors: Dexter Charles M. , Corley Ronald B.
Published: March 2007

The expression of IgG Fc receptor (FcR) molecules was examined on Ly‐1+ B cells and B cells tumors. FcR molecules were found on a representative Ly‐1+ B cell lymphoma of the pre‐B and as well as of the mature cell B phenotypes. The expression of the FcR did not change on these cells during their differentiation to B cells or to antibody‐secreting cells, respectively. Ly‐1+ B cells were found at low frequency (∼2%) in the spleens of normal mice, and in the peritoneal cavity where their representation was greater. The frequency of Ly‐1+ B cells declined after birth, although their numbers increased in both organs. These Ly‐1+ B cells expressed the FcR molecule throughout ontogeny. Furthermore, the amount of FcR expressed on Ly‐1+ B cells was similar to the levels expressed on their “conventional” (Ly‐1−) B cell counterparts. The FcR was also found on Ly‐1+ B cells from autoimmune mice. The significance of the expression of FcR molecules on Ly‐1+ B cells is discussed.
Activation of T cells by cross‐linking an anti‐CD3 antibody with a second anti‐T cell antibody: mechanism and subset‐specific activation
Authors: Walker Christoph , Bettens Florence , Pichler Werner J.
Published: March 2007

Binding of anti‐CD3 antibody BMA030‐F(ab′)2 to T cells is not able to induce T cell proliferation even after additional cross‐linking by plastic‐bound goat anti‐mouse Ig antibodies (panning) and addition of either interleukin (IL)1 or IL2. In search for agents able to complement the signals provided by BMA030‐F(ab′)2, several antibodies directed against CD2, CD4, CD5, CD6 and CD8 were used. Neither of these caused T cell proliferation either by themselves or when simply added together with BMA030‐F(ab′)2. However, if BMA030‐F(ab′)2 and any other of these anti‐CD2, CD4, CD5, CD6 or CD8 antibodies were cross‐linked, then proliferation of T cells occurred. The mitogenic effect of cross‐linking BMA030‐F(ab′)2 and a second anti‐T cell antibody is dependent on the presence of monocytes or exogenously added IL2. The enhancing effect of monocytes could not be replaced by addition of IL1, suggesting that other soluble factors or monocyte contact might be involved in induction of activation signals.

The mechanism leading to this mitogenic effect is dependent on combining cross‐linking of BMA030 with the second anti‐T cell antibody, whereby the second antibody appears to act as an “anchor” for the CD3 antibody, controlling and/or changing the signal transduction via the CD3 structure. This “anchor” hypothesis could be substantiated by the following observations: (a) the best stimulatory signals were obtained at a BMA030/anti‐T cell antibody ratio of 1:1; (b) the effect is independent of the antibody isotype and the epitope recognized by the second antibody; (c) the binding of BMA030 and separate cross‐linking of the anti‐T cell antibody was not sufficient to trigger T cell mitogenesis; (d) immobilization by direct binding of the CD3 antibody alone or of the CD3 and second membrane structure/antibody complex separately was not sufficient to stimulate T cell proliferation.

A further interesting aspect of this finding is the fact that a selective T cell stimulation is possible, since cross‐linking BMA030‐F(ab′)2 plus anti‐CD4 antibodies induced a selective stimulation of T4 cells, while cross‐linking BMA030‐F(ab′)2 plus anti‐CD8 activated solely T8 cells. It is thus possible to selectively activate T cell subsets in vitro.
Anti‐HLA‐class II monoclonal antibodies inhibit polyclonal B cell differentiation in vitro at the accessory cell level
Authors: De Rie Menno A. , Kabel Peter , Sauerwein Robert W. , Van Lier Rene A. W. , von Dem Borne Albert E. G. Kr. , Melief Cornelis J. M. , Miedema Frank
Published: March 2007

We produced and characterized a panel of anti‐HLA class II monoclonal antibodies (mAb) to study the inhibition of T cell‐dependent B cell differentiation. The newly produced mAb belonged to different isotypes and subclasses, and recognized at least two different nonpolymorphic HLA class II determinants. We found that anti‐HLA class II mAb inhibited the pokeweed mitogen (PWM)‐ and interleukin 2‐driven T cell‐dependent IgM synthesis, but did not affect the phorbol myristate acetate‐, phytohemagglutinin‐, anti‐T3‐ or PWM‐induced T cell proliferation. Moreover, when the mAb were tested in a monocyte‐independent B cell differentiation assay in which T cells are activated by insoluble anti‐T3 mAb in the absence of accessory cells, no effect was seen on IgM synthesis. These data indicate that inhibition of T cell‐dependent B cell differentiation by anti‐HLA class II mAb is caused by interference with an accessory cell function essential for B cell differentiation.
Limiting dilution assay for human B cells based on their activation by mutant EL4 thymoma cells: total and anti‐malaria responder B cell frequencies
Authors: Wen Li , Hanvanich Mattana , Werner‐Favre Christiane , Brouwers Nicolette , Perrin Luc H. , Zubler Rudolf H.
Published: March 2007

