Suppression of delayed-type hypersensitivity to syngeneic testicular cells in mice: involvement of suppressor T cells which act at the induction stage

Suppressor cells for delayed footpad reaction (DFR) against syngeneic testicular cells (TC) were detected in the spleen cells of donor mice immunized intravenously (iv) with viable syngeneic TC. Cyclophosphamide (CY)-pretreated recipients were given spleen cells from donors iv, immunized subcutaneously (sc) with syngeneic TC, and the footpad reaction at 24 hr was elicited with syngeneic TC 6 days after immunization. DFR in the recipients was suppressed by the transfer of spleen suppressor cells. The suppressor cells induced were Thy-1+, CY-sensitive, adult thymectomy (ATx)-resistant and act only at the induction stage. They directly suppress the generation of effector T cells for delayed-type hypersensitivity (DTH). When mice pretreated with CY were actively immunized with syngeneic TC, DFR could be provoked to a measurable level only when they were immunized sc. However, peritoneal exudate cells of those tolerant mice immunized sc without CY pretreatment or immunized iv with CY pretreatment also passively transferred DFR locally, suggesting the existence of effector T cells for DTH even in tolerant mice.     

T-T cell interaction in the induction of delayed-type hypersensitivity (DTH) responses: vaccinia virus-reactive helper T cell activity involved in enhanced in vivo induction of DTH responses and its application to augmentation of tumor-specific DTH responses

The role of antigen-specific helper T cells in augmenting the in vivo development of delayed-type hypersensitivity (DTH) responses was investigated. C3H/HeN mice were inoculated i.p. with vaccinia virus to generate virus-reactive helper T cell activity. These vaccinia virus-primed or unprimed mice were subsequently immunized subcutaneously (s.c.) with either trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self), vaccinia virus-infected spleen cells (virus-self), or cells modified with TNP subsequent to virus infection (virus-self-TNP). Seven days later, these mice were tested for anti-TNP DTH responses either by challenging them directly with TNP-self into footpads or by utilizing a local adoptive transfer system. The results demonstrated that vaccinia virus-primed mice failed to generate significant anti-TNP DTH responses when s.c. immunization was provided by either virus-self or TNP-self alone. In contrast, vaccinia virus-primed mice, but not unprimed mice, could generate augmented anti-TNP DTH responses when immunized with virus-self-TNP. Anti-vaccinia virus-reactive helper activity was successfully transferred into 600 R x-irradiated unprimed syngeneic mice by injecting i.v. spleen cells from virus-primed mice. These helper T cells were found to be antigen specific and were mediated by Thy-1+, Lyt-1+2- cells. DTH effector cells enhanced by helper T cells were also antigen specific and were of the Thy-1+, Lyt-1+2- phenotype. Furthermore, vaccinia virus-reactive helper T cell activity could be applied to augment the induction of tumor-specific DTH responses by immunization with vaccinia virus-infected syngeneic X5563 tumor cells. T-T cell interaction between Lyt-1+ helper T cells and Lyt-1+ DTH effector T cells is discussed in the light of the augmenting mechanism of in vivo anti-tumor-specific immune responses.     

Influence of 2'-deoxyguanosine upon the development of DTH effector T cells and suppressor T cells in vivo

Subcutaneous (s.c.) immunization of mice with allogeneic spleen cells can induce delayed-type hypersensitivity (DTH) to both major and minor histocompatibility antigens. Intravenous immunization with allogeneic spleen cells, however, induces a poor state of DTH. Furthermore, i.v. immunization with allogeneic spleen cells, especially if they have been irradiated, induces suppressor T lymphocytes. These suppressor T cells are capable of suppressing the host-vs-graft (HvG) DTH reactivity that normally arises after s.c. immunization. Moreover, they can suppress the development of anti-host DTH effector T cells during graft-vs-host (GvH) reactions. These models for HvG and GvH DTH reactivity were used to study the influence of 2'-deoxyguanosine (dGuo) and guanosine (Guo) on the generation of DTH-reactive T cells and suppressor T cells in vivo. It was found that daily i.p. administration of 0.01 mg dGuo to mice immunized i.v. partially prevented the generation of suppressor T cell activity, whereas daily administration of 0.1 or 1 mg dGuo resulted in a complete abolition. Administration of dGuo has no effect on the anti-host DTH reactivity by spleen cells from nonsuppressed donors except for when a daily dose of 10 mg is administered. This dose proved to be toxic for precursors of DTH effector T cells. Daily i.p. injection of Guo had no effect on the generation of suppressor T cells nor on the generation of DTH effector T cells. The effect of dGuo was found to be due to a direct effect on suppressor T cells and not to the induction of contrasuppressor cells. These data suggest a differential sensitivity of DTH-reactive T cells and suppressor T cells for dGuo. Because suppressor T cells and DTH-reactive T cells require proliferation for expressing maximal functional activity in the systems used, both cell types probably have different enzyme activities involved in the purine metabolism and similar deoxycytidine kinase activities, but have different nucleotidase (5'NT) activities, those in suppressor T cells being the lowest. If so, suppressor T cells will accumulate deoxyguanosine triphosphate, which causes an inhibition of the ribonucleotide reductase activity and thus of the DNA synthesis by these cells.     

