Similarity between natural and recombinant human alpha-fetoprotein as inhibitors of estrogen-dependent breast cancer growth.

Alpha-fetoprotein (AFP) isolated from rodent amniotic fluid or human cord sera, upon incubation with a molar excess of estradiol, is converted to a form which inhibits estrogen-stimulated tissue growth. The purpose of this study was to determine whether recombinant human AFP produced in an E. coli expression system retained this function. The recombinant protein was similar to the natural protein isolated from pooled human cord sera in all functional aspects evaluated. It was detected by monoclonal and polyclonal antibodies to the natural protein. Following exposure to estradiol, it was converted to an inhibitor of estrogen-stimulated growth of immature mouse uterus yielding a dose/response curve similar to that of the natural protein. It inhibited the growth of estrogen-dependent (MCF-7) but not estrogen-independent (MDA-MB-231) breast cancer xenografts with the same schedule dependency and resultant histological changes as the natural protein. Availability of large quantities of homogeneous, biologically active recombinant human AFP will facilitate further studies of structure/function, mechanism, and therapeutic potential of this agent as a regular of breast cancer growth.

Purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in Escherichia coli.

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function(s) remains unclear. A more complete analysis of the physiological activities of this oncofetal protein has, until now, been severely limited by the lack of an appropriate source from which to obtain pure AFP in any sizeable quantity. In the present investigation, we obviate this problem by cloning and efficiently overexpressing mature mouse and human AFP cDNA's in Escherichia coli. For recombinant mouse AFP (rMoAFP), large segments of the coding region were excised from the preexisting plasmids pAFP1 and pAFP2, which together encompass 90% of the AFP sequence. The mouse cDNA was made complete by the addition of N- and C-terminal encoding oligonucleotides. Mouse AFP cDNA was expressed directly as a full-length molecule in vector pTrp4 or as fusion proteins in plasmids pMALc and pRX1 under the transcriptional control of trp or tac promoters. Accumulation of rMoAFP was significantly increased in protease-deficient E. coli strains over nonprotease-deficient strains, > or = 10% of total cell protein. Of the gene fusion proteins examined, none offered significant advantage over the direct expression product in terms of recombinant protein stability, overall levels of synthesis, or facilitated purification. Recombinant AFP polypeptides expressed by pTrp4 were as expected, deposited in bacterial inclusion bodies. Subsequent to resolubilization/refolding, rMoAFP was first enriched by passage over Q-Sepharose resin followed by final purification using immobilized copper-chelate affinity chromatography. Protein sequencing of the N-terminus revealed that purified rMoAFP had a deletion of the first nine amino acids coded for by the full-length mouse AFP cDNA. Similar N-terminal deletions are observed with AFP isolates originating from natural sources. A complete human AFP cDNA was generated from a fetal liver cDNA library and was cloned into vector pTrp4. Recombinant human AFP (rHuAFP) was expressed under the identical conditions employed for rMoAFP but purification had to be modified to include preparative Mono Q anion exchange chromatography. N-terminal sequencing, amino acid compositional analysis, and electrospray mass spectrometry revealed that purified rHuAFP was intact and unaltered and that the initiator methionine was completely removed. The biological activity of recombinant AFP, as judged by its inhibitory effects on in vitro lymphocyte proliferation, was equivalent to that of the native protein. The availability of large quantities of mouse and human recombinant AFP molecules should now permit detailed structure-function analyses of this important oncofetal protein to proceed in a manner unimpeded by previous limitations in both quantity and quality of the native proteins.

Decidua-associated suppressor cells in abortion-prone DBA/2-mated CBA/J mice that release bioactive transforming growth factor beta2-related immunosuppressive molecules express a bone marrow-derived natural suppressor cell marker and gamma delta T-cell receptor.

