An analysis of B cell memory. I. Interaction of helper and suppressor effects in the in vitro expression of IgM memory.

The pfc response of Srbc primed IgM memory cells has been characterized by limiting dilution analysis in vitro, in which LPS was used to maximize the response of spleen cells to Srbc. The analysis suggested that, even under these conditions, expression of B cell memory was not directly assayed and cell collaboration effects were still basic to the system.Two types of cells, as defined by function, appeared necessary to elicit optimal clonal proliferation of IgM B memory cells: firstly, helper T cells were essential for B cell proliferation even with LPS present in culture. Under appropriate conditions, helper activity could be provided by normal thymus cells. Secondly, activated T cells were required for the maximal conversion of normal thymus cells to helper cells. A third activity, T cell-dependent suppression, was observed at high cell doses. The implications of these results and the need for a comprehensive analysis of in vitro conditions for each individual type of experiment is discussed.     

Ontogeny of cell-mediated cytotoxicity: induction of CTL in early postnatal thymocytes.

In this study we characterized the time when cytotoxic T lymphocytes (CTL) can be induced in the thymus and spleen from their immediate CTL precursors (CTL-P). In contrast to fetal or newborn thymus, the thymus of 1 to 2-day-old C57BL/6 mice contained cells that, after cultivation in vitro with allogeneic DBA/2 stimulating cells, exhibited high levels (as great or greater than that induced in adult thymocytes) of CTL activity as measured by the ability to lyse P815 (DBA/2) tumor target cells. However, CTL activity induced in spleen cells remained how during the first 5 days of life, increased sharply between 6 to 9 days, and reached adult levels at 11 to 20 days. Furthermore, early postnatal spleen cells did not suppress the adult splenic CTL response. These results suggest 1) that the full potential to generate CTL in response to an allogeneic stimulus commences in the thymus on the first day after birth and 2) a different temporal appearance of immediate CTL precursors in the thymus and spleen.     

In Vitro Reconstitution of Anti-Sheep Erythrocyte Antibody Response of T Cell-Depleted Spleen Cells by Allogeneic T Cells or by Factors Derived from Them

Restoration of the impaired antibody response to sheep erythrocytes (SRBC) in cultures of mouse spleen cells, which were deprived of thymus-derived lymphocytes (T cells) by treatment with anti-mouse brain-associated θ (BAθ) antiserum and complement, was studied by adding a small portion of syngeneic or allogeneic normal spleen cells in vitro. Allogeneic spleen cells had a far greater effect than syngeneic spleen cells on the restoration, as far as the normal spleen cells added were able to recognize the alloantigens on the anti-BAθ serum-treated spleen cells (bone marrow-derived lymphocytes). Treatment of the allogeneic spleen cells with mitomycin C did not affect their activity in the restoration of the impaired antibody response. The possibility that the role of T cells in the antibody response to SRBC may be replaced by a nonspecific mediator derived from T cells reacting with allogeneic cells was proven by the finding that supernatant of the mixed allogeneic spleen cell cultures restored the impaired anti-SRBC antibody response of the T cell-depleted spleen cells. The effect of such culture supernatant on the restoration of the antibody response was greatest when it was added to the T cell-depleted spleen cell cultures one day after cultivation with SRBC, suggesting that the effectiveness may result from triggering of the proliferation and differentiation of antibody-forming cell precursors, which have already reacted with the antigen, to antibody-forming cells.     

T mitogens trigger LPS responsiveness in mouse thymus cells.

Mouse thymus cells are essentially unresponsive to lipopolysaccharide (LPS) stimulation. However, when cultured with minimally mitogenic levels of concanavalin A or submitogenic ratios of mitomycin-treated allogeneic spleen cells, in combination with LPS, they demonstrate levels of DNA synthesis de novo or greater than those induced by the T cell mitogen alone. Dose-response kinetics were characteristic of LPS. The subpopulation containing the LPS responsive cells was of low net buoyant density. Neither phytohemagglutinin nor pokeweed mitogen acted synergistically with LPS in this model to trigger thymus cells. The data suggest that LPS triggering may involve interaction with a T cell subpopulation.     

