Purification and physicochemical characterization of murine T cell replacing factor (TRF)

Murine T cell replacing factor (TRF) was purified from a cellfree supernatant of a T cell hybridoma (B151K12) that constitutively produces TRF. Two assay systems for TRF activity were employed: 1) induction of anti-DNP IgG PFC responses in cultures of splenic B cells from DNP-KLH-primed BALB/c mice, and 2) induction of IgM PFC in chronic B cell leukemic cells (BCL1). The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, gel permeation with fast protein liquid chromatography (FPLC), and disc polyacrylamide gel electrophoresis. Overall, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity, and the specific activity of the purified TRF was approximately 9.6 X 10(4) U/mg. The TRF that is active in these systems is distinct from the other lymphokines such as IL 1, IL 2, BCGFI (now known as BSFp1), and gamma-interferon. The TRF is extremely hydrophobic, with an apparent m.w. of 50,000 to 60,000 on gel permeation chromatography and 18,000 on SDS-PAGE under reducing conditions. Highly purified B151-TRF abrogated the activity by treatment with trypsin but not with RNase. Moreover, it bound to lima bean agglutinin-Sepharose specific for N-acetylgalactosamine residues, indicating that B151-TRF is a glycosylated glycoprotein containing N-acetylgalactosamine residues. The role of N-acetylgalactosamine residues on TRF activity was additionally substantiated by the fact that the addition of appropriate amounts of N-acetylgalactosamine in the assay systems for TRF preferentially induced a profound suppression for TRF-mediated PFC responses.     

Primed lymphoid cell tolerance. II. In vivo tolerization of highly tolerogen-sensitive hapten-primed, potentially IgG-producing B cells

The relative ease of tolerizing IgM-bearing versus IgG-bearing B cells was investigated. Previous work had shown that IgG-bearing trinitrophenyl (TNP)-specific B cells from mice primed and boosted with TNP-keyhole limpet hemocyanin (TNP-KLH) are highly susceptible to tolerization in vitro by TNP presented on an unrelated carrier. TNP-OVA was used as tolerogen, as it may represent a more general class of tolerogens than those which are nonmetabolizable or immunoglobulin containing. This study showed that highly primed B cells are tolerizable in vivo using TNP-OVA, with the IgG response to TNP-KLH easier to tolerize than the IgM response. To determine if the ease of tolerization of the IgG response in vivo was due to intrinsic differences in B-cell precursors of the IgM and IgG responses, tolerance was performed in vitro with B cells of defined surface isotypes. A T-independent antigen, TNP-endotoxin, was employed to minimize T-cell effects. At least 10 times as much TNP-OVA was required to tolerize B cells bearing the IgM surface isotype than those with the IgG surface isotype. Thus, the ease of inhibition of the IgG response as compared to the IgM response in vivo by preexposure to TNP-OVA may be at least partially explained by inherent differences in IgM and IgG B-cell precursors.     

Regulation of IgG1 and IgG2 subclass expression by adjuvant-activated splenic suppressor T cells

Spleen cells from mice immunized with different adjuvants are able to suppress secondary in vitro IgG plaque-forming cell (PFC) responses. The suppressive effect is mediated by Lyt-2-positive T cells. IgG subclasses are affected differentially depending on the number of T suppressor (Ts) cells added in the assays. At low Ts cell concentration IgG2a and IgG2b PFC responses are selectively inhibited. At higher Ts cell concentration IgG1 responses could also be completely inhibited, but IgA and IgM responses are not affected. Suppressor cells responsible for IgG1, IgG2a, and IgG2b suppression are never found in the draining lymph nodes of adjuvant-immunized animals.     