Tumor bearer T cells suppress Bacillus Calmette-Guérin-potentiated antitumor responses. III. Identification of an auxiliary efferent suppressor-T-cell population

T cells (Ts-eff) induced in BALB/c mice by subcutaneous (sc) growth of syngeneic Meth A tumors can adoptively suppress the effector phase of delayed-type hypersensitivity (DTH) in Bacillus Calmette-Guérin (BCG)-primed and unprimed recipients which have been sensitized with irradiated Meth A cells but they do not inhibit the augmented DTH response in recipients inoculated with cyclophosphamide (CY) 2 days prior to sensitization. By reconstituting CY-treated immunized recipients with selected spleen cell populations, it has been demonstrated that Ts-eff suppress DTH by interacting with a second or auxiliary suppressor cell population present in immune but not normal spleens. These auxiliary suppressor cells (Ts-aux) are Thy+, Lyt 1-2+ and I-J+, phenotypically similar to Ts-eff. Their activity is not influenced by B-cell depletion. Unlike Ts-eff, Ts-aux do not bear receptors specific for Meth A cells. Ts-aux and Ts-eff share similar sensitivity to irradiation and high dose (100 mg/kg) CY but unlike Ts-eff, Ts-aux are cortisone sensitive, nondividing, nonadherent cells which are absent from the thymus. The phenotype and mechanism of action of Ts-aux resemble those of the auxiliary or Ts3 cells defined in models of contact sensitivity, DTH to simple haptens, and in vitro antibody responses.     

Regulation of the intensity of delayed-type hypersensitivity to sheep erythrocytes by the K-region of the major histocompatibility complex in mice

The injection of 6 x 10(9) sheep red blood cells (SRBC) to mice suppressed the delayed type hypersensitivity (DTH) in situ and activated spleen T cells which prevent sensitization of syngeneic recipients. Similar effect was obtained when suppressor cells induced in F1 hybrids were transferred to parental mice. Suppression was also reached in allogeneic strain combination if suppressor cells of donors and recipients shared the major histocompatibility complex (MHC). Studied performed with recombinant and mutant strains revealed that the prerequisite for interaction of DTH suppressors and effectors was the identity of K-region of MHC. Passive transfer of DTH to SRBC was also possible if donors and recipients were identical in K-region of MHC. It is believed that interaction between DTH suppressors and effectors is restricted by a region of MHC whose product takes part in antigen representation.     

Activation of T cells by glutaraldehyde-fixed erythrocyte antigens: radiosensitivity of cells mediating delayed-type hypersensitivity reactions in the mouse.

Mice immunized with glutaraldehyde-fixed sheep red blood cells (G-SRBC) show delayed-type hypersensitivity (DTH) reactions to G-SRBC or SRBC. The specificity of the DTH reaction of mice sensitized with glutaraldehyde-fixed antigens is similar to that found after sensitization with unfixed antigens. The dose-response curve for sensitization by glutaraldehyde-fixed SRBC was very different from the curve for normal SRBC. At low doses, both antigens were effective in sensitizing to show DTH but neither induced an antibody response. However, at high antigen doses, only the glutaraldehyde-fixed antigen was efficient in sensitizing to show DTH and it failed to raise an antibody titer. Spleen cells of mice sensitized with fixed RBC can transfer DTH locally but if the donor cells are irradiated (500 R), the transfer is abrogated. In contrast, the transfer of DTH by spleen cells of mice immunized with unfixed antigen is not affected by 500 R. The transfer of DTH by spleen cells of mice immunized with fixed antigen can be blocked by “in vitro desensitization” while the transfer of DTH by spleen cells from mice primed with normal antigen is resistant to “in vitro desensitization.” These results suggest that immunization of mice with different physical states of the same antigen can result in the activation of antigen-specific T cells which exhibit markedly different properties.     