LPS-induced autoantibody response. I. Ontogenic development of PFC response to bromelain-treated syngeneic erythrocytes

Normal adult mice have been shown to contain a large number of cells secreting antibodies against bromelain-treated syngeneic erythrocytes (Br.MRBC) and the numbers remarkably increase by the stimulation with LPS. In this report development of the anti-Br.MRBC response during ontogeny was examined and it was shown that on the injection of LPS suckling mice responded little to generate splenic plaque-forming cells (PFC) against Br.MRBC in vivo and in vitro. The responsiveness of suckling mice to produce anti-Br.MRBC was shown to be less developed than the anti-TNP response or the mitotic response to LPS. The low responsiveness of suckling mice was analyzed in terms of suppressor activity in the spleen cell population, proliferative capacities of the precursors of anti-Br.MRBC PFC, and their frequencies in the spleen. In the coculture experiment of suckling and adult spleen cells or culture of anti-brain-associated Thy 1-treated, macrophage-depleted spleen cell population, no evidence was obtained to show that suckling spleen cells contained suppressor cells. Kinetic profiles studied in vitro showed that anti-Br.MRBC PFC in the suckling spleen did not increase during the culture as those in the adult spleen. Studies on the precursor frequencies revealed that spleen cells of 15-day-old mice contained precursors of anti-Br.MRBC PFC amounting to 20.5% of the adult precursors whereas the PFC response in vitro by the former was only 4% of the latter. From these experimental data, it was concluded that the low responsiveness of suckling mice was partly due to the low frequency of the precursors in the spleen and, in addition, to the defective nature of the precursors in proliferating to differentiate into PFC.     

Immunorestoration of old mice by injection of thymus extract: Enhancement of T cell-T cell cooperation in the in vitro antibody response

The effect of injecting a thymus extract (TP-1 or Thy-5) into immunodeficient old mice on the in vitro antibody response of their spleen cells was investigated by techniques suitable for dissecting out T- and B-cell reactivities. The anti-TNP antibody response of HRBC-primed spleen cells from old mice uninjected or injected with TP-1 or Thy-5 was elicited in vitro by TNP-HRBC or TNP-Ficoll. Treatment with TP-1 or Thy-5 was found to induce only a slight increase in the anti-TNP antibody response to both immunogens. The helper activity of HRBC-primed spleen cells from untreated or treated old mice was titrated by adding graded numbers of these primed cells to cultures containing a constant number of normal spleen cells from young mice and the immunogen TNP-HRBC. Under these conditions it was found that both thymus extracts are very effective in restoring T cell-T cell cooperation in the generation of helper cell activity.     

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.     

Graft-versus-host induced immunosuppression: depressed T cell helper function in vitro.

The cause of graft-versus-host (GVH) induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) was investigated by in vitro restoration experiments employing a double compartment culture vessel. The two culture compartments were separated by a cell impermeable membrane. Restoring cells were placed in one chamber and responding GVH spleen cells plus SRBC were placed in the other chamber. It was demonstrated that thymus, lymph node, and spleen cells restored the PFC response whereas bone marrow cells did not. Treatment of the restoring cells with anti-theta serum plus complement abrogated restoration. Supernatants obtained from antigen free cell cultures restored nearly as well as whole cell suspensions. The degree of restoration was not increased by allogeneic or xenogeneic antigenic stimulation of the restoring cells. Thymus and lymphoid cells obtained from animals experiencing a GVH reaction restored as well as normal cells, however spleen cells were unable to restore by day 5 post-GVH induction. The results suggest that GVH induced immunosuppression of the PFC response is due, at least in part, to a depressed T cell factor production by splenic T cells.     

The origin of cytotoxic T lymphocyte precursors (CTL-P): MHC-restricted and alloreactive CTL-P in the spleen during regeneration after a sublethal dose of cyclophosphamide (Cy)

The regenerating spleen 8 days after an injection of a sublethal dose (300 mg/kg) of cyclophosphamide (Cy) had a defective capacity to give rise to cytotoxic T lymphocytes (CTL) in response against allogeneic cells, whereas the cytotoxicity against 2,4,6-trinitrophenyl-(TNP) modified syngeneic cells was at the normal level. Alloresponse was first obtained 2 wk after the Cy treatment. The limiting dilution analysis showed this at the clonal level: the frequency of anti-TNP-specific CTL precursors (CTL-P) in the spleen treated with Cy 8 days previously was the same as the frequency in the normal spleen. The defective alloresponse was due to a decreased number of allospecific CTL-P that was later increasing. The regenerative capacity was not abolished by adult thymectomy or treatment of the mice with a bone marrow-seeking isotope, 89Sr, suggesting that these CTL-P are derived from Cy-resistant splenic precursors rather than from the thymus or bone marrow. These precursors have probably been under thymic education: the dominance of H-2k-restricted CTL over H-2d-restricted CTL in the response of (H-2k X H-2d)F1 mice to TNP-self is known to be influenced by the H-2 genotype of the thymus, and this dominance was also demonstrated with anti-TNP CTL derived from these F1 mice pretreated with Cy. The CTL-P in the regenerating spleen (day 8) were not hydrocortisone sensitive, and nylon wool-purified T cells from this stage had a lactate dehydrogenase (LDH) isoenzyme pattern of the mature T cell type (rather than of the thymocyte type). Thus, in these aspects the T cells of the regenerating spleen resembled normal splenic cells. These data suggest that the Cy-resistant spleen population contains cells that can give rise to CTL-P that have a defective specificity repertoire at the beginning of the regeneration, but later mature to a normally alloreactive population.     

