Antigen-initiated B lymphocyte differentiation. IX. Characterization of memory AFC progenitors by buoyant density and sedimentation velocity separation.

The characteristics of memory B cell antibody-forming cell (AFC) progenitors from long-term hapten-primed CBA mice were investigated by using sedimentation velocity and buoyant density separation to isolate physically distinct B cell sub-sets. The isolated fractions were assayed by the adoptive immune response to NIP-POL antigen, under conditions where neither T cells nor other accessory cells were limiting the IgM or IgG AFC responses. The results were compared to previous studies on the IgM AFC-progenitors of unprimed adult mice. Splenic IgM and IgG memory AFC-progenitor activity was largely found among the typical B cells of slow to medium sedimentation rate, in contrast to the fastre sedimenting IgM AFC-progenitor activity of unprimed animals. Splenic IgM and IgG memory AFC-progenitor activity was found among the medium to light density cells, and so resembled by this parameter the IgM AFC-progenitor activity in unprimed animals. Thoracic duct lymphocytes from hapten-primed mice also exhibited memory IgM and IgG AFC-progenitor activity in the slow-medium sedimentation range. However, in contrast to spleen, the IgM and IgG memory AFC-progenitor activity in lymph was found among very dense B cells. Two physically distinct sub-populations of memory B cells have thus been identified, namely: i) small, medium-light density, presumably tissue-resident B lymphocytes found in spleen; and ii) small, dense, presumably recirculating B lymphocytes found in lymph. Both physical forms include IgM and IgG progenitors. Both forms are distinct from the larger, medium-light density "virgin" AFC-progenitors in the spleen of unprimed adult mice.     

Antigen-initiated B lymphocyte differentiation. XIV. Nonspecific effects of antigen stimulation cause proliferation in the "pre-progenitor" subset of primary B cells.

The effect of specific and nonspecific stimuli on the cycle status of subsets of primary B lymphocytes was assessed by preinjecting donor CBA mice 1 to 2 days previously with various substances, and then incubating the isolated spleen cells with high specific activity 3H-TdR before assay. AFC-progenitor activity was assessed as a response to NIP-POL antigen, either by adoptive transfer to irradiated recipients or by cell culture. Previous studies showed these assays reflected the activity of different subsets of B cells, termed "pre-progenitors" (adoptive assay) and "direct progenitors" (culture assay). Most functional primary B cells, whether assayed in culture or by adoptive transfer, were not initially in rapid cell cycle in normal adult mice. However, nonspecific stimulation for 1 day caused NIP-specific adoptive transfer IgM AFC-progenitors to enter rapid cell cycle. This effect was independent of T cells and not related to the antigenicity of the stimulus: particulate peritoneal irritants were the most effective stimulants. In contrast to adoptive transfer results. AFC-progenitors assayed in cell culture were unaffected by nonspecific stimuli, but were activated into cell cycle by specific antigen.     

Antigen-inhibited B-lymphocyte differentiation. X. Sedimentation velocity characterization of antigen-binding cells and antibody-forming-cell progenitors in the in vitro microculture immune response of unprimed CBA mice to NIP--POL antigen.

The B lymphocytes serving as antibody-forming-cell (AFC) progenitors have been investigated using two different types of separation methods. Sedimentation velocity fractionation was used to separate subsets of B lymphocytes differing primarily in size. Fractionation on a 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP)-gelatin matrix was used to separate NIP-binding cells, a population highly enriched for cells with surface Ig receptors specific for the NIP hapten. Assessment of the functional capacity of the separated B cells was by culture at limiting dilution in the presence of thymus “filler” cells, using the T-independent antigen NIP-POL (polymerized bacterial flagellin) to induce antibody formation. Splenic AFC-progenitors from both adult conventional and neonatal germ-free mice were a physically heterogeneous population, with activity in small, medium, and large lymphocytes. The cells enriched by NIP-gelatin binding, whether isolated and counted directly or isolated and assayed as AFC-progenitors, were no less heterogeneous. These NIP-binding cells resembled in sedimentation characteristics the overall B-cell and overall NIP-specific AFC-progenitor populations, except for some relative enrichment of medium-sized cells (S value, 5.5 mm/hr). The small (S value, 3.4 mm/hr) B-cell region of adult mouse spleen contained both NIP-binding cells and cells responsive as AFC-progenitors in the microculture assay. This contrasts with the results of the in vivo adoptive immune assay, where the smaller B-cell region is unresponsive in unprimed adult animals.     

