Depressed T-cell reactivity and suppressor activity of lymphoid cells from cyclophosphamide-treated mice

A study was made of the in vitro proliferative activity of thymus-derived lymphoid cells from cyclophosphamide-treated mice (Cy-mice) and the relationship between this and some in vivo immunological responses. The proliferative response to phytohaemagglutinin (PHA) and allogeneic cells was depressed for up to 3 weeks after drug treatment in spleen and lymph node cells, responsiveness recovering more rapidly in lymph node cells. Cell concentration in culture was shown to be important in such measurements as cells from some Cy-mice were able to inhibit their own proliferation and that of normal lymph node cells. No stable soluble factor responsible for this effect could be isolated. It was shown that in vitro proliferative activity is not a good indicator of in vivo T-cell capability as indicated by the very rapid recovery of ability to reject skin grafts and the fairly rapid recovery of ability to produce cytotoxic cells compared to the slower recovery of in vitro T-cell activities.     

Modulation of lymphocyte functions by group A streptococcal membrane

Protoplast membranes isolated from group A streptococci suppress functions of mouse B cells in vivo and in vitro. Intraperitoneal injection 24 or 72 hr (but not 12 hr) before collection of lymphoid cells results in a selective decrease in the mitogenic response of bone marrow cells to dextran sulfate (DS). The response of bone marrow cells to lipopolysaccharide (LPS), and spleen cells to both DS and LPS, is unaltered. In vitro exposure of lymphocytes to membranes concomitantly with mitogen reduces the response to both DS and LPS, however, the DS response is more susceptible to low doses of membrane. Suppression of the response to DS in vitro is not mediated by cells bearing Thy 1.2 antigen. Neither the phytohemagglutinin (PHA)-responsive cells nor the adherent cells participate in suppression of the LPS response in vitro. In contrast to the suppression of B-cell functions neither the PHA nor concanavalin A (Con A) response of mouse bone marrow, spleen, or thymus cells is altered by streptococcal protoplast membranes injected 24 hr before collection of cells. In vitro exposure of spleen cells to a limited range of concentrations of membrane results in an enhanced proliferative response of spleen cells stimulated by suboptimal doses of PHA. This synergism is not mediated by the adherent cells. Addition of membranes to spleen cell cultures in vitro has no effect upon the response of spleen cells to suboptimal doses of Con A or to optimal doses of either Con A or PHA. Higher concentrations of membranes reduce the proliferative response of both control and mitogen-stimulated cells. This nonselective suppression by high doses of membranes is not due to toxicity. Delayed hypersensitivity to sheep erythrocytes is potentiated by injection of membranes. These studies suggest that streptococcal membranes preferentially suppress the immature B cells and enhance certain T-cell functions.     

A bicellular mechanism in the immune response to chemically defined antigens. 3. Interaction of thymus and bone marrow-derived cells

The role of thymus and bone marrow-derived cells in the in vitro response to the dinitrophenyl (DNP) determinant was studied using the millipore filter well technique for spleen organ cultures. Antibodies to DNP were assayed by the technique of inactivation of DNP-coupled T-4 bacteriophage. It was found that spleens of mice total-body irradiated at 750 R, treated with bone marrow and thymus cells after exposure and immunized against rabbit serum albumin (RSA) were able to produce antibodies to DNP when challenged in vitro with DNP-RSA. Such a response was not produced by spleen explants from x-irradiated mice treated with either thymus or bone marrow cells. Neither were antibodies to DNP produced by spleens of animals repopulated with thymus and bone marrow cells, but not immunized with the carrier. This carrier effect was manifested when the irradiated mice were treated with RSA and thymus cells 6–8 days before administration of the bone marrow cells. Yet, such an effect was not observed when the RSA and bone marrow cells were given 6–8 days before injection of the thymus cells. Thus, the thymus-derived cells appear to play the role of cells sensitive to the carrier (RSA), whereas the bone marrow seems to be involved in the production of antibodies.     

