John M. Mansfield

Genetics of resistance to the African trypanosomes: IV. Resistance of radiation chimeras to Trypanosoma rhodesiense infection

The cellular bases of resistance to the African trypanosomes were examined in inbred mice. As part of these studies, reciprocal bone marrow cell transplants were performed between H-2 compatible mice which differ in relative resistance to Trypanosoma brucei rhodesiense infection. Survival times, parasitemias and IgM antibody responses to the surface antigen of the infecting variant type were measured in these semiallogeneic bone marrow chimeras. Relatively resistant C57BL/10 mice, intermediate A.By mice, and least resistant C3H.SW mice that were reconstituted after lethal irradiation with syngeneic bone marrow cells displayed resistance and immunity characteristic of the homologous donor strain. When C57BL/10 mice were reconstituted with C3H.SW mouse bone marrow cells they retained the ability to produce antibodies to trypanosome surface antigen but the antibody titers were significantly reduced. Control of parasitemia and mean survival time were reduced in these chimeras, but differed significantly from C3H.SW mice. A.By mice that received cells from C57BL/10 donors exhibited antibody responses and survival times similar to the C57BL/10 mice. Survival times of A.By mice given syngeneic cells or C3H.SW cells were the same, but the antibody responses of A.By mice given C3H.SW cells were lower than those of A.By mice given syngeneic cells. C3H.SW mice reconstituted with C57BL/10 bone marrow cells were capable of making antibodies and controlling parasitemia, in marked contrast to the absence of such responses in C3H.SW mice reconstituted with syngeneic cells. Survival times, however, were indistinguishable from those of C3H.SW mice given syngeneic cells. Thus, resistance to T. b. rhodesiense was shown for the first time to depend on donor bone marrow derived cells as well as upon radiation-resistant cells/factors associated with host genetic background. Also, parasite-specific IgM antibody responses seem to be regulated by a mechanism which does not depend on bone marrow derived cells alone, and the presence of such immune responses is not linked to survival time.

Morphological changes in Trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo.

The effect of alpha-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3-4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.

Characteristics of the splenic suppressor cell-target cell interaction in experimental African trypanosomiasis.

Abstract We have examined further the relationship between immunosuppression and suppressor cell activity in experimental African trypanosomiasis. In the present study we describe the nature of the interaction between splenic suppressor macrophages from Trypanosoma rhodesiense -infected C57BL/6 mice and target effector cells in the primary in vitro PFC response to SRBC. Suppressor cell potential was expressed only when cell-cell contact of a noncytolytic nature was established between infected spleen cells and normal splenic responder cells. Isolation of suppressor cells from responder cells by a cell-impermeable membrane completely abrogated suppression. Similarly, supernatant fluids from infected spleen cell cultures could not passively transfer suppression. Suppressor cells did not act via prostaglandin synthesis in that indomethacin failed to restore responsiveness to infected spleen cells or to passively suppressed normal cultures. Inhibition of DNA synthesis by irradiation of mitomycin C treatment did not block suppressor cell function, but suppressor cell effects were inhibited by exposure of infected spleen cells to silica particles or to heat treatment. We conclude that suppressor cell effects in experimental African trypanosomiasis are consistent with a suppressor macrophage acting via a noncytolytic cell-cell interaction with responder target cells.

CHARACTERIZATION OF A RELATIVELY RARE CLASS B, TYPE 2 TRYPANOSOME VARIANT SURFACE GLYCOPROTEIN GENE*

The variant surface glycoprotein (VSG) gene of Trypanosoma brucei rhodesiense LouTat 1.5, a defined African trypanosome variant antigenic type, was cloned and sequenced. Southern blot analysis revealed 2 DNA restriction fragments in both VSG 1.5 expressor and nonexpressor populations, suggesting that there are 2 genomic copies of the VSG 1.5 gene and no expression-linked copies. Pulsed-field gel electrophoresis followed by Southern blot analysis showed that each copy of the VSG 1.5 gene exists on a separate large chromosome in both the expressor (approximately 3.5- and 4-megabase (Mb) chromosomes) and nonexpressor (approximately 4- and 5.7-Mb chromosomes) populations. Thus, VSG genes may be present on larger chromosomes than previously reported. Sequence analysis and alignments revealed that the VSG 1.5 molecule is a class B VSG with 12 cysteine residues in the N-terminus and is classified as a type 2 VSG based on C-terminus motifs. This classification shows that the VSG 1.5 molecule represents a relatively rare VSG class and type. Taken together, these studies provide additional information on VSG genes and proteins and supply the foundation for structure-function analysis of the VSG 1.5 surface antigen expressed by trypanosomes of the LouTat 1 serodeme.