Suppressors of the graft vs graft reaction in tolerance to alloantigens

Immune response and suppressor cell activity of CBA (H-2k) mice made tolerant to allogeneic C57B1/6 (H-2b) heart graft were studied in graft-versus-graft reaction (GvGR). Intact CBA spleen cells inhibited response of (CBA X C57B1/6)F1 cells to antigenic stimulus (sheep red blood cells--SRBC), when injected together into lethally irradiated (CBA X C57B1/6)F1 mice. Spleen cells of tolerant mice were unable to decrease immune response of (CBA X C57B1/6F1 lymphocytes to SRBC and suppressed specifically the inhibition induced by intact CBA spleen cells. Spleen cells from tolerant mice were also capable of suppressing GvGR induced by CBA lymphocytes immune to C57B1/6 cells. Pretreatment of tolerant spleen cells with rabbit antithymocyte globulin and complement before adoptive transfer diminished markedly the suppression. The results obtained in the study suggest that suppression of transplantation immunity in this model is mostly due to T suppressor cells.     

Antigen-initiated B lymphocyte differentiation. V. Electrophoretic separation of different subpopulations of AFC progenitors for unprimed IgM and memory IgG responses to the NIP determinant.

Spleen cells from CBA mice were separated by continuous, free-buffer film cell electrophoresis, and the capacity of cells in different fractions to mount an adoptive immune response specific for the NIP hapten determined. Experimental conditions were such that AFC progenitor B cells were measured, rather than helper or suppressor T cells. The IgM response of unprimed animals (a virgin or antigen inexperienced population) and the IgG response of long-term hapten-primed animals (a B memory cell population) were compared. The results indicated physical and biological heterogeneity in splenic B cells, with AFC progenitors for unprimed IgM and memory IgG responses being extensively separated.AFC progenitors for a primary IgM response in normal, germ-free and athymic mouse spleen, and bone marrow, separated into three distinct populations. Two of these were of much higher mobility than the typical splenic B cells and separated in the T cell zone. These cells produced a relatively early peak response of AFC after stimulation.AFC progenitors for a secondary IgG response were predominantly typical low-mobility B cells. Three regions of activity were separated, one overlapping part of the IgM progenitors. The slowest migrating activity peaks corresponded to the mobility of some recirculating B cells. These cells produced a more delayed AFC response after stimulation.AFC from the spleens of immunised mice separated as a single, broad, mediummobility peak distinct from most B cells and AFC progenitors. IgM and IgG (memory) AFC had similar electrophoretic characteristics.     

Isogeneic lymphocyte interactions (ILI) in CBA/N mice: Presence of ILI-Type 2 and absence of ILI-Type 1

The ability of spleen cells from immune defective CBA/N mice to stimulate two types of isogeneic lymphocyte interactions (ILI) was studied. In normal mice adult splenic cells induce proliferation of syngeneic neonatal thymocytes in Type 1 ILI and of syngeneic adult lymph node cells in Type 2 ILI. We have shown that CBA/N spleen cells are inoperative in ILI-Type 1 because the stimulating antigen, murine differentiation antigen 1 (MDA-1), is absent. Murine differentiation antigen 2 (MDA-2), however, is present and Type 2 ILI is evoked by CBA/N spleen cells. The results suggest that MDA-1 and MDA-2 are present on different subsets of B cells. In previous studies in other mice we have shown MDA-1 to be a strong contender for the role of receptor for T-cell signals. Our present findings lend further support to that hypothesis.     

Synergistic effects of the xid gene in X chromosome congenic mice. I. Inability of C3.CBA/N mice to respond to thymus-dependent antigens in adoptive transfer assays