The analysis of human B cell responses at the clonal level (limiting dilution assay) is still technically difficult. In the present study we report on a culture system that leads to activation, proliferation and differentiation into antibody‐secreting cells (ASC) of about 90% of B cells from peripheral blood or spleen. In this system, B cells are cultured in the presence of (a) a mutant subclone of the mouse thymoma EL4 for B cell activation and (b) human T cell plus macrophage supernatant as source of proliferation and differentiation factors. ASC precursors generating clonal responses of IgM only, IgM plus IgG, or IgG only occurred at a ratio of about 6:3:1. The mean clone size was 380 cytoplasmic Ig+ cells; the mean amount of Ig secreted per clone was 20 ng.

Furthermore, it has been found using this system that a considerable proportion of peripheral blood B cells from individuals with a history of malaria infection could generate clones of anti‐malaria (Plasmodium falciparum) ASC (range of 0.1 to 1%, n = 6). In a control group of blood donors the corresponding frequencies were 10 times lower (range of 0.01 to 0.1%, n = 9). These results show that the EL4 culture system can be applied to the investigation of the human B cell specificity repertoire and of priming effects such as result from infectious disease.
Mutual tolerization of histoincompatible lymphocytes
Authors: Rammensee Hans‐Georg , Bevan Michael J.
Published: March 2007

T lymphocytes react strongly against foreign major histocompatibility complex encoded class I antigens by destroying incompatible tissue in vivo, and by generating cytotoxic T lymphocytes (CTL) in vitro. The absence of reactivity against self antigens may be due to clonal deletion of self‐reactive T cells during their ontogeny in the thymus. The functional clonal deletion of mature T cells in the periphery was described recently: CTL recognizing antigen on other CTL are eliminated (Rammensee et al., Immunol. Today 1985. 6: 41). Hence, in a normal immune system only autoreactive cells would be eliminated. Here we show that injection of lymphocytes into class I‐incompatible mice leads to abrogation of host anti‐donor as well as donor anti‐host reactivity, leaving a mixed population of host and donor T cells reactive against third‐party antigens. The results demonstrate the existence of a peripheral failsafe mechanism for the elimination of autoaggressive CTL. Whether this failsafe mechanism is actually used under physiological conditions is a different question.
“In vivo” activated splenic T cells are refractory to interleukin 2 growth “in vitro”
Authors: Bandeira António , Larsson Eva‐Lotta , Forni Luciana , Pereira Pablo , Coutinho António
Published: March 2007

Naturally activated T lymphocytes present in normal mouse spleen were studied for direct reactivity to interleukin 2 (IL 2) and for binding of anti‐IL 2 receptor (IL 2R) antibodies or radiolabeled IL 2. The majority of large‐sized splenic T lymphocytes are IL 2R−; thus, at the most one third of large L3T4+ T cells and of large Lyt‐2+ T lymphocytes bind (weakly) anti‐IL 2R antibodies; furthermore, most IL 2R+ cells in the normal spleen are actually Lyt‐2−, L3T4−. Total large splenic lymphocytes do not express more than an average of 150 high‐affinity IL 2R/cell. Such cell populations, as well as large T cells enriched from them, failed to proliferate exponentially in the presence of recombinant IL 2, but did not suppress the IL 2‐dependent proliferative responses of small T cells activated in the same cultures. In vivo activation of small T cells, in concanavalin A‐treated irradiated syngeneic hosts, also results in refractoriness to growth in recombinant IL 2. In addition, these cells remain insensitive to ligands such as concanavalin A, phorbol myristate acetate and ionomycin, in the presence or absence of IL 2. These results question the role of IL 2 as a growth factor of in vivo activated splenic T lymphocytes, or else indicate that most of these are terminally differentiated cells.
Synthesis and cell surface display of class II determinants by long‐term propagated rat T line cells
Authors: Reske Konrad , Möhle Uta , Sun Deming , Wekerle Hartmut
Published: March 2007