The decidua of allopregnant mice contains a novel population of Thy1 Lyt1 CD4 CD8 asialoGM1- non-B small lymphocytic suppressor cells that release transforming growth factor (TGF) SS2-related suppressor molecules. The "null" phenotype of this cell population is similar to some bone marrow-derived natural suppressor cell (NSC) populations, and the latter may release TGF(beta)s. We now report that the TGF beta2-producing suppressor cells in the uterine decidua of DBA/2-mated CBA/J female mice-linked to prevention of abortions-are inactivated effectively by 1E5/B5.1 but not by 2C1.1 rat monoclonal antibodies to murine pregnancy-associated splenic NSC in the presence of complement. Immunostaining of a subpopulation of cells in decidua with 1E5/B5.1 but not with 2C1.1 was shown by flow cytometry. Release of suppressor factor was also abrogated by 1E5/B5.1 + complement but not by 2C1.1 + complement, and the suppressor factor was specifically neutralized by anti-TGF beta2 and not by anti-TGF beta3. Splenic pregnancy NSC are susceptible to 2C1.1, produce TGF beta1, and express CD3 and alpha beta T-cell receptor (TcR) chains. Release of suppressor factor by the decidual NSC was abrogated by treatment with anti-CD3 (145 2C11) and anti-TcR gamma delta (GL4) monoclonal antibodies + complement, but not by anti-TcR alpha beta (H57) + complement; and cells sorted using anti-TcR gamma delta (GL3) released suppressive activity in vitro. Slightly more suppressive activity was released by implantation-site decidua where there was no epithelium than from epithelialized inter-implantation-site decidua; no significant activity was released from placental tissue, but combining implantation-site tissue with placental tissue led to release of enhanced levels of immunosuppressive activity. There appear to be subtypes of bone marrow-derived TcR+ NSC with different phenotypes and tissue localization patterns in pregnancy. The previously reported dependence of decidual NSC activity on the presence of soluble signals from fetal trophoblast may be explainable by the ability of cells bearing TcR gamma delta to recognize and react to placental trophoblast cell antigen.

Evidence that immunosuppression is an intrinsic property of the alpha-fetoprotein molecule.

Among the proteins that comprise the albumin family, alpha-fetoprotein (AFP) is the only member which exhibits immunoregulatory properties. However, some investigations have argued that AFP-mediated immunosuppression is not an inherent property of the molecule itself, but is instead, hypothesized to be either a function of a low molecular weight inhibitor bound to AFP or to a post-translational modification of the protein. AFP cannot be isolated from natural sources in quantities sufficient for the detailed biochemical and functional analyses required to resolve these issues. We have therefore produced recombinant forms of the protein (rAFP) by cloning the cDNA's for mouse and human AFP in both eukaryotic and prokaryotic expression systems. As described in this report, we were able to abundantly express rAFP's in bacterial, baculovirus and yeast expression systems. Recombinant proteins derived from each expression system were recognized by polyclonal and monoclonal anti-AFP antibodies as determined by immunoblot analysis. Pure recombinant protein samples, as characterized by polyacrylamide gel analyses, N-terminal sequencing and FPLC and HPLC chromatography, were evaluated for their immunoregulatory properties in murine and human in vitro immunological assays. The results of these studies establish that rAFP is functionally equivalent to natural fetal derived AFP molecules. Importantly, the data reported here demonstrate that AFP-mediated immunoregulation is an activity intrinsic to the molecule itself and cannot be attributed to either putative non-covalently bound moieties or to post-translational modifications such as glycosylation and sialylation. These studies provide a basis for initiating detailed investigations into the potential clinical usefulness of AFP as an immunotherapeutic agent.

Reactivity of monoclonal antibody 1E5.B5 with a novel phenotypic marker expressed on a murine natural suppressor cell subset.

Natural suppressor (NS) cells are antigen-nonspecific, MHC-independent immunoregulatory cells that are typically found in murine bone marrow (BM), newborn (NB) mouse spleen, and in splenic tissue of adult mice during pregnancy and following cyclophosphamide (CY) treatment. There has been a pressing need for the development of NS cell-specific monoclonal antibodies (mAb) since NS cells are generally described as null cells which lack the usual phenotypic markers of mature T cells, B cells, and macrophages. Here we present evidence that mAb 1E5.B5, which was raised in rats against murine splenic pregnancy-associated NS (SPANS) cells, recognizes a unique antigenic marker expressed by some, but not all, murine NS cells. In the presence of complement, mAb 1E5.B5 effectively eliminates SPANS activity, and diminishes NS activity of CY-treated spleen cells in mixed lymphocyte reactions (MLR). However, cytotoxic pretreatment with mAb 1E5.B5 had minimal effects on NS activity of BM and NB spleen cells. We also show that pregnancy spleen cells and CY-spleen cells with moderate NS activity in MLR can be positively selected for by "panning" with mAb 1E5.B5. In contrast, only weakly inhibitory cells are isolated from BM and NB spleen by this procedure. Cellular ELISA and flow cytometry confirm that mAb 1E5.B5 has specificity for pregnancy spleen cells and CY-spleen cells, as well as for NB spleen and BM cell preparations. Western blot analysis reveals that mAb 1E5.B5 reacts with a novel 50 kDa NS cell-associated antigen which we have termed NS-1. The NS-1 antigen is not present on other null cells such as natural killer (NK) cells and natural cytotoxic (NC) cells since cytotoxic pretreatment of pregnancy spleen cells with mAb 1E5.B5 does not affect antibody-dependent cell-mediated cytotoxicity, NK or NC activity.