A history of theories of acquired immunity

Alloimmune mouse spleen cells are capable of carrying out nonspecific cell-mediated cytolysis of syngeneic target cells when incubated in the presence of lectins such as Con A or PHA (lectin-dependent cell-mediated cytotoxicity). In the present study plant lectins from a variety of sources were examined for their ability to participate in alloimmune-LDCC. Reactivity was then compared to mitogenic activity and the ability to activate cytotoxic effector cells in vitro. Of the lectins tested only those reported to be T-cell mitogens were capable of participating in alloimmune-LDCC. Agglutinating but nonmitogenic lectins (e.g., WGA) or mitogens such as LPS or PWM failed to yield positive LDCC. Of the T-cell mitogens demonstrating positive reactivity in the alloimmune-LDCC assay, only a portion were able to generate cytolytic activity when incubated with normal spleen cells in vitro (Con A, GPA, lentil). Crude PHA, purified erythroagglutinin, or leukagglutinin failed to generate cytotoxic effector cells in this system even though these were mitogenic and demonstrated positive alloimmune-LDCC. The results suggest that T-cell mitogens interact with cytotoxic effector cells in a manner which specifically triggers cytolysis. The relationship of this interaction to other lymphocyte-lectin interactions is discussed.     

T-cell mediated suppression in the MRL mouse

MRL/lpr mice possess an autosomal recessive gene, lpr, which is associated with lymphoproliferation and acceleration of autoimmune disease. Lymphoproliferation has been ascribed to a single gene defect predominantly affecting the T-lymphocyte component of the immune system. MRL/++ mice do not possess the lpr autosomal recessive mutation and do not develop early lymphadenopathy. T-lymphocyte functional activity was studied in these mice using the polyclonal T-cell mitogens PHA and Con A. Our results indicated a significant suppression of the spleen and lymph node response of MRL/lpr mice to these polyclonal mitogens as compared to the MRL/++ response noted as early as 6 weeks of age. In addition, there was a progressive decline in the MRL/lpr spleen and lymph node cell mitogenic responses with increasing age. Spleen and lymph node cells from 20-week MRL/lpr mice were also relatively unresponsive in the mixed lymphocyte culture reaction as compared to cells from MRL/ ++ or BALB/c mice. The in vitro proliferative response of the MRL mice was further examined with respect to possible accessory cell modulation by both macrophages and T cells. It was found that in 20-week MRL/lpr lymph nodes a significant degree of suppression of lymphocyte proliferation could be mediated by the MRL/lpr T cell. Increased lymphocyte proliferation to a mitogenic signal could only be demonstrated in those MRL/lpr mice 3 weeks of age.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. II. Antigen-dependent enhancement by exogenous prostaglandins of the E series.

The spleen cells from CFW/D mice injected with dimethylbenzanthracene-induced leukemia virus exhibited a progressive decline in the in vitro response to heterologous erythrocyte antigens in parallel with tumor growth. Cell transfer experiments revealed that this immunodepressed state may involve a B-cell defect rather than extrinsic factors in the cellular environment since: (i) nonresponsiveness could be transferred to irradiated non-tumor-bearing mice with spleen cells, and (ii) T cells from tumorbearing mice cooperated with normal bone marrow cells, but bone marrow from tumorbearing mice did not cooperate with normal T cells. In addition, T cells from the thymic tumor could cooperate with normal bone marrow cells upon transfer to irradiated recipients. TL 485-2 cells, a T-cell line derived from the tumor, could be specifically activated with SRBC thereby indicating that the virus transformed T cells were immunocompetent. Suppressor cells, which appeared in the spleen concomitant with immunodepression and tumor development, may directly raise B-cell thresholds for T-dependent triggering signals since the antibody response of spleen cells from tumor-bearing mice could be restored by adding agents such as LPS, 2 mercaptoethanol, or T cells exogenously preactivated in normal animals. The suppressor cell could be enriched by adherence to plastic and was removed by treatment with carbonyl iron. In addition, it was unlikely that the suppressor cell was a virus-infected cell since transformed, virus-infected cells from the tumor or TL 485-2 cells were not suppressive when added to spleen cells in vitro but rather resulted in a marked, polyclonal enhancement of the PFC response. The interaction of TL 485-2 cells and normal spleen cells resulted in the release of a stimulatory factor which increased DNA synthesis in resting cells as well as increasing PFC. The role of these enhancing factors and suppressor cells in controlling tumor growth remains to be elucidated.     