We have investigated the capacity of the encephalitogenic BS rat T cell line bs 83 and its variant clone bs83.III.C6 to synthesize and express RT1.B‐specific class II molecule subsets defined by monoclonal antibodies (mAb) MRC‐OX6 and MRC‐OX3. Earlier studies had indicated that mAb MRC‐OX6 recognizes three distinct molecular species: an immature oligomeric polypeptide chain complex comprised of the polymorphic subunits α,β and the invariant proteins of the γ group; a biosynthetic intermediate composed of post‐translationally modified α,β and γ chain (denoted p35) and a fully glycosylated α,β two‐chain complex derived from the plasma membrane. MRC‐OX3 was shown to recognize a serologically distinct α,β two‐chain complex that coexists with the MRC‐OX6‐specific heterodimer at the cell surface. Here we show that premutant bs83 cells were unable to synthesize class II molecules of either set. In contrast endogeneous synthesis by mutant cells of MRC‐OX6‐specific molecules was demonstrated. Unlike control spleen cells variant cells failed to synthesize the mature MRC‐OX3‐reactive class II subset. Instead a three‐polypeptide chain complex comprised of the terminally glycosylated subunits α,β and invariant chain p35 was present at the cell surface. This complex appears to represent the preserved biosynthetic intermediate that failed to release invariant chain p35 upon its transit into the plasma membrane. These latter observations support our notion of γ chain‐induced epitope diversification during post‐translational maturation of RT1.B‐specific class II molecules.
Functional diversity of Ia epitopes on macrophages
Authors: Kedar Igal , Steinberg Alfred D.
Published: March 2007

A panel of monoclonal antibodies directed against various epitopes of mouse Iak was used to block functions mediated by Iak on CBA/J macrophages in two different assay systems. The first system was the recently described proliferation inhibition of an H‐2k T cell clone (8.1.11.2) by a combination of CBA/J (nonactivated) macrophages and anti‐Iak (Kedar, I. et al. J. Immunol. 1986. 136: 3166). The second system was the well known macrophage cytotoxicity induced by interferon against various transformed cells. We found that anti‐Iak antibodies directed against serological specificity number 2 (on the α chain of IA) blocked the proliferative stimulus of the macrophage Iak for the 8.1.11.2 T cell clone. Both an IgG and an IgM antibody with this specificity were effective, whereas seven other anti‐Iak (including several for other specificities on the α chain of IA) were not. Conversely, two different monoclonal anti‐Iak antibodies directed against serological specificity number 17 (on the β chain of IA) inhibited the cytolytic activity of the activated macrophages for the 8.1.11.2 T cell clone as well as the P815 (H‐2d) mastocytoma as target, whereas four other antibodies were ineffective in this regard. In addition to providing evidence that Ia may participate in the cytolytic activity of activated macrophages, these studies demonstrate that different epitopes of Iak on macrophages are differentially involved in different biological functions.
B lymphocyte differentiation in the rat: production and characterization of monoclonal antibodies to B lineage‐associated antigens
Authors: Kroese Frans G. M. , Wubbena Auk S. , Opstelten Davine , Deenen Gerrit Jan , Schwander Edwin H. , De Leij Loe , Vos Hans , Poppema Sibrand , Volberda Johanna , Nieuwenhuis Paul
Published: March 2007

Three mouse monoclonal antibodies (mAb) directed against rat B lineage antigens were produced. The mAb, designated HIS14 (IgG1), HIS22 (IgM) and HIS24 (IgG2b), were characterized for binding to lymphoid and nonlymphoid tissues by immunoperoxidase staining of frozen sections and by (double‐) immunofluorescence staining of single cell suspensions from lymphoid organs. HIS14 recognized a pan B cell determinant: it reacted with virtually all cells of each anatomic B cell compartment and with about 95% of surface (s)Ig+ cells in thoracic duct lymph and in suspensions of spleen and lymph nodes. HIS22 and HIS24 detected B lineage‐associated antigens expressed by major subpopulations of B cells. HIS22 predominantly stained the lymphocyte corona, but not (or weakly) the germinal centers and splenic marginal zones, whereas HIS24 reacted with both corona and germinal center and not (or weakly) with marginal zone. In accordance with this, substantial proportions of sIg+ cells in spleen cell suspensions did not express HIS22 or HIS24 determinants (20% and 27%, respectively). In bone marrow the vast majority of cytomplasmic μ+ pre‐B cells were HIS14+ and HIS24+, and up to one third also HIS22+, indicating an appearance of the determinants early in B lymphocytopoiesis. The antigens recognized by HIS14, HIS22 and HIS24 are lost during the final stage of B cell differentiation: none of the mAb bound to plasma cells.