Pregnancy-associated suppressor cells in mice: functional characteristics of CD3+4-8-45R+ T cells with natural suppressor activity.

Natural suppressor (NS) cells are MHC-unrestricted regulatory cells with non-specific inhibitory activity for immune responses. In adult mice, NS cells are characteristically found in bone marrow and in splenic tissue following total lymphoid irradiation and cyclophosphamide treatments. Recently, we have shown that the spleens of pregnant mice harbour a population of lymphocytes which resemble NS cells in terms of phenotype and inhibitory activity. In this study, we use positive and negative selection techniques to further characterize splenic pregnancy-associated NS (SPANS) cells as predominantly 'double negative' T cells (CD3+4-8-) bearing receptors for the lectins wheat germ agglutinin and soybean agglutinin, as well as expressing CD45R and the heat-stable J11d.2 antigen. Taken together, these findings lead us to conclude that SPANS cells belong to an immature T cell lineage. In keeping with their T cell phenotype, SPANS cells do not express the natural killer (NK) cell-specific markers NK2.1 and asialoGM1 and do not mediate lytic activity against NK-sensitive YAC-1 cells, although natural cytotoxic activity against WEHI-164 cells was found to co-purify with SPANS cells. Suppressive activity of SPANS cells in mixed lymphocyte reactions (MLR) is abolished by treatment with mitomycin C, suggesting that natural suppression in this system is a proliferation-dependent phenomenon. Preincubation of SPANS cells with conditioned medium from Con A-stimulated T cell cultures results in augmented NS activity, indicating that SPANS cells respond to T cell signals. Our data suggest that SPANS cells mediate suppression via the elaboration of a soluble suppressor factor since SPANS cells do not require cell-cell contact to mediate suppression and supernatants from short-term cultures of SPANS cell-enriched SBA+ pregnancy spleen cells inhibit MLR. We believe that SPANS cell may be important in regulating hematopoiesis and maternal anti-fetal immunity during murine pregnancy.

Analytical- and preparative-scale separation of molecular variants of alpha-fetoprotein by anion-exchange chromatography on Monobead resins.

A rapid and reliable purification procedure is described that is useful for both analytical detection and quantitative recovery of milligram amounts of individual molecular variants of mouse alpha-fetoprotein (AFP). The appropriate separation conditions were developed with an analytical-size Mono Q anion-exchange column linked to an automated Fast Protein Liquid Chromatography system. Effective separations of fetal-derived AFP variants was accomplished within 20 min under mild conditions with an L-histidine buffer. Employing the optimal separation conditions established on the Mono Q HR 5/5 column we upscaled the procedure by using a preparative Mono Q HR 16/10 column in order to obtain milligram quantities of each molecular variant of AFP. Seven distinct isomeric forms of AFP could be recovered on the preparative anion exchanger in a highly reproducible manner. Each of the seven protein peaks eluted from the Mono Q column were confirmed to be distinct isoforms of AFP by isoelectric focusing and Western blotting developed with monospecific anti-AFP antisera. This method in its scaled up version offers the benefit of providing milligram quantities of immunochemically pure AFP isomers for structure and function studies.

Isolation and characterization of a distinct immunoregulatory isoform of alpha-fetoprotein produced by the normal fetus.