Suppression of polyclonal human B cell activation by IgG binding factors: interference with the maturation of Ig-containing cells into Ig-secreting cells

Human IgG binding factors (IgG BF) were prepared by immunopurification on IgG immunosorbents from cell-free supernatants of unstimulated peripheral blood mononuclear cells (PB MNC). The suppressive effects of IgG BF was studied using PB MNC stimulated by pokeweed mitogen or by nocardia delipidated cell mitogen. At the end of the culture three parameters of B cell activation were measured: (1) the numbers of IgM-, IgG-, or IgA-containing cells (CC) using direct immunofluorescence, (2) the numbers of IgM, IgG, or IgA plaque-forming cells (PFC) using a Protein A hemolytic plaque assay, and (3) the concentrations of IgM, IgG, or IgA in culture supernatants using an enzyme-linked immunosorbent assay. Addition of IgG BF at the third day of culture resulted in a selective decrease of IgG CC, while IgM CC and IgA CC were increased or unchanged. Conversely, IgG BF induced a nonselective diminution of the number of PFC and of the amount of secreted Ig of the three major Ig classes. Therefore the results demonstrate two distinct effects of IgG BF: (1) an isotype-specific suppression of cells producing IgG, demonstrated by the parallel decrease of IgG CC and IgG PFC, and (2) a blocking of the late stages of B cell maturation evidenced by the discrepancy between normal or elevated Ig CC and decreased Ig PFC of the IgM and IgA classes.     

Defective primary and secondary IgG responses to thymic-dependent antigens in autoimmune PN mice

Palmerston North (PN) mice spontaneously develop autoimmune disease resembling SLE. Because immune responsiveness has not been defined in this strain, a study was designed to assay primary splenic plaque-forming cell (PFC) responses to thymus-dependent (TD) and thymus-independent (TI) Ag. Initial surveys of PN mice inoculated with the TD Ag SRBC showed adequate production of IgM PFC, but small numbers of IgG PFC were developed with polyspecific antiserum. In contrast, H-2-compatible DBA/1 control mice gave the expected responses to SRBC (IgG plaques elevated twofold compared with IgM plaques). PN mice had the usual responses to Ag that are largely TI; both PN and DBA/1 mice had active IgM and modest IgG responses to TNP-LPS and TNP-Ficoll. Additional experiments determined that PN mice had similar patterns of defective IgG responses to several different TD Ag (SRBC, horse RBC, and DNP-keyhole limpet hemocyanin). In each instance, the usual predominance of IgG1 plaques was absent, and total numbers of plaques developed with antisera specific for IgG isotypes were suppressed. Defective PN IgG production was evident as early as 3 wk of age, was not influenced by aging to 43 wk, and was not corrected by increasing the antigenic challenge 10-fold. PN spleen cells treated with monoclonal anti-Thy-1.2 and C were injected with pools of DBA/1 T cells into 850-rad irradiated (DBA/1 x PN)F1 hybrids. These recipients expressed low IgG1 responses to SRBC, suggesting that the B cell-containing fraction that was not lysed by anti-Thy-1.2 transferred the PN defect. PN mice, which do not respond to TD Ag with active IgG production, contradict the proposal that autoimmunity is associated with hyper-responsiveness to TD and TI Ag.     

Regulation of T15 idiotype dominance. II. Genes unlinked to the Igh locus regulate T15 dominance of the secondary adoptive transfer response to phosphocholine

We have studied the idiotype and fine specificity of the secondary immune response to phosphocholine (PC) in C57BL (B10, B10.D2, and B.C8) and BALB (BALB/c, BAB-14, and C.B20) congenic strains of mice. In vivo IgM responses of mice from these two genetic backgrounds differed in their T15 idiotypic representation. BALB strains expressed the T15 idiotype on greater than 90% of their IgM, PC-specific plaque-forming cells (PFC), whereas C57BL strains expressed the T15 idiotype on approximately 50% of their IgM PFC. All strains examined expressed greater than 75% PC-inhibitable, VHPC idiotype-positive, IgM PFC. The IgG3 and IgA memory responses were similar to the IgM memory response; BALB strains produced a higher proportion of T15+ PFC than C57BL strains; however, the majority of IgG3 and IgA PFC in all strains were VHPC+, and PC-inhibitable. In contrast, the IgG1 memory response was not dominated by T15+, VHPC+, PC-inhibitable PFC in any of the strains tested. The IgG1 PFC required nitrophenylphosphocholine (NPPC) for efficient inhibition. The IgG2 memory response generally mimicked the IgG1 response with respect to idiotype and specificity. These data demonstrate that the representation of the T15 idiotype in the anti-PC immune response is determined by genes outside both the MHC and Igh genetic loci. Control of T15 expression in secondary IgM, IgG3, and IgA anti-PC responses was examined by using a cell-mixing protocol with primed T and B cells from BALB/c and B10.D2 mice. T15 representation in these responses was determined by the genotype of the B cell, not by the genotype of the helper T cell. Similarly, the B cell genotype was responsible for the idiotypic profile of a primary, in vitro, T-dependent, anti-PC response.     

