Role of L-alanine in the response of human lymphocytes to PHA and Con A

The response of umbilical cord blood lymphocytes (UCBL) to PHA and Con A is strongly dependent upon the presence of dialyzable plasma components. The impaired response of human cells in the presence of dialyzed human plasma was restored by calf thymus extract. In the present experiments we have further purified calf thymus extract by means of paper electrophoresis and chromatography on thin-layer cellulose plates. We reached the conclusion that the active material in this model is a single amino acid, alanine. When the different isomeric forms of alanine were tested, we found that L-alanine is the only biologically active material. In addition, we have observed here that the allogeneic response of UCBL is also dependent upon the presence of dialyzable plasma components. This impaired allogeneic response of UCBL in the presence of DHP was restored by addition of L-alanine. The present findings suggest that, under in vitro conditions, for human cells to respond to mitogenic or allogeneic stimulation L-alanine is essential.     

Role of thymic hormone (THF) and of a thymic plasma recirculating factor (TPRF) in the modulation of human lymphocyte response to PHA and Con A.

The results presented here point to the possibility that calf thymus extracts contain, in addition to the thymic hormone (THF), a second component: thymic plasma recirculating factor (TPRF). THF, which is involved in the process of T cell maturation and has been characterized as a protein of m.w. 3000 eluted in the void volume of a G-10 Sephadex column (G-10-I), caused an increased level of intracellular cAMP in umbilical cord blood lymphocytes (UCBL). This is in agreement with our previous observation that THF plays a major role in the differentiation of T cells. The second active material, TPRF, also isolated from thymic extract, is of a molecular size below 500 and was eluted in a G-10 Sephadex column at the fourth protein peak; it seems to circulate in the blood. Previously, we had observed in impaired response of UCBL to PHA and Con A stimulation in the presence of dialyzed human plasma (DHP). Our present results indicate that this impaired response is restored exclusively by TPRF. A factor with TPRF-like activity was also isolated from the plasma of normal donors; yet it was not detected in the plasma of thymectomized patients suffering from myasthenia gravis (MG). This suggests that TPRF from plasma is thymus dependent. TPRF does not affect the level of intracellular cAMP in UCBL.     

Effect of various thymic and nonthymic factors on in vitro antibody formation by spleen cells from nude mice

Thymosin and thymic humoral factor (THF), both prepared from whole thymus tissue, increased the in vitro anti-SRBC response of spleen cells from nu/nu mice. Lymphoid and nonlymphoid control preparations did not influence the response. Standard isolation procedures for thymosin and THF were employed to prepare factors from thymus lymphocytes (TL) separated from calf thymus and from thymus tissue enriched in thymus epithelium (TE) by preirradiation of the calves. The activity of the TE preparations was greater than that of the total thymus (TT) preparations. The TL preparations were marginally effective. However, the activity of the TE preparations could not be attributed exclusively to epithelium-derived factors, since spleen and lymph node extracts from the irradiated calves contained stimulatory material of an unknown origin which may also be present in the TE preparations.     

Autologous mixed lymphocyte reaction in man. I. Relationship with age and sex

Coculture of normal human T lymphocytes with autologous cells enriched in B lymphocytes results in increased DNA synthesis. In our system, the responder cells are mononuclear cells stimulated by irradiated autologous non-T cells in the presence of 20% inactivated autologous plasma. The results indicate that the proliferative response is age and sex related; significantly higher levels of thymidine incorporation are observed with female peripheral blood lymphocytes and, moreover, it appears that this strong stimulation is more pronounced in older female subjects. In contrast, the proliferative responses against allogeneic cells are not modified irrespective of the age and sex group considered. The involvement of autorosette-forming cells (A-RFC) in such autologous mixed lymphocyte reaction (A-MLR) is demonstrated by the marked impairment of the A-MLR after removal of A-RFC from responding cells. These data suggest that older healthy subjects, particularly females, exhibit a higher incidence of in vitro autologous reactions.     

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.     

Effect of cell-free murine liver extract on lymphocyte blastogenesis in vitro.

The effect of cell-free liver extract (LE) on the proliferation of spleen cells in vitro was examined using [³H]thymidine incorporation. LE inhibited the blastogenic response of murine lymphocytes stimulated with plant mitogens, phytohemagglutinin, and concanavalin A and in the mixed lymphocyte reaction (MLR). Suppression of cell proliferation occurred whether the LE was syngeneic or allogeneic to the responding cells. This effect was observed only when LE was present in cultures, as preincubation of cells with LE did not impair their capacity to respond to stimulation. Profound suppression of proliferation was achieved with the addition of LE to the culture up to 48 hr after the onset of stimulation. However, the inhibitory effect was readily reversible upon removal of LE from the culture. Furthermore, although LE was capable of suppressing the generation of cytotoxic lymphocytes, LE did not interfere with their capacity for cytolysis. These findings indicate the presence of a potent inhibitor of lymphocyte proliferation in a cell-free extract of murine liver.     

Terminal deoxynucleotidyl transferase: characterization of extraction and assay conditions from human and calf tissue.