In this report, we examine the functional significance of the molecular microheterogeneity of alpha-fetoprotein (AFP). In doing so, we have taken the direct approach of purifying the naturally occurring isomeric forms of fetal-derived AFP using a preparative anion exchange column linked to an automated fast protein liquid chromatography (FPLC) system followed by parallel testing of each isolated molecular variant for in vitro immunoregulatory activity. The data obtained demonstrate the presence of seven distinct variants of AFP as defined by their retention volumes on FPLC elution profiles, by their pIs on analytical IEF gels, and by Western blot analysis. Molecular mass determination by SDS-PAGE showed each isomer to be equivalent in size to 69,000-dalton native unfractionated AFP molecules. All the immunosuppressive activity of AFP was localized to a single variant representing only 6% of the total composition of native AFP. The immunoregulating isomer termed AFP-1 was the least acidic of the seven isolated variants with a pI of 5.1 and displayed a sialic acid content of 1 mol/mol of protein. The inhibitory activity of AFP-1 could be readily measured on T cell-dependent antibody synthesis, Con A-induced stimulation of Lyt-1+23- thymocyte DNA synthesis, and lymphokine-activated NK cell activity. All other isomers were without effect in these test systems. The immunosuppressive AFP-1 isomer also displayed the strongest growth-promoting influence on cultured bone marrow lymphocytes. There was no correlation between functional activity and degree of expression of sialic acid residues on the AFP molecules. These findings demonstrate that the immunoregulating function of AFP is confined to a distinct and relatively small subpopulation of native AFP molecules and should therefore contribute to the resolution of outstanding questions regarding the structure/function relationship of this onco-fetal glycoprotein.

Specific maternal anti-fetal lymphoproliferative responses and their regulation by natural immunosuppressive factors.

Maternal immune responses to autologous and/or oncodevelopmental antigenic determinants expressed on fetal tissues may constitute a potential hazard to fetal survival distinct from the well-studied maternal immune reactivity directed against paternally-derived fetal alloantigens. Splenic T cells with a helper phenotype (Lyt 1+2-) obtained from primiparous CBA/J mice pregnant by syngeneic matings were found to proliferate in response to co-culture with syngeneic Lyt 1-2- fetal thymus cells. In contrast, splenic T cells from virgin animals failed to react against fetal stimulator cells, suggesting that autosensitization to fetal gene products is a pregnancy-associated phenomena. Addition of anti-Ia alloantiserum at initiation of culture virtually abrogated the blastogenic response by maternal T cells, indicating that this is an Ia-dependent reaction. The addition of natural suppressor (NS) cells or alpha-fetoprotein (AFP), both of which are naturally occurring pregnancy-associated immunoregulatory factors, was found to markedly inhibit in-vitro maternal anti-fetal autoreactivity. NS cells and AFP may play an important role in maintaining homeostasis in the fetal-placental environment during murine pregnancy.

Altered immune response patterns in murine syngeneic pregnancy: presence of natural null suppressor cells in maternal spleen identifiable by monoclonal antibodies.

Expression of certain autologous lymphocyte-activating antigenic determinants on the developing embryo is known to provide a stimulus for maternal anti-fetal autoproliferative responses. If left unregulated these responses could exert negative influences on the reproductive process by converting to autoaggressive forms of immune reactivity. In normal circumstances, immunological reactions of this nature are therefore likely to be under the control of pregnancy-associated immunoregulatory elements found within the maternal/fetal environment. In the present investigation we describe a naturally occurring splenic inhibitory cell type devoid of conventional T, B, and macrophage surface markers associated with syngeneic murine pregnancy that is capable of exerting potent immunosuppressive effects on an in vitro expression of fetal/newborn T cell autoreactivity, namely the autologous mixed lymphocyte reaction (AMLR). Maternal spleen cells inhibitory for AMLR were found to be highly resistant to cytotoxic pretreatment with a panel of conventional antisera directed against T cell-specific antigenic determinants. The non-T nature of the natural splenic suppressor cell was further indicated by experiments showing that purified spleen T cells had no inhibitory activity. Pregnancy spleen cell populations that were effectively depleted of macrophages retained full ability to inhibit AMLR. Maternal suppressor activity could be localized to the spleen cell population bearing receptors for the B cell-specific lectin, soybean agglutinin (SBA). A panel of monoclonal antibodies prepared against enriched populations of suppressor cells was screened and selected for specific reactivity using an ELISA against glutaraldehyde-fixed SBA+ spleen cell subpopulations from pregnant versus virgin animals. Several of the monoclonals developed against suppressor-enriched spleen cell populations from isopregnant as well as allopregnant animals were effective in reducing or eliminating suppressor cell activity following cytotoxic pretreatment in the presence of complement. The novel set of anti-suppressor monoclonal antibodies described here should prove useful in furthering the isolation and characterization of pregnancy-associated suppressor cells and in determining their relationship to natural suppressor cell populations described in other systems.

