A comparison of serum-free media for the support of in vitro mitogen-induced blastogenic expansion of cytolytic lymphocytes

The high cost and potential dangers of including human AB serum in incubation media used to expand lymphocyte populations in vitro for adoptive immunotherapy have stimulated efforts to develop defined media which can support both the expansion and induction of lymphocytes with tumor cytolytic activity in the absence of serum. Lymphocyte proliferation following exposure to either PHA or the combination of phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore, ionomycin, was evaluated. Although the media tested, X-Vivo 10, HB-104, AIM V, and HL-1, supported the generation of comparable levels of LAK activity after 3–5 days incubation with 10³ U human recombinant interleukin-2 (rIL-2)/ml, there were striking differences in the ability of each medium to support mitogenically stimulated lymphocytes in the absence of serum, with cells in AIM V and X-Vivo 10 showing the highest levels of DNA synthesis. In long-term cultures (17 days) of blood MNC stimulated by PDBu and ionomycin, X-Vivo 10 and HB-104 yielded the greatest numbers of cells. The addition of 2% AB serum greatly enhanced the ability of each medium to support cell proliferation to equivalent maximum levels. The results indicate that while all four serum-free media were suitable for lymphocyte culture and support the development of LAK activity, they differ in their capacity to support expansion of lymphocyte populations in response to polyclonal mitogenic activation. This latter characteristic should be considered before choosing a particular serum-free formulation as its constituents may affect mechanistic interpretations regarding signal transduction events.     

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.     

Primary in vitro antibody response from human peripheral blood lymphocytes.

A method for the induction of a primary in vitro antibody response from human peripheral blood lymphocytes is presented. Upon cultivation with trinitrophenyl conjugated polyacrylamide beads (TNP-PAA), an anti-TNP response can be obtained as indicated by the appearance of direct plaque-forming cells from day 5 of culture, with a reproducible peak on day 8. These plaques correspond to cells actively producing antibody of the IgM type, as shown by their inhibition by cycloheximide and by anti-human IgM serum, but not by anti-human Fc gamma serum. Their specificity for the TNP hapten can be demonstrated by the effector cell blockade phenomenon, with highly substituted TNP-human IgG. Although the anti-TNP response induced by TNP-PAA in mouse spleen cell cultures appears T independent the same response in human PBL may involve in addition the participation of T cells, since E-RFC depletion before culture led to a markedly decreased number of plaque-forming cells. A significant response could be obtained from the PBL of all of the 30 normal individuals tested. Importantly, the response was reproducible in its magnitude in the six individuals tested in at least three different experiments. Thus, the in vitro stimulation of human PBL by TNP-PAA can be proposed as a reliable test for the study of human B cell function in a specific primary antibody response.     

Cellular immunity in the mouse. I. In vitro lymphocyte reactivity

The mixed lymphocyte culture (MLC) and antigen-mediated proliferative response represent important correlates to the in vivo phenomena of allograft rejection and delayed hypersensitivity. This study defines an in vitro model to measure mouse lymphocyte responsiveness to allogeneic cells, antigen (tuberculoprotein), and nonspecific mitogens. Results describe optimal cells concentration, time and conditions of culture. Optimal conditions include the use of high cell concentration, flat-bottomed vials, RPMI-1640 medium, and fresh human serum. Peripheral blood lymphocytes demonstrated greater proliferation than lymph node lymphocytes, which in turn demonstrated greater activity than splenic lymphocytes. Significant proliferation occurred in serum-free media, dialyzed against fresh serum and supplemented with hydrocortisone and carrier protein. The MLC response in the mouse appears dependent on multiple subpopulations of cells and on soluble substances produced by them.     

Cellular origin of cytotoxic effectors and secondary educated lymphocytes in human mixed leukocyte reaction

Human lymphocytes sensitized in vitro during a mixed leucocyte reaction (MLR) against an allogeneic-stimulating cell respond by blast transformation and generation of specific cytotoxic effector cells. Both proliferation and cytotoxicity are maximum on Days 6 and 7 of culture. On Day 14, no more dividing cells or cytotoxic cells are detected in such primary cultures. Restimulation by the specific priming cell triggers a secondary proliferative response and rapid reappearance of specific cytotoxic effector cells. The velocity sedimentation cell separation method which separates cells according to their size was applied to human lymphocytes sensitized in vitro during an MLR on Day 7 of culture. Blast cells were separated from nondividing small lymphocytes. It was shown that: (1) cytotoxic effectors generated at the peak of a primary response are exclusively present in the isolated blast population; (2) highly cytotoxic secondary effector cells are induced to reappear mainly from the blast-derived population upon restimulation; and (3) secondary educated proliferative cells mainly derive from the blast population. Conversely, the blast-depleted small lymphocyte population is operationally depleted of cells able to respond by proliferation to the priming cell while responding normally against third party control cells. HLA-D region specificity of the secondary proliferative response is suggested.     