As far as detectable, neither cells of myeloid and erythroid lineages in bone marrow nor thymocytes were stained by HIS14, HIS22 or HIS24. In suspensions of peripheral lymphoid organs (spleen and lymph nodes) but not in thoracic duct lymph, HIS14 and HIS24 labeled a small proportion (12% and 14%, respectively) of Ig− cells. HIS22 did not bind to Ig− peripheral lymphocytes. Reactivity of HIS14, HIS22 and HIS24 with nonlymphoid tissues was virtually absent; HIS22 stained the high endothelial venules in lymph nodes and Peyer's patches.

As determined by immunoblotting, the antigenic determinants on lymph node cells recognized by HIS14, HIS22 and HIS24 were present on molecules with an apparent molecular mass of 205 kDa, 210 (and 175) kDa and 205 kDa, respectively, which is similar to the molecular mass of the B cell form of the rat leukocyte common antigen. In addition, the antigens recognized by HIS14, HIS22 and HIS24 co‐capped with the leukocyte common antigen. This suggests that each of the three mAb recognize determinants present on the B cell form of the leukocyte common antigen.
Monoclonal antibodies identify three epitope clusters on the mouse p55 subunit of the interleukin 2 receptor: relationship to the interleukin 2‐binding site
Authors: Moreau Jean‐Louis , Nabholz Markus , Diamantstein Tibor , Malek Thomas , Shevach Ethan , Thèze Jacques
Published: March 2007

A new rat monoclonal antibody (5A2) directed against the mouse interleukin 2 receptor (IL2R) is described. This antibody binds specifically to IL2R+ cells, competes with both cold IL2 and 125I‐labeled IL2 for the IL2‐binding site and inhibits IL2‐induced T cell proliferation. This reagent was compared to five previously characterized rat monoclonal antibodies directed against the p55 subunit of the mouse IL 2R. The capacity of all the antibodies to inhibit IL2‐induced T cell proliferation was assessed. The relationship of these eight antibodies to each other and to the IL2‐binding site was studied by cross‐inhibition assays, and by Scatchard analysis of the data.

The results indicate that the six monoclonal antibodies recognize epitopes in three areas of the p55 subunit of the mouse IL2R. Antibodies against two of the clusters affect IL2 binding. One cluster defined by 3 antibodies is probably located in the area of the IL 2‐binding site as there is competitive inhibition between IL2 and antibodies against this cluster. A single antibody directed against an epitope outside this cluster appears to inhibit IL2 binding by inducing a conformational change in the p55 subunit of the IL2R. Two other antibodies identify a third region which is not involved in the formation of the binding site.
The immune response of the mouse to lymphocytic choriomeningitis virus V. High numbers of cytolytic T lymphocytes are generated in the spleen during acute infection
Authors: Moskophidis Demetrius , Assmann‐Wischer Ursula , Simon Markus M. , Lehmann‐Grube Fritz
Published: March 2007

In the spleens of C57BL/6J (B6) and CBA/J (CBA) mice undergoing acute infection with lymphocytic choriomeningitis (LCM) virus, lymphocytes with the ability to develop in vitro into LCM virus‐specific cytolytic clones were enumerated by use of the limiting dilution method. At intervals after virus inoculation, defined numbers of cells were cultivated with virus‐infected syngeneic stimulator cells and T cell growth factor in multiple wells of microculture plates. After 7 days, individual cell cultures were tested for their ability to cause release of 51Cr from infected and uninfected syngeneic target cells. In cultures seeded with spleen cells from uninfected mice or from mice infected 3 days previously, no cytolytic activity was observed. On day 5, cells developing into LCM virus‐specific cytolytic effector cells were detected. They rose in numbers, and on days 8 to 9 after infection, values of approximately 1/10 and 1/200 in B6 and CBA mice, respectively, were calculated. A low proportion of micro‐cultures proved cytolytic also for noninfected syngeneic target cells, but the counts thus released were consistently much lower than the counts set free from infected targets, and no regular dose‐response relationships existed between seeded cells and positive cultures. Determination of cell surface antigens of responder cells by negative and positive selection procedures disclosed that they were predominantly T lymphocytes and expressed Lyt‐2 but not L3T4 surface markers. Lysis by the great majority of LCM virus‐specific clones was restricted by products of the major histocompatibility gene complex (MHC), but a few lysed, in addition, allogeneic infected or uninfected targets; however, a consistent pattern of alloreactivity was not observed. Furthermore, cells of a proportion of the cultures also lysed uninfected YAC cells. Probably this natural killer‐like activity was acquired by T lymphocytes during prolonged cultivation. We conclude that most spleen cells that during acute infection with LCM virus attained the ability to develop in vitro into LCM virus‐specific cytolytic clones were derived from MHC‐restricted Lyt‐2+, L3T4− antigen‐specific cytolytic T lymphocytes and their activated precursors.
The role of monocyte lymphocyte function‐associated antigen 1 (LFA‐1) in accessory cell function
Authors: Dougherty Graeme J. , Hogg Nancy
Published: March 2007

