Resistance of neonatal tolerance in mice to abrogation by normal immunocytes

The mechanisms of neonatally induced tolerance in CAF₁ mice to bovine serum albumin were investigated with cell transfer experiments and primary interaction between radiolabeled antigen and antisera for antibody measurements. Adoptive transfer of normal syngeneic splenocytes, of thymocytes, or of thymus-bone marrow cell combinations did not break this tolerance though proved effective for breaking irradiation-caused tolerance. Similar transfer of spleen cells taken from immunized mice did abrogate neonatal tolerance; but spleen cells transferred from immunized mice that subsequently also had been desensitized did not, even though they could provide nontolerant recipients with anamnestic responses. Neither allogeneic nor xenogeneic spleen cells broke the tolerance.Thus, the neonatal tolerance studied appears to suppress primary immunologic response but not an anamnestic response. It appears more likely to be an active suppressive phenomenon than one of clone loss.     

Factors associated with the development of neonatal tolerance after the administration of a plasmid DNA vaccine

A plasmid DNA vaccine encoding the circumsporozoite protein of malaria (pCSP) induces tolerance rather than immunity when administered to newborn mice. We find that this tolerance persists for >1 yr after neonatal pCSP administration and interferes with the induction of protective immunity in animals challenged with live sporozoites. Susceptibility to tolerance induction wanes rapidly with age, disappearing within 1 wk of birth. Higher doses of plasmid are more tolerogenic, and susceptibility to tolerance is not MHC-restricted. CD8+ T cells from tolerant mice suppress the in vitro Ag-specific immune response of cells from adult mice immunized with pCSP. Similarly, CD8+ T cells from tolerant mice transfer nonresponsiveness to naive syngeneic recipients. These findings clarify the cellular basis and factors contributing to the development of DNA vaccine-induced neonatal tolerance.     

Different requirements for the adoptive transfer of oral tolerance and its indirect effects assessed by DTH and antibody responses in mice

Oral tolerance inhibits T-cell dependent reactions to antigens previously contacted by oral route. Parenteral re-exposure to orally-tolerated antigens inhibits immune responses to unrelated antigens, a phenomenon we have called “indirect effects” of oral tolerance. We examined the requirements of previous irradiation of C57BL/6 and BALB/c recipients to successful transfer of oral tolerance and its indirect effects using 1 × 10⁸ splenocytes. When DTH reactions were evaluated, irradiation was not required to transfer both oral tolerance and its indirect effects. C57BL/6, but not BALB/c recipients, required irradiation to adopt suppressed antibody responses to tolerizing antigen. In BALB/c recipients, the indirect effect was transferred only if serum from the tolerant donors was added to the transferred splenocytes. CFSE labeled donor cells were not eliminated from non-irradiated C57BL/6, although unable to suppress antibody responses. Our results provide further evidences on the existence of a functional barrier in immunocompetent recipients that hinders the adoptive transfer of different immunological activities. Interactions between cells and serum components may be necessary to bypass this barrier.     

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.     

Regulatory T cells induced by 1 alpha,25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance

1alpha,25-dihydroxyvitamin D3, the active form of vitamin D3, and mycophenolate mofetil, a selective inhibitor of T and B cell proliferation, modulate APC function and induce dendritic cells (DCs) with a tolerogenic phenotype. Here we show that a short treatment with these agents induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. Peritransplant macrophages and DCs from tolerant mice express down-regulated CD40, CD80, and CD86 costimulatory molecules. In addition, DCs from the graft area of tolerant mice secrete, upon stimulation with CD4+ cells, 10-fold lower levels of IL-12 compared with DCs from acutely rejecting mice, and induce a CD4+ T cell response characterized by selective abrogation of IFN-gamma production. CD4+ but not CD8+ or class II+ cells from tolerant mice, transferred into naive syngeneic recipients, prevent rejection of donor-type islet grafts. Graft acceptance is associated with impaired development of IFN-gamma-producing type 1 CD4+ and CD8+ cells and an increased percentage of CD4+CD25+ regulatory cells expressing CD152 in the spleen and in the transplant-draining lymph node. Transfer of CD4+CD25+ cells from tolerant but not naive mice protects 100% of the syngeneic recipients from islet allograft rejection. These results demonstrate that a short treatment with immunosuppressive agents, such as 1alpha,25-dihydroxyvitamin D3/mycophenolate mofetil, induces tolerance to islet allografts associated with an increased frequency of CD4+CD25+ regulatory cells that can adoptively transfer transplantation tolerance.     