The xid gene, which causes a B lymphocyte immune defect in CBA/N mice, has been bred onto the C3H/HeN background. The resulting X chromosome congenic mice (C3.CBA/N) exhibit immunologic defects that are much more profound than the defect exhibited by CBA/N mice; thus, the B cells from C3.CBA/N mice not only fail to respond to thymus-independent (TI) type 2 antigens such as TNP-Ficoll, but they fail to respond in vitro to TI-type 1 antigens such as TNP-Brucella abortus (BA) and B cell mitogens such as LPS and Nocardia water-soluble mitogen. In this paper we show that the synergistic defect seen in C3.CBA/N B cells is also elicited in adoptive transfer assays to thymus-dependent (TD) antigens such as TNP-KLH and PC-KLH, antigens to which both parental strains respond. Thus, the secondary adoptive transfer response of C3.CBA/N spleen cells is generally less than 5% of the immune response produced by CBA/N or C3H/HeN spleen cells. This synergistic defect is restricted to the C3.CBA/N B cells, since C3.CBA/N T cells can provide help to CBA/N B cells that is equivalent to the help obtained with CBA/N T cells. The low responsiveness of C3.CBA/N spleen cells to TD antigens, which is elicited in adoptive transfer assays, is not seen when the intact animal is immunized with antigen in CFA; this, intact C3.CBA/N mice produce anti-PC-KLH and anti-TNP-KLH responses only slightly lower than the responses of CBA/N mice to these same antigens. In contrast, when these mice are immunized with phenol-extracted LPS, a TI-type 1 antigen, their antibody responses are severely depressed. These data suggest that under conditions in which T cell help may be limiting or in which the intact physiology of the T and B cells has been disrupted, C3.CBA/N B cells demonstrate profound immunologic impairment; however, when adequate T cell help is available and the splenic architecture is not disrupted, their immune responses appear to progress in a normal fashion.     

Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.     

Mechanism of the intensification of the immune response to Staphylococcus in mice after the transplantation of primed donor splenocytes

Experiments on CBA, C57Bl/6 mice and (CBA X X C57Bl/6)F1 hybrids were made to study the mechanism of stimulation of the immune response to staphylococci after injection of primed splenocytes. The stimulating action of immune splenocytes was reversed after their in-vitro treatment with anti-immunoglobulin serum and complement. The stimulant effect was also seen in a semi-allogeneic system (adoptive transfer of CBA mice immune cells to (CBA X C57Bl/6)F1 recipients). Preincubation of splenocytes with CBA-anti-C57Bl/6-serum and complement prior to demonstration of antibody-forming cells did not influence their number in the spleen of hybrid recipients injected with immune cells carrying parent genotype but decreased this indicator of the immune response in control mice. It is concluded that stimulation of the immune response to staphylococci after transplantation of primed splenocytes is due to the anamnestic response of donor's cells repeatedly stimulated by antigen in the recipient's host.     

Functional activity of cells suppressing the humoral immune response in internal irradiation

In the adoptive transfer system of (CBA X C57BL/6)F1 mice, the estimation was made of the function of splenic cells, suppressors of the humoral immune response to sheep erythrocytes 1 and 6 months following the injection of 125I and 131I. Low absorbed doses of the radioactive isotopes were shown to stimulate the activity of the suppressors generated in mouse spleen.     

INFLUENCE OF AGE AND ANTIGENIC STIMULATION ON GRANULOCYTE AND MACROPHAGE PROGENITOR CELLS IN THE MOUSE SPLEEN

The frequency and proliferative activity of granulocytic and macrophage progenitor cells were determined in the spleens of C₅₇BL, BALD/c, NZB and CBA mice. These cells were detected by their capacity to form granulocytic and/or macrophage colonies ( in vitro colony-forming cells, CFC) in agar culture. In vitro CFCs were low in frequency in the adult spleen (4–28/10⁵ cells) compared with the bone marrow (180–280/10⁵ cells). However, the neonatal spleen, both in germfree and conventional mice, contained high levels of in vitro CFCs. From the low suiciding index with tritiated thymidine and the small numbers of cluster-forming cells in relation to colony numbers, many in vitro CFCs in the adult C₅₇BL spleen appear to be in a non-cycling state. The level and activity of in vitro CFCs were extremely low in the spleen of adult germfree CBA mice but were greatly increased in conventional mice following the injection of a bacterial antigen.     

Phenomenon of parental resistance and its genetic regulation]

Lymphocytes of mice F1 (CBA X M523) and F1 (A X M523) transplanted to 1000 R irradiated CBA or A mice responded to the test antigens--SRBC or S. typhi Vi-antigen--by formation of 100--1000 times less antibody forming cells than in syngeneic recipients. An intermediate result is achieved when the lymphoid cells are transplanted to the irradiated M523 mice. Lymphocytes of mice F1 (A X CBA), F1 (CBA X C57Bl/6), or F1 (A X A.CA) developed a similar immune response in the irradiated syngeneic mice and in both parental lines. The ability of parental line M523 to respond to SRBC was the same as in the other lines studied when examined in situ or in adoptive transfer experiments. The stem hemopoietic cells of mice F1 (CBA X M523) develop in the spleen of CBA mice 2--2.5 times less hemopoietic colonies than in the spleen of syngeneic animals. A conclusion was drawn that mutation M523 in CBA mice inhibited the proliferation and differentiation of hemopoietic and lymphoid cells in the irradiated nonsyngeneic recipients.     