Monoclonal antibodies directed against the common β subunit shared by lymphocyte function‐associated antigen‐1 (LFA‐1), CR3 and p150,95 or the α subunit of LFA‐1, inhibited the T lymphocyte proliferative response to tetanus toxoid, pokeweed mitogen‐1 and OKT3 but had little or no effect on the response to the leukoagglutinating lectins pokeweed mitogen‐2 and phytohemagglutinin. The LFA‐1 molecule was shown to participate in the adhesive interaction between monocytes and T lymphocytes that was required for the initiation of an immune response. Both monocyte and T lymphocyte LFA‐1 were found to contribute to this interaction. These results further define the part played by the LFA‐1 molecule in the activation of T lymphocytes.
Phenotype of stromal cell‐associated thymocytes in situ is compatible with selection of the T cell repertoire at an “immature” stage of thymic T cell differentiation
Authors: Kyewski Bruno A. , Momburg Frank , Schirrmacher Volker
Published: March 2007

Murine thymocytes interacting with cortical macrophages, cortical epithelial cells and medullary dendritic cells in situ express T cell receptors at low to intermediate density. They co‐express the lineage markers Lyt‐2 and L3T4 and are not enriched for cells expressing high‐density interleukin 2 and lymph node homing receptors. Thus, recognition of stromal cells in situ in distinct thymic microenvironments occurs at a common stage of T cell maturation which is phenotypically intermediate between intrathymic precursor cells and mature medullary‐type thymocytes. The surface phenotype of dendritic cell‐associated thymocytes indicates the presence of thymocytes with a “cortical” phenotype within the antigen‐exposed (“nonsterile”) phase of T cell maturation in the medulla.
Alternative pathway of complement activation by stimulated T lymphocytes I. Binding of C3 fragments
Authors: Sármay Gabriella , Ramos Oscar F. , Klein Eva , Kai Chieko , Gergely Janos
Published: March 2007

Human blood lymphocytes cultured for 3 days with concanavalin A (Con A), phytohemagglutinin or pokeweed mitogen, in mixed lymphocyte culture with added interleukin 2 and stimulated by a lymphoblastoid cell line were found to activate and bind C3 molecules when exposed to human serum. The split products of C3 were detected in the supernatants and on the surface of the activated cells. The surface‐attached C3 fragment on the Con A blast was identified as C3b by immune adherence i.e. binding of CR1 carrying human erythrocytes. In the Con A‐stimulated population the majority of cells that activated and bound C3 were CD3 and Fcγ receptor (CD16)‐positive but complement receptor‐negative blasts. In this cell subset both CD4 and CD8‐positive cells were detected but their frequency suggested that a proportion of them carried both markers.
Internalization of human T lymphocyte receptors
Authors: Telerman Adam , Amson Robert B. , Romasco Franco , Wybran Joseph , Galand Paul , Mosselmans Roger
Published: March 2007

Monoclonal antibodies specific to human T lymphocyte receptors are currently being used to define the biochemical structure of these proteins as well as of functionally distinct cell subsets. Since one of the antibodies (OKT3) recognizing the T3 (CD3) receptor mimics vital physiological processes involved in the activation of the immune system and has been successfully used as a therapeutical agent, we investigated one of the mechanisms underlying this antibody‐receptor interaction. Our results show that after binding of OKT3, the complex (OKT3‐T3) disappears rapidly from the cell surface. Using electron microscopy, we found that this down‐regulation is due to the internalization of the complex. Parallel experiments performed on the T11 (CD2) and T4 (CD4)/AIDS retrovirus receptor indicate that the same mechanism applies for the down‐regulation of those molecules. These data suggest that the T3, T11 and T4 receptors have a behavior comparable to other well characterized, hormonal and viral receptors; they provide information on the metabolization pathway of surface receptors and on the possible intracellular penetration of ligands like the HTLV‐III/LAV agent in human T lymphocytes.
L3T4+ T cells able to mediate parasite‐specific delayed‐type hypersensitivity play a role in the pathology of experimental Chagas' disease
Authors: Hontebeyrie‐Joskowicz Mireille , Said Gerard , Milon Geneviève , Marchal Giles , Eisen Harvey
Published: March 2007