Induction of suppressor cells by a tumor-derived suppressor factor

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.     

Difference in thymus dependency between effector and suppressor T cells for delayed footpad reaction to sheep erythrocytes in mice

Effects of thymectomy at various times after birth on effector and suppressor T cells for a delayed footpad reaction were determined in 6-week-old mice immunized intraperitoneally (ip) with sheep erythrocytes (SRBC). Mice thymectomized 1 day after birth (Tx-1 mice) gave delayed footpad reactions weaker than those of mice thymectomized 7 days after birth (Tx-7 mice) or sham operated (SH mice) after immunization with a low dose of SRBC. After immunization with a high dose of SRBC, on the other hand, Tx-1 mice showed reactions stronger than those of Tx-7 or SH mice. Pretreatment with cyclophosphamide (CY) augmented the delayed footpad reaction in Tx-7 or SH mice, but not in Tx-1 mice, immunized with a high dose of SRBC. The presence of T cells suppressive for the delayed footpad reaction in the spleen of Tx-7 or SH mice was confirmed by cell transfer experiments. These results suggest that effector T cells responsible for a delayed footpad reaction to SRBC are less thymus dependent and require the presence of the thymus for a shorter period in their development compared to suppressor T cells.     

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.     

Suppression and contrasuppression in athymic nude mice: nude mice produce the antigen-specific component of a T suppressor factor that inhibits the late 24-hr phase of DTH but do not generate suppression nor contrasuppression of the early initiating phase of DTH.

Previous studies demonstrated that the initiation of murine delayed-type hypersensitivity (DTH), as exemplified by contact sensitivity induced by picryl chloride (PCI) or oxazolone (OX), is due to antigen-specific, T cell-derived, DTH-initiating factors called, respectively, PCl-F and OX-F. These factors participate in the extravascular recruitment of CD4+, Th-1, DTH effector T cells in the elicitation of DTH. Related factors also participate, together with nonantigen binding factors derived from CD8+ T cells, to constitute an antigen-specific T cell-derived suppressor factor (TsF) that can down regulate the ability of Th-1 effector T cells to mediate DTH. Since it was shown recently that athymic nude mice can make antigen-specific, DTH-initiating T cell factors, the current study tested whether nude mice also could produce the antigen-specific component of the TsF that suppresses DTH effector T cells. We found that antigen-specific factors from nu/nu mice could complement the nonantigen-binding subfactor produced in normal mice to constitute the whole antigen-specific TsF. Additional studies showed that the successful adoptive cell transfer of DTH-initiating T cell activity from nude mice into normal mice required cyclophosphamide treatment of the recipient. In contrast, transfer of DTH-initiating cell activity from nu/+ mice did not require cyclophosphamide treatment of the recipients. We hypothesized that nude mice lacked contrasuppressor cells. Although nude mice were able to manifest the early, initiating phase of DTH, we found that there was no suppression of early DTH-initiating T cells in nude mice, compared to nu/+. Therefore the production of DTH-initiating T cell factor could be boosted in nude mice. The ability to boost DTH-initiating cells in nude mice should facilitate the development of cell lines and clones with the ability to initiate DTH.     

The inhibitory effect of T-2 toxin on tolerance induction of delayed-type hypersensitivity in mice.