Generation of cytotoxic lymphocytes to syngeneic tumor by using co-stimulator (Interleukin 2)

Cytotoxic lymphocytes (CL) highly active against the syngeneic mastocytoma, P815, were generated from spleen cells of DBA mice cultured with co-stimulator (Interleukin 2) and P815. More CL activity was generated from spleen cells of P815 tumor-bearing mice than from spleen cells of normal mice. Thymus cells from tumor-bearing mice, however, did not produce increased CL activity. Most of the CL were Thy 1 and Ly 1 positive. The optimal culture conditions and kinetics were similar to those for the generation of allogeneic cytotoxic T lymphocytes. The cytotoxic activity against syngeneic P815 was similar in magnitude to the response of DBA spleen cells to allogeneic tumor lines and to the response of allogeneic CBA spleen cells to P815. Although CL generated from tumor-bearing mice did not lyse normal DBA cells, they did lyse, to a much lesser degree, a number of tumor cell lines other than the sensitizing P815. This nonspecific lysis was not H2 restricted nor was it restricted to tumors of lymphoid origin. Generation of nonspecific cytolytic activity was antigen independent, occurring in the presence of co-stimulator alone.     

Biological characterization of T and B lymphocytes separated from normal mouse spleen by a physical adherence column method

The capacity of spleen cell populations enriched for T and B lymphocytes by a physical adherence column method to respond in vitro to phytomitogens and allogeneic lymphocytes was determined. Column filtrate cells (T lymphocytes) responded well to phytohaemagglutinin- and mitomycin-C-treated allogeneic spleen cells, but poorly to pokeweed mitogen. Adherent cell populations from the column (B and some T lymphocytes) responded well to pokeweed mitogen, but poorly to phytohaemagglutinin- and mitomycin-C-treated allogeneic cells.Purified peripheral T lymphocytes prepared from normal mouse spleen by the column method reconstituted the depleted in vitro antibody response to the thymic-dependent SRBC antigen of all B lymphocyte sources tested, namely, spleen cells from congenitally athymic mice, neonatally thymectomized mice, and adult thymectomized mice which had been reconstituted with bone marrow, and a lymphocyte population prepared by incubating spleen cells with anti-θ serum and complement. When transferred with sheep erythrocytes to congenitally athymic mice, purified peripheral T cells restored the in vivo IgM and IgG responses of these animals. These results confirm that the column filtrate is a thymus derived subpopulation of cells capable of cell-mediated immunity and cooperation with B lymphocytes in humoral immunity both in vitro and in vivo.     

Suppression of in vitro antibody responses by Listeria primed spleen cells.

Spleen cells from mice primed with virulent Listeria monocytogenes do not develop an anti-SRBC plaque forming cell response to SRBC in culture. Furthermore, when Listeria primed spleen cells are co-cultured with normal spleen cells and SRBC, the anti-SRBC response of the normal cells is suppressed. Listeria primed spleen cells from T cell depleted donors are equally effective at immunosuppression. The immunosuppressive effect does not appear to be due to the presence of the bacterium or its products per se in the cultures. Furthermore, the effect cannot be transferred across a 0.45 μm pore membrane. Kinetic studies show that the immunosuppressive effect develops by 2 days post-Listeria inoculation and peaks by Day 6. Low doses of Listeria are not immunosuppressive and produce some enhancement effect. From these results, it is suggested that a population of non-T cell dependent cells develop in Listeria primed hosts that nonspecifically suppress the response of B cells to an unrelated antigen in culture.     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

T-cell development in the absence of a thymus: the number, the phenotype, and the functional capacity of T lymphocytes in nude mice