During the chronic phase of infection with the parasite Trypanosoma cruzi, mice develop inflammatory lesions in the heart and skeletal muscles, as well as in peripheral nerves and the liver. We demonstrated the presence, in the blood of chronically infected mice, of L3T4+ T cells able to transfer a specific T. cruzi delayed‐type hypersensitivity (DTH) reaction. Transfer of the chronic inflammatory lesions was obtained by injecting Lyt‐2+‐depleted lymphocytes from either lymph node or blood of infected mice. T cell lines were established from the chronically infected mice by culturing peripheral blood lymphocytes or lymph node cells with either T. cruzi extracts (TC) or mouse peripheral nerve extracts (PN). Those cell lines that presented an L3T4+ phenotype were also able to specifically transfer a local DTH reaction to naive recipients. Examination of the antigen specificities of these TDTH lines revealed three types: those that mediated a DTH reaction to TC, those that responded to both TC and PN and those that provoked a DTH response when injected with subinflammatory doses of an irrelevant antigen. Some of the lines, when injected into the sciatic nerve of naive recipients, provoked demyelination of the type observed in chronically infected animals. These results suggest that T. cruzi and the host nervous system share common epitopes that can be recognized by TDTH cells.
Cytotoxic T lymphocyte recognition of a xenogeneic major histocompatibility complex antigen expressed in transgenic mice
Authors: Bluestone Jeffrey A. , Pescovitz Mark D. , Frels William I. , Singer Dinah S. , Hodes Richard J.
Published: March 2007

Introduction of a porcine major histocompatability complex (MHC) class I gene (PD1) into the genome of a C57BL/10 (B10) mouse has been shown to lead to cell surface expression of the porcine MHC antigen, SLAPD1 in a transgenic mouse. The PD1 product expressed on spleen cells from the transgenic mice stimulated B10 spleen cells in a mixed lymphocyte culture to generate PD1‐specific cytotoxic T lymphocytes (CTL). The CTL were PD1 specific since they lysed transgenic splenic blast cells and PD1‐transfected L cells, but not B10 blasts or control L cells. The CTL were L3T4−, Lyt‐2+ and their activity was partially inhibited by either anti‐Lyt‐2 antibody or by anti‐swine MHC alloantibodies. The repertoire of responding B10 anti‐transgenic CTL was assessed by examining their cross‐reactivity on a series of murine allogeneic targets. The B10 anti‐transgenic CTL showed some cross‐reactivity on conventional allogeneic targets, but reacted strongly on a series of mutant H‐2Kbm blast cells. In addition, B10 anti‐B6.cH‐2bm6 CTL cross‐reacted extensively on the transgenic target cells. These results demonstrated that normal B10 CTL possess a repertoire specific for the products of the xenogeneic class I gene PD1, that this repertoire is cross‐reactive with the conventional alloreactive CTL repertoire, and that there exists an unanticipated relationship between PD1‐specific CTL and CTL specific for Kb mutant determinants.
Germinal centers develop oligoclonally
Authors: Kroese Frans G. M. , Wubbena Auk S. , Seijen Hendrik G. , Nieuwenhuis Paul
Published: March 2007

As part of our studies into the role of germinal centers, we investigated whether each de novo generated germinal center (GC) develops from one single GC precursor cell (GCPC, monoclonal development), a few GCPC (oligoclonal development) or from many GCPC (polyclonal development). Thus, lethally (9 Gy) X‐irradiated AO (RT1u) rats were reconstituted with 108 thoracic duct lymphocytes (TDL) containing mixtures of AO and AO × BN cells in various ratios. The AO TDL were tolerant for AO × BN cells by using TDL from AO →(AO × BN)F1 (RT1u/n) X‐irradiation bone marrow chimeras. To induce GC formation in the spleen of TDL‐reconstituted rats, animals were i.v. injected with 109 sheep red blood cells. Five days after reconstitution and antigenic challenge spleens were taken for analysis of cellular make up of de novo generated GC. Spleen sections were immunohistochemically stained with monoclonal antibody F17‐23‐2, recognizing major histocompatibility complex class II antigens of the RT1n haplotype but not the RT1u haplotype, to discriminate between B cells of AO and AO × BN origin. Analysis of the GC in spleens of rats reconstituted with a mixture of AO and AO × BN TDL revealed three types of GC: GC entirely composed of AO cells, GC entirely composed of AO × BN cells and GC containing a mixture of both. The relative frequencies of these three types of GC indicated that in our experimental system, de novo GC developed oligoclonally from one to three GCPC. These data strongly suggest that GC are sites of antigen‐driven expansion of peripheral B cells to very large clones.
Curing BB rats of freshly manifested diabetes by short‐term treatment with a combination of a monoclonal anti‐interleukin 2 receptor antibody and a subtherapeutic dose of cyclosporin A
Authors: Hahn Hans J. , Lucke Silke , Klöting Ingrid , Volk Hans D. , Baehr Rüdiger V. , Diamantstein Tibor
Published: March 2007

