The Journal of Experimental Medicine

Buerstedde, J M; Pease, L R; Nilson, A E; Bell, M P; Chase, C; Buerstedde, G; McKean, D J

doi: 10.1084/jem.168.3.823pmid: 3049906

A panel of mutant class II genes have been constructed using site-directed mutagenesis and DNA-mediated gene transfer. Using this technique, Ak beta polypeptides have been altered by substituting one or more Ad beta-specific residues at polymorphic positions in the beta 1 domain. Transfection of M12.C3 B lymphoma cells with most mutant Ak beta* genes results in the expression of Ak beta* Ad alpha molecules on the cell surface. However, the substitution of a single d allele residue at position 78 or 86 in the Ak beta polypeptide results in either the complete absence or very low levels, respectively, of cell surface expression of the Ak beta* Ad alpha molecule, but does not alter Ak beta* Ak alpha expression. The T.86 Ak beta* Ad alpha is expressed primarily in an intracellular compartment while the T.78 Ak beta* molecule does not appear to be produced. The core-glycosylated T.78 Ak beta* polypeptide does, however, form a complex intracellularly with the core-glycosylated Ii polypeptide. Substitution of the combination of d allele residues at Ak beta polymorphic positions 9, 12, 13, 14, and 17 results in the absence of Ak beta* Ak alpha cell surface expression but does not alter the expression of this mutant Ak beta* polypeptide with the Ad alpha polypeptide. These allele-specific expression mutants demonstrate that substitution at certain beta 1 domain positions may result in the alteration of Ia cell surface expression and that the transport of Ia molecules from the Golgi apparatus to the cell surface may be regulated by signals that are determined by the interaction of polymorphic residues in both the alpha and beta polypeptides.

Ishida, Y; Chused, T M

doi: 10.1084/jem.168.3.839pmid: 2971755

Calcium management differs in T and B lymphocytes. Ca2+i elevation in response to calcium ionophores is up to 10 times greater in T cells than B cells. There is no difference between them in ionophore uptake. T cells, but not B cells, possess a calcium-sensitive potassium channel which produces membrane hyperpolarization at Ca2+i above 200 nM. This alters T cell density providing a rapid and easy method of cell separation. In contrast, B cells depolarize when Ca2+i is increased. Isolated B cell membrane vesicle ATP-dependent calcium pump activity is higher than T cell vesicles. Membrane depolarization reduces the Ca2+i response to ionomycin, most dramatically in T cells because they are hyperpolarized by increased Ca2+i. The most likely basis of this behavior is an effect of membrane potential on lymphocyte membrane calcium pump activity. This mechanism provides an explanation of the inhibitory effect of membrane depolarization on T lymphocyte responses.

Lebman, D A; Coffman, R L

doi: 10.1084/jem.168.3.853pmid: 3049907

Although it has been established that IL-4 enhances both IgG1 and IgE secretion in LPS-stimulated B cell cultures, these studies failed to determine whether IL-4 preferentially induces isotype switching or preferentially allows for the maturation of precommitted precursor cells. To distinguish between these possibilities, it is necessary to ascertain the effect of IL-4 on the isotypes secreted by individual precursor cells during clonal expansion. Therefore, clonal cultures of B cells stimulated with a Th2 helper cell line specific for rabbit Ig and rabbit anti-mouse IgM were established. The majority of B cells are capable of undergoing clonal expansion under these conditions. To vary the level of IL-4 present, either IL-4 or anti-IL-4 was added to cultures. In the presence of IL-4 there was an increase in the proportion of clones that secreted IgE and a decrease in the proportion of clones that secreted IgM. The addition of IL-4 to cultures also increased the amount of IgE secreted by individual clones. Thus, these experiments definitively prove that IL-4 causes specific heavy chain class switching to IgE in Th2-stimulated B cell cultures. In contrast, IL-4 does not affect the proportion of clones secreting IgG1, suggesting that other consequences of Th cell-B cell interactions play a role in the generation of an IgG1 response.