In vitro proliferation of rabbit bone marrow-derived and thymus-derived lymphocytes in response to vaccinia virus

Peripheral blood leukocytes from rabbits immunized with vaccinia virus were incubated in vitro with vaccinia antigen, and resultant lymphocyte proliferation was measured by incorporation of tritiated thymidine into acid-insoluble material. Significant lymphocyte stimulation was observed at a time when antiviral antibody was being synthesized in vivo. The extent of proliferation by bone marrow-derived lymphocytes after culture with viral antigen was determined by simultaneous detection of complement receptor lymphocytes (CRLs have been shown to be B cells) and uptake of tritiated thymidine in these CRLs by radioautography. The results indicate that both bone marrow-derived and thymus-derived lymphocytes participate in the in vitro proliferative response of rabbit peripheral blood lymphocytes to vaccinia antigen.     

Acceleration of human lymphocyte activation after preliminary culture

Human peripheral blood mononuclear cells which are precultured in tissue culture medium containing 5% fetal calf serum (FCS) have markedly augmented reactivity to a suboptimal concentration of phytohemagglutinin (PHA). The augmentation of response is due in part to an acceleration of lymphocyte activation. Precultured cells also respond in an accelerated manner to allogeneic cells. The augmentation is apparent after 24 hr of preliminary culture, and reaches a maximum at 4 to 7 days. Kinetic studies of the culturally augmented PHA response indicate that an increased number of reactive lymphocytes rather than an increased rate of proliferation accounts for the effect. Adding fresh cells to the precultured cells does not suppress their augmented response. When human serum is substituted for FCS in the preliminary culture, the augmentation of response does not occur. Serum-mixing experiments indicate a suppressive effect of human serum. These studies may be relevant for understanding immunologic mechanisms related to transplantation and autoimmunity.     

The response of human peripheral blood lymphocytes to phytohemagglutinin: determination of cell numbers.

Optimization and definition of conditions for studying lymphocyte function in vitro resulted in exponential proliferation of lymphocytes from day 2 to day 5 with an average doubling time of 20 hr. The number of cells in culture on day 5 was 5–10 times as great as the number initially planted and 10–20 times as great as the number surviving in culture on day 2. An improved pronase-cetrimide technique was used to determine the number of viable lymphocytes as a function of time after addition of PHA. The volume changes in nuclei, obtained after cetrimide treatment, were quantitated using a curve-fitting computer program.The response could be described in terms of an induction phase (0–2 days) characterized by a decrease in cellularity and an increase in nuclear volume, a proliferation phase (2–5 days) characterized by an exponential proliferation and a continued increase in the number of cells having a large nuclear volume, and a lysis phase (5–14 days) characterized by a decrease in cellularity and a decrease in nuclear volume. The results reported here suggest that the ratio of the number of cells cultured to the volume of culture medium was crucial for optimal transformation and proliferation, 10⁵ cells/ml producing far better responses than 10⁶ cells/ml.     

Age-related alterations in cell division and cell cycle kinetics in control and trimethyltin-treated lymphocytes of human individuals

Trimethyltin chloride induced age-related suppression of cell division and cell cycle kinetics in human peripheral blood lymphocytes cultured in RPMI 1640 culture medium supplemented with human AB serum, phytohemagglutinin and bromodeoxyuridine. A high frequency of M₁ (first metaphase) cells was seen in cultures treated with a high dose (C ₁ = 1.0 g per culture) and in lymphocytes from donors in the age range 40–70 years. The delay in cell division and cell cycle kinetics may indicate a longer duration in DNA synthesis induced by trimethyltin chloride in aged lymphocytes.     

An in vitro model for induction of immunological unresponsiveness to turkey gamma-globulin in primed mouse spleen cells. I. The secondary in vitro response and induction of unresponsiveness.

Spleen cells from mice previously immunized with turkey γ-globulin (TGG) were shown to give a vigorous secondary response in vitro when challenged in Mishell-Dutton cultures with TGG covalently coupled to pig erthrocytes (TGG-PRBC). However, 90–100% of the response could be abrogated by the incorporation of soluble TGG (sTGG) into the culture medium at concentrations greater than 1 mg/ml. Unresponsiveness, as measured by the absence of plaque-forming cells (PFC) in cultures receiving sTGG, was found to be antigen specific in that these cultures were still able to give normal PFC responses to sheep or burro erythrocytes. Spleen cells incubated with sTGG for short periods of time were shown to remain unresponsive after removal of sTGG from the culture and addition of TGG-PRBC. A 1-hr exposure period resulted in greater than 70% Unresponsiveness and a complete unresponsive state required only 8 hr of exposure. In contrast to the continued Unresponsiveness of sTGG-treated cells in vitro, spleen cells incubated with sTGG for 24 hr were fully responsive to an immunogenic challenge with alum-precipitated TGG when they were transferred into irradiated syngeneic mice. These data suggest that the readily induced unresponsive state in cultures of TGG primed cells may involve either a reversible antigen blockade of antigen-sensitive lymphocytes or a peripheral inhibition of reactive cells by suppressor lymphocytes.     

Specific activation of human T lymphocytes by Robinia pseudoacacia seeds' lectin.

Human peripheral blood and tonsil lymphocytes were fractionated into T and B cells by centrifugation after rosetting with native sheep erythrocytes and tested with Robinia pseudoacacia lectin. The purity of B- and T-enriched populations was checked by E-rosette formation or heterologous antisera specific for B or T lymphocytes. The proliferative response of T cells to Robinia lectin from all the donors tested was not found to differ from that of unfractionated cells, whereas no response of highly purified B cells could be observed to the lectin even with different concentrations of the lectin