The BioBreeding (BB) rat develops spontaneously a syndrome resembling human type I diabetes mellitus. The short‐term treatment of newly diagnosed diabetic BB rats with the anti‐interleukin 2 receptor (IL2R) monoclonal antibody ART‐18 in combination with a subtherapeutic dose of cyclosporin A for 10 days, a treatment shown previously to eliminate specifically antigen‐activated IL 2R+ T lymphocytes by sparing suppressor cells, resulted in (a) normoglycemia in 70% of the animals, (b) the maintenance of B cell volume density and (c) an increase in pancreatic insulin content. Moreover, the glucose tolerance of successfully treated animals was not significantly different from that of normoglycemic BB rats during the whole observation period (up to 120 days after the end of the therapy). This is the first report on successful treatment of a spontaneous autoimmune disease by a short‐term and specific immunosuppressive regimen with limited side effects.
The induction of helper and suppressor cells with secondary anti‐hen egg‐white lysozyme B hybridoma cells in the absence of antigen
Authors: Kawahara Douglas J. , Miller Alexander , Sercarz Eli E.
Published: March 2007

The results presented in this report define a dominant T cell‐recognized public idiotype (SRId) expressed on monoclonal anti‐chicken egg‐white lysozyme (HEL) antibodies produced by hybridomas derived from secondary response lymphocytes. This Id mediates interactions between SRId+ B cells and SRId‐recognizing T cells. In the absence of exogenous antigen, irradiated secondary anti‐HEL B hybridoma cells (B‐Hyb) of nonoverlapping specificity can be used to induce a helper T cell population capable of specifically stimulating an in vitro anti‐HEL plaque‐forming cell (PFC) response. Importantly, similar immunizations using carbodiimide‐treated secondary anti‐HEL B‐Hyb cross‐primed for a suppressor T cell population capable of suppressing this in vitro anti‐HEL PFC response. That is, suppression was seen not only to the response induced by the homologous B‐Hyb but to other B‐Hyb which express anti‐HEL monoclonal antibody of nonoverlapping specificity. This evidence is consistent with the presence of a pre‐existent regulatory Id network involving SRId in antigennaive animals. After immunization with HEL, regulatory cells exert a strong selective pressure which leads to a secondary anti‐HEL B population, of varying fine specificity, but uniformly positive for SRId.
Myasthenic sera recognize the human acetylcholine receptor bound to thymopoietin
Authors: Morel Evelyne , Garabedian Béatrice Vernet‐Der , Raimond Françoise , Audhya Tapan K. , Goldstein Gideon , Bach Jean‐François
Published: March 2007

The thymic hormone, thymopoietin (Tpo), from human (HTpo), bovine (BTpo) and from synthetic (sHTpo) origins bound to the acetylcholine receptor (AChR) solubilized by Triton 1.5% from human muscle. This binding was demonstrated either by inhibition of formation of radiolabeled α bungarotoxin (αBgt)‐AChR complexes measured after precipitation by ammonium sulfate or by a myasthenic serum containing a high concentration of anti‐AChR antibodies, or directly by incubating the human AChR with radiolabeled sHTpo or BTpo. The 125I‐labeled αBgt‐AChR complexes were totally inhibited by 10−6 M sHTpo or BTpo. The complexes formed by AChR and the radiolabeled Tpo were recognized specifically by sera containing anti‐AChR antibodies from myasthenic patients. The active pentapeptide derivative of Tpo, thymopentin, another thymic hormone, thymulin, as well as bovine insulin did not interfere with the specific binding of αBgt to human AChR. Tpo and anti‐AChR antibodies could participate together in the inhibition of neuromuscular conduction with Tpo modulating the depressive effect of the antibodies on the neuromuscular junction in myasthenia gravis.
Activation of memory and virgin B cell clones in hyperimmune animals
Authors: Berek Claudia , Jarvis John M. , Milstein Cesar
Published: March 2007