Surface IgG-bearing cells retain the capacity to secrete IgM

Our previous studies indicated that a large proportion of surface IgG+-primed B cells give rise to clones secreting IgM antibodies. Due to the implications of this result relative to molecular mechanisms of class switching, it was important to document that the surface IgG had been endogenously synthesized by the surface IgG+ cells and was not present as a result of cytophilic IgG. Therefore, spleen cells from immunized mice were treated sequentially with anti-immunoglobulin and protease which removed greater than 99% of surface immunoglobulin. After overnight incubation to allow resynthesis of surface immunoglobulin, the treated cells were sorted for surface IgG-bearing cells and were transferred to carrier-primed, irradiated adoptive recipients for analysis in the splenic focus assay. It was found that the majority of antibody-secreting clones derived from these surface IgG+ B cells still synthesized IgM. These data are discussed relative to current concepts of molecular mechanisms of immunoglobulin class switching.     

Regulation of T15 idiotype dominance. III. Phosphocholine-specific B-cell activation in vivo requires major histocompatibility complex-restricted T-cell help

We have examined the requirement for major histocompatibility complex (MHC)-restricted T-cell help in the secondary in vivo antibody response to phosphocholine (PC). The memory response to PC has been demonstrated previously to be comprised of T15-dominant IgM and IgG3 plaque-forming cells (PFC) derived primarily from the Lyb-5+ B-cell subset, and IgG1 and IgG2 PFC, few of which bear the T15 idiotype and are predominantly derived from the Lyb-5- B-cell subset. Using carrier-primed (A X B)F1 T cells which have matured in a parentA chimeric environment so that "self" recognition is of the MHC determinants of parentA but not parentB, we have found that parentA PC-primed B cells, but not parentB PC-primed B cells, are activated. Even in the presence of an ongoing parentA anti-PC response, parentB PC-primed B cells were not activated, indicating that the restriction was between the helper T cell and the B cell, not between T-helper and accessory cells. MHC-restricted T-cell help was required by B cells producing T15+ and T15- IgM, IgG3, IgG1, and IgG2 responses.     

Extracellular signal-regulated protein kinase 2 is required for efficient generation of B cells bearing antigen-specific immunoglobulin G

Activation of extracellular signal-regulated protein kinase (ERK) has been implicated in proliferation as well as differentiation in a wide variety of cell types. Using B-cell-specific gene-targeted mice, we report here that in T-cell-dependent immune responses, ERK2 is required to generate efficient immunoglobulin G (IgG) production. In its absence, the proportion of antigen-specific surface IgG1-bearing cells and the subsequent number of IgG1 antibody-secreting cells were decreased, despite apparently unimpaired class switch recombination. Notably, this defect was countered by overexpression of the antiapoptotic factor Bcl-2. Together, our results suggest that ERK2 plays a key role in efficient generation of antigen-specific IgG-bearing B cells by promoting their survival.     

Selective elimination of a B cell subset having acceptor site(s) for T cell-replacing factor (TRF) with biotinylated antibody to the acceptor site(s) and avidin-ricin A-chain conjugate

