Doug Redelman

Studies of rheumatoid synovial fluid lymphocytes. II. A comparison of their behavior with blood mononuclear cells in the autologous mixed lymphocyte reaction and response to TCGF.

Synovial fluid lymphocytes (SFL) and peripheral blood lymphocytes (PBL) from patients with rheumatoid arthritis were compared for their response to lectin stimulation and for their behavior in the autologous mixed lymphocyte reaction (AMLR). The SFL proliferative response to phytohemagglutinin (PHA), as measured by tritiated thymidine incorporation at 72 hr, was lower than that of PBL (P less than 0.001). When T-cell growth factor (TCGF) was added to the medium, there was an increase in the SFL proliferative response to PHA (P less than 0.05). In contrast, TCGF did not alter significantly the PBL proliferative response to PHA. Mixing experiments were performed to determine whether the poor SFL proliferative response was due to passive absorption and removal of in situ-generated TCGF by suppressor cells. When cultured together, SFL did not suppress the PBL proliferative response to PHA, suggesting that decreased production of TCGF rather than competitive binding of TCGF results in the poor SFL proliferative response to lectin stimulation. In the AMLR, synovial fluid non-T cells were found to be more stimulatory to peripheral blood T cells than were peripheral blood non-T cells (P less than 0.001). In comparison to peripheral blood T cells, synovial fluid T cells were poor responders in the AMLR. Repetitive in vitro autologous stimulation of peripheral blood T cells resulted in proliferative responsiveness analogous to that of SFL, i.e., a relatively poor proliferative response in the AMLR and a poor response to PHA. The latter could be augmented by TCGF. The SFL requirement for exogenous TCGF is consistent with a state of immune activation. In vivo stimulation by non-T cells may play an important role in the immune activation which characterizes rheumatoid SFL.

In vitro studies of the rabbit immune system: II. Functional characterization of rabbit T and B populations separated by adherence to nylon wool or lysis with anti-thymocyte serum and complement

Abstract Rabbit spleen lymphocytes with functions analogous to murine T and B cells have been separated by treatment with goat anti-rabbit thymocyte serum plus complement (ATS + C) or passage over columns of nylon wool (NW). ATS + C lysis of spleen cells from sheep erythrocyte (SRBC)-immunized rabbits removes 40–60% of the total cells, but 80–100% of the fully differentiated B cells (PFC) are recovered. Conversely, the primed spleen cells which are nonadherent to NW contain ~5–20% of the applied cells but only 0.05–1.0% of the applied PFC. Neither cell population is capable of producing an anti-SRBC PFC response when cultured individually. However, the two cell types complement each other and act synergistically to reconsitute the in vitro anti-SRBC PFC response. Thus, the rabbit spleen contains separable lymphocyte populations with functions equivalent to T and B cells as defined in the murine system.

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

Abstract The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro . Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBLs) and 50% of the spleen cells while passage over NW yields 40% of the applied PBLs and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBLs respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBLs that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.

The mechanism of cell-mediated cytotoxicity: II. The apparent biochemical requirements for cytolysis are influenced by the source and frequency of murine cytotoxic T lymphocytes☆

Abstract Cytotoxic T lymphocytes (CTL) were generated in allogeneic mixed-lymphocyte cultures (MLC) with the responder cells from immunized mice (secondary (II∘) MLC.) These CTL could be maintained as proliferating cells for 2 to 4 weeks by culturing in a medium with T-cell growth factor (TCGF) activity and were 10 to 30 times as active as effectors obtained directly from immunized mice or those generated in primary (I∘) MLC. Cytolysis by CTL from II∘ MLC was refractory to inhibition by azide, cyanide, or cycloheximide in contrast to previous reports that these agents were inhibitory. However, cytolysis by CTL from II∘ MLC was sensitive to azide when sufficient normal spleen cells were added to reduce the apparent CTL frequency to approximate that of the less active populations. Cycloheximide (Cx) partially inhibited cytolysis by CTL from immune spleen cells as shown earlier by others. However, CTL from II∘ MLC remained refractory to Cx even when diluted with normal spleen cells. These data show that CTL populations from various sources have both qualitative and quantitative differences that may alter their apparent biochemical requirements for CML. These considerations must be taken into account when attempting to define the minimal requirements for CTL-mediated killing.