Tolerance to a xenotransplant in an adoptive system

Splenocytes of mice tolerant to rat neonatal heart graft were unable to respond to rat blood cells (RBC) when transferred adoptively to lethally irradiated syngeneic recipients 10 or 30 days after tolerogenic treatment. Early after induction of tolerance spleen cells of experimental mice were also unable to respond to sheep red blood cells. However, they responded vigorously to goose red blood cells. Later on (30 days after treatment) tolerance was found to be strictly RBC-specific. Cells suppressing anti-RBC response of intact cells were detected in the spleen of mice both 10 and 30 days after the induction of tolerance. Their suppressive activity was strictly RBC-specific. The results obtained show that early after tolerogenic treatment experimental mice are unable to respond due both to the deficiency of T-helpers involved in the response to mammalian blood cells and to activation of RBC-specific I-J+ T-suppressors. Thirty days after treatment tolerance is maintained solely by RBC-specific T-suppressor cells.     

Immunologic tolerance to HGG in mice. I. Suppression of the HGG response in normal mice with spleen cells or a spleen cell lysate from tolerant mice.

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-gamma-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-gamma-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.     

Mechanisms in immune tolerance. I. A specific block of immunological memory in HSA-tolerant mice

Evidence is provided indicating that the transient immune response detected during the induction of tolerance to HSA in mice, results in immunological memory which persists in an immunosuppressed state in the tolerant animals. The mechanism which blocks this memory is antigen-specific and can be selectively inactivated by total body irradiation in the range of 650–900 R. Consequently tolerant mice, irradiated within this range and restored with normal syngeneic spleen cells, respond better to the tolerizing antigens than do similarly treated normal mice. The tolerizing block can be transmitted from “tolerant” to normal spleen cells when a mixture of both is transferred to normal irradiated recipients. Since similar inhibitory activity is also demonstrated by fresh sera of tolerant mice, it is suggested that the blocking mechanism depends on inhibitory cells which act via a humoral inhibitor.     

Tolerance induced by grafting semi-allogeneic adult skin to larval Xenopus laevis: Possible involvement of specific suppressor cell activity

Major histocompatibility complex (MHC)-homozygous Xenopus laevis were rendered tolerant to semi-allogeneic antigens by grafting skins of adult frogs during larval stages (larvally induced tolerance), and this tolerant state was compared with the tolerance induced in early thymectomized frogs by the grafting of semi-allogeneic nonlymphoid thymuses (thymus-reconstituted tolerance). In contrast to a total inability of thymus-reconstituted frogs both to reject skins and to exhibit a mixed leukocyte reaction (MLR) against the semi-allogeneic donor, larvally induced tolerant frogs showed a strong MLR against leukocytes of the tolerizing skin donor (split tolerance). Breakdown of the tolerant state in thymus-reconstituted frogs were easily accomplished by inoculation with syngeneic splenocytes, but this breakdown was extremely difficult to achieve in frogs with larvally induced tolerance. The injection of splenocytes from larvally induced tolerant frogs into normal frogs significantly suppressed semi-allogeneic graft rejection in the latter group; no suppression was obtained when splenocytes from thymus-reconstituted frogs were used. In addition, in the thymectomized frogs, recovery of allograft rejection capacity against the pertinent semi-allogeneic antigens were suppressed by the injection of splenocytes from larvally induced tolerant frogs, with the degree of suppression depending on the splenocyte dose. These results indicate that the larvally induced tolerant state is maintained by specifically induced suppressor cells affecting the in vivo allograft response but not the MLR.     

Tolerance mechanism in experimental ovarian and gastric autoimmune diseases.