Expression of domains of mouse alpha-fetoprotein in Escherichia coli.

A mouse alpha-fetoprotein complementary DNA containing the coding region for amino acids 256 to 548 was fused to the lac transcriptional and translational control elements contained on the expression vector pOP203-28. The expression of a 35 kD hybrid lac Z'- alpha-fetoprotein polypeptide in Escherichia coli was demonstrated by the chloramphenicol release assay and by immunoprecipitation using rabbit anti-mouse alpha-fetoprotein antibodies as probe.

Modulation of the immune system by Neisseria meningitidis.

The immunomodulatory potential of Neisseria meningitidis was investigated. Spleen cells from mice injected intraperitoneally with low to moderate doses of meningococci (10(4)-10(7)) were found to display enhanced responses to the mitogens lipopolysaccharide (LPS), phytohaemagglutinin (PHA), and concanavalin A (Con A). In contrast, high doses of meningococci (10(8)-10(9)) caused a marked decrease in mitogenic reactivity. Meningococci-injected mice also displayed a dose-dependent suppression of a primary anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) response. The timing between the injection of SRBC and of meningococci appeared to play an important role in the induction of suppression by the organisms. Thus, decreased PFC responses were observed only when the bacteria were injected prior to the antigen. When meningococci were injected at the same time or after SRBC, normal or even increased PFC responses developed. Kinetic experiments showed that the onset of suppression of both mitogen and antibody responses by meningococci was very rapid, so that by 6-7 h after injection of the bacteria, mice showed markedly reduced mitogen responses and became essentially unable to mount an antibody response against SRBC. Suppression of mitogen responses was relatively transient, since reactivity returned to normal after 48 h. However, the ability of infected animals to mount a normal anti-SRBC response did not fully return until 12 days after the infection. Spleen cells from meningococci-infected mice also showed markedly depressed PFC responses when stimulated with SRBC in vitro but failed to suppress the response of normal spleen cells in mixed cultures. These observations indicate that putative suppressor cells, if they exist at all, are too insignificant in terms of numbers and/or efficiency to account for the observed immunosuppression. A more likely explanation for the inhibition, which is supported by our data, presented here and elsewhere, is that certain surface components of meningococci are capable of imparting immunosuppressive signals directly onto target lymphocytes.

Monoclonal antibodies against murine neonatal and pregnancy-associated natural suppressor cells induce resorption of the fetus.

Pregnant and neonatal/fetal mice have been shown to harbour naturally occurring inhibitory cells of both T and non-T type. Non-T suppressor cells present in the spleen of primiparous pregnant and newborn animals inhibit proliferative responses in autologous and allogeneic mixed lymphocyte reactions. Such cells can be positively selected for by agglutination with the B cell-specific lectin soybean agglutinin (SBA). We generated rat IgG monoclonal antibodies against unique cell surface structures on the non-T inhibitory cells, and cytotoxic pretreatment of spleen cells from pregnant or neonatal/fetal mice largely abrogates their suppressive activity on proliferative responses. Furthermore, in vivo administration of such antibodies to pregnant inbred and outbred mice results in termination of the pregnancy or decreased litter size. Since injection of anti-T cell IgG monoclonal antibodies does not interfere with the delivery of normal sized litters it is evident from these studies that the non-T immunoregulatory cells, in contrast to T-inhibitory cells, are of great importance in ensuring immunological homeostasis in the fetal-placental environment during pregnancy in mice.

Experimental myasthenia gravis induced in mice by passive transfer of human myasthenic immunoglobulin. Evidence for an ameliorating effect by alpha-fetoprotein.