Methods of extraction and assay of terminal deoxynucleotidyl transferase (TdT) from human lymphoblasts and calf thymus were compared. A high salt concentration was mandatory for complete enzyme extraction, while dialysis of the crude extract resulted in a major loss of enzyme activity. In addition, TdT was partially purified from lymphoblasts of patients with acute lymphoblastic leukemia. The K_m for the monomer, deoxy-guanosine 5′-triphosphate (dGTP), is high (~0.1 mm) in the presence of either Mg²⁺ or Mn²⁺, whereas the K_m for the initiator, poly(deoxyadenylic acid $\text{[poly(d(pA)}\text{50}\text{)]}$, with an average chain length of 50 residues, is 2.5 μm in the presence of Mg²⁺ and 0.3 μm in the presence of Mn²⁺. The maximum velocity is higher for the calf thymus TdT in the presence of Mg²⁺ than in Mn²⁺. Human TdT catalyzes the polymerization of dGTP at a higher rate in the presence of Mn²⁺ than with Mg²⁺. These data illustrate that partially purified human TdT differs in catalytic properties from the purified calf thymus enzyme. Therefore, optimal conditions for assay of TdT in extracts from calf and human tissues differ.     

Further characterization of suppressor lymphocytes induced by fetal calf serum in murine lymphoid cell cultures: comparison with in vitro-generated cytotoxic lymphocytes.

Nonspecific suppressor cell (SPC) activity has been induced in vitro by preculturing splenocytes from normal mice in the presence of fetal calf serum (FCS) for 3 days or more. In adoptive transfer experiments in vivo, these precultured SPC were shown to reduce the humoral response of mice to SRBC and the cell-mediated cytotoxic (CMC) response to allogeneic tumor cells. In mixing experiments in vitro, using freshly explanted splenocytes, the precultured splenocytes abrogated the generation of specific cytotoxic lymphocytes (CL) in primary mixed lymphocyte-tumor cell cultures (MLTC). By contrast, secondary cytotoxic response was only marginally affected. Supernatants of precultured cells were also inhibitory, although to a lesser degree than whole cells. The induction of suppressive activity was abolished by addition of mitogenic amounts of concanavalin A to the preculturing medium.By the use of cell fractionation techniques it was found that both specific CL and nonspecific SPC lack an Fc receptor, do not adhere to nylon wool, and cannot be separated from each other by density sedimentation on a discontinuous BSA gradient. However, precursors of SPC and CL differed in their susceptibility to cyclophosphamide, hydrocortisone, and irradiation. The data presented does not exclude the possibility that suppressive activity exerted by FCS-induced SPC is mediated through a cytotoxic effect.     

Endogenous Thymic Factors Regulating Cell Proliferation and Analysis of Their Mechanism of Action

Endogenous factors inhibiting the proliferation of T-lymphocytes were investigated which may function as modulators of T-lymphocyte production within the thymus. an extract from calf thymus (T4) enriched in lymphocyte chalone arrests rat thymocytes at the G₁ S boundary and in the S phase of the cell cycle in short-term cultures. It also inhibits the proliferative response of human peripheral blood lymphocytes to PHA-P in a time-dependent manner, as well as the spontaneous proliferation of in vitro cultured human chronic leukaemic lymphoblasts. This crude extract contains two active moities which can be isolated by molecular filtration on Sephadex G-75 column. A species non-specific, cell line selectivity inhibitory effect is characteristic of the high molecular weight fraction (mol. wt. > 40,000). This activity is resistant to moderate heat treatment and trypsin but is sensitive to mild alkaline hydrolysis and to RNase A digestion. About ten protein components and a toluidine blue positive substance can be detected by analytical polyacrylamide gel electrophoresis. the active inhibitor, a proposed protein-RNA complex, might be identical with the chalone. The low molecular weight, non-dialysable factor (T4–4) inhibits [³H]thymidine incorporation into acid insoluble DNA in a cell non-specific manner. A possible relationship between the two activities is discussed.     

The effects of crude and purified human gonadotropin on in vitro stimulated human lymphocyte cultures.

Crude human chorionic gonadotropin (hCG) was found to be several fold more immunosuppressive than purified hCG in human peripheral blood lymphocyte cultures stimulated by phytohemagglutinin, pokeweed, purified protein derivative and allogeneic cells in vitro. Immunosuppression by crude hCG was consistently noted at levels less than 1000 IU/ml and usually 80% inhibition was achieved with doses of 5000–10,000 IU/ml, whereas 40–50% inhibition or less was observed by purified hCG at 10,000 IU/ml. In two crude hCG preparations subjected to Sephadex G-100 chromatography, the fractions that inhibited lymphocyte cultures appeared in the eluate after the major peak of hCG activity. These data indicate that inhibitory substance(s) other than hCG are responsible for most of the immunosuppressive properties of first trimester pregnancy urine. Both crude and purified hCG were stimulatory to human lymphocytes when used alone without mitogens when cultured in fetal calf serum.     