Mice injected intravenously with 1 X 10(9) sheep red blood cells (SRBC) showed no delayed-type hypersensitivity (DTH) response to SRBC and were unresponsive to DTH induction by sc injection of an optimal dose of SRBC. However, when treated with T-2 toxin, a mycotoxin, 2 days after the iv injection, mice became to show significant DTH response and to be responsive to the DTH induction by the sc injection. When the spleen cells of the mice receiving the iv injection were transferred to unsensitized syngeneic recipients, the DTH response of the recipients to SRBC was suppressed. However, the suppressor activity of the spleen cells was decreased by T-2 toxin treatment. By the iv injection, cell population of the spleen was increased and that of the thymus decreased. In contrast, by T-2 toxin treatment 2 days after the iv injection, cell population of the spleen was not increased and that of the thymus was markedly decreased. The ratio of theta-bearing cells was increased in the spleen by the iv injection. However, such increase was not observed after the T-2 toxin treatment. The ratio of Ig-bearing cells in the spleen was not changed by the iv injection and the T-2 toxin treatment after the iv injection. T-2 toxin seems to interfere with generation of suppressor cells for the DTH response.     

Prophylactic immunization against experimental leishmaniasis. V. Mechanism of the anti-protective blocking effect induced by subcutaneous immunization against Leishmania major infection

The responsiveness of BALB/c mice to protective i.v. immunization with 150,000-rad irradiated or heat-killed Leishmania major promastigotes can be totally suppressed by prior subcutaneous (s.c.) injection of the same "vaccine." Induction of this effect is leishmania specific for although prevention of protection against L. major infection can be obtained with either homologous or Leishmania donovani promastigotes, it does not follow s.c. administration of an immunogenic Trypanosoma cruzi epimastigote preparation. Multiple s.c. injections of irradiated L. major promastigotes do not inhibit the subsequent antibody response of any major isotype to i.v. immunization, but rather induce some priming. The same s.c. injections induced delayed-type hypersensitivity (DTH) reactivity that could be transferred locally or systemically, although it was weaker than in mice with cured infections. Parallel cell-mediated immunity (CMI) responses were also reflected in vitro in specific lymphocyte transformation assays. Despite this evidence of a DTH/helper type of T cell response, transfer of 5 X 10(7) viable T cell-enriched spleen cells from 4 X s.c. immunized donors to normal recipients completely abrogated the protective response to i.v. immunization. Conversely, T cell-depleted (anti-Thy-1.2 + C treated) cells were without effect. The inhibitory T cells were defined by monoclonal antibody pretreatment as possessing an Lyt-1+2-,L3T4+ phenotype. T cells from s.c. immunized donors were also shown, by mixed transfer experiments, to counteract completely the protective effect of T cells from i.v. immunized donors in 550-rad irradiated recipients. They were as potent as suppressor T cells from donors with progressive disease both in this capacity and in abrogating the prophylactic effect of sublethal irradiation itself. The similarities and differences between suppressor and immune effector T cells induced by s.c. or i.v. immunization and those arising in response to leishmanial infection itself are discussed.     

Cellular cooperation in the expression of murine delayed-type hypersensitivity (DTH). 1. A normal accessory cell population enhances DTH produced by immune peritoneal exudate T lymphocytes

A murine system for local passive transfer of delayed-type hypersensitivity (DTH) has recently been described. It was determined that untreated and T-lymphocyte-enriched (nylon-wool-nonadherent) fractions of peritoneal exudate (PE) cells from immunized donors could be transferred with soluble antigen to normal recipient footpads to efficiently produce a local DTH response. Untreated spleen or lymph node (LN) cell populations were strikingly less capable in this regard. It is now reported that addition of normal untreated PE cell populations to immune T-enriched PE cells markedly enhanced the DTH response transferred by the latter. Specific swelling was dose dependent with respect to each cell type. Removal of T lymphocytes from the normal PE cell population did not affect its enhancement of DTH. By cotransfer of 1 X 10(7) normal PE cells, significant specific swelling was obtained using 1-3 X 10(5) T-enriched immune PE cells. This represented a three- to seven-fold reduction in the requirement for the latter cell type. This scheme of DTH enhancement was employed to evaluate the mechanisms for decreased capability of immune LN and spleen for DTH transfer when compared to PE. No evidence was found that either adherent or nonadherent suppressor cells are operative at the time of DTH expression. Cotransfer of a DTH-enhancing population failed to equalize DTH expression by LN and spleen with that of PE. It is concluded that DTH effector-T-cell activity is enriched in immune peritoneal exudate and that non-T-cell population(s) from that source actively enhance DTH expression.     

