Adoptive Secondary Response Research Articles

Antigen transport II. The significance of the localization of antigen-laden cells in the spleen

Previous studies have demonstrated that macrophage-like cells transporting antigen, e.g., human serum albumin (HSA) appear in thoracic duct lymph and blood shortly after antigen injection. The in vivo migration of these antigen-laden (Ag-L) cells from the blood stream was examined systematically by transferring Ag-L cells bearing 125I-labelled HSA into syngeneic rats. There was no evidence autoradiographically that Ag-L cells migrated into lymph nodes, but the localization in the spleen followed a defined pattern: within the first hours after transfer, a majority of radiolabelled cells were identified in the marginal zone; by 3 hr and up to 4 days later, 60–80% of labelled cells were resident in the red pulp; Ag-L cells failed to migrate into the white pulp in significant numbers. Ag-L cells which had localized to the spleen, when examined 3 and 18 hr after transfer using combined autoradiography and immunoperoxidase staining, did not express la determinants in situ. The ability of Ag-L cells to stimulate an adoptive secondary response was tested in splenectomized, irradiated recipients receiving HSA-specific memory cells. Removal of the spleen before transfer severely reduced the antibody response evoked by Ag-L cells transporting HSA, thus indicating the functional importance of antigen transport to the spleen. Since Ag-L cell migration was primarily into the red pulp, we have considered whether the red pulp may provide a relevant microenvironment for lymphocyte/ antigen interaction.

Identification of an idiotypic marker of a major regulatory T cell of the immune response in B10.BR mice to ferredoxin. The relationship of idiotypic regulation to conventional hapten-carrier effects.

An anti-idiotypic antiserum was raised in rabbits to a monoclonal antibody (Fd-1) with specificity for one (the N epitope) of the two antigenic epitopes found on the ferredoxin (Fd) molecule. The anti-idiotypic antiserum (anti-Fd-1) was used to demonstrate that the Fd-1 idiotype was expressed at significant levels in most anti-Fd antisera raised in B10.BR mice. Examination of antisera raised in other mouse strains demonstrated that expression of this idiotype mapped to the IgH gene complex and was found in the antisera of all mouse strains examined with the Ig-1 allotype. When splenocytes from Fd-immune B10.Br mice were treated with anti-Fd-1 and transferred to irradiated syngeneic recipients, the adoptive secondary response was significantly higher in animals receiving treated cells as opposed to control animals, which received normal rabbit serum-treated cells. This response produced a net increase in antibody to both determinants, and the relative amount of Fd-1 idiotype was not significantly altered. Further studies with separated cell populations showed that the overall increase of anti-Fd antibody produced was attributable to the effects of the anti-idiotypic serum on a population(s) of T cells. Treatment of mice with the Fd-1 monoclonal antibody (which should react with anti-idiotypic cells) had an analogous effect to that of the anti-idiotype, in that mice so treated produced higher concentrations of anti-Fd antibodies when they were immunized and these antibodies exhibited net increases to both determinants. A model is presented to explain these results.

The relationship between surface immunoglobulin isotype and the immune function of murine B lymphocytes. V. High affinity secondary antibody responses are transferred by both IgD-positive and IgD-negative memory B cells.

We examined the adoptive secondary anti-DNP responses restored by surface IgD+ and IgD- memory B cells. Several parameters that might affect the affinity and magnitude of the adoptive responses were studied: 1) time after priming of cell donors, 2) source of anti-IgD antibodies used for immunofluorescent cell staining, 3) adjuvant used for priming, 4) carrier protein used for priming, 5) amount of antigen used for the challenge of adoptive hosts, and 6) the strain of ice used as donors and recipients. In contrast to previous reports, the present results demonstrate that the selection of cells with high affinity antigen receptors can occur to the same extent in both the delta + and delta - memory cell pools. This suggests that the loss of surface IgD is not a necessary intermediate stage in the maturation of memory B cells.

Cross-Priming of Murine B Cells with TNP Conjugates of Hemocyanin and Ficoll: Characteristics of Primed B Cells Responding to Both Antigens