The mechanism of cell-mediated cytotoxicity. III. Protease-specific inhibitors preferentially block later events in cytotoxic T lymphocyte-mediated lysis than do inhibitors of methylation or thiol-reactive agents.

Highly active mouse cytotoxic T lymphocytes (CTL) generated in secondary mixed-lymphocyte responses were used to examine the manner in which adenosine derivatives, thiol-specific reagents, or protease-specific probes affected CTL-mediated lysis (CML). The adenosine deaminase inhibitor deoxycoformycin (dCF) enhanced inhibition by adenosine (AR) or by deoxyadenosine (AdR), but not by 7-deazaadenosine (tubercidin). L-Homocysteinethiolactone (L-Hcy) acted synergistically with AR, but not with AdR or tubercidin, to block CML. Thus, AR derivatives may act both by affecting cellular methylation reactions, as demonstrated by the synergism between AR and L-Hcy, and by inhibiting other events required for CML. Conditions were then established to determine whether these reagents preferentially affected either the Ca2+-independent initial stage of cytolysis or the subsequent Ca2+-dependent events. Methylation inhibitors blocked lysis most effectively if added before effector-target binding. Similarly, the nonpenetrating thiol-specific reagent quaternary ammonium monobromobimane (qBBr) was more inhibitory when added prior to the Ca2+-dependent stage. Protease inhibitors such as alpha-1-antichymotrypsin and protease substrates such as acetyltyrosine ethyl ester (ATEE) or tyrosine ethyl ester (TEE) also inhibited CML. But, in contrast to qBBr or methylation inhibitors, neither TEE nor ATEE was more effective when added prior to the initial effector-target interaction. Furthermore, TEE did not appreciably affect CTL binding to target cells at concentrations that nearly abrogated CML. Thus, the implicated protease step is unique in that it does not appear to participate in recognition or binding.

In vitro studies of the rabbit immune system: I. Hemolytic plaque-forming response of dissociated spleen cells from normal and primed rabbits☆

Abstract The conditions for the in vitro generation of primary and secondary immune responses by rabbit spleen cells to sheep red blood cell (SRBC) antigen have been examined. Spleen cells from many normal and all previously immunized rabbits are capable of producing in vitro plaque-forming cell (PFC) responses when cultured as dissociated cell suspensions in the presence of antigen. Primed spleen cells generate approximately 100 times the number of PFCs obtained in normal cultures with a shorter lag period. Both types of cultures demonstrate a period of exponential increase in PFCs during which the doubling time is 12–14 hr. This increase occurs after 1 day of culture of spleen cells from primed rabbits and after 4 days of culture of spleen cells from unprimed rabbits. The PFCs which arise in cultures of primed cells appear not to be the progeny of those generated in vivo but to be derived from an increased number of PFC precursors. Repeated immunization of the spleen cell donor is required to produce significant numbers of indirect (IgG) PFC or indirect precursors; most of the PFC found after a single immunization in vivo or in vitro are direct (IgM). There is no evidence for conversion of IgM to IgG PFC in vitro . This system should provide a means for further identification of the cellular interactions involved in the immune response of the rabbit.

The mechanism of cell-mediated cytotoxicity: IV. K-76 COONa, Which inhibits the activity of factor I and of C5, inhibits early events in cytotoxic T-lymphocyte-mediated cytolysis and in T-lymphocyte activation☆

K-76 COONa is a derivative of a fungal product which blocks complement (C)-mediated lysis by combining with C5 and preventing its activation to C5b. K-76 COONa can also combine with Factor I and inhibit its ability to hydrolyze C3b to iC3b. The inclusion of K-76 COONa at concentrations similar to those which inhibit C lysis blocked both murine cytotoxic-T-lymphocyte (CTL)-mediated lysis (CML) and the lectin-stimulated proliferative response of murine and human T lymphocytes. A modified cation pulse procedure has been used to determine which phases of CML were most sensitive to the drug. K-76 COONa was inhibitory when it was added to CML prior to the early Mg+2-dependent binding phase, but was much less effective when it was added at any time after the formation of CTL-target conjugates. The principal effect of the drug on the proliferative response was also exerted during an early phase of the response. K-76 COONa did not appreciably decrease the production of T-cell growth factor (TCGF), but it did inhibit the induction of TCGF receptor expression by both functional criteria, i.e., induction of responsiveness to TCGF, and by morphological criteria, i.e., the expression of the Tac antigen. Later events, such as the TCGF-dependent proliferation of cycling T cells, were less sensitive to the drug. Evidence is discussed suggesting that molecules similar to Factor I and to C3 may be involved both in the early events of CML and of T-lymphocyte activation.