Prophylactic immunization against experimental leishmaniasis. III. Protection against fatal Leishmania tropica infection induced by irradiated promastigotes involves Lyt-1+2- T cells that do not mediate cutaneous DTH

Protective immunity against fatal L. tropica infection in genetically vulnerable BALB/c mice can be induced by prophylactic immunization with irradiated promastigotes even when heat-killed. Such immunity is adoptively transferable transiently into intact or durably into sub-lethally irradiated (200 or 550 rad) syngeneic recipients by splenic T but not B cells. The effector T cells are of the Lyt-1+2- phenotype, devoid of demonstrable cytotoxic activity. The immune splenic T cell population expresses specific helper activity for antibody synthesis. A causal role for helper T cells in this capacity, however, seems unlikely, because it was shown in the accompanying paper that antibody does not determine the protective immunity against L. tropica. The immunized donors show no detectable cutaneous DTH or its early memory recall in response to live or killed promastigotes or a soluble L. tropica antigen preparation. Spleen, lymph node, and peritoneal exudate cells from protectively immunized donors similarly fail to transfer DTH locally or systemically. These cells also lack demonstrable suppressive activity against the expression or induction of DTH to L. tropica. Thus, protection against L. tropica induced by prophylactic i.v. immunization with irradiated promastigotes appears to be conferred by Lyt-1+2- T cells that are distinguishable from T cells mediating either both DTH and T help, or cytotoxicity.     

Effects of pertussis toxin on delayed-type hypersensitivity responses and on the activity of suppressor T cells on the responses

Experiments were performed on mice to investigate the effects of pertussis toxin (PT) on delayed-type hypersensitivity (DTH) to ovalbumin (OA) and on the activity of suppressor T cells on the DTH (DTH-Ts). Mice immunized with alum-precipitated ovalbumin showed a transient DTH, which was determined as footpad swelling which disappeared 2 weeks after immunization. Maximal footpad swelling was observed 24 hr after DTH elicitation. On the other hand, when mice received PT (2 micrograms/mouse) at the time of immunization, the transient DTH became an enhanced and persistent DTH, which persisted for at least 4 weeks. In addition, the time of maximum footpad swelling was delayed from 24 to 48 hr after DTH elicitation. The immune spleen T cells from PT-treated mice showed a persistently high ability to transfer DTH into syngenic naive mice. DTH-Ts was induced in spleens of mice injected iv with OA-coupled syngeneic spleen cells. However, when these mice received PT at the time of suppressor induction, their spleen cells revealed considerably reduced suppressor activity. The activity of DTH-Ts was also reduced when DTH-Ts were either treated in vitro with PT or transferred into PT-injected recipient mice. From these results, interference with the suppressor function of DTH-Ts from PT was considered to be, at least in part, as an enhancing mechanism of DTH.     

Direct in vitro evidence for different susceptibilities to 4-hydroperoxycyclophosphamide of antigen-primed T cells regulating humoral and cell-mediated immune responses to sheep erythrocytes: a possible explanation for the inverse action of cyclophosphamide on humoral and cell-mediated immune responses

Suppressor T cells of humoral immune responses, effector T cells mediating DTH, suppressor T cells of DTH, and helper T cells of humoral immune responses, all with specificity to SRBC, were produced in mice. The biologic activity was tested in adoptive transfer experiments. In vitro treatment with different doses of 4-hydroperoxycyclophosphamide (4-HPCy) yielded the result that the various activities tested were not uniformly sensitive to the action of this drug: Suppressor T cells of humoral immune responses and effector T cells mediating DTH were resistant to doses of 4-HPCy that eliminated the activities of suppressor T cells of DTH and helper cells of the humoral immune response. These findings help to explain the various effects cyclophosphamide has on the in vivo immune response and may help to form a basis for the rational manipulation of the immune response by drugs that selectively affect different subgroups of immune cells.     

Regulatory mechanism of delayed-type hypersensitivity in mice. I. Properties of memory cpells and suppressor cells for delayed-type hypersensitivity against ovalbumin.