Mita, S; Harada, N; Naomi, S; Hitoshi, Y; Sakamoto, K; Akagi, M; Tominaga, A; Takatsu, K

doi: 10.1084/jem.168.3.863pmid: 3262707

T cell-replacing factor (TRF)/IL-5 is a glycosylated polypeptide that acts as a key factor for B cell growth and differentiation. Since IL-5 action is probably mediated by specific cell surface receptor(s), we have characterized the binding of IL-5 to cells using biosynthetically 35Smethionine-labeled IL-5 and 125I-IL-5 that had been prepared using Bolton-Hunter reagent. The radiolabeled IL-5 binds specifically to BCL1-B20 (in vitro line) (a murine chronic B cell leukemic cell line previously shown to differentiate into IgM-secreting cells in response to IL-5) within 10 min at 37 degrees C. There are two classes of binding sites with high affinity (Kd = 66 pM) and low affinity (Kd = 12 nM) for IL-5 and an average number of binding sites for high affinity and for low affinity were 400 and 7,500 per cell, respectively. The specificity of binding of radiolabeled IL-5 has been confirmed by demonstrating that only unlabeled IL-5 and anti-IL-5 mAb but not by IL-1, IL-2, IL-3, IFN-gamma, and GM-CSF inhibit radiolabeled IL-5 binding to BCL1-B20 cells. Treatment of surface-bound radiolabeled IL-5 with bivalent crosslinkers identified a membrane polypeptide of Mr 46,500 to which IL-5 is crosslinked. A variety of cell types have been surveyed for the capacity to bind specifically radiolabeled IL-5 with high affinity. BCL1 cells MOPC104E (murine myeloma cell line) expressed IL-5-R, whereas BAL. 17 and L10 A (B cell lymphoma) did not. T cell line, mastocytoma cell line, or macrophage tumor cell line did not display detectable levels of IL-5-R. were hardly detectable on normal resting B cells but were expressed on LPS-activated B cells, fitting the function of IL-5 that acts on activated B cells for their differentiation into Ig-secreting cells. Intriguingly, early B cell lines (J-87 and T-88) that grow in the presence of IL-5 expressed significant but low numbers of high-affinity binding sites for IL-5. The biological effects of IL-5 were mediated by high-affinity binding sites. The identification and characterization of IL-5-R should provide new insight into the apparent diverse biological activities of IL-5.

Koike, K; Nakahata, T; Takagi, M; Kobayashi, T; Ishiguro, A; Tsuji, K; Naganuma, K; Okano, A; Akiyama, Y; Akabane, T

doi: 10.1084/jem.168.3.879pmid: 3049908

We investigated the effects of B cell stimulatory factor 2/interleukin 6 (BSF-2/IL-6) on the development of murine hemopoietic progenitors using serum-containing culture and serum-free culture. In serum-containing culture, BSF-2 mainly supported multipotential blast cell colonies from spleen cells of normal and 5-fluorouracil (5-FU)-treated mice. In serum-free culture, no colony growth was seen in the presence of BSF-2. Addition of BSF-2 to the serum-free culture containing IL-3 resulted in a significant increase in the number of colonies formed from multipotential progenitors in spleen cells and bone marrow cells of 5-FU-treated mice, whereas no effects were seen on the number of single or oligolineage colonies formed by the spleen cells of normal mice. These results suggested that BSF-2 and IL-3 act synergistically on the multipotential progenitors but not on the maturer progenitors. When BSF-2 was added to a culture containing low concentrations of IL-3 (1 U/ml, 4 U/ml), which had little effect on colony formation, the number of total colonies formed by the spleen cells and bone marrow cells of 5-FU-treated mice increased significantly. The combination of BSF-2 and 40 U/ml of IL-3 resulted in a significant enlargement of GMM colonies. Thus, BSF-2 appears to enhance the sensitivity of multipotential hemopoietic progenitors to IL-3.

Benaroch, P; Georgatsou, E; Bordenave, G

doi: 10.1084/jem.168.3.891pmid: 2902183

We report here the successful induction of allotype suppression in homozygous Ighb/b mice (CB20 or C57BL/6) by neonatal injection of T splenocytes from Igha congenic sensitized mice (BALB/c or BC8, respectively). The sensitization of the T cell donors was achieved by two intravenous injections of B splenocytes from Ighb congenic mice. Treated homozygous Ighb/b mice developed, as of 16-24 wk of age, a chronic suppression of Igh-1b expression (IgG2a of Ighb haplotype). The other productions tested (IgM, IgD, and IgA) of Ighb haplotype were unaffected. In vivo treatment with cytotoxic anti-CD4 or anti-CD8 mAb of mice subjected to chronic Igh-1b suppression clearly showed that CD8+ lymphocytes (suppressor or cytotoxic cell) were essential for the maintenance of the suppression. The suppression was indeed abrogated after a 1-wk treatment with anti-CD8 mAb containing culture supernatant, whereas, the anti-CD4-treated mice continued to be subjected to suppression. This anti-CD8 in vivo treatment was shown to have no effect on thymus but to severely reduce the percentages of CD8+ cells in spleen and in peripheral blood without affecting the percentages of CD4+ cells, leading to a large and rapid Igh-1b expression (up to 0.5 mg per ml of serum, the day after the end of the treatment). This suppression abrogation, and thus the Igh-1b expression, was either transient or permanent. When it was transient, a second 1-wk treatment with anti-CD8 mAb containing culture supernatant induced once again a rapid and significant production of Igh-1b (up to 0.3 mg of Igh-1b per ml of serum).