Trypanosoma cruzi: the T-cell dependence of the primary immune response and the effects of depletion of T cells and Ig-bearing cells on immunological memory

The effects of T-cell depletion on primary infection with Trypanosoma cruzi and on immunological memory to this parasite were studied in a syngeneic mouse system. Exacerbation of T. cruzi infections occurred in thymectomized, irradiated, bone marrow-reconstituted (TX) C57BL/6J mice compared to sham thymectomized, irradiated, bone marrow-reconstituted (STX) mice. Reconstitution of TX mice with thymocytes restored the resistance to a level equivalent to that of STX mice. Immunological memory against T. cruzi present in spleen cells in mice recovered from T. cruzi infections could be ablated by treatment with rabbit anti-brain-associated theta serum but not with rabbit anti-mouse immunoglobulin serum prior to adoptive transfer of immune spleen cells into TX mice. These experiments suggest that modulation of the primary immune response and memory against T. cruzi depends largely on the thymus-derived lymphocyte. The possible implications of this T-cell regulation on previously reported effector mechanisms againt this parasite are discussed.     

In vitro growth of murine T cells. IV. Use of T-cell growth factor to clone lymphoid cells

Murine bone marrow cells can suppress the in vitro primary antibody response of normal spleen cells without apparent cytotoxicity. The bone marrow cells suppress the response to both T-dependent (SRBC) and T-independent (DNP-Ficoll) antigens. When bone marrow cells are fractionated on a sucrose density gradient, the suppressive activity is found in the residue rather than the lymphocyte fraction. The suppressive activity is either unaffected or enhanced by treatment with anti-T- and anti-B-cell serums. Pretreatment of mice with phenylhydrazine which reduces the number of pre-B cells did not reduce the suppressive activity of their bone marrow cells. Suppressive activity is abolished by irradiation of the marrow cells in vitro with 1000 R prior to assay. The activity is present in the marrow of thymus deficient (nude) mice, infant mice, and mice which have been made polycythemic by transfusion. Furthermore, the suppressor cell can phagocytize iron carbonyl particles, is slightly adherent to plastic and Sephadex G-10, and can bind to EA monolayers. We conclude that the suppressor cell is not a mature lymphocyte or granulocyte nor a member of the erythrocytic series, but is likely to be an immature cell possibly of the myeloid series. We speculate on the physiologic role of this cell.     

Macrophage activation during experimental murine brucellosis: II. Inhibition of in vitro lymphocyte proliferation by brucella-activated macrophages

During infection of CBA mice with Brucella abortus strain 19, there is a massive accumulation of macrophage-like cells in the spleen with resultant gross splenomegaly. In vitro cultures of cells from these spleens show a reduced proliferative response to brucellin and to other mitogens (phytohemagglutinin, concanavalin A, and lipopolysaccharide). The effect could be overcome by the addition of high concentrations of mitogen. Removal of adherent cells from spleen populations derived from 20-day infected mice abrogated the suppressive effect. Conversely, adherent cells from the spleens of 20-day infected mice inhibited proliferation of normal spleen cell cultures. Inhibition of responsiveness of normal spleen cells by cells from the spleens of infected mice occurred even when the two populations were separated by dialysis membranes. Although proliferation was measured by uptake of tritiated thymidine, inhibition in this system was not due to the release of unlabeled thymidine from macrophages.     