To study the long‐term memory response, BALB/c mice were allowed to rest for over a year after a secondary immunization with the hapten 2‐phenyl‐oxazol‐5‐one (phOx). For the tertiary immunization two different protocols were used. In one protocol mice were injected i.v. and 3 days later spleen cells were fused to a nonproducing hybridoma line. PhOx‐specific hybridomas were established and the sequence of the heavy and light chain mRNA was determined. This tertiary response resembled the diversity pattern of the secondary response with a further increase both in somatic mutations and in the average dissociation constant. The high number of somatic mutations demonstrates the persistence of memory B cell clones over a long time period. In the second protocol mice were boosted with an i.p. injection of alumprecipitated antigen phOx and 7 or 14 days later spleen cells were fused. Sequence analysis of heavy and light chain mRNA showed that these tertiary response antibody molecules had surprisingly few somatic mutations, indicating an activation of virgin B cell clones in these hyperimmunized animals. The maturation of these newly stimulated B cell clones seems to follow somewhat similar rules to those found for the primary response. It appears therefore that the two immunization protocols reflect the response of memory and virgin B cells, respectively.
The role of L3T4+ cells in the pathogenesis of lupus in lpr‐bearing mice. I. Defects in the production of interleukins 2 and 3
Authors: Santoro Thomas J. , Lehmann Kathrin R. , Batt Rebecca A. , Kotzin Brian L.
Published: March 2007

Mice which bear the lpr gene spontaneously develop autoimmune syndromes characterized by massive expansion of an unusual T cell subset which is phenotypically Thy‐1+, L3T4−, Lyt‐2−, B220+. The mutant T cells are refractory to stimulation with mitogenic lectins and, by implication, are thought to be solely responsible for the defects in lymphokine production manifested by lpr mice. The contribution of the remaining L3T4+ T cell subset to the latter derangements has not been previously examined and is the focus of this study. We found that abnormalities in concanavalin A‐induced interleukin 2 and 3 production in the spleens of MRL‐lpr/lpr and C57BL/6.lpr mice occurred in the presence of limited infiltration with B220+, L3T4− T cells. Mixing experiments indicated that B220+ T cells were not suppressive. Furthermore, lpr spleen cells enriched for L3T4+ cells and depleted of sIg+, B220+ and Lyt‐2+ cells demonstrated reductions in lymphokine production which were comparable to those seen in unfractionated preparations. Spleen cells from C57BL/6.lpr mice, enriched for L3T4+ cells, were also markedly impaired in a mixed leukocyte reaction in response to stimulator cells from the class II major histocompatibility complex mutant bm12. The results indicate that the aberrations in lymphokine production and proliferation in the spleen cells of lpr mice involve not only B220+ T cells but also L3T4+ cells and suggest a potential role for the L3T4+ subset in the pathogenesis of lupus in lpr‐bearing mice.
Comparison of the clonal diversity of the B cell repertoires in adult mice that differ in the expression of cell surface IgD
Authors: Fultz Marion J. , Finkelman Fred D. , Metcalf Eleanor S.
Published: March 2007

To determine whether the expression of surface IgD (sIgD) influences the extent of the expressed B cell repertoire, the clonal diversity of the B cell population in mice treated chronically with anti‐IgD (delta) antibodies has been compared with the B cell repertoire observed in control animals, using the splenic focus limiting dilution B cell assay. The results show that the phosphorylcholine (PC)‐specific B cell precursor frequency in anti‐delta antibody‐treated mice is increased when compared with that of control mice. Isotype and idiotype (T15) analyses of PC clonal products from anti‐delta antibody‐treated and control mice revealed no distributional differences. Analyses of the 2,4‐dinitrophenyl (DNP)‐ and fluoresein isothiocyanate‐specific B cell repertoires confirmed that the equal or increased precursor frequencies observed in anti‐delta antibody‐treated mice are not specific for the PC antigen. The increased precursor frequency of B cells from anti‐delta antibody‐treated mice was not the result of increased homing of B cells from anti‐delta antibody‐treated mice to recipient spleens, since B cells from control mice homed twice as well to recipient spleens as did B cells from anti‐delta antibody‐treated mice. Other studies demonstrated that (a) on average, antibody‐secreting clones were generated more slowly when B cells from anti‐delta antibody‐treated mice were used as a source of precursors than B cells of control mice and (b) both sIg− spleen cells and sIg+ spleen cells from anti‐delta antibody‐treated mice generated a higher frequency of specific antibody‐secreting clones than did the corresponding populations from control mice. These observations suggest that a population of sIgM+sIgD− B cells exists that resembles sIgD+ B cells rather than neonatal or xid B cells in its ability to generate responses to PC and suggests that the sIgM+ sIgD− B cells from anti‐delta antibody‐treated mice are more responsive than are sIgM+IgD+ B cells, regardless of antigenic specificity, to the stimuli provided in the splenic focus system. Finally, this study suggests that the expression of sIgD does not influence the extent of the expressed B cell repertoire.