Interaction of the lymph node and splenic lymphocytes of mice in inactivation of allogeneic hematopoietic stem cells

A study was made of interaction of mouse spleen and lymph node lymphocytes in inactivation of allogeneic stem cells. It was established that T lymphocytes of the lymph nodes and spleen lymphocytes do not interact on combined administration; their action is of additive nature. B lymphocytes of the lymph nodes have a regulating activity both in respect to T lymphocytes of the lymph nodes and lymphocytes of the spleen. The stem cells serve as target. Depending on the stem cells/B lymphocytes ratio B lymphocytes are capable of exerting either helper or suppressor action.     

Cryopreservation of hemopoietic tissue aimed at reducing immunological reactivity

A method has been developed of decreasing immunologic activity of lymphocytes from spleen, lymph nodes, and bone marrow (freshly collected or frozen) with a high percentage of intact stem cells. In experiments in vivo on lethally irradiated mice, it was demonstrated that during combined transplantation to the recipients of preserved bone marrow from two genetically different donors, a rapid decrease or absence of the effect of inactivation of hemopoietic stem cells under the influence of allogeneic lymphocytes was observed in the mixed graft. When it is necessary to transplant large quantities of bone marrow from several genetically different donors, the use of cryopreserved bone marrow is preferable to freshly drawn marrow due to the higher proliferative activity and the decreased risk in the development of immunological reactions.     

New Ideas on Amphibian Immunity: The Lymph Gland: A Generator of both T and B Cells

Anuran amphibian larvae of the genus Rana possess three majorlymphoid organs: the thymus, the lymph gland, and the spleen.Other accumulations of lymphocytes are present in the kidney,liver, and intestine. The thymus develops lymphocytes firstfollowed by peripheral differentiation of other lymphoid centers.Since bone marrow is absent in larvae, there must be an alternativesource of stem cells that differentiate into T cells under thymicinfluence or B cells under the influence of some other organ(s).We are concerned with the source of B cells after bullfrog larvaebecome immunologically mature. Such an organ should possessstem cells, and, to qualify further, it should play a majorrole in antibody synthesis. The lymph gland seems to fulfillthis requirement. Contrary to our expectations, lymph glandscan restore not only antibody synthesis but also transplantationimmunity in a significant number of larvae. Thus, the lymphgland may house stem cells that can differentiate into B cellsand T cells.     

Two subpopulations of stem cells for T cell lineage

An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells, the generation of donor-derived T cells being observed in two out of 14 recipients transferred with as few as 1.5 X 10(4) cells. The stem cell activity of spleen cells was estimated to be about 1% of that of bone marrow cells, and no activity was found in thymus cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. Spleen cells showed a markedly high level of activity 7 days after the reconstitution, followed by a decline, whereas the activity of bone marrow cells was very low on day 7 and increased crosswise. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells. Such patterns of compartmentalization of stem cells in the spleen and bone marrow of irradiated recipients completely conform to the general scheme of the relationship between restricted stem cells and less mature stem cells, including pluripotent stem cells, which became evident in other systems such as in the differentiation of spleen colony-forming cells or of stem cells for B cell lineage.     

Inactivation of the colony forming cells in bone marrow by allogenic T-lymphocytes]

Bone marrow cells together with allogeneic cortisone-resistant thymocytes or the lymph node cells of intact mice treated with antilymphocytic T or O-antisera were transplanted to the lethally irradiated mice. The antisera eliminated the capacity of T lymphocytes to inactivate allogeneic stem cells. Cortisone-resistant thymocytes displayed a strong inactivating action. The principal role in the processes of inactivation of genetically foreign stem cells was played by T lymphocytes. B lymphocytes possessed no properties of killer cells. The presence of B lymphocytes in the population of killer cells apparently failed to serve as a determinant one for the development of inactivation processes.     