Plasmodium berghei: Reduction of the mouse's specific lymphoproliferative response in relation to corticosterone and pregnancy

Spleen cells from mice immune to Plasmodium berghei exhibited a significantly increased in vitro proliferative response to parasitized reticulocytes compared to spleen cells from normal mice. The specific response to malaria antigen was decreased in spleen cells from pregnant immune mice in contrast to the nonspecific response to the mitogen phytohemagglutinin. Addition of mouse serum to spleen cell cultures of immune mice depressed both the phytohemagglutinin and the specific proliferative response, whereas serum of pregnant mice exerted an even stronger inhibition than serum of nonpregnant mice. Charcoal adsorption of mouse sera for the elimination of steroid hormones removed the serum dependent immunosuppression from normal as well as pregnant serum. Corticosterone added to the spleen cell cultures depressed also the proliferative response. These findings demonstrate that the response to malaria antigen is decreased in immune mice during pregnancy. The possible effect of serum corticosterone on the depression of the immune response is discussed.     

Effect of Anti-Ia Serum In Vitro on Lipopolysaccharide-Induced Proliferative Responses of Murine Bone Marrow and Spleen Cells

The in vitro proliferative response of murine bone marrow cells and spleen cells to bacterial lipopolysaccharide (LPS) and the effect of anti-Ia serum on the response were studied. The incorporation of [³H]thymidine into cells prepared from bone marrow increased in the presence of LPS, but the addition of anti-Ia serum to the cultures reduced the incorporation. Pretreatment of bone marrow cells with anti-Ia serum and complement did not abolish the ability of the cells to respond to LPS, while the same pretreatment destroyed this ability in spleen cells. These results suggest that cultures of Ia-negative bone marrow cells generate Ia-positive cells during the culture period, and the Ia-positive cells are responsive cells to LPS. The proliferative response of 1- or 2-week-old spleen cells was easily suppressed by anti-Ia serum when compared with that of 4-week-old spleen cells. Furthermore, the responses of spleen cells obtained from γ-irradiated and syngeneic bone marrow cell-reconstituted mice were prominently suppressed by anti-Ia serum in comparison with that of normal adult spleen cells. These findings suggest that LPS-responsive lymphocytes in the developmental stage are quite sensitive to anti-Ia serum. The effect of anti-Ia serum on the maturation of bone marrow-derived lymphocytes was discussed.     

In vitro immune blastogenesis during contact sensitivity in tumor-bearing mice: I. Description of progressive impairment and demonstration of splenic suppressor cells

T-cell responsiveness was measured by the DNA response of disassociated spleen and lymph node cells when exposed to antigen in vitro. Sensitized splenic lymphocytes from fibrosarcoma-bearing mice immunized with 2,4-dinitro-1,5-difluorobenzene (DN2FB) demonstrated a progressive decrease in T-cell responsiveness to the haptenprotein conjugate DNP-BSA. Hyporesponsiveness to the dinitrophenylated-protein conjugate appeared in the spleens but not lymph nodes of tumorous animals. Normal host lymph node cells (LNC) responded strongly 24 to 48 h after sensitization and subsequently declined with a corresponding increase in responsiveness in the spleen. Tumor-bearing hosts (TBH) had similar LNC kinetics during immunization, however, spleen cells were significantly suppressed when compared to normal BALB/c mice sensitization kinetics. Spleen cells from TBH were also capable of suppressing the in vitro response of normal primed lymphocytes to DNP-BSA when admixed. Results from these experiments suggest that in vitro measurement of contact sensitivity was affected by suppressor cells/products existing in the spleens but not lymph nodes of fibrosarcoma-bearing mice.     

Immunologic unresponsiveness of mouse spleen sensitized to allogenic tumors.

CS7BL/6 mice were sensitized with an ip injection of allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the tumor allosensitized mice were cultured and tested for their responsiveness to mitogens and alloantigens, and for their ability to generate cytotoxic cells in vitro. The results indicate that 15 day tumor-sensitized spleen cells are hypo-responsive in mixed lymphocyte culture (MLC) with DBA/2 or AKR as stimulating spleen cells. The cells which are hypo-responsive in MLC can proliferate in response to mitogens and they also can generate cytotoxic cells in vitro. MLC reactivity recovers in about 2–3 months which is $\text{1}\text{1}\text{2}\text{–2}\text{1}\text{2}$ months after the mice have rejected their tumors. The mechanism of MLC hypo-responsiveness was investigated. The results suggest the presence of a suppressor cell which does not appear to be a macrophage or a B-cell. The suppressor cell can be separated from the cytotoxic cell and therefore appears to be a noncytotoxic T-cell.     