Abstract There is strong evidence that separate subsets of B cells respond to primary challenge with TNP-Ficoll (TI.2) and with TNP-KLH (TD). Although TD antigens can prime for responses to TI antigens, it has been suggested that, at least in vitro, distinct subsets respond to TI.2 and TD antigens even after such priming. The present experiments on adoptive secondary immune responses in LAF1 mice suggest that a single population of primed cells may respond to both antigens. This conclusion is based on the findings that 1) High-affinity 7S antibody-producing cells primed by TNP-KLH can be challenged by TNP-Ficoll even after removal of T cells; 2) Priming with TNP-Ficoll induced 2- to 4-fold increased responsiveness to itself and to TNP-KLH, although the maturation with respect to affinity distribution of these IgG responses is significantly less than after priming with TNP-KLH; 3) TNP-KLH primed-cells never give higher responses upon challenge with both antigens simultaneously than to one of these antigens injected alone; 4) Primary and secondary responses to both antigens are reduced to a similar degree after pretreatment of cells with anti-Ia and C; 5) Receptor blockade of TNP-KLH primed cells by incubation with TNP-SIII reduces responses to TNP-KLH by 77 to 90% and to TNP-Ficoll by 60 to 70%. The findings are interpreted as supporting the view that B cell subpopulations responding to TI.2 and TD antigens represent stages of differentiation of a single B cell lineage that are overlapping rather than distinct subsets in vivo.

Immunologic tolerance in the chicken: II. Isolation and characterization of a soluble factor from a macrophage-like cell from chickens tolerant to bovine serum albumin

Macrophage-like cells harvested from 14-day-old chickens rendered tolerant at hatching with bovine serum albumin have been shown to release a soluble product which suppressed the adoptive secondary response to BSA. In addition, this suppressor factor suppressed the in vitro response to the non-cross-reacting antigen sheep red blood cells. The suppressor factor was found, by affinity chromatography, not to be composed of immunoglobulin but yet had the capacity to bind to the tolergen. Also, anti-line 3 antiserum, which is directed toward the major histocompatibility complex, removed the suppressive activity from macrophage supernatants.

Adaptive Differentiation of Murine Lymphocytes

Abstract Responses to the synthetic terpolymer L-glutamic acid, L-lysine, L-phenylalanine (GLΦ) and hapten derivatives thereof are controlled by two complementing H-2-linked Ir genes in the mouse. F1 hybrids derived from two different nonresponder strains (one of which possesses the α and the other β Ir-GLΦ gene) are phenotypic responders to GLΦ and 2,4-dinitrophenyl (DNP)-GLΦ. Moreover, spleen cells from DNP-GLΦ-primed F1 mice can adoptively transfer secondary anti-DNP antibody responses to irradiated F1 recipients that have been challenged with DNP-GLΦ. When, however, GLΦ-primed F1 helper T cells are transferred together with the DNP-specific F1 B cells that had been primed in separate mice altogether by DNP coupled to an unrelated protein carrier, such mixtures failed to develop adequate adoptive secondary anti-DNP responses to DNP-GLΦ. This contrasted with the ability of the same GLΦ-primed F1 T cells to provide helper activity for DNP-primed B cells from responder recombinant B10.A (5R) mice. More important, the apparent defect of GLΦ-primed F1 T cells in providing help for DNP-primed F1 B cells (primed to a DNP-protein conjugate) could be readily overcome by using DNP-primed B cells from donor F1 mice primed with DNP-GLΦ. As discussed herein, these results suggest that interacting T and B lymphocytes pair off into partner cell sets, any pair of which interact optimally when a “best fit” reciprocal self-recognition occurs between them.

An immunoregulatory factor associated with spleen cells from tumor‐bearing animals.: III. Characterization of the factor's target cells

An immunoregulatory factor associated with spleen cells from tumor-bearing mice was found to potentiate the generation of antibody-producing cells (APC). In an attempt to characterize the target cell of this enhancing factor (EF), its activity in mice devoid of mature T lymphocytes was tested. Levels of anti-SRBC APC were augmented when EF was injected together with SRBC to nude mice and "B" mice. In addition, EF potentiated the antibody response against the IgM-inducing T-independent pneumococcal plysacchardide SIII antigen. These results suggest that EF most probably exerts its enhancing influence directly on B lymphocytes. In a different line of experiments adoptive secondary responses were performed. Mice were immunized with SRBC as carrier or with the NIP-chicken erythrocytes (as a source of NIP-specific primed B cells) in the presence or absence of EF. Various combinations of spleen cells from the donor immunized mice were transferred in a mixture with NIP-SRBC to lethally irradiated recipient mice, EF did not exert any potentiation effect on helper function, except when primed B cells were used. In contrast, a clear activation or clone expansion of hapten-specific B cells was observed. These findings indicate that the enhancing factor from tumor-bearing animals directly affected the antigenic triggering of B lymphocytes and their subsequent proliferation and differentiation to antibody-producing cells.