A small but definite proportion of T-lymphocyte-like cells have been reported in nu/nu (nude) mouse spleen despite the congenital absence of a thymus in these animals. We have determined the number and the characteristics of such cells using flow cytometry. The level of T-like cells increased with age. In 4-month-old nu/nu CBA spleen, 14% of all cells expressed some Thy 1 antigen. However, only 4% expressed mature T-cell levels, and only the 2% with the highest Thy 1 also showed a normal distribution of Ly 1 and Ly 2 antigens. These T-like cells were slightly larger than normal nondividing T lymphocytes. We have assessed the total functional capacity of T-like cells in nu/nu CBA spleen using a high-cloning-efficiency limit-dilution culture system. Almost all precursor cells capable of forming clones when stimulated with concanavalin A in the presence of irradiated spleen cells and growth factors, and almost all precursors of those clones that were cytolytic in a lectin-mediated tumor-cell-lysis assay, were within this 2% subpopulation of nu/nu spleen cells with mature T-cell markers. Increased levels of purified interleukin 2 failed to induce further precursor function, indicating that maturation of pre-T cells was not obtained. However the nu/nu spleen cells bearing mature T-cell markers displayed only 10-30% of the cloning efficiency of normal splenic T cells. The majority of nu/nu spleen T-like cells, even within this phenotypically "normal" subset, appeared to be nonfunctional. We conclude that the absence of a thymus leads to qualitative, as well as quantitative, deficiencies in the T-cell population, and various interpretations are discussed.     

Antigen-Specific Proliferative Response of Peritoneal Exudate Lymphocytes Primed with Antigen and Bacterial Lipopolysaccharide: The Roles of Ia+ Accessory Cells and IL-2

In vitro antigen-specific proliferation was investigated in a lymphocyte population that had been taken from the peritoneal exudate cells (PEC) of C3H/HeN mice (Ia^k) primed in vivo with both bacterial lipopolysaccharide (LPS) and horse red blood cells (HRBC) and had been purified by passage through a nylon fiber column (Nfc). The proliferative response of the Nfc-passed lymphocytes primed with HRBC and LPS [T(HRBC + LPS) cells] depended on the dose of antigen in the cultures, and the response was higher than that of cells prepared from mice primed with HRBC alone [T(HRBC) cells]. No response was seen in the cells prepared from the LPS-primed mice [T(LPS) cells] or normal mice [T(N) cells]. The response of the T(HRBC) cells was abolished by previous treatment of the cells with anti-Ia^k antibody and complement (C), whereas the response of the T(HRBC + LPS) cells was retained after the same treatment, indicating that the Ia^– T(HRBC + LPS) cells can proliferate in response to antigen in spite of Ia+ accessory cell-depletion. Supernatants from the cultures of Ia^– T(HRBC + LPS) cells in the presence of HRBC showed abundant IL-2 activity, while those of Ia^– T(HRBC) cells did not. The IL-2 should be produced by the L3T4 cell population in T(HRBC + LPS) cells in response to antigen, since the previous treatment of the cells with anti-L3T4 antibody and C abrogated the production. On the other hand, the Ia^– T(HRBC + LPS) cells as well as the Ia^– T(LPS) cells could respond to IL-2 dose-dependently when recombinant IL-2 was added into the cultures, but the response of Ia^– T(HRBC) cells to IL-2 was very weak. The cell population responding to IL-2 in the T(HRBC + LPS) cells as well as T(LPS) cells must be AsGM1-positive or natural killer (NK) cells, since previous treatment of the cells with anti-AsGM1 antibody and C abrogates the response. Together these results suggest that L3T4 lymphocytes capable of producing IL-2 in response to HRBC antigen without Ia⁺ accessory cells are generated in the PEC of the mice after priming with LPS and antigen together, and the IL-2 produced by the L3T4 lymphocytes induces the proliferation of the LPS-primed AsGM1+ cells.     

Suppression of the in vitro secondary response to syngeneic tumor and of in vivo tumor therapy with immune cells by culture-induced suppressor cells.

Mice with advanced disseminated syngeneic tumor can be successfully treated with a combination of chemotherapy and adoptively transferred syngeneic immune cells. We have previously demonstrated that in vivo primed cells secondarily sensitized in vitro became more effective in tumor therapy, whereas primed cells cultured for 5 days without tumor stimulation became less effective than an equal number of uncultured fresh primed cells. Therefore, we examined stimulated and unstimulated cultures of tumor-primed cells for the presence of culture-induced suppressor cells, and determined whether in vivo tumor therapy with immune cells could be inhibited by concurrent inoculation of immune effector cells and cultured normal spleen cells, which contain culture-induced suppressor cells but are devoid of additional effector cells. The in vitro primary allogeneic response was suppressed by cultured normal spleen cells, or tumor-primed spleen cells previously cultured for 5 days with or without tumor stimulation. In vitro secondary sensitization to syngeneic tumor was suppressed by normal or tumor-primed cells that had previously been cultured for 5 days without stimulation. The majority of this suppression was mediated by T cells in the cultured populations. The efficacy of fresh tumor-primed cells, as well as primed cells secondarily sensitized in vitro, in adoptive chemoimmunotherapy of advanced tumor was diminished by concurrent inoculation of cultured normal cells. The cells mediating suppression of in vivo therapy required previous in vitro culture for induction, and were radiation sensitive.     