Studies of the T-lymphocytes resident in the spleens of thymectomized, irradiated, bone marrow-reconstituted (TXB) mice.

The T-lymphocytes resident in the spleens of thymectomized, lethally irradiated mice that had been reconstituted with syngeneic bone marrow (TXB) were characterized. Both recently reconstituted N-TXB, (approximately 3 weeks after bone marrow injection) and aged (>6 months after reconstitution) A-TXB animals were studied. The T-lymphocytes from spleens of recently reconstituted N-TXB donors did not respond to PHA but did react significantly to Concanavalin A (Con A). The lack of PHA sensitivity was not due to dilution of reactive cells by other cell types. Removal of adherent cells, likewise, did not restore N-TXB spleen cell PHA responsiveness. N-TXB splenic T-cells were cortisone resistant. N-TXB spleen cells by themselves did not cause a graft vs host response. However, N-TXB spleen cells amplified the graft vs host response of normal lymph node cells but not N-TXB lymph node cells. Addition of cyclic GMP enhanced [³H]thymidine uptake of N-TXB spleen cells caused by Con A. N-TXB spleen cells were exclusively spleen seeking. The Con A reactive cell within N-TXB spleens was demonstrated to be of donor origin. Fetal liver as well as syngeneic bone marrow contained cells capable of reconstituting the Con A response. Spleen cells from aged. (>6 months) A-TXB were found to be PHA sensitive. Competitive inhibition assays measuring θ expression in A-TXB spleen cells indicate a significant increase in the θ positive lymphocyte population occurred with time. The data indicate that considerable reconstitution of θ positive cells had occurred in A-TXB donors. The results also suggest that the T-lymphocyte population of the TXB spleen may be a unique subpopulation of T-lymphocytes that resides exclusively in spleen and bone marrow.     

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.     

Appearance of lymphocyte surface markers and functional responses in neonatal and young rabbit spleens

We examined spleen cells from newborn to 1-month-old rabbits for easily detectable surface immunoglobulin, complement receptors, and for in vitro proliferative responsiveness to anti-immunoglobulin antisera and several mitogens. From birth through the first month of life about 15% of the cells from rabbit spleens had easily detectable surface immunoglobulin while about 45% had C3 receptors. In adults as many as 77% of the spleen cells had easily detectable surface Ig but the proportion with C3 receptors remained about 45%. The proliferative response to anti-allotype antisera was present at birth, and was at adult levels by 1 month of age. The proliferative response to pokeweed mitogen was low when cells were obtained during the first week of life but was comparable in magnitude to the response of adult cells by 2 weeks of age. In vitro responsiveness to concanavalin A was present at low levels at birth and increased sharply during the first week. We did not observe significant stimulation of spleen cells from neonatal to 4-week-old rabbits by lipopolysaccharide from Salmonella typhosa. Our data suggest that lymphocyte surface markers and functional responses appear asynchronously in spleen cells of developing rabbits.     

Role of the H-2I region in the generation of an antiviral cytotoxic T-cell response in vitro

The participation of H-2I gene products in generating virus-specific proliferative and/or cytotoxic T-lymphocyte (CTL) responses was investigated. Spleen cells from mice infected with vaccinia virus were restimulated secondarily in vitro with syngeneic virus-infected peritoneal exudate cells (PEC) and then restimulated in tertiary cultures with virus-infected PEC from syngeneic and partially histoincompatible strains of mice. Based on the finding that comparable proliferative responses resulted when stimulating the responding cells were histocompatible at the H-2K, I, or D region of the major histocompatibility complex (MHC), the additively enhanced, but not potentiated, proliferation caused by identity at two or three H-2 regions was analyzed. Enhancement of proliferation followed when the H-2K/D components plus virus and the H-2I components plus virus were either on the same, or alternatively on two, stimulating cells. This suggests that H-2K, D, and I plus virus trigger distinct T-cell subsets. A virus-specific CTL response was generated in vitro when spleen cells from virus-primed mice and even unprimed mice were stimulated with cells sharing only H-2K and/or H-2D of the MHC. Identity at the H-2I region did not stimulate a CTL response, nor did it influence the magnitude of the $\text{K}\text{D}$ restricted response. Nevertheless, the presence of anti-Ia antiserum in cultures of syngeneic stimulators and responders inhibited the cytotoxic response to a great extent. Therefore, H-2I region products seem to participate in the generation of virus-specific CTL in vitro.     