Abrogation of neonatally induced permanent transplantation tolerance in mice: quantitative aspects.

Neonatal transplantation tolerance was induced in CBA (H-2k) mice by the intravenous injection of 20 million (CBAxA)F1 spleen cells to the transplantation antigens of the A mouse strain. Those mice which carried an A (H-2a) skin allograft without any sign of rejection for at least 120 days, were considered to be permanently tolerant and were selected for further experiments. Abrogation of permanent transplantation tolerance was achieved by injecting the tolerant mice with different doses (50, 100 and 200 millions, respectively) of normal syngeneic (CBA) lymphoid (spleen) cells. Dynamics of the rejection of the test skin allografts tolerated so far revealed well reproducible dose-response curves. Further groups of tolerant CBA mice were given 10, 50, 100, or 200 million "sensitized" (G + 16) CBA spleen cells: "sensitization" by A-skin allografting was performed 16 days before. The sensitized spleen cells abolished the state of tolerance more vigorously and effectively than the normal CBA spleen cells did. In a third group of experiments, the abrogating capacity of 50 million sensitized CBA spleen cells 16, 120, 240, or 360 days after sensitization was compared. The efficacy of the sensitized cells in abolishing the state of tolerance decreased continuously, but, even 360 days after sensitization a remarkably strong immunologic memory was demonstrable. The excellent quantitative correlations found between the number of the injected lymphoid cells and the dynamics of the abrogation of tolerance offer a highly promising new possibility for studying the immunological activity, the immunologic memory, etc., of the different lymphoid cell (sub)populations in performing the transplantation immune reactions.     

Studies on the mechanism of transplantation tolerance: I. Do suppressor cells play a role in the induction and maintenance of neonatal transplantation tolerance?

Neonatal transplantation tolerance was induced in CBA (H-2^k) mice to A (H-2^a) mice by injection of (CBA × A)F₁ spleen cells. Animals carrying an A-skin test allograft for more than 4 months without any visible sign of rejection were considered to be permanently tolerant. Permanently tolerant CBA mice were given normal syngeneic spleen cells to abrogate the state of tolerance. Abrogation of tolerance was greatly facilitated by antithymocyte serum (ATS) treatment of tolerant mice prior to the normal syngeneic cell transfer. Survival of A allografts on normal, adult, ATS-treated CBA mice was significantly prolonged (and in many cases “adult” tolerance was achieved) by transfer of spleen cells of syngeneic mice made permanently tolerant at neonatal age. The possible role of the F₁-cell “contamination” in the tolerance-inducing effect of the transferred “tolerant” spleen cells was excluded. The results indicate that ATS-sensitive suppressor cells play a definite role in the induction, maintenance, and transfer of neonatally induced transplantation tolerance.     

Generation of cytotoxic T lymphocytes during coxsackievirus tb-3 infection. II. Characterization of effector cells and demonstration cytotoxicity against viral-infected myofibers1.

This report describes studies characterizing the virus-specific cytotoxic effector cells which are present in the spleens of mice 7 days after infection with Coxsackievirus B-3. An in vitro 51Cr assay employing eyngeneic virus-infected neonatal fibroblasts was used to measure cytotoxic activity. Treatment of immune cells with (anti-thy-1.2) and complement abolished dtheir cytotoxic activity, but no reduction occurred when B cells were removed by incubation with anti-Ig and complement or macrophages eliminated by adherence depletion. The findings therefore imply that the cytotoxic reaction was mediated by sensitized T cells and that B cells and macrophages did not play an important role. Reciprocal assays performed with BALB/c and CBA/J cells showed that Coxsackievirus-immune spleen cells lysed infected syngeneic targets but not allogeneic targets, providing further evidence that cytotoxicity was mediated by effector T cells. In addition and in vitro assay system employing neonatal myocardial cells was developed and used to demonstrate that Coxsackievirus-infected myofibers were susceptible to destruction by immune spleen cells. The evidence suggests that mice infected with Coxsackie B viruses are able to mount a cell-mediated immune response with production of cytotoxic T cells which have the capacity to damage tissues infected with these agents.     