The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

In vitro immune response to the 2,4,6-trinitrophenyl determinant in aged C57BL/6J mice:changes in the humoral immune response to, avidity for the TNP determinant and responsiveness to LPS effect with aging.

An in vitro anti-TNP response of the spleen cells from aged C57BL/6J mice showed approximately 4-fold less PFC than did that from young adult mice. Anti-theta serum-treated young spleen cells gave an anti-TNP response that was definitely greater than the response of the anti-theta serum-treated aged spleen cells in the presence of the exogenous activated thymus cells as helper cells. These results suggest that the deficits in B cells may be partly responsible for the imparied anti-TNP response of the aged spleen cells. To examine further the capacity of stem cells in the bone marrow to generate B cells responsible for anti-TNP response in the spleen, we injected i.v. 1.5 to 2.0 times 10(7) bone marrow cells from young or aged mice into lethally irradiated syngeneic recipients that had previously been thymectomized. Four to 6 weeks later, 10(7) spleen cells from the two groups of these recipient mice were immunized with TNP-SRBC in the presence of the exogenous activated thymus cells and assayed for anti-TNP PFC. The response of the aged marrow-derived B cells was approximately one-half of that of the young marrow-derived B cells.The avidity for TNP determinant of the antibodies produced by the PFC was determined by the plaque-inhibition technique. The avidity of the antibodies produced by the aged mice was approximately 33 times lower than that by the young mice. Anti-TNP response of the young spleen cells were markedly enhanced by the addition of LPS to the cultures, whereas no or little enhancement of the response was induced in the aged spleen cells even in the presence of high concentration of LPS. In contrast, DNA synthesis of both the young and aged spleen cells was comparably stimulated by 1 mug/ml and 10 mug/ml of LPS, however, it was rather less in the aged spleen cells at a concentration of 100 mug/ml. Mechanisms responsible for the changes in avidity and responsiveness to LPS with aging are discussed.     

Cell-Cooperation in Anti-Sheep Red Blood Cell Antibody Response in Mouse Spleen Cell Cultures

Restoration of impaired antibody response to sheep red blood cells (SRBC) in spleen cell cultures from mice treated with heterologous antilymphocyte globulin (ALG) was studied by adding normal cells from various sources, to explore the problems of cell-cooperation in anti-SRBC antibody response and the target of ALG. When spleen cells from ALG-treated mice were separated into macrophage-rich and lymphoid cell-rich subpopulations, only the latter was found to be impaired in the ability for anti-SRBC antibody response. Addition of even a small number of normal allogeneic spleen cells sufficiently restored the impaired anti-SRBC antibody response of the spleen cells from ALG-treated mice. By use of allo-antisera, most hemolysin plaque-forming cells (PFC) generated in such cultures were proved to be derived from the cells of ALG-treated mice. Restoration was also achieved by adding thymus-derived cells, which were obtained from spleens of mice heavily irradiated and repopulated with syngeneic thymus cells, or lymphoid cells directly collected from thymuses. All results indicate that ALG selectively depletes the thymus-derived antigen reactive cells (ARC) in the spleen cell population, and that ARC supplied from normal spleen or thymus can interact with plaque-forming cell precursors (PFCP) that remain intact in the spleen cell population of ALG-treated mice. The results also suggest that a single ARC interacts with more than one PFCP and makes them develop into PFC.     

Antigen-specific protection against graft-vs-host induced immune deficiency

A severe, antigen-nonspecific, and long-lasting immune-deficient state can be induced in healthy, adult immune-competent F1 hybrid mice by a single i.v. injection of parental T lymphocytes. The present report demonstrates that this graft-vs-host-induced immune deficiency (GVHID) can be prevented in an antigen-specific way by immunization of the F1 mice with allogeneic cells before induction of GVHID. Thus, spleen cells from (A X B)F1 mice primed with allogeneic cells from strain C and then injected with parental spleen cells from A did not generate cytotoxic T lymphocyte responses to trinitophenyl-modified self cells or to allogeneic cells from third party strains D or E. However, spleen cells from the same mice generated normal levels of cytotoxic T lymphocyte activity to allogeneic cells from C, the strain used for immunization. Furthermore, mice exposed to murine cytomegalovirus before induction of GVHID were resistant to a subsequent challenge with murine cytomegalovirus, whereas GVHID mice that received only the murine cytomegalovirus challenge all died. These findings are discussed with respect to the possibilities that primed and unprimed T helper cells may be differentially susceptible to the suppressive effects of GVH.