Studies on the proliferation of thymus cells injected into syngeneic or alogeneic irradiated mice

Thymus cells from normal isologous or allogeneic animals were injected intravenously into lethally irradiated mice. With the aid of a particle size analyser it was observed that blast cells appeared in the spleens of mice injected with allogeneic thymus cells. By means of specific alloantisera against the infused cells or host, it was established that these cells were derived from the injected cells. Evidence is presented indicating that the blast transformed cells were derived from a specific population of cortisone resistant thymic cells.     

"Self-reactive" T cells. III. In vitro restimulation of T cells, "responding" in vivo or in vitro to syngeneic lymphoid cells

Polyclonally activated lymphoblasts, transferred to syngeneic recipient mice, elicited a host T-cell-mediated "response" in vivo. These T cells, which "responded" in vivo to syngeneic lymphoblasts (i.e. in vivo primed "responder" T-cell population), acquired the capacity to "stimulate" a "response" of syngeneic T cells in vitro in a syngeneic one-way mixed lymphocyte culture, S-MLC (i.e. in vitro primed "'responder" T-cell population). We now describe the presence of memory and specificity in these two types of "self-reactive"responder" T-cell populations. This is investigated in in vitro "restimulation" experiments with mitomycin-blocked syngeneic and allogeneic lymphoid cells of various origin. "Self-reactive" T cells could be restimulated repeatedly (over many weeks) with mitomycin-blocked syngeneic lymphoid-cell populations, but not with mitomycin-blocked allogeneic normal spleen cells. "Self-reactive" T cells "responded" to syngeneic large activated lymphoblasts, as well as to syngeneic small resting lymphocytes. We found no "responder" T-cell reactivity specific for the mitogen that induced syngeneic "stimulator" cell populations. Both populations of "self-reactive" T cells displayed reactivity to mitogen-induced allogeneic lymphoblasts.     

Cytotoxicity of temporarily adherent human mononuclear blood cells in monocyte monolayers

Adherent monolayers of freshly isolated human peripheral blood mononuclear cells consist mostly (greater than 90%) of monocytes. The initially adherent nonmonocytic cells detach during the first day of in vitro culture. Concomitant with cell detachment the cytotoxicity mediated by adherent monolayers is reduced. This is in part due to medium exchange with removal of initially adherent cells from the monolayers, cells that are potent mediators of cytostasis and cytolysis. The temporarily adherent cells consist of 10% monocytes, 35% T lymphocytes, 10% B lymphocytes, and 45% non-T, non-B lymphocytes. In addition, 45% of these cells stain positively with the monoclonal antibody OKT 10. The reduction in monolayer-mediated cytoxicity from day 0 to day 1 of in vitro culture is less pronounced if the temporarily adherent cells are kept within the culture during the cytotoxic assay time. When the temporarily adherent cells are returned to adherent monocyte monolayers an additive effect on cytolysis is demonstrated with nonactivated, lymphokine-activated and alpha-interferon-activated effector cells, and in antibody-dependent cell-mediated cytotoxicity. The heterogenous population of temporarily adherent cells in freshly isolated monolayers of human mononuclear blood cells seems to be responsible for some of the cytotoxic effects regularly ascribed to freshly isolated human blood monocytes.     

The self-sustaining capacity of lymphoid cells in the irradiated thymus

The self-sustaining capacity of thymus lymphoid cells was studied in mice exposed to sublethal doses of X-rays. During exposure various parts of the organism were protected by lead shielding. When the thymus was protected and the rest of the organism exposed to radiation an initial cellular depletion of the thymus not exceeding 70 per cent was followed by a complete cellular regeneration of the organ within 3 weeks. This regeneration was shown to proceed without any cellular support from the bone marrow. When the thymus was included in the radiation field and/or damaged by cortisone treatment resulting in a depopulation exceeding 70 per cent, no or only partial regeneration occurred. The results indicate that the cell population in the thymus of mice can be self-sustaining for at least 3 weeks.     

The role of thymus on autosensitization against syngeneic normal and malignant cells.

Mouse lymphocytes were exposed to syngeneic fibroblasts and tumor cells in Millipore chambers inserted into the peritoneal cavity of intact and thymectomized mice. Autosensitization to fibroblasts occurred only if the chambers were carried by thymectomized mice. Sensitization to tumor-specific antigens also took place in intact mice. If thymic extract was administered to thymectomized mice autosensitization in the chambers was inhibited.     

Reconstitution of a thymus cell-deprived immune system by syngeneic and allogeneic thymocytes in vitro

Mouse spleen cell suspensions deprived of their T cells by treatment with anti-Θ-serum and complement exhibit a drastically reduced immune response to sheep red blood cells in vitro. Syngeneic thymocytes generally fail to reconstitute the system. A full response can, however, be obtained by the addition of thymocytes from syngeneic mice pretreated with hydrocortisone, or by the addition of allogeneic thymocytes.     

MHC restriction of murine T lymphocyte reactivity analysed by growth of bone marrow cells in vitro on thymus epithelial monolayers.