Delayed-type hypersensitivity (DTH) response in mice induced by sc injection of alum-absorbed ovalbumin (OA) was accelerated and enhanced by priming sc with a low dose of urea-denatured ovalbumin (UD-OA), 2 or more days earlier, whereas it was suppressed by priming sc with a high dose of UD-OA, 0 or more days earlier. The ability in primed mice to accelerate or suppress the DTH response could be transferred antigen specifically into cyclophosphamide (CY)-pretreated recipients or normal recipients by spleen cells from primed mice, but not by the T-cell-depleted spleen cells. Furthermore, the ability of spleen cells to transfer the acceleration or the suppression appeared transiently around 7 or 4 days after priming, although the acceleration or the suppression in donor mice persisted for a much longer time. Pretreatment with CY abolished the suppression of DTH response in high dose-primed mice and resulted in the acceleration of DTH response. These results suggest that the activity of DTH-related memory T cells which accelerate and enhance the response can be inhibited by suppressor T cells for the DTH response.     

Different time course patterns of local expression of delayed-type hypersensitivity to sheep red blood cells in mice.

The delayed-type hypersensitivity (DTH) reaction, a peripheral expression of cell-mediated immunity is still a crucial in vivo immunological test. Nevertheless, the biological significance of its time course remains unclear. Thus, an exhaustive study of DTH was undertaken in mice immunized with increasing doses of sheep red blood cells (SRBC) inoculated intravenously (iv) or subcutaneously. The results showed that overall DTH reactions peaked at 18 hr except in mice iv immunized with the lowest doses (10(5) and 10(6)) and elicited at Day 4. The protracted DTH reaction was shown to be associated with an histological picture of tuberculin-type reaction. A part of the 18-hr DTH reaction is mediated by serum in mice inoculated with large doses of SRBC; nevertheless, numeration by limiting dilution analysis of circulating DTH cells showed that the frequency of these cells correlates with the 18-hr DTH level. The protracted DTH shown at 42 and 48 hr, 4 days after immunization with 10(5) and 10(6) SRBC, could not be transferred in naive recipients with immune spleen cells; it was independent of the antigen life span and did not result from immunization modulation at the bone marrow level on recruitable cells.     

Requirement for a bacterial flora before mice generate cells capable of mediating the delayed hypersensitivity reaction to sheep red blood cells.

A delayed-type hypersensitivity (DTH) reaction can be elicited by an injection of 10(8) sheep red blood cells (SRBC) into a rear footpad of conventional (CV) mice previously immunized with small doses of SRBC. In contrast, immunization of germ-free (GF) mice with the same doses of SRBC produced no DTH when immunization was by the intravenous (i.v.) route, and only weak reactions when immunization was by the subcutaneous (footpad) route. Varying the immunizing dose of SRBC, or the time at which DTH was elicited, did not produce a state of DTH responsiveness in i.v. immunized GF mice. However, the transfer of lymphocytes from CV mice, immunized 4 to 5 days previously with SRBC, into GF mice, conferred on GF mice the capacity to express DTH. Although DTH was not readily demonstrable in GF mice immunized with SRBC, they nevertheless produced normal levels of hemagglutinating antibody to SRBC. Finally, it was shown that GF mice could generate a normal DTH response to SRBC if they were first monoassociated with a Gram-negative bacterial flora.     

Antigen-specific augmentation factor involved in murine delayed-type footpad reaction. IV. Effect of delayed-type hypersensitivity augmentation factor on in vitro induction of DTH

We found an antigen-specific factor capable of augmenting delayed-type hypersensitivity (DTH) in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR), or in the culture supernatant of the mixture of sensitized T cells and specific antigens. This factor (DTH augmentation factor; DAF) was confirmed to augment DTH in transferred recipients. In this paper, such an activity of DAF was further investigated using the system with in vitro induction and local transfer of DTH. DAF also augmented the primary in vitro induction of DTH, when spleen cells from mice transferred with the DAF-containing serum 12 hr previously or spleen cells incubated with the DAF-containing serum on ice for 2 hr were cultured with heterologous erythrocytes. DAF acted on the induction phase of DTH and augmented a typical DTH which was dependent on Thy-1-positive T cells. DAF showed antigen specificity, but was not assigned to conventional immunoglobulin. The activity of DAF was detected when nylon-wool nonadherent cells were incubated with DAF prior to the culture of those cells and antigens, but not detected when only nylon-wool adherent cells were incubated with DAF. Thus, DAF exerted its effect through binding to acceptor cells which were included in nylon-wool nonadherent spleen cells from normal mice.