In vitro studies of the rabbit immune system. VIII. The production of rabbit T cell growth factor (TCGF) and its relationship to mouse and human TCGF

Rabbit spleen cells (1 X 10(6)/ml) stimulated with Con A (2.5 micrograms/ml) begin producing T cell growth factor (TCGF) activity within 2-3 h at 37 degrees C and reach a plateau after 20-24 h. Lymphocytes from the mesenteric lymph nodes (MLN) also produce TCGF activity but require higher lectin and/or cell concentrations for optimal production. TCGF generation by MLN can be improved by including a small proportion of spleen cells (10-40%). Rabbit lymphocytes also produce TCGF in serum-free, protein-free medium, but the Con A concentration must be reduced to 0.5-1.25 micrograms/ml. Rabbit TCGF activity eluted from gel filtration columns at a volume equivalent to approximately 15,000 molecular weight, similar to human TCGF activity. However, when rabbit, mouse and human TCGFs were compared for their ability to support proliferation by activated cells from the 3 species, rabbit and mouse TCGFs appeared more similar. In all cases, the homologous combinations of cells and TCGF were most efficient. Human TCGF would support some proliferative activity in all 3 cell types but rabbit and mouse TCGFs failed to support the growth of human cells.

Studies on rabbit lymphocytes in vitro: XX. Demonstration of cells reactive to more than one mitogen by mixed sequential stimulation

Abstract Rabbit peripheral blood lymphocyte (PBL) cultures stimulated by ConA and then blocked by the addition of competing sugar or antiserum after 6–15 h of ConA prestimulation respond to restimulation by PHA or PWM to a much greater extent than to continuous stimulation or delayed stimulation with PHA or PWM. This effect of mixed lectin sequential stimulation indicates that many of the same PBLs will respond to more than one mitogen, but that some cells require preactivation by one mitogen in order to respond fully to another mitogen. Thus, the number of PBLs which respond to PHA or PWM alone is much less than the number which respond following pretreatment with ConA when the pretreatment effect of ConA alone is blocked. Rabbit PBLs do not respond to LPS and preactivation by ConA does not prepare rabbit PBLs to respond to LPS.

Mixed lymphocyte reactions in the rabbit using peripheral blood cells: The effects of cell preparation and skin greating☆

Abstract The magnitude of mixed lymphocyte reactions using rabbit peripheral blood cells is subject to a number of variables including the method of cell preparation, the culture conditions employed, the genetic relationship of the cell donors, the number of bleedings immediately preceding the test bleed and the length of time that the cells are cultured. Mixed lymphocyte reactions (MLR) were carried out in microcultures and optimal responses were obtained when cultures contained 1 × 10 6 cells and were fed daily. Both two-way and one-way MLR were done using irradiation or mitomycin-C to inhibit the response of one donor population. The responses between various pairs of rabbits fell into three categories: high responders with 6,000–9,000 cpm 125 IUdR incorporated into DNA, low responders with 1,000–3,000 cpm incorporated and non-responders with 20–70 cpm incorporated. In all cases the background incorporation was less than 70 cpm. There was no MLR prior to the third day of culture; the response peaked on the fifth or sixth day and declined thereafter. Repeated bleeding of the rabbits at short intervals resulted in decreased MLR. Lymphocytes derived from defibrinated blood were primarily responding cells whereas lymphocytes from heparinized blood were stimulatory cells. The responding cells were sensitive to killing by anti-thymocyte serum (ATS) and complement. There was some evidence that the magnitude of MLR is affected by genetic relatedness. The duration of skin graft survival in these studies was not related to the magnitude of MLR.