Raff, H V; Siscoe, P J; Wolff, E A; Maloney, G; Shuford, W

doi: 10.1084/jem.168.3.905pmid: 3049909

Group B streptococcal (GBS) infections cause significant mortality and morbidity among infants. Passive antibody immunotherapy has been proposed as treatment for infected infants. To this end, two human mAb-secreting cell lines were produced by EBV immortalization of human B cells. The mAbs were specific for the group B polysaccharide and bound to strains of all five serotypes as demonstrated by ELISA and crossed immunoelectrophoresis. The mAbs reacted and opsonized 100% (132/132) of the clinical isolates tested which represented all four capsule types. Both prophylactic and therapeutic protection with these mAbs were demonstrated in neonatal rats given lethal infections of types Ia and III human clinical isolates. These data indicate that a single human mAb directed against the group B carbohydrate can protect against GBS infections caused by the different serotypes. This antibody may be useful in the passive immunotherapy of infants infected with GBS.

Borysiewicz, L K; Hickling, J K; Graham, S; Sinclair, J; Cranage, M P; Smith, G L; Sissons, J G

doi: 10.1084/jem.168.3.919pmid: 2844952

CTL are held to be an important host defense mechanism in persistent herpes-virus infections. We have therefore studied the nature and specificity of human cytomegalovirus (HCMV)-specific CTL in normal persistently infected individuals. This was achieved by using vaccinia recombinants encoding viral genes expressed at different stages of the virus replicative cycle, a structural glycoprotein gB (vac.gB) and the major 72-kD immediate early nonstructural protein (vac.IE) of HCMV, combined with limiting dilution analysis of the CTL response. In two subjects, 43 and 58% of HCMV CTL precursors (CTLp) lysed vac.IE-infected cells, in contrast to less than 6% lysing gB-infected cells. HCMV-specific CTL could also be generated by secondary in vitro stimulation with vac.gB- but not vac.IE-infected autologous fibroblasts. The high frequency of 72-kD IE protein-specific CTL suggests that this is at least a major recognition element for the HCMV-specific CTL response in asymptomatic persistently infected individuals, and CTL with this specificity may be important in maintaining the normal virus/host equilibrium.

Tokunaga, K; Saueracker, G; Kay, P H; Christiansen, F T; Anand, R; Dawkins, R L

doi: 10.1084/jem.168.3.933pmid: 3171479

The genomic organization of the human MHC was examined in multiple examples of six different supratypes using pulsed field electrophoresis (PFGE) after digestion of genomic DNA with infrequency cutting restriction endonucleases. Differences in restriction fragment length and band intensity were shown to be specific for each supratype. Mapping of the MHC revealed that each supratype contains previously undescribed deletions and insertions between HLA B and DQ regions.

Gottlieb, A B; Luster, A D; Posnett, D N; Carter, D M

doi: 10.1084/jem.168.3.941pmid: 2459293

The pathologic features of psoriatic plaques are inflammation and increased epidermal turnover. IP-10, a cytokine the expression of which is induced by gamma-interferon, is a member of a family of soluble mediators with inflammatory and growth-promoting activities. IP-10 protein was detected in keratinocytes and the dermal infiltrate from active psoriatic plaques using an affinity-purified rabbit anti-IP-10 antibody in immunoperoxidase studies. Successful treatment of active plaques decreased IP-10 expression in plaques. These results were corroborated by Northern blot analysis with an IP-10 cDNA probe. We have previously detected activated T cells and HLA-DR keratinocytes in active psoriatic plaques. Since IP-10 is detected in delayed cellular immune responses, the present study further points to the role of ongoing cellular immune responses in the pathogenesis of psoriasis.