PHA activation of encephalitogenic T cells: in vitro line selection overcomes splenic suppression

In this study we compared myelin basic protein (MBP) and phytohemagglutinin (PHA) for their ability to induce proliferation and experimental autoimmune encephalomyelitis (EAE) transfer activity in mixed cell cultures obtained from spleen and lymph nodes versus highly selected MBP-specific T cell lines and clones. Established MBP-specific cells derived initially from immune lymph nodes attained both proliferative and EAE-transfer activities after in vitro activation with either MBP or PHA. In contrast, PHA was unable to induce immune spleen cells to transfer EAE, in spite of its potent mitogenic activity. On the basis of these results, we evaluated the in vitro proliferation and differentiation responses of MBP-specific T cells during the line selection process using cells derived from both immune lymph node and immune spleen. During the initial selection process with MBP, proliferation of MBP-specific T cell precursors from immunized spleen populations was reduced relative to lymph node cells. After antigen-dependent selection the encephalitogenic cells from either organ exhibited identical in vitro response characteristics. Freshly isolated immune spleen cells were potent suppressors of MBP-specific T cell proliferation suggesting that the in vitro differences between the two organs was due to splenic suppression of the encephalitogenic cells.     

Reutilization by maternal and embryonal cells of nuclear materials from H3-thymidine labeled lymphocytes introduced into the lumen of intestine of rats

Summary H³-thymidine labeled lymphocytes from thymus and lymph nodes of donor rats were washed and injected in to the intestine of recipient rats on the 11th and 19th day of gestation; subsequent labeling of maternal and embryonal cells was studied autoradiographically 24 hours after injection. In 12-day embryos, numerous stem cells or hemocytoblasts were labeled frequently intensely. In 20-day embryos, stem cells or hemocytoblasts scattered throughout the liver were often labeled. In other fetal tissues at this stage, cells in thymus, spleen, mesenteric lymph node and intestine were labeled but scarcely and weakly. In mothers, labeling in lymphoid tissues was scarce but definite, in thymus, mesenteric lymph node and spleen. These results suggest that nuclear materials from lymphocytes emigrated into the intestinal canal of the mother could be reutilized by maternal and embryonal cells.     

A splenic requirement for the generation of suppressor T cells.

Tolerance to contact sensitization with DNFB may be induced by DNBSO3. This specific unresponsiveness may occur via one or both of two mechanisms--production of suppressor T cells or clone inhibition. We investigated the role of the spleen in this unresponsiveness. Splenectomized mice may be tolerized by i.v. injection of DNBSO3, but they are incapable of serving as donors of lymph node cells for transfer of tolerance to normal recipients. Kinetic studies indicated that the spleen must be present at least three days after tolerization in order to permit development of a significant number of suppressor cells in the peripheral lymph nodes. We interpret these results to indicate that 1) clone inhibition does not require the spleen, 2) the generation of suppressor T cells is dependent on the presence of the spleen, and 3) it is likely that tolerogens in this system induce suppressor cells in the spleen and some of these cells or their products leave the spleen to reach the peripheral lymph nodes.     

MHC-linked Ir gene control of T cell responses: GAT-specific proliferating T cells and helper T cells are elicited in nonresponder mice immunized with soluble GAT

The immune response to the synthetic terpolymer GAT is controlled by MHC-linked Ir gene(s). We show in this paper that antigen-presenting cells and T cells from mice belonging to two nonresponder strains (SJL and DBA/1) can present and recognize GAT, respectively. This has been measured with a T cell proliferation assay of GAT-primed lymph node cells. In order to detect T cell proliferation among GAT-primed lymph node cells from DBA/1 mice, it is necessary to treat the cells with monoclonal anti-Lyt-2 antibodies and complement (C) before the assay. These conclusions were further verified with SJL mice, when a T cell line derived from LN cells was used. We have shown that after immunization with GAT, specific T helper cells can be generated in the lymph nodes of SJL mice but not in the lymph nodes of DBA/1 mice. Furthermore, GAT-specific T helper cells can be detected in the spleen of SJL mice after immunizations with GAT, provided these spleen cells are pretreated with monoclonal anti-Lyt-2 antibodies + C or mild irradiation. Together, these results support the general idea that nonresponsiveness can be explained by a regulatory imbalance rather than by discrete cellular "defects."     