Neonatal splenocytes, neonatal thymocytes, or phenotypically mature adult thymocytes, transferred from normal BALB/c mice to syngeneic athymic nu/nu (or SCID) mice, led to autoimmune oophoritis and autoimmune gastritis, with corresponding serum autoantibodies, in the recipients. The overall disease incidence was 73%; the pathology ranged from mild to severe, with complete loss of ovarian follicles and gastric parietal cells. CD4+ neonatal spleen cells and CD4+ CD8- adult thymocytes were required for autoimmune disease induction. Adult spleen cells did not elicit disease, but they prevented disease when co-transferred with neonatal spleen cells. However, in confirmation of an earlier report by Sakaguchi et al., (J. Exp. Med. 161:72, 1985), a subset of adult splenic T cells expressing a low level of CD5 molecules elicited similar autoimmune diseases. Thus, self-reactive T cells responsible for autoimmune disease of the stomach and ovary are not effectively deleted in the thymus, and they exist in the peripheral lymphoid organs of normal mice. We conclude that the functional expression of the self-reactive T cells is ontogenetically regulated; whereas T cells in the neonatal mice readily elicited autoimmune diseases in nu/nu recipients, regulatory cells may render self-reactive T cells in the normal adults unresponsive.     

Suppressor T cells induced by soluble immune complexes can adoptively transfer inhibition of cytophilic antibody receptors on macrophages.

Immune complexes (soluble antigens of L1210 and antibody to L1210) when given to allogeneic C3H mice generated suppressor cells that inhibited receptors for cytophilic antibody on macrophages. Thymocytes or nylon-nonadherent splenic T cells (4 × 10⁷) from immune-complex-treated mice transferred this suppressive activity when injected into normal syngeneic mice. Maximal suppression of macrophages occurred 4 to 6 days after transfer. In contrast, even 5 × 10⁷ nylon-adherent, non-T spleen cells from immune-complex-treated (“suppressed”) mice failed to induce macrophage suppression in the syngeneic recipients. When T-cell-depleted “B” mice were used as recipients, neither thymocytes nor splenic T cells from suppressed mice were able to transfer suppressive activity. However, the admixture of 2 × 10⁷ normal syngeneic thymocytes with 4 × 10⁷ thymocytes from suppressed mice restored the latter's ability to elicit suppression of macrophages in T-cell-deprived recipients. Peritoneal monocytes from recipients of suppressor thymocytes (to L1210) could not attach cytophilic antibody to L1210 but could attach cytophilic antibody to EL-4 and sheep erythrocytes. Thus, suppressor T cells induced by immune complexes can transfer immunologically specific macrophage suppression (inhibition of cytophilic antibody receptors) to syngeneic recipients. The suppressor cells required the cooperation of normal T cells, suggesting either recruitment of suppressor cells from, or a helper effect by, the normal T cells, in order to produce their effect.     

Cellular events in protein-tolerant inbred rats. III. Antigen-reactive B cells in rats tolerant to human serum albumin.

Rats tolerant to human serum albumin (HSA) were injected with selected lymphocyte populations and challenged with HSA plus adjuvant to test for loss of tolerance. Thoracic duct lymphocytes (TDL) from normal or immune rats, either untreated or depleted of Ig-bearing cells or HSA-binding cells by affinity chromatography were all equally effective in restoring the HSA antibody response in previously tolerant recipients. In contrast, recirculating B cells (TDL from B rats) were not effective. The results indicated that unresponsiveness to HSA was a lesion of the T- but not the B-cell compartment. However, antibody affinity failed to mature to a high level in tolerant rats that were restored with T cells, and the response of transferred primed B cells into unresponsive recipients was inhibited, suggesting that the tolerant state was not merely due to a T-cell deletion.     

Loss of carrier-determined tolerance in vitro with loss of receptor blockade.