The induction of experimental autoimmune myasthenia gravis (EAMG) was studied by the passive transfer of gamma-globulin from myasthenia gravis (MG) patients to C57BL/6 mice. Muscular weakness and electromyographic decrements (EMG) could be consistently induced in all mice injected with gamma-globulin from certain selected MG patients. There was, however, no correlation between the antiacetylcholine receptor antibody titre in the donor gamma-globulin and the ability to induce EAMG. The possible beneficial effects of immunoregulatory alpha-fetoprotein (AFP) treatment were investigated employing the passive EAMG model. Mice were protected against the onset of severe symptoms provided the AFP was administered before and after passive transfer. The exaggerated fatigue characteristics associated with murine EAMG as detected by EMG could be alleviated by AFP treatment. These findings raise the possibility that AFP may be of some therapeutic value in the control of MG.

Identification of macromolecular insoluble cold globulin (MICG) as a new marker shared by NK and ADCC cells, but not expressed by NC cells.

We report here that cytotoxic pretreatment of spleen cells from six different strains of young adult mice with a monospecific rabbit antiserum against macromolecular insoluble cold globulin (MICG) effectively abrogates spontaneous NK activity directed towards YAC-1 tumour cells. MICG is a 225,000 molecular weight glycoprotein that is present in the plasma membrane of adult thymocytes and peripheral T cells, as well as in embryonic prothymocytes, but absent in granulocytes and B lymphocytes. The diminished NK activity in lymphocyte populations selectively depleted of MICG+ cells could not be restored by in vitro exposure to the NK boosting agents interleukin-2 (IL-2) and interferon. Lymphokine-activated spleen NK cells, generated by 48 hr preculture with IL-2 or interferon, expressed high levels of MICG surface antigen, moderate amounts of Thy 1.2 and, in striking contrast to spontaneous NK, very low to negligible amounts of AsGM1. Likewise, spontaneous NK cells in bone marrow were also shown to be both MICG+ and AsGM1+, while lymphokine-activated bone marrow NK cells remained MICG+, but lacked AsGM1. Thus, a clear distinction could be observed between spontaneous and activated NK cells with respect to differential expression of MICG and AsGM1. MICG was also detected on ADCC effector cells, whereas no surface MICG could be found on NC cells. These data are in line with the view that at least certain types of NK cells develop along a common lineage with T lymphocytes in the mouse.

Selective impairment of B cell function by Neisseria meningitidis.

Spleen cells from CBA/J mice infected with Neisseria meningitidis displayed depressed in vitro plaque-forming cell (PFC) responses to T-dependent (sheep red blood cell; SRBC) and T-independent (TNP-LPS, TNP-Ficoll) antigens. The inhibition was observed over a wide range of antigen concentrations. The decreased responsiveness of splenocytes from infected mice was due to a selective impairment of B-cell function since helper-T-cell activity was intact in infected mice as shown by the ability of T-enriched lymphocytes to cooperate with normal B-enriched lymphocytes in the generation of an anti-SRBC response, accessory macrophage function was preserved since adherent spleen cells from bacteria-injected mice were shown to produce normal or increased levels of IL-1 and were able to cooperate with normal non-adherent spleen cells in the generation of PFC against SRBC. Addition of peritoneal cells from normal animals or extraneous IL-1 both failed to restore normal PFC responses in cultures of splenocytes from infected mice. Finally, B-enriched lymphocytes from infected mice produced poor anti-SRBC responses when cultured with either Con A supernatant or T-enriched lymphocytes from normal or infected mice. Cell-mixing experiments failed to detect the presence of suppressor cells in cultures of unfractionated spleen cells or B-enriched lymphocytes from infected mice. Therefore, the immunological unresponsiveness associated with a Neisseria meningitidis infection was attributed to a meningococcus-induced defect(s) in B-cell function. In vivo polyclonal B-cell activation leading to clonal exhaustion did not play a major role in the depression of humoral responses since meningococcal infection induced little or no polyclonal Ig secretion.

Murine neonatal spleen contains natural T and non-T suppressor cells capable of inhibiting adult alloreactive and newborn autoreactive T-cell proliferation.