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

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

Temporary characteristics of the process of stem cell inactivation by allogenic lymphocytes]

The time during which transplated lymphocytes block proliferation and differentiation of non-syngeic stem cells has been determined by retrasplantation of immuno-competent cells from one lethally irradiated recipient to another one. It was established that process of inactivation of CFU by allogeneic lymphocytes proceeds itwo stages. At the first stage, the colonization of recipient's tissues takes place. The colonization of tissues and processes of early recognition are completed during the first hours after transplantation of cell mixtures. At the second stage, the processes of redistribution of injected cells occur and a complete inactivation of stem cells take place. These events are completed in bone marrow and spleen 4-5 days after transplantation of cells mixture, possibly with the participation of lymphocytes sensibilized with the target-cells.     

Rat T lymphocyte-specific antigens and their cross-reactivity with mouse T cells.

Anti-rat T lymphocyte serum (ATLS)2 was prepared by immunizing rabbits with purified T cells from rat mesenteric nodes and absorbed with rat red cells and syngeneic sarcoma cells. The specificity of ATLS for rat T cells was confirmed by the following reasons: a) ATLS was not toxic for bone marrow cells but lysed most of the thymocytes and a number of spleen and lymph node cells, which were inversely correlated to the percentage of cells with B cell characteristics in respective organs; b) anatomical localization of ATLS-reactive cells in lymphoid organs coincided to the thymus-dependent areas, i.e. the paracortex of lymph node and the periarteriolar region of spleen; c) spleen cells treated with ATLS and complement failed to respond to phytohemagglutinin but normally responded to bacterial lipopolysaccharide; d) those cells treated with ATLS and complement could not induce a graft-vs-host reaction in F1 hosts, whereas the same treatment did not affect direct plaque-forming cells. All of these data confirm the specificity of ATLS and indicate that ATLS recognizes rat T lymphocyte-specific antigens (RTLA). Absorption studies showed that RTLA were present in higher concentration on medullary thymocytes and peripheral T cells than on cortical thymocytes, but absent from bone marrow, liver, and brain tissues. When the cross-reactivity of RTLA with mouse T cells was studied by C-dependent cytotoxicity and immunofluorescence, it was found that mouse T cells shared at least one determinant of RTLA with rat T cells, and that distribution pattern of the cross-reacting antigens in mouse lymphoid tissues was essentially the same as that of RTLA in rat lymphoid organs.     

Role of B-lymphocytes in inactivating allogenic stem cells]

Antisera to membrane antigens of B lumphocytes eliminated the capacity of lymphocytes to inactivate allogenous stem cells by 60%; however, lymphocytes from the lymph nodes of B mice possessed no inactivating capacity. T-lymphocytes were the main criteria inactivating allogenous stem cells. Cooperating with T-lymphocytes, B-lymphocytes probably contributed to inactivation of precursor cells realized by T-lymphocytes. However, the presence of B-lymphocytes in the killer cells population was not a determinant, since T-lymphocytes were capable of inactivating allogenous stem cells without any participation of B-lymphocytes.     

T and B lymphocyte migration into syngeneic tumors.