A covalent conjugate of avidin with ricin subunit A-chain (avidin-RA) was prepared by using N-succinimidyl 3-(2-pyridyldithio)propionate as a coupling agent. Selective cytotoxic activity after the combined treatment of spleen cells with biotinylated antibody and avidin-RA was demonstrated by the fact that the responsiveness to LPS was selectively abrogated by pretreatment of the cells with biotinylated rabbit anti-mouse immunoglobulin (MIg) antibody, but not with biotinylated anti-Thy-1.2 antibody. Neither the biotinylated antibody alone nor avidin-RA alone was effective in decreasing the responses to mitogens. Moreover, a high anti-DNP PFC response elicited by DNP-KLH-primed BALB/c mouse spleen cells stimulated in vitro with DNP-KLH was mostly abrogated by the pretreatment of the cells with biotinylated anti-MIg antibody and avidin-RA. Again, neither the biotinylated antibody alone nor avidin-RA alone was effective in decreasing the anti-DNP PFC response. This cell-killing method with the use of biotinylated antibody and avidin-RA was applied and evaluated in experimental systems in which the helper action of T cells on B cells was mediated by T cell-replacing factor (TRF) or was performed by the direct interaction of T cells with B cells (cognate interaction). When DNP-KLH-primed splenic B cells, pretreated with biotinylated F(ab')2 fragment of DCF1 male anti-BALB/c-B IgG antibody against acceptor site(s) for TRF followed by treatment with avidin-RA, were stimulated with DNP-OVA in the presence of monoclonal TRF, the anti-DNP PFC response was significantly decreased, whereas the same treated B cells responded well to stimulation with DNP-PPD in the presence of Tbc-primed T cells (cognate interaction). These results indicate that B cells responsible for the cognate interaction and those having TRF acceptor site(s) belong to a distinct subpopulation of B cells, and that the cytocidal action of the noncovalent conjugate of the antibody and RA formed from the biotinylated antibody and avidin-RA via an avidin-biotin complex has immunologic selectivity, eliminating only the latter subset of B cells recognized by the antibody.     

Idiotypic properties of the murine anti-arsonate antibody response: B- and T-cell influences

In a previous report characterizing the arsonate (ABA)-specific plaque-forming cell (PFC) responses of A/J mice induced by ABA-KLH, two interesting characteristics of the idiotypic (Id) profile were noted: (1) an apparent Id selectivity in the isotype switch since the earliest appearing IgG PFC in the primary response were significantly more "cross-reactive Id" (CRI)-dominant than the IgM PFC population, and, (2) a temporal waning of CRI dominance with time among IgG PFC, from 75-100% CRI+ PFC to about 25-45% CRI+ PFC in secondary responses. Experiments were performed to determine whether these effects are largely attributable to T or to B cells. Mice were immunized with a T-independent (TI) form of ABA (ABA-Brucella abortus) and apparent Id selectivity was observed; the earliest IgG PFC averaged 75% CRI+ while IgM PFC were only 39% CRI+. Due to the TI nature of the Ag, this provides suggestive, but not conclusive, evidence that the Id asymmetry in the isotype switch may be attributable to the direct interaction of Ag with B cells. Other studies addressed the temporal shift in CRI dominance. First, it was found that preexposure of mice to either KLH or to ABA (on an irrelevant carrier) resulted in diminished CRI dominance in subsequent "primary" responses to ABA-KLH. Secondly, adoptive transfer experiments with B and T cells from virgin mice (Bv, Tv) or ABA-KLH-primed mice (Bp, Tp) showed that recipients of Bv + Tp or Bp + Tv generated anti-ABA PFC responses with intermediate CRI levels. The Tv cells had some preferential tendency to activate CRI+ clones in the Bp population. The results demonstrate that CRI levels are jointly determined by the immune status of both B and T cells. A simple model is offered which accounts for early Id dominance and its gradual decline and has as its central postulate the assumption that CRI+ B cells in the virgin ABA-specific repertoire have an affinity advantage over CRI- clones.     

IFN-gamma stimulates IgG2a secretion by murine B cells stimulated with bacterial lipopolysaccharide