In vitro sensitization to transplantation antigens. VII. Enhanced graft-vs-host activity of in vitro allosensitized lymphoid cells

We have previously demonstrated that C57BL/6J lymphoid cells sensitized in vitro to C3H/He transplantation antigens, present on macrophage monolayers, can transfer an accelerated C3H allograft response to recipient C57 mice. The present report indicates that C57 lymphoid cells sensitized to C3H alloantigens, present on macrophage monolayers, can also mediate a graft-versus-host (GVH) reaction in (C3H × C57) F₁ newborn mice. This GVH reaction is of greater magnitude than that produced by noncultured C57 cells. The magnitude of the augmented GVH reaction produced by cultured C57 cells is dependent on the source of lymphoid cells: lymph node, spleen, and bone marrow cells are consistently more active than cultured thymus cells—the reduced capability of cultured thymus cells to mediate the GVH reaction parallels their reduced ability to transfer allograft immunity. To test whether monolayers, other than macrophages, can sensitize lymphoid cells in vitro we incubated C57 lymphoid cells on C3H-derived L cells. Lymph node cells incubated with L cells demonstrate an increased GVH reaction in newborn mice. The in vitro sensitization of spleen and bone marrow cells on L cells is less consistent. Thymus cannot be sensitized by L cells. Monolayers of L cells are therefore not as efficient a sensitizing source as macrophages.     

Immunologic significance of nonspecific suppressor cells in spleens of tumor-bearing mice.

Spleen cells from mice bearing a progressively growing syngeneic tumor failed to respond to stimulation with mitogens in vitro. This lack of reactivity was due to the presence of nylon wool-adherent cells in the population that could inhibit the mitogen response of normal lymphocytes. Paradoxically, at times when strong suppressor cell activity could be detected in tumor-bearing mice, the animals responded normally to in vivo immunization with sheep erythrocytes and allogeneic tumors, and to in vitro sensitization with allogeneic tumor cells. Regression of a highly antigenic syngeneic tumor also was unaffected by the presence of these suppressor cells. Thus, the occurrence of nonspecific suppressor cells in the spleens of tumor-bearing mice did not influence the overall immunologic competence of these animals.     

Studies of murine lymphocytes and alloantigens: I. Ly-6 mitogen and MLR responses

Antisera to the mouse lymphocyte surface alloantigens Ly-6.1 and Ly-6.2 were used to further study the functional distribution of these antigens. After selective depletion with antiserum + rabbit complement (RC), lymph node or spleen cells from Ly-6 congenic (C3H and C3H.B6-Ly-6^b) and noncongenic strains of mice were tested for: (a) their proliferative responses to T- and B-cell mitogens; and (b) their proliferative responses to alloantigens, or ability to stimulate in the MLR. Lymphoid cells required in the proliferative responses to the mitogens leucoagglutinin, concanavalin A (Con A), lipopolysaccharide (LPS), and pokeweed mitogen (PWM) were Ly-6⁺. Lymph node responder cells in the mixed lymphocyte reaction (MLR) were also Ly-6⁺, whereas spleen stimulator cells were Ly-6^?. Treatment of lymph node cells with anti-Ly-6 sera in the absence of RC had no specific blocking effect on the response to any of these mitogens. The studies indicate that the Ly-6 antigen is a potentially valuable marker for distinguishing between functionally distinct Ly-1⁺ T-cell subsets.