Mature mouse T lymphocytes, derived from long-term culture of bone marrow cells on thymus epithelial monolayers, were analysed with respect to their ability to co-operate with B cells (for antibody production) or T cells (in the generation of cytotoxic cells) when the bone marrow T precursor cells and the thymus epithelial cells differ at defined regions of the major histocompatibility complex. A pool of more mature bone marrow T-cell precursors gave rise to T cells interacting only with T/B lymphocytes sharing MHC determinants with the strain of origin of the bone marrow cells used. In contrast, a more immature bone marrow T-cell precursor pool produced T lymphocytes which had acquired MHC restriction (in terms of co-operativity with T/B cells) as defined by the MHC determinants of the thymus epithelium, and not those MHC determinants of the cultured bone marrow population. In addition, some evidence was obtained for Ir gene control (mapping in the MHC region) in the development of the repertoire of T cells involved in production of cytotoxic responses in vitro to TNP-modified self antigens.     

In vitro activation of murine Kupffer cells by lymphokines or endotoxins to lyse syngeneic tumor cells.

Murine Kupffer cells (RC) were isolated in sufficient number and purity to allow in vitro investigations of their tumoricidal capabilities. The identity of the adherent cells as KCs was established by morphologic, histochemical, and functional criteria. The yield of KCs varied from young (high) to old (low) mice but was not affected by the mouse strain. KCs activated in vitro by either endotoxins (lipopolysaccharide) or lymphokines were rendered highly cytotoxic against syngeneic melanoma or fibrosarcoma target cells. These studies indicate that KCs may indeed play a role in destruction of tumor cells in vivo and thus be important in host defense against developing hepatic cancer metastases.     

Immunoglobulin-secreting cells in various maternal lymphoid tissues during syngeneic and allogeneic murine pregnancy

The effects of syngeneic (CBA×CBA) and allogeneic ($\text{CBA}\text{×}\text{C}\text{57}\text{B}\text{1}$) pregnancy on immunoglobulin (Ig)-secreting cells in various maternal organs/tissues have been investigated by using the protein A plaque assay. The following organs/tissues were examined: $\text{a}$) spleen, $\text{b}$) cervical nodes, $\text{c}$) inguinal nodes, $\text{d}$) mesenteric nodes, $\text{e}$) paraaortic (uterus-draining) nodes, $\text{f}$) Peyer's patches, and $\text{g}$) bone marrow (femur). The changes observed were similar and of the same magnitude in all pregnant animals, irrespective of the type of mating. At mid-gestation (day 14) a distinct increase in Ig secretors was observed, predominantly in the spleen. At the end of pregnancy (day 20) the para-aortic nodes contained dramatically increased numbers of plaque forming cells. A slight increase in both IgM and IgG-secreting cells was also seen in bone marrow at the very end of pregnancy, while Peyer's patches and the nodes of neck and legs appeared to be unaffected throughout the period of gestation.     

Asymmetric division of immature lymphoid cells of the embryonic mouse thymus

Cell division of thymus lymphoid cells from 15- to 19-day-old embryonic mice, as well as from 2- and 7-day-old mice was investigated on cell smears stained with Giemsa or with the Feulgen reaction followed with malachite green. Asymmetrically dividing cells were observed during the development. At the anaphase and telophase of such cells, the nucleus of one of two immature daughter cells was apparently smaller and more condensed than that of the counterpart cell. The asymmetry was already apparent at the level of sister chromosomes at anaphase. Sometimes a difference also existed in the cytoplasm: the cytoplasm of the smaller immature daughter cell with condensed nucleus was stained with Giemsa more deeply than that of the counterpart. The frequency of cells displaying apparent asymmetric division among total dividing cells at the telophase was around 10% between 15 and 19 days of gestation, and decreased to 2% on days 2 and 7 after birth. Asymmetrically dividing cells belonged to immature lymphoid cells of larger size. The biological implications of the present phenomenon are discussed.     

Age dependent reaction of mouse thymus cells with autologous and syngeneic erythrocytes.

The age dependent events influencing the rosette forming capacity of Balb/c thymus and spleen cells against autologous or syngeneic erythrocytes were examined. A large number of autologous and syngeneic rosette forming cells (RFC) were observed in normal Balb/c mice in vitro. RFC were significantly greater in the thymus than in the spleen. The rosette forming T-cells (T-RFC) have the following characteristics: newborn Balb/c thymus has T-cells which react syngeneic erythrocytes from older donors. The T-RFC showed broad cross-reactivity with erythrocytes from other mouse strains but low reactivity with human or sheep erythrocytes. The auto and syngeneic T-RFC could be enhanced by non-specific serum proteins (FCS or BSA) or EDTA but was effectively inhibited by normal mouse serum. T-RFC resided in the cortisone sensitive population. The data indicate that the development of autologous and syngeneic rosette formation of thymus cells is dependent on the age of the erythrocyte donor. The age dependent change on the erythrocyte surface occurred relatively early in the life of the animal. The results also imply that certain subpopulations of thymus lymphocytes are capable of recognizing possible surface antigenic changes of the erythrocytes.     