The migration of splenic T and B lymphocytes into syngeneic tumors undergoing immunologic rejection was investigates. Spleen cells were obtained from normal BALC/c mice or BALB/c mice bearing tumors induced by murine sarcoma virus (MSV). Either whole spleen cells or immunoabsorbent purified T and B cells were radiolabeled with sodium chromate-51 and injected i.v. into normal or MSV inducted-tumor bearing syngeneic recipients. Twenty-four hours later the recipient mice were sacrificed and radioactivity was assessed for tumor, contralateral normal muscle, the lymph nodes draining the tumor and contralateral draining lymph nodes, peripheral lymph nodes, spleen, and liver. Both T and B lymphocytes from either normal or MSV tumor-bearing animals show greatly increased migration into the tumor when compared with normal muscle. Migration of T cells from both normal and MSV tumor bearers was 30 times that of migration to normal muscle. B cells from tumor-bearing mice, on the other hand, localized in the tumor itself only 50% as frequently as did B cells from normal animals. In addition, T cells from MSV tumor bearers were found in the highest proportion in the lymph node draining the tumor site. We conclude that T and B lymphocytes from either normal or tumor-bearing mice migrate to a syngeneic tumor undergoing immunologic rejection. In contrast, the migration of both T and B cells from tumor-bearing animals was decreased to the peripheral lymph nodes at the time of maximum tumor growth.     

Transfer of immunity to Babesia microti of human origin using T lymphocytes in mice

Lymph node and spleen cells from mice infected with Babesia microti of human origin developed the ability to transfer adoptive immunity to naive mice within 25 days after infection. This protective activity was greater in cells obtained at 32 days than in cells obtained at 25 days postinfection and remained stable up to 52 days postinfection. Recipients of lymph node cells and spleen cells displayed similar peak parasitemias although 2 days after peak parasitemia, immune spleen cell recipients had significantly lower parasitemias than immune lymph node cell recipients. Strong protective activity was demonstrated when cells were transferred 1 day postinfection, while equal numbers of cells, transferred 3 days postinfection did not confer significant protection over nonimmune cells. There was also a suggestion that the number of immune spleen cells necessary for significant protection was directly related to the number of parasites inoculated. The subpopulation of lymphocytes responsible for the transfer of adoptive immunity to B. microti of human origin was then studied in BALB/c mice depleted of T lymphocytes by thymectomy and lethal irradiation. One day after infection with B. microti, T-cell-depleted mice were given complement-treated immune spleen cells, anti-θ serum-treated immune spleen cells, nonimmune spleen cells, or no cells. Similar experiments were performed comparing the effects of anti-immunoglobulin serum-treated and unfractionated immune spleen cells on B. microti parasitemia. Treatment with anti-θ serum abrogated the protective activity of immune spleen cells while anti-immunoglobulin serum treatment had no effect. These results suggest that immunologic memory of B. microti in BALB/c mice is modulated by T rather than B lymphocytes.     

Interaction of allogeneic lymphocytes and precursor cells during the primary and secondary immune response]

A mixture of lymph node cells from CBA mice and spleen cells from C57Bl/6J mice stimulated by the cheep erythrocytes fro the first or second time was transplanted in the lethally irradiated mice (CBA X C57Bl/6j)Fl. The interaction of allogenic cells during the secondary immune response was accompanied by the complete inactivation of antibody producents. Under the ratio of interacting cell elements 1 : 1-1 : 2, 93-96% of precursor cells and 98% of antibody forming cells were inactivated. Under the ratio 1 : 5, the index of inactivation of precursor cells fell down to 35%. During the primary response, under the ratio 1 : 1, only 20-48% of precursor cells and 68% of antibody forming cells were inactivated. Under the ratio 1 : 2, no inactivation of precursor cells was observed and, under the ratio 1 : 10, the antibody formation was stimulated. Following the delayed by 1-3 days transplantation of CBA lymphocytes, the cooperative effect was registered with respect to the spleen cells from C57Bl/6J mice stimulated by the erythrocytes for the first time. The interaction of allogenic cells resulted in the 3-4-fold increase in the number of antibody forming cells.     

B-Lymphocyte differentiation in lethally irradiated and reconstituted mice. II. Recovery of humoral immune responsiveness.