The spleen of neonatal mice is known to be a rich source of cells capable of suppressing a variety of immune functions of adult lymphocytes in vitro. From such observations has emerged the concept that the gradual development in ability to express immune functions after birth is due in part to the parallel normal physiological decay of naturally occurring regulatory suppressor cells. There is, however, some confusion in the literature as to the exact nature of the newborn of the newborn inhibitory cell type(s). In contrast to most previous reports which detect only a single type of neonatal suppressor cell, usually a T cell, we show here that newborn spleen harbors both T and non-T inhibitory cells. Both types of suppressor cells could be shown to suppress the proliferative response of adult spleen to alloantigens as well as newborn T cells reacting against self-Ia antigen in the autologous mixed lymphocyte reaction (AMLR). Newborn suppressor T cells were characterized as being non-adherent to Ig-anti-Ig affinity columns, soybean agglutinin receptor negative (SBA-), and susceptible to lysis by anti-T-cell specific antiserum plus complement. Non-T suppressor cells were identified as non-phagocytic, SBA receptor positive (SBA+), and resistant to cytotoxic treatment with anti-T-cell antibodies and complement. The apparent controversy surrounding previous reports as to the T versus non-T nature of newborn suppressor cells can be reconciled by the present observation that both types of inhibitory cells coexist in the spleen. Furthermore, the demonstration that newborn suppressor cells can effectively regulate T-cell proliferative activity mediated by other newborn cells provides more direct support for the contention that such inhibitory cells play a physiological role in controlling immune responsiveness during early ontogeny.

Suppression by alpha-fetoprotein of murine natural killer cell activity stimulated in vitro and in vivo by interferon and interleukin 2.

Natural killer (NK) cells are 'spontaneously' cytotoxic cells thought to be involved in surveillance against tumour cells, rejection of virally infected cells, and regulation of haematopoietic stem cell differentiation and antibody synthesis. Fetus-derived alpha-fetoprotein (AFP) has been shown to regulate certain T cell-mediated immune reactions in vitro and in vivo. The lack of NK activity in newborn mice with high endogenous levels of AFP, together with the presence of cells expressing NK surface markers, also suggests that AFP may regulate NK activity. In this study we compared the effects of AFP on spontaneous versus activated murine NK activity. The lytic ability of both freshly prepared splenic NK cells and those arising after incubation for 24 h with interferon, Poly I:C, or T-cell growth factor (TCGF) was not affected by AFP if the latter was present only during the killing phase. However, if AFP was added at the beginning and retained for the duration of the 24-h in vitro lymphokine stimulation, the subsequent NK activity induced by interferon, Poly I:C, and TCGF was found to be significantly suppressed. This inhibition is both dose- and time-dependent. Delayed addition experiments showed that when AFP is present during the first 6 h of in vitro stimulation it will suppress interferon and TCGF-boosted NK activity by 50-80%. The AFP-mediated inhibitory effect on lymphokine-stimulated NK activity is not the result of increased death of effector cells nor, in the case of interferon and polyribonucleotides, of non-specific binding of AFP to the enhancing agents. In vivo injections of Poly I:C or TCGF failed to increase neonatal NK function, while administration of interferon did cause slightly higher levels of NK activity. However, spleen cells from newborn animals cultured for 24 h in the presence of lymphokines resulted in markedly elevated NK function and this in vitro activation could be suppressed by purified fetus-derived AFP. Thus, the in vivo pattern of NK activation in newborns with high endogenous levels of AFP was very similar to that of adult NK stimulation in vitro when exogenous AFP was added.

In vitro activation of bone marrow-derived T-and non-T-cell subsets by alpha-fetoprotein.

Alpha-Fetoprotein (AFP) is a major serum glycoprotein during embryonic and early postnatal life. A number of diverse biologic functions have been attributed to AFP, including osmotic and carrier function and immunosuppressive activity. In this study we demonstrate that AFP selectively stimulates in vitro proliferation of two distinct subsets of adult murine bone marrow cells. One population of AFP-reactive bone marrow cells expresses surface receptors for soybean agglutinin (SBA) lectin. SBA+ bone marrow cells are resistant to cytotoxic pretreatment with T-cell-specific antisera and are not retained on Ig-anti-Ig affinity columns. The absence of conventional T- and B-cell markers, coupled with the presence of SBA receptors, suggests that AFP-activated non-T bone marrow cells may belong to an immature set of B lymphocytes. A second population of AFP-responsive bone marrow cells expresses the Thy-1+ Lyt 1+2- phenotype characteristic of conventional mature adult T helper cells. The potential physiological relevance of the mitogenic effects of AFP on bone marrow cells with respect to immunoregulatory processes in the fetal/newborn environments is discussed.