Restitution of the thymus in lethally irradiated mice after transplantation of syngeneic or allogeneic bone marrow

The early restitution of the thymus of bone marrow chimeras was investigated by the immunoperoxidase technique using monoclonal antibodies against Thy-1 and Lyt-1, Lyt-2, Lyt-3. Within two weeks, normal thymus histology was restored in mice which received untreated syngeneic BM or syngeneic or allogeneic BM pretreated with SAL (specificed antilymphocytic serum). Irradiation depleted the thymic cortex of small Thy-1+, Lyt-1+2+3+ cells but did not affect a medullary population of medium sized weakly stained Thy-1+, strongly stained Lyt-1+ cells. Preceded by the appearance of an increasing number of large Thy-1+, Lyt-1- blasts (days 2 and 4), the thymic cortex was repopulated (beginning on day 6) by smaller Thy-1+ cells which acquired Lyt-1, Lyt-2 and Lyt-3 though, obviously not in a strictly sequential manner. Simultaneously, the medullary radioresistant cells disappeared, nd the medulla was subsequently repopulated (beginning on day 8) by thymocytes of a mature phenotype. Early restitution of the thymus in radiation control mice was similar to the bone marrow chimeras. The results indicate that the histological restitution of the thymus originates substantially from radioresistant precursors of host origin. Graft-versus-host reaction induced by untreated allogeneic bone marrow cells prevented normal thymic restitution. A delayed localized cortical repopulation with small Thy-1+, Lyt-1+2+3+ cells, progressive destruction of thymic architecture and almost no restoration of the medullary immunocompetent thymocytes were noted. T cell differentiation obviously was seriously affected by the injuries to the thymic microenvironment due to alloreactive T cells.     

Induction of "allogeneic effect"-like reaction by syngeneic TNP-modified lymphoid cells

TNP-substituted SRBC-immune spleen cells, when injected into cyclophosphamide-treated recipients, are recognized by T lymphocytes and produce, in the presence of specific antigen (SRBC), significantly more PFC than nonsubstituted cells. Labeling of immune B cells is more important in producing the augmented responses than is the labeling of immune T cells. TNP determinant has to be bound directly to the transferred immune cells to produce enhanced antibody responses, as when recipients were injected with non-substituted immune cells and TNP-substituted non-immune cells simultaneously, no increase in PFC number was noted (lack of a bystander effect). When recipients were rendered tolerant to TNP, two separate effects were observed, dependent on the mode of inducing unresponsiveness. In mice which were treated with TNP over an extended period of time, lack of recognition of TNP was demonstrated, such that TNP-substituted cells failed, when transferred, to produce an augmented response. When a short-term tolerogenic regime was used, the adoptively transferred TNP-labeled cells gave a very poor response (greater than 95% inhibition) due to in vivo suppression and/or killing. These results, together with the lack of influence of tolerance induced to unrelated hapten (DNP or OX), confirm the antigen specificity of the phenomenon. The reaction observed by us shows a striking resemblance with, but not identical to, the "allogeneic effect" produced by MHC encoded alloantigens. Our results extend the list of analogous immune reactions induced by MHC encoded alloantigens and TNP-derivatized self.     

Pattern of distribution of syngeneic spleen cells in the lymphoid organs of neonatally thymectomized mice

Newborn mice were thymectomized and intraperitoneally inoculated with syngeneic spleen cells on days 0, 3, 7 and 20 after birth. Donor cells were chromosomally distinguishable from host cells (T6). Cytological analysis performed between days 2 and 118 post-natal revealed greatly increased proportions of donor cells in host lymph nodes if comparison were made with similarly injected non-thymectomized hosts. The effect is highly specific, since the proportions of donor cells in the host spleen and bone marrow remained similar to those in control, non-thymectomized mice (syngeneic standard). The mechanism behind this phenomenon is unclear.     

Experimental myasthenia in mice. The role of the thymus and lymphoid cells

A possible function of the thymus gland with regard to developing neuromuscular block after immunization with thymus extract is discussed.

Inbred female C57BL/6 mice were immunized with thymus extract from female C57BL/6 (syngeneic), male C57BL/6 mice, AKR mice (allogeneic) and guinea-pigs and calves (xenogeneic). Two weeks later, the waning phenomenon was observed from the electromyography (EMG) in xenogeneically, allogeneically and syngeneically immunized mice. The waning reverted to a normal pattern following an intraperitoneal injection of neostigmine methylsulphate. EMG tracings revealed a decrease in the incidence of the waning phenomenon in those mice which were immunized after adult thymectomy.

The passive transfer of the waning phenomenon to non-thymectomized recipients could be achieved by the intravenous administration of lymph node cells obtained from the mice that had developed a myasthenic neuromuscular block after immunization. This finding was also observed in the recipients when the donors of lymph node cells had not developed the block.

On the other hand, administration of sera from mice with the waning phenomenon after immunization resulted in a neuromuscular block in both normal and adult-thymectomized mice. However, sera obtained from mice without waning did not have this feature.