Experiments were done to determine whether carrier-determined tolerance is reversible and whether the loss of tolerance is accompanied by the loss of receptor blockade. Spleen cells from mice made tolerant with DNP-isologous IgG remained tolerant when transferred to irradiated syngeneic mice. If these same tolerant spleen cells were incubated for 24 hr or more before transfer the tolerance was lost. Autoradiology was done on the tolerant cells with either 125I anti-DNP or 125DNP-KLH, before and after incubation in vitro. When the cells were tolerant the number of DNP ABC was decreased whereas cells having DNP on their surface were increased. When the cells lost tolerance after in vitro incubation, the hapten-bearing cells were no longer present although the number of cells free DNP receptors increased to normal. These data suggest that in carrier determined tolerance the reactivation of tolerant lymphocytes may involve reversible receptor blockade.     

Regulatory CD8+ T cells control neonatal tolerance to a Th2-mediated autoimmunity

Exposure of newborn animals to a foreign Ag may result in immunological tolerance to that specific Ag, a phenomenon called neonatal tolerance. We have previously reported that neonatal administration to Brown-Norway rats of mercury, a heavy metal toxicant, induces a dominant tolerance, specific for the chemical otherwise responsible for Th2 cell-mediated autoimmune responses in this susceptible strain of rats. Neonatal exposure to Ags can prime immunity, rather than inactivate or delete responses, and sustain regulatory functions effective against autoreactive T cells. Here, we address whether such a tolerant response is due to the generation of regulatory cells. The results suggest that the CD8(+) T cell subset is involved in neonatal tolerance to mercuric salt-induced Th2 autoimmune disease. Thus, we demonstrate that in vivo CD8 depletion breaks tolerance following mercury recall in animals under a neonatal tolerance protocol. Furthermore, adoptive cotransfer of splenocytes from naive and tolerant rats as well as transfer of CD8(+) T cells from tolerant animals prevent naive syngeneic rats from developing pathologic Th2 immune responses. These observations indicate that CD8(+) T cells are endowed with regulatory functions in neonatal tolerance and mediate active suppression. Moreover, neonatal tolerance induced the expansion of CD8(+)CD45RC(high) T cells and the emergence of a high percentage of IFN-gamma-synthesizing CD8(+) T cells, which probably reflects the implication of regulatory Tc1 cells. Thus, in vivo induction of neonatal tolerance suppresses Th2 autoimmune responses via generation of a CD8(+) cell-mediated regulatory response.     

Cell cooperation in abolition of tolerance of xenogeneic tumor.

Thymectomized, lethally irradiated mice reconstituted with syngeneic bone marrow cells are tolerant to xenogeneic Yoshida ascites sarcoma (YAS). The tolerance was abolished by an injection of syngeneic normal spleen, thymus, or lymph node cells given simultaneously with YAS. Allogeneic and semiallogeneic spleen cells were ineffective. The YAS-rejecting mice produced specific anti-tumor antibodies. The serum of these mice transferred to tolerant T-cell-deficient mice protected the latter from inoculated YAS cells. These serum-protected mice were not able to resist the reinoculum of the tumor cells as the mice restored with lymphoid cells did. The latter mice rejected the YAS at the time when donor cells were practically absent in their lymphoid tissue. The low effective ratio of injected syngeneic lymphoid to tumor cells, efficiency of injected thymus cells, and other data led to the conclusion that transferred lymphoid cells did not act directly on tumor cells but through cooperation with host lymphoid cells. The cooperation of donor T- and host B-lymphocytes enabled the activation of the latter, and YAS cells were rejected.     

Neonatal splenic suppressor cells in the chicken. I. In vivo suppression of the immune response to bovine serum albumin by normal and tolerized neonatal spleen cells

The presence of active splenic suppressor cells in neonatal chickens, either normal or tolerant to bovine serum albumin (BSA), was examined by assessment of their effect on both primary and adoptively transferred secondary responses to BSA or sheep red blood cells (SRC). Both normal and BSA tolerized spleen cells were shown to be highly suppressive of secondary anti-BSA responses generated by specifically primed adult spleen cells in inert recipients. Suppression of the secondary anti-BSA response by normal spleen cells was slightly less effective than that seen with BSA tolerant spleen cells. Transfer of BSA tolerant spleen cells into normal recipients, followed by BSA challenge, prevented any significant primary anti-BSA response. In contrast, transfer of normal spleen cells into normal recipients, followed by BSA challenge, failed to show any suppression of the resulting primary response. Neither normal nor BSA tolerant neonatal spleen cells were capable of suppressing either primary or secondary responses to SRC. Thus, chickens tolerized to BSA have suppressor cells specific for the tolerizing antigen. We present evidence that both the tolerance associated suppressors and the suppressors detected in normal neonatal chickens are T cells.     