Effect of age of mouse recipients on the functional activity of transplanted hematopoietic and lymphoid cells]

When transplanting the bone marrow cells from adult C57BL mice to the lethally irradiated (CBA X C57BL) F1 hybrids of different age, the decrease of the colony forming activity of the stem haemopoietic cells was observed in the spleen of the older recipients, as compared with the 3 months old ones. The joint transplantation of the bone marrow and thymus cells resulted in both the cases in the stimulation of the growth of colonies. The number of endogenous colonies of haemopoietic cells arising in the spleen of animals following the sublethal irradiation was greater in younger hybrids. After the induction of the "transplant versus host" reaction by the lymph node or spleen cells from the CBA mice, the relative weight of spleen and regional lymph node, respectively, in the older recipients exceeded those in the younger ones.     

Regulatory T cell subpopulations in pregnancy. I. Evidence for suppressive activity of the early phase of MLR.

Previous in vivo experiments have provided evidence of suppressive activity induced by multiple allogeneic pregnancies. The reactivity of maternal spleen cells toward paternal strain alloantigens was investigated by use of MLR microculture technique. A study of the kinetics of the MLR showed an early peak of reactivity (48-hr culture) followed by a decline leading to a decreased reactivity by 96 hr when spleen cells from allogeneically pregnant mice were compared to those of virgin or even isogeneically pregnant mice, suggesting the possible action of MLR regulatory cells. A strong suppression of a H-2k (CBA) anti-H-2a (A/J) or anti-H-2d (C57BL/Ks) MLR was observed when mitomycin-treated spleen cells from CBA mice multiparous by A/J or C57BL/Ks (but not CBA) males were added to the culture. This suppression was abolished by treating the regulatory cell population with anti-theta serum plus complement or replacing the 1% normal mouse serum in the medium by a proper antiidiotypic mouse serum.     

Requirements of NK cells and proinflammatory cytokines in T cell-dependent neonatal autoimmune ovarian disease triggered by immune complex

A model of neonatal autoimmune disease has been described recently in which an epitope-specific autoantibody to murine zona pellucida 3 induces severe ovarian disease in neonatal, but not adult, mice (neonatal AOD). The autoantibody forms immune complex with endogenous ovarian zona pellucida 3, and a pathogenic CD4(+) T cell response is triggered. The basis for the predominant neonatal susceptibility has not been clarified. In this study innate immunity, including neonatal NK cells, in neonatal AOD was investigated. Neonatal spleen contained readily detectable NK1.1(+)TCRVbeta(-), but not NK1.1(+)TCRVbeta(+), cells. Ab depletion of NK1.1(+)TCRVbeta(-) cells inhibited neonatal AOD development. Moreover, in adoptive transfer of neonatal AOD, recipient disease was ameliorated when either donor or recipient NK cells were depleted. Thus, NK cells operate in both induction and effector phases of the disease. IFN-gamma was produced by neonatal NK cells in vivo, and it may be important in neonatal AOD. Indeed, ovaries with neonatal AOD expressed high levels of IFN-gamma and TNF-alpha which correlated with disease severity, and the disease was inhibited by IFN-gamma or TNF-alpha Ab. Importantly, disease was enhanced by recombinant IFN-gamma, and treatment of T cell donors with IFN-gamma Ab also significantly reduced adoptive transfer of neonatal AOD. Finally, neonatal AOD was ameliorated in mice deficient in FcgammaRIII and was enhanced in FcgammaRIIB-deficient mice. We conclude that neonatal NK cells promote pathogenic T cell response at multiple stages during neonatal autoimmune disease pathogenesis. Also operative in neonatal AOD are other mediators of the innate system, including proinflammatory cytokines and FcgammaRIII signaling.     

Analysis of certain mechanisms of antigen-specific suppression of the immune response

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.     

Mendelian sergregation of a tolerance-inducing self-antigen in the spleen

Others have reported that CBA/J neonatal thymus cells possess mixed lymphocyte culture reactivity against adult spleen cells from the same strain. This activity is not present in adult thymus. In this paper we describe experiments which show that C3H/ HeJ, a closely related strain, lacks the spleen antigen which CBA possesses. The antigen segregates as a simple Mendelian dominant trait in reciprocal backcross experiments. Only animals that possess the antigen lose their ability to react with it. These observations make this system a promising one to determine the mechanism of self-tolerance in nonchimeric individuals.