Effects of Theophylline Treatment on Mouse B-16 Melanoma Cells in vitro

The effects of theophylline treatment on mouse B-16 melanoma cell growth, metabolism, and membrane antigen expression in vitro were studied. Theophylline treatment inhibited DNA synthesis and the cell growth rate, and caused an elevation of intracellular cAMP levels. Cells treated with theophylline became elongated and assumed a normal fibroblast-like morphology. Theophylline treatment of B-16 cells also reduced the levels of tumor specific antigen and H-2 antigen detectable on the cell membrane.

Incorporation of 3H-Thymidine into PHA-Stimulated Rabbit Peripheral Blood Lymphocytes. Kinetics of the Response

Summary Rabbit PBL were shown to incorporate predictably high levels of 3H-TdR following stimulation with PHA. The conditions necessary for optimal stimulation of rabbit PBL were: (a) separation of lymphocytes from whole blood by the Hypaque-Ficoll technique; (b) 2 × 106 PBL per culture in RPMI-1640 containing 5–10% heat-inactivated, autologous plasma; (c) 48 hr culture period; (d) 5 μg/ml PHA-P; and (e) 1 μCi/ml 3H-TdR added to cultures 22 hr before termination. Under these conditions, individual and group variations in the response to PHA were slight, and PBL responsiveness to other mitogens as well as to antigen were good.

Lymphocyte function in experimental African trypanosomiasis: VII. Loss of antigen-nonspecific suppressor-T-cell activity☆

The extent of immunosuppression occurring in mice infected with the pathogenic African trypanosomes was studied. Spleen cells from Trypanosoma rhodesiense-infected C57BL/6J mice were tested for antigen-nonspecific suppressor-T-cell (Ts) activity after concanavalin A (Con A) treatment in vitro. After exposure to Con A, control and infected mouse spleen cells were added to responder spleen cell cultures stimulated with sheep erythrocytes (SRBC). Assays for the resultant plaque-forming cell responses to SRBC revealed that antigen-nonspecific Ts activity was lost during the first week of infection. Changes in infected mouse T-cell subpopulations, including a terminal loss of Lyt 2.2+ cells, accompanied but did not precede the demonstrable loss of Ts function. Splenic suppressor macrophages which arise during infections with T. rhodesiense also did not seem to be associated with the loss of antigen-nonspecific Ts activity. It is concluded that the generalized immunosuppression associated with experimental African trypanosomiasis extends to the mitogen-induced Ts population.

Immunobiological properties of a concanavalin a derivative.

Abstract A monovalent subunit of concanavalin A (Con A) was tested for mitogenic effects on murine splenic lymphocytes in vitro . In contrast to the effects of intact Con A, the monovalent derivative was not mitogenic at any concentration tested. Furthermore, prior exposure of splenic lymphocytes to monovalent Con A rendered the cells refractory to stimulation by the unmodified lectin.

Lymphocyte function in experimental African trypanosomiasis: VIII. Loss of suppressor T cell function in lymph nodes☆

The immunosuppression that occurs in mice experimentally infected with African trypanosomiasis has been examined further. In the present study we have examined lymph node cells from Trypanosoma rhodesiense-infected C57Bl/6J mice for the ability to produce mitogen induced antigen-nonspecific suppressor T cells (Ts). Inguinal, mesenteric, and brachial lymph node cells were harvested from uninfected control mice and from mice at different periods of infection. These cells were cultured with or without concanavalin A (Con A) for 48 hr to induce Ts activity. After stimulation, the control and infected lymph node cells were passed over Sephadex G-10 columns to remove suppressor macrophages that arise during the infection from Con A-induced Ts. The column passed cells were then added to normal mouse responder spleen cells in a primary in vitro antibody response culture system with sheep erythrocytes (SRBC) as antigen. The resultant plaque-forming cell responses to SRBC indicated that Ts function was not induced in infected lymph node cell populations. However, early in the infection, a stimulatory signal was provided by both the untreated and Con A-treated infected lymph node cells, which was lost in the terminal stage. Determinations of T cell subpopulations revealed that the infected Lyt 2.2-bearing subpopulation was not significantly altered from normal controls. We conclude that T. rhodesense infected mice fail to mount normal lymph node cell antigen nonspecific Ts responses and that this loss of activity may be due to an intrinsic dysfunction in the suppressor T cell population.

Production of factors with immunosuppressive activity by a murine lymphoblastoid tumor cell line.

Summary A clone of L1210 cells has been shown to spontaneously release an MIF-like factor in vitro. Culture supernatant fluids containing such MIF activity are immunosuppressive when injected with or prior to an antigenic stimulus in syngeneic mice, and are also suppressive to a lesser degree when administered with antigen in a primary in vitro antibody response system. The secretion of immunosuppressive factors) by L1210 cells may represent yet another example of tumor cell associated mechanisms which alter host immune system function.