rIFN-gamma strikingly enhances the secretion of IgG2a by murine splenic B cells stimulated with bacterial LPS in vitro and concomitantly suppresses the production of IgG3, IgG1, IgG2b, and IgE while sparing IgM secretion. IFN-gamma stimulates highly purified B cell populations to secrete IgG2a, strongly suggesting that it acts directly on B cells. It increases the frequency of precursors of IgG2a-expressing soft agar colonies and enhances the number of IgG2a+ cells in colonies indicating that it both increases the frequency of precursors of IgG2a+ cells and enhances the number of IgG2a+ daughter cells emerging from each precursor. IFN-gamma completes its action within the first 24 to 48 h of a 6-day culture with LPS and its addition cannot be delayed beyond the first 48 h. Preincubation of resting B cells in the presence of IFN-gamma leads to a time dependent increase, up to 42 h, in IgG2a secretion upon subsequent addition of LPS. IFN-gamma can exert this action on resting B cells that have been selected for absence of membrane IgG expression by cell sorting. The promotion of IgG2a secretion appears to be a specific property of IFN-gamma in that IFN-alpha, IFN-beta, IL-1, IL-2, IL-3, IL-4, IL-5, granulocyte-macrophage-CSF, granulocyte-CSF, and CSF-1 fail to enhance IgG2a secretion by LPS-stimulated B cells.     

Regulation of B cell maturation and differentiation. I. Suppression of pokeweed mitogen-induced B cell differentiation by tumor necrosis factor (TNF)

Growth and differentiation of B cells into Ig-secreting plasma cells is regulated by both T cells and macrophages and/or their secreted factors. Although the regulatory role of various cell-derived factors has been examined, the involvement of the macrophage-derived factor, TNF, in human B cell growth and differentiation has not yet been investigated. In the present study we examine the role of rTNF in polyclonal B cell response of human PBL induced by PWM. The addition of rTNF at the initiation of the culture resulted in the dose-dependent inhibition of the generation of both IgG and IgM PFC. Inhibition of PFC development followed the same dose response as rTNF-mediated cytotoxicity against a TNF-sensitive tumor target. The mechanism of rTNF-mediated suppression was examined in different experimental systems. Recombinant TNF did not affect the viability or proliferation of either the T cell or B cell subpopulations, suggesting that TNF does not mediate its suppressive effect by cytotoxic mechanisms. Kinetic studies in which rTNF was added at different times after initiation of culture indicated that inhibition can be observed as late as 4 days of culture and suggested that TNF acts at a late phase of the growth and differentiation pathway of B cells. In further studies we examined the cellular level of TNF-mediated suppression. The addition of rTNF to supernatants containing helper factors and enriched B cells resulted in no inhibition, suggesting that TNF does not act at the B cell level. This was confirmed by demonstrating that rTNF does not inhibit spontaneous PFC development by the CESS B cell line. The effect of TNF on T cell subpopulations was examined by using normal or irradiated T cells, which inactivate suppressor cells. Addition of rTNF to B cells combined with either T cell population suppressed both IgG and IgM PFC development, indicating that the target cell for suppression is the T helper cell but not ruling out an effect on macrophages or the T suppressor cells. Combined, the observed results demonstrate that rTNF suppresses PWM-induced B cell differentiation without affecting B cell proliferation. TNF appears to mediate the suppression by acting directly on T helper cells or else by regulating the production of factors controlling T cell activation and lymphokine secretion.     

Human recombinant IL-4 induces activated B lymphocytes to produce IgG and IgM

In this report, we describe a novel biologic activity of IL-4 namely, its ability to induce activated human B cells to produce IgM. Staphylococcus aureus Cowan I-activated blasts prepared from high density tonsil B cells were found to secrete IgG and IgM, but no IgE, when cultured in the presence of rIL-4. The differentiating activity of rIL-4 was totally blocked by a neutralizing anti-IL-4 antiserum, therefore demonstrating that the IgG/IgM-inducing activity of rIL-4 was an intrinsic property of IL-4. rIL-4 was only minimally inducing Ig production of blasts prepared from low density B cells, whereas it induced B cell blasts prepared from high density B cells to secrete a high amount of Ig. Delayed additions of the neutralizing anti-IL-4 antiserum demonstrated that a 48-h contact between IL-4 and B cell blasts was required for optimal Ig production. The IL-4-mediated IgG and IgM production was neither suppressed by IFN-gamma nor by anti-CD23 mAb 25, whereas these agents have been shown earlier to inhibit IgE production of enriched B cells cultured in the presence of IL-4. These data indicate that the IgG/IgM-inducing activity of IL-4 is not regulated like the IL-4-induced IgE production by enriched B cells.     