Antigen-induced in vitro lymphocyte blastogenesis in the rabbit: a T-cell response.

In vitro lymphocyte stimulation of sensitized rabbit lymphocytes to specific antigen (ovalbumin) was found to depend on thymic-dependent lymphocytes. This conclusion is based on an enhanced response upon enrichment with T lymphocytes by passage of lymphocytes through nylon wool, and on the elimination of the response after treatment of lymphocytes with complement and an antiserum to rabbit thymus cells prepared in a goat. Specificity of the antiserum was demonstrated by elimination of in vitro T-cell function and retention of in vitro B-cell functions.     

Spontaneous adherence and rosette formation of lymphocytes to Leydig cells: an in vitro technique

A spontaneous rosette phenomenon occurs in vitro when suspensions of mouse testicular cells are mixed with suspensions of lymphocytes. The core cell of these rosettes are the Leydig cells, and the coronas are formed by lymphocytes. No other testicular cell, either seminal or Sertoli, participates in lymphocyte binding.Autogeneic, syngeneic, allogeneic, and xenogeneic lymphocytes from thymus, spleen, and peripheral blood similarly react with mouse Leydig cells. Lymphocytes from spleens of athymic nude mice and from chicken bursa also give Leydig rosettes. It is suggested that mouse Leydig cells carry membrane receptors spontaneously recognized by lymphocytes.     

Selective T cell killing of human lymphocytes by ultraviolet radiation

The effects of ultraviolet radiation (uv) on human B and T lymphocytes were studied. In vitro studies showed that T lymphocytes were more sensitive to uv than B lymphocytes as assessed by eosin-dye exclusion. Following uv exposure, the viable lymphocytes responded to mitogens (PHA, PWM), and functional B lymphocytes were present at a time when no viable T cells were detected. Varying doses of uv were required to abrogate different in vitro responses (proliferative response to antigen or allogeneic cells, MIF production, and cell-mediated lympholysis). In vivo, uv was able to diminish an established cutaneous delayed hypersensitivity response. In vitro uv treatment of parental mouse spleen cells eliminated a graft-versus-host reaction in F₁ recipients as determined by the spleen index. The basis for the differential effect of uv on B and T lymphocyte viability and functional responses is unknown.     

Identification of human lymphocyte-derived lymphotoxins with binding and cell-lytic activity on NK-sensitive cell lines in vitro

Supernatants obtained from lectin-restimulated, preactivated, human peripheral blood lymphocytes rapidly released (5–24 hr) high levels of lymphotoxin (LT) activity in vitro. Peripheral blood lymphocytes were preactivated by coculturing with either fetal calf serum or with allogeneic continuous B-cell lines (LCCL) which were treated with mitomycin C. These Supernatants contained a population of L-929 cell-lytic LT forms which also selectively bind to the NK-sensitive K-562 cell. However, lytic LT forms for L-929 cells from cPBL and LCCL cultures did not bind to the NK-sensitive MOLT-4 or NK-resistant Raji cells. Additional studies reveal these supernatants contain a second set of LT forms which have cell-binding and cell-lytic activity detectable on MOLT-4 and K-562 cells in a 12 to 18 hr ⁵¹Cr-release assay. Cell-lytic form(s) for the MOLT-4 and K-562 cells were not stable for more than a week at ?20°C. These findings indicate that materials with LT activity are heterogeneous with respect to their capacity to recognize common and discrete cell-surface components on different types of target cells in vitro.     

EVALUATION OF THE SPECIFICITY OF A LYMPHOID CHALONE

An extract from calf spleens, injected into mice, was found to inhibit their lymphocyte proliferative response to PHA-M and PWM in vitro. Despite the ability of the spleen extract to inhibit the response of lymphocytes to stimuli in vitro, no effect was observed on the repopulation of lymphocytes in the peripheral blood of sublethally irradiated mice. The data suggest that the spleen extract acts as a specific inhibitor of the immune competent cells since neither the precursors of lymphocytes nor other haematopoietic cells were affected.     

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.     

Cellular immunity in the mouse. IV. Altered thymic-dependent lymphocyte reactivity in the chronic graft vs host reaction and leukemia virus activation.

The lymphocytes obtained from several F₁ strains undergoing chronic GVH reactions were studied for in vitro alterations of thymic-dependent lymphoid function. Spontaneous blastogenesis was increased. The in vitro response to nonspecific mitogenic stimuli (PHA and CON-A) and specific antigenic challenge (SRBC and allogeneic cells) was initially increased and subsequently impaired. The degree of alteration was related to the severity of the observed disease and dependent upon the F₁-parental combination employed. Thymic-dependent lymphocytes obtained from animals with GVH disease possessed the ability to suppress actively the response of normal mouse cells in vitro to various T-cell mitogenic stimuli and this suppressive activity was present in the supernatant culture fluid from such cells. The mechanism of this altered in vitro T-cell reactivity is not yet completely understood, but may in part be related to the immunologic activation of murine leukemia virus from mouse cells undergoing allogeneic stimulation.     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.