Studies on the mechanism of suppression by T cells in an adoptive secondary response

Suppression of antibody synthesis by lymphocytes was studied using an adoptive secondary response model in which human serum albumin (HSA)-primed lymphocytes (memory cells) from the thoracic ducts of inbred rats were inhibited in irradiated recipients by nonimmune lymphocytes after mixed cell transfer. This investigation extended earlier work and formally showed that the suppressor cells were peripheral thymus-derived lymphocytes, which could rapidly recirculate from the blood to lymph, were present in spleen but not in bone marrow, and that primed T cells lacked this property to inhibit. The suppressive effect was independent of antigen dose but was markedly influenced by the form of antigen used for challenge in that suppression was significantly abrogated with aggregated HSA or with soluble HSA in the presence of specific antibody. Suppressor cells were found to exert their effect maximally at the time of antigen injection, but became ineffective by 40 hr following challenge. The results are considered within a larger framework of cellular regulation of antibody synthesis.

Induction and Mechanism of Tolerance to Bovine Serum Albumin in Mice Given Total Lymphoid Irradiation (TLI)

Abstract BALB/c mice given total lymphoid irradiation (TLI) were injected i.p. with bovine serum albumin (BSA) in saline, and challenged with DNP-BSA in complete Freund's adjuvant 6 weeks later. The latter animals made no anti-DNP antibody response as measured by a modified Farr assay, but made a normal anti-DNP response after challenge with DNP-BGG in adjuvant. Normal mice or mice given whole body irradiation were not tolerized by the i.p. injection of BSA in saline. Spleen cells from unresponsive mice (TLI + BSA in saline) suppressed the adoptive secondary anti-DNP response of sublethally irradiated syngeneic hosts given BSA-primed T cells, DNP-BSA-primed B cells, and DNP-BSA in saline. The suppressor cells were antigen specific, and were inactivated by in vitro treatment with anti-Thy 1.2 antiserum and complement. The findings suggest that soluble antigens administered to mice after TLI evoke a state of tolerance that is maintained by antigen-specific suppressor T cells. A similar mechanism may be involved in the maintenance of tolerance to allografts. These findings may have important clinical implications for patients treated with TLI for lymphoid malignancies.

Immunologic Tolerance in the Chicken

Abstract Serum from 14-day-old chickens rendered tolerant at hatching with 1250 mg bovine serum albumin (BSA)/KBW was found to suppress an adoptive secondary response to BSA and an in vitro response to sheep red blood cells (SRBC). Chromatographic separation resulted in three fractions: I, II, and III. The suppressive activity was found to be contained in fraction I (m.w. >1 × 106) and fraction III (m.w. <2.5 × 104). Analysis of fraction I by affinity chromatographic techniques revealed that it was composed of soluble BSA-antibody complexes and their removal removed the suppressive activity. Analysis of peak III revealed that the suppressive activity could only be removed by BSA and anti-Line 3 antiserum columns.

Regulation of IgE Antibody Production by Serum Molecules

Abstract IgE antibody production in mice of high and low IgE responder phenotypes, respectively, can be appreciably enhanced in magnitude after low dose whole body X-irradiation. Such enhanced responses, as well as adoptive secondary IgE responses, can be markedly suppressed by passive transfer of CFA-immune serum in low responder strains, but not in high responder strains. The studies presented here demonstrate that the suppressive activity of CFA-immune serum on IgE antibody production is strain specific. This is true even in reciprocal combinations of low IgE responder SJL and C57BL/6 mice, in which it was shown that serum capable of suppressing mice of the isologous strain was ineffective in diminishing IgE antibody production in the other low responder strain. Absence of suppressive activity in CFA-immune sera obtained from H-2 congenic donors which differed in their H-2 haplotypes while sharing many similarities in the background genome and, conversely, effective suppressive activity of H-2 congenic donor sera when H-2-identities between donor and recipient mice existed, strongly suggested a role, at least in part, of H-2 genes in dictating the strain specificity of such suppressive activity. Additional experiments provided evidence for a possible role of macrophages in catabolism of the active molecules in CFA-immune sera. These observations, together with those presented in the preceding paper, may provide valuable insight toward successful development of appropriate manipulations that could ultimately convert high IgE responder individuals into low responders.