The recovery of humoral immune responsiveness was studied in lethally irradiated, fetal liver-reconstituted mice. By means of both membrane fluorescence and antibody formation to sheep red blood cells (SRBC) as a functional assay, the rate of recovery of the compartments of B and T lymphocytes was determined in various lymphoid organs. The recovery of the immunoglobulin-positive (B) cell compartment after irradiation and reconstitution started in the spleen. This organ was also found to be the first in which the recovery of the B-cell population was completed. The interval between the recovery of the B-cell population in the spleen and that in the other organs tested was found to increase when the irradiated mice were reconstituted with spleen colony cells instead of fetal liver cells. This proved to be caused by the number and nature of the reconstituting hemopoietic stem cells. The immunoglobulin-positive (B) cells were found to appear before SRBC-reactive B cells could be demonstrated in spleen, lymph nodes, and Peyer's patches. The appearance of T lymphocytes in the various lymphoid organs required even more time. By means of cell transfer experiments, a sequential appearance of the precursors of anti-SRBC IgM-, IgG-, and IgA-plaque-forming cells could be demonstrated in spleen, bone marrow, lymph nodes, and Peyer's patches.     

20alpha-hydroxysteroid dehydrogenase: a T lymphocyte-associated enzyme.

20alpha-Hydroxysteroid dehydrogenase (20alpha-SDH), an enzyme which reduces progesterone to 20alpha-dihydroprogesterone, was found to be associated with T lymphocytes. 20alphaSDH activity was present in spleen cells bearing theta antigen, spleen cells nonadherent to nylon wool (T lymphocyte-enriched population), and in thymocytes. Almost no enzymatic activity was found in bone marrow cells from normal mice and in spleen cells from neonatally thymectomized or athymic nude mice. T cell mitogens (PHA and Con A), but not the B cell mitogen LPS, induced high levels of enzymatic activity 48 hr after addition to spleen cell cultures. The level of 20alphaSDH activity in lymphocytes was age dependent. At the age of 4 weeks 20alphaSDH activity in thymocytes, spleen cells, and lymph node lymphocytes was 3 to 5 times higher than at 8 and 16 weeks. Progesterone (5.0 X 10(-7) M) was found to inhibit thymocyte proliferation after exposure to mitogens, but not 20alpha-dihydroprogesterone (10(-6) M). 20alpha SDH may protect the embryonic thymocytes against high concentrations of progesterone.     

T cell differentiation in lethally irradiated and reconstituted mice: stem cell influx demonstrated with immunoperoxidase technique.

Using both an anti-stem cell serum and an anti-T cell serum the influx of stem cells in mouse thymus and spleen after lethal irradiation and reconstitution was determined by immunoperoxidase staining. In both organs a rapid influx was observed reaching a maximum on Day 5 after irradiation and cell transfer. Thereafter a decline of stem cells took place while the number of T cells in the thymus increased gradually, reaching a maximum on Day 12. T cells could only be detected in the spleen after 3 weeks.     

Immunologic responses to a murine mammary adenocarcinoma: in vitro production of specific killer cells is dependent on active T lymphocytes.

The mode of production of specifically armed monocytic killer cells was investigated with the T1699 mammary adenocarcinoma in syngeneic DBA/2 mice. After overnight in vitro incubation of cells from the spleen but not from the lymph nodes, blood, or from the peritoneal cavity produced specific killer cells. The activation of spleen cells was inhibited by pretreatment with anti-theta serum and C; however, already activated specific killer cells were not sensitive to the same treatment. Removal of phagocytic cells did not significantly affect the cytotoxicity of the splenic killer cells whereas removal of rayon-wool adherent cells greatly reduced both the total cytotoxicity, and to a lesser extent, the cytotoxicity indices. Overnight co-cultivation of normal peritoneal-exudate cells with the lymph node cells from tumor-bearers, although neither class of cells alone was cytotoxic to T1699 cells in vitro, produced specific monocytic killer cells, through steps dependent on active T lymphocyte function. Culture spupernatants of tumor-bearer's spleen cells also contained factor(s) which induced cytotoxicity mediated by normal peritoneal-exudate cells against T1699 cells in vitro; and the production of the factor(s) was also inhibited by pretreatment of the spleen cells with anti-theta serum but not by anti-mouse IgG or anti-mouse whole gamma-globulins serum and C.