Abrogation of the capacity of delayed-type hypersensitivity responses to alloantigens by intravenous injection of neuraminidase-treated allogeneic cells

BALB/c or C3H/He mice were inoculated i.v. with allogeneic spleen cells untreated or treated with neuraminidase. Appreciable or potent anti-allo-delayed-type hypersensitivity (DTH) responses were observed when mice were inoculated i.v. with untreated allogeneic cells or inoculated i.v. with those cells followed by s.c. immunization with untreated allogeneic cells. In contrast, i.v. inoculation of neuraminidase-treated allogeneic cells (presensitization) not only failed to induce any significant anti-allo-DTH responses but also abolished the capability of the animals to develop DTH responses after s.c. immunization, indicating the tolerance induction. This tolerance was alloantigen-specific, and rapidly inducible and long lasting. The induction of suppressor cell activity was demonstrated in tolerant mice. However, this activity was associated only with the tolerant state around 4 to 7 days after the i.v. presensitization, but was no longer detected in mice more than 14 days after the presensitization, although these mice exhibited complete tolerant state. When spleen cells from such tolerant mice were transferred i.v. into 600 R x-irradiated syngeneic recipient mice alone or together with normal syngeneic spleen cells, these tolerant spleen cells themselves failed to induce DTH responses but did not exhibit suppressive effect on the generation of DTH responses induced by normal spleen cells co-transferred. These results indicate that i.v. administration of neuraminidase-treated allogeneic cells results in the induction of alloantigen-specific tolerance which is not always associated with the induction of suppressor cell activity but rather with the elimination or functional impairment of alloantigen-specific clones.     

The induction of tolerance to DNFB contact sensitivity by using hapten-modified lymphoid cells. III. Effects of hapten concentration on the ability of MLS-disparate cells to induce rapid unresponsiveness

We investigated genetic restrictions in the induction of immediate tolerance to DNFB contact sensitivity in mice. Using spleen cells from various donor strains haptenated at 500 micro M DNFB, we were unable to detect any restrictions in tolerance induction in recipients that were either syngeneic or allogeneic to the donor strain. However, if the concentration of hapten used in the in vitro labeling was decreased (from 500 micro M to 2.5 to 5 micro M DNFB), differences in tolerogenesis between the various donor strain haplotypes were found. Haptenated spleen cells labeled with 5 micro M DNFB produced a profound level of unresponsiveness in allogeneic recipients but produced minimal tolerance in syngeneic animals. This tolerant state was shown to be antigen-specific and was not produced by unmodified allogeneic cells alone. Further genetic analysis demonstrated that an efficient tolerant state was produced when the donor of the tolerogen and recipient differed at the MLS locus rather than at either the MHC or minor regions. This phenomenon required viable, Thy 1-bearing cells in the haptenated donor population for efficient tolerogenesis to DNFB contact sensitivity.     

Tolerance induced by TNP-derivatized syngeneic erythrocytes: evidence for cooperation between hapten-specific T and hapten-specific B lymphocytes in the immune response.

Tolerance to the DNP haptenic determinant was induced with a single i.v. injection of trinitrophenylated syngeneic red blood cells. The tolerant state lasted 1 month and was stable on transfer to irradiated thymectomized syngeneic recipients. Suppressor activity was found soon after injection of tolerogen but was lost before the termination of tolerance. The unresponsive state could be reversed by adding normal thymus cells to tolerant spleen cells but not by normal bone marrow cells. LPS when given with immunogen restored the normal immune response in tolerant mice. Thus the injection of TNP-MRBC induced partial immune unresponsiveness which was characterized by the induction of T cell suppressor activity and by a hapten-specific helper T cells tolerance. Finally, these studies suggest a cooperative interaction between DNP-specific T lymphocytes and DNP-specific B lymphocytes in the immune response to DNP-BGG.