The relationship between surface immunoglobulin isotype and immune function of murine B lymphocytes. III. Expression of a single predominant isotype on primed and unprimed B cells

We determined whether primed and unprimed B cells in the spleen of (BALB/c x C57BL/Ka)F(1) mice contain subpopulations that express a predominant surface Ig isotype. Spleen cells were stained for surface isotypes and sorted on the fluorescence-activated cell sorter (FACS) in order to obtain B cells bearing predominantly IgM (mup cells), IgD (deltap cells), or IgG (gammap cells). Each population was assayed for its capacity to restore the adoptive primary and secondary anti-bovine serum albumin (BSA) antibody response in irradiated syngeneic recipients. In addition, the adoptive response restored by isotype-predominant cells was compared to that restored by isotype- positive cells (B cells bearing a given surface isotype alone or in combination with others). The experimental results show that mup cells restore the adoptive primary and secondary IgM and IgG responses to BSA, and gammaP cells restore only the primary and secondary IgG response. Deltap Cells restored the adoptive secondary IgG response, but failed to restore the adoptive primary response at the cell doses tested. GammaP Cells but not deltap cells suppressed the IgM response of the mu(+) and delta(+) cells. The contribution of isotype-predominant cells to both the adoptive primary and secondary anti-BSA response was smaller than that of B cells bearing a combination of surface isotypes. Differences in the Ig isotype pattern expressed on the surface of primed and unprimed B cells are discussed.

The Role of Antigen-Antibody Complexes in Mediating Immunologic Unresponsiveness in the Chicken

A significant suppression of an adoptive secondary response to either BSA or sheep red blood cells (SRBC) resulted when chicken spleen cells were incubated with BSA-antibody (Ab) complexes formed in antigen excess (molar ratio Ag1Ab1.1). Serum from 14-day old birds rendered unresponsive at hatching with large doses of BSA also produced suppression. In addition the in vitro response to SRBC was suppressed by the above treatment. Complexes formed at equivalence either had no effect on the subsequent responses of primed cells or produced an enhancement. The fact that both the homologous and heterologous responses were inhibited could indicate that both were suppressed via a common pathway.

The relationship between surface immunoglobulin isotype and immune function of murine B lymphocytes. I. Surface immunoglobulin isotypes on primed B cells in the spleen.

We investigated the ability of IgM-, IgD-, and IgG-bearing cells from the spleens of (BALB/c x C57BL/Ka)F1 mice primed to dinitrophenyl-bovine serum albumin (DNP-BSA) to restore the adoptive secondary anti-BSA and anti-DNP antibody responses. A rabbit anti-mouse IgD antiserum was prepared and the specificity documented by radioimmunoprecipitation, and cell surface staining. Purified populations of IgM-, IgD-, and IgG-bearing cells were prepared by immunofluorescent staining with isotype-specific reagents, and sorting on the fluorescence activated cell sorter. Bright or dull cells were transferred to irradiated syngeneic recipients which were challenged with DNP-BSA in saline. Unfractionated spleen cells restored an adoptive secondary serum antibody response which was all IgG (2-mercaptoethanol resistant). Purified IgM- or IgD-bearing cells restored both the secondary IgM and IgG antibody response. IgG-bearing cells restored only the IgG response. In addition, the IgG-bearing cells appear to suppress the adoptive secondary IgM response, since depletion of IgG-bearing cells from transferred spleen cells results in a marked increase in the adoptive IgM response.

Maturation of B Lymphocytes in Rats

Abstract We examined the ability of large and small thoracic duct cells obtained from Lewis rats primed to DNP to restore the adoptive secondary anti-DNP response in irradiated syngeneic hosts given an excess of T helper cells. The large cells were four to five times more active on a per cell basis than were the small cells. However, the large cells constitute only 7 to 8% of the thoracic duct cells and, therefore, make a minor contribution to the restorative activity of the unfractionated cells. Pretreatment of thoracic duct cell donors with high specific activity 3H-TdR for 48 hr before cannulation markedly reduced the memory B cell activity of the large cells, but had little effect on that of small cells. In addition, the activity of the large cells diminished with time after primary immunization, but that of the small cells remained stable. Immunofluorescent staining of the large and small cells for surface IgM and IgG, and subsequent sorting on the fluorescence-activated cell sorter (FA CS), showed that surface IgM was present on large memory B cells, and that IgG was present on small memory B cells. The experimental results suggest that two subpopulations of memory B cells in the thoracic duct lymph differ by size, rate of turnover, persistence after primary immunization, and class of surface IgG.

Demonstration that IgG memory is carried by IgG-bearing cells.

Memory B cells which give rise to IgG antibody-producing cells were generally assumed to be IgG-bearing cells. However, recent studies indicating that very few IgG-bearing cells exist in lymphoid tissue brought this assumption into question. In this study, we examined directly the question whether IgG-bearing cells contain functional precursors of IgG antibody-producing cells. Using the adoptive secondary immune response, we demonstrated that Ig-1 b-bearing cells, isolated with the fluorescence-activated cell sorter (FACS), are the functional precursors of Ig- 1 b-producing cells. Further, we have enriched IgG2 and IgG1 memory B cells using the FACS. The results show that IgG2-bearing cells are the functional precursors of the IgG2 antibody-producing cells. Likewise, the IgG1-bearing cells are the functional precursors of IgG1 antibody-producing cells. Thus, IgG memory cells have surface IgG which indicates the class and allotype commitment of the memory cell and its progeny antibody-forming cells.