Keyhole limpet hemocyanin-propagated Peyer's patch T cell clones that help IgA responses

Four T cell clones, isolated from Peyer's patches of keyhole limpet hemocyanin (KLH)-primed BALB/c mice, were selected on the basis of their ability to help IgA responses by TNP-KLH-primed BALB/c mouse B cells. Two were KLH-dependent both in terms of their own proliferative response and in terms of their help for that of B cells. The other two were autoreactive and helped B cells proliferate independently of the presence of Ag. Both primed and unprimed B cells proliferated to some extent when helped by the KLH-reactive clones in the presence of high concentrations of either KLH or TNP-KLH. Much higher proliferation was, however, induced when primed, but not unprimed, B cells were exposed to the T cells in the presence of low concentrations of TNP-KLH but not KLH, i.e., under conditions favoring direct, cognate interaction between the T and B cells. Only modest IgM, and no IgG or IgA plaque-forming cell (PFC) responses were generated by TNP-primed B cells upon interaction with either autoreactive T cells in the absence of Ag or KLH-reactive T cells in the presence of high concentrations of KLH. For high IgM responses as well as for the appearance of IgG and IgA PFC responses, TNP-KLH was required whatever the source of the T cell help. The isotype ratios depended on the TNP-KLH concentration; IgA responses were highest and IgM responses lowest at the lowest TNP-KLH concentrations suggesting that the precursors of the IgA PFC have higher average affinity for TNP than the precursors of IgM PFC. Overall, the results are compatible with the idea that the precursors of IgA and IgG PFC and many of the precursors of IgM PFC in the long term primed B cell populations used in these experiments require engagement of their Ag-receptors before they express sufficient class II Ag and/or receptors for "switch" and differentiation factors for cognate interaction with T cells leading to PFC responses.     

Evidence for existence of two distinct TNP-Ficoll-responsive B cell subpopulations preferentially reactive either to a T cell-replacing factor (B151-TRF) or to a signal from accessory cells

The ability of B cells to respond to TNP-Ficoll has been shown to correlate with their ability to respond to T cell-replacing factor (TRF). The present study analyzed the relationship of TNP-Ficoll-responsive B cells to a TRF-responsive B cell subpopulation. The B cells from normal, unprimed mice responded to TNP-Ficoll in the presence of accessory cells. Such responses were notably augmented by the addition of TRF derived from a monoclonal T cell hybridoma, B151K12(B151-TRF). Interestingly, B cells of mutant X-linked immunodeficient DBA/2Ha which failed to respond to B151-TRF gave anti-TNP PFC responses to TNP-Ficoll comparable to those of normal mice, depending on the presence of accessory cells. However, under this condition, the addition of B151-TRF did not augment the TNP-Ficoll responses. One explanation of the augmentation of TNP-Ficoll response by TRF for the B cells from nondefective mice was that two distinct B cell subpopulations exist which differ in their respective activation requirement for TRF and accessory cells. To examine this possibility, syngeneic accessory cells were pulsed with TNP-Ficoll and were assayed for their ability to activate normal B cells in the presence or absence of B151-TRF. The results revealed that TNP-Ficoll-pulsed accessory cells were able to induce primary anti-TNP PFC responses in normal B cells to the same magnitude as soluble TNP-Ficoll. However, these B cell responses induced by the TNP-Ficoll-pulsed accessory cells were not augmented by the addition of B151-TRF to the culture. These results support the notion that two distinct TNP-Ficoll-responsive B cell subpopulations exist; one requires accessory cell-B cell interaction to be activated by TNP-Ficoll but fails to respond to TRF, and the other can be activated by TRF in a totally accessory cell-independent manner.     

Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

TRF requirements for in vitro PFC responses to SRBC and R36a. I. TRF is distinct from IL 2 but indistinguishable from polyclonal BCSF

In vitro PFC responses to the thymus-independent (TI) antigen Streptococcus pneumoniae R36a require T cell replacing factor(s) (TRF). This requirement for TRF is as significant as for the thymus-dependent (TD) antigen SRBC. TRF is shown to be distinct from IL 2 by the following observations: 1) culture supernatants from the cloned T cell line L2, collected over an 8-day period after allogeneic stimulation, transiently contain IL 2 activity but maintain high levels of TRF activity throughout 192 hr; 2) L2V, a variant subclone of L2, produces much higher levels of TRF activity than the parental line but no detectable IL 2 activity; 3) the addition of IL2+, TRF- supernatants from the T cell hybridoma FS6-14.13 does not affect the L2V SF-driven PFC responses to R36a or SRBC; and 4) the addition of contaminating T cells to cultures containing T cell-depleted spleen cells, L2V SF, and antigen does not affect the PFC response. TRF does appear to be indistinguishable from polyclonal B cell stimulating factor (BCSF), which stimulates polyclonal PFC responses in the absence of antigen, mitogen, or anti-Ig. The TRF and BCSF activities of L2V SF could not be separated by ion-exchange, hydrophobic-interaction, and gel-filtration chromatography. TRF and BCSF have an apparent m.w. of approximately 40,000.     

Variations in the responses of C57BL and a mice to sheep red blood cells: II. Analysis of plaque-forming cells

The number of direct and indirect plaque-forming cells (PFC) and the serum hemolytic activity was determined for A/He, C57BL/6J, and B6AF1 mice responding to multiple injections of sheep red blood cells (SRBC). Although the kinetics of the primary response differed, all mice had high numbers of both direct and indirect PFC and low-titered 2-mercaptoethanol (2-ME) sensitive serum antibody. Following multiple SRBC injections, the A/He spleens contained predominantly IgG producing PFC. Their serum antibody activity was resistant to 2-ME signifying the presence of IgG. The serum activity of both the C57BL/6J and B6AF1 mice was sensitive to 2-ME (IgM antibody) over the course of immunization, and although there was a definite IgM PFC memory response, the presence of 7S memory PFC was questionable. The results are discussed in terms of the maturation of the antibody response to SRBC and of the question of the postulated IgM and IgG switch.     

Isotype-specific immunoregulation. Systemic antigen induces splenic T contrasuppressor cells which support IgM and IgG subclass but not IgA responses

In the present study, we have isolated and characterized the Lyt-1+, -2- T contrasuppressor (Tcs) cells from mice systemically primed with SRBC. Adoptive transfer of splenic Tcs cells from these mice abrogates oral tolerance and supports IgM and IgG anti-SRBC plaque-forming cell (PFC) responses; however, unlike the responses seen after transfer of Tcs cells derived from orally primed mice, low IgA responses were seen. Mice systemically primed with lower SRBC doses (0.01 to 1%) exhibited contrasuppression only within the L3T4- T cell subset, whereas mice primed with a high dose of SRBC (10%), harbored Lyt-1+, -2- Tcs cells in both the L3T4+ and L3T4- subsets. Both the L3T4- and L3T4+ Tcs cell subsets supported IgM and IgG responses when adoptively transferred to orally tolerized mice, and when added to tolerized spleen cell cultures. Splenic Tcs cells from systemically primed mice supported mainly IgG1 and IgG2b subclass anti-SRBC PFC responses, a pattern also seen with Tcs cells derived from orally primed mice. Both L3T4+ and L3T4- Tcs cells from systemically primed mice exhibited well established characteristics of contrasuppressor cells including binding to Vicia villosa lectin and expression of I-J. The splenic effector Tcs cells which support IgM, IgG1 and IgG2b anti-SRBC PFC responses are antigen-specific, since both L3T4- and L3T4+ Tcs cells from spleens of mice primed with 10% SRBC reverse tolerance to SRBC, but not to horse erythrocytes (HRBC). Further, both L3T4- and L3T4+ Tcs cells from HRBC-primed mice reverse tolerance to IgM and IgG anti-HRBC, but not to anti-SRBC responses. Isolation of T3-positive Lyt-1+, -2- and L3T4- Tcs cell subsets by flow cytometry followed by adoptive transfer, showed that effector Tcs cells express T3 and presumably contain an Ag-R (TCR-T3 complex). These studies show that systemic priming with heterologous RBC induces splenic Ag specific Tcs cells in a dose-dependent manner, which support IgM and IgG subclass responses, but not IgA responses.