Inhibition of adoptive secondary responses by lymphoid cell populations

The ability of normal and tolerant lymphoid cells to inhibit the adoptive secondary response was investigated in order to delineate the influence which the host can exert on a memory cell population as the host recovers from the effects of irradiation. LBN rats were irradiated with 500R whole body X-irradiation, reconstituted with either normal or tolerant lymphoid cells, then they were given immune spleen cells and challenged with soluble antigen (DNP-BGG). Cells capable of suppressing the adoptive secondary responses were shown to possess the following characteristics: 1) in nonimmune donors they were found in the greatest concentration in lymph nodes, followed by spleen and bone marrow and were practically absent in the thymus; 2) their numbers were not increased in donors rendered tolerant by long term treatment with deaggregated DNP-BGG plus cyclophosphamide nor in donors given large doses of DNP-BGG 48 to 72 hr before sacrifice; 3) in animals rendered tolerant by long term antigen treatment alone some enhancement of suppressor function was seen; 4) the suppressor cells could be shown among both glass wool adherent and nonadherent cells and 5) the nonantigen specific suppressor cells did not affect the kinetics of antibody formation nor the affinity of the antibody which was produced. These results are discussed in terms of the nature and source of the suppressor cell populations and their relevance to the control of secondary responses in intact animals.

Transition in the character of immunological memory in mice after immunization. II. Memory in T and B cell populations.

Teh immunological memory in antibody response of mice to bovine serum albumin (BSA) was investigated at the level of antibody-producing cells or their precursor B cells and thymus-dependent helper T cells. Spleen cells obtained from mice previously primed with alum-precipitated BSA at various times were transferred to irradiated syngeneic mice. Spleen cells from mice immunized 8 days or 64 days before presented a high degree of adoptive secondary response, whereas the adoptive response of cells from mice immunized 2 days previously was found to be inferior even to that of unprimed spleen cells. Primed spleen cells treated with anti-mouse thymocyte rabbit serum plus complement were supplemented with normal thymus cells and the restoration of the responsiveness was examined. It was suggested that the memory was carried mainly by T cells in the earlier phases of the immunological memory (2 days or 8 days after the primary immunization). On the other hand, the immunological memory in the B-cell population was shown to grow gradually toward the later phase (later than 40 days).

The requirement for C3 receptors on the precursors of 19S and 7S antibody-forming cells.

The main conclusion from this study is that C3 receptors are not required for the generation from B cells of a thymus-dependent 7S antibody response. The requirement for C3 receptors on the precursors of antibody-forming cells was studied in an adoptive transfer system using thoracic duct lymphocytes (TDL) from primed rats as a source of precursors and irradiated recipients as hosts. 7S precursors were found in both the CR+ and the CR- fractions of TDL and it was established that the response transferred by CR- cells did not arise from either a raidoresistant B cell in the host or from CR+ cells contaminating the CR- population. Thus, the C3 receptor is not obligatory for B-cell-T-cell cooperation in the 7S response. The precursors of 19S antibody-forming cells were found only in the CR+ subpopulation. The CR-Ig+ subpopulation was shown to contain all the B blasts in rat TDL and a very small number (approximately 1% of all TDL) of small lymphocytes. This latter population contained the CR- 7S precursors and contributed approximately 20% of the total adoptive secondary 7S response transferred by CR+ and CR- subpopulations combined. This observation suggests that the percentage of rat TDL committed to carry 7S memory is small, a conclusion which is confirmed and extended in the following paper.

The allogeneic effect in inbred mice: V. Analysis of B cell immunoglobulin classes influenced by non-specific and carrier-specific T cell stimuli

The studies presented here show that the allogeneic effect on adoptive secondary responses to homologous and heterologous conjugates affects the plaque-forming cell response of immunoglobulin classes IgM, IgG, IgG 1, IgG 2a + 2b and IgA. We have demonstrated that the increase in PFC resulting from the allogeneic effect varies among the immunoglobulin classes analyzed, most probably reflecting the relative frequencies of precursor B cells in a particular primed cell population, and not a preferential stimulation of one class of B lymphocytes. A comparison of the allogeneic effect and the syngeneic cooperative response between carrier-primed T cells and hapten-specific B cells showed parallel responses of DNP-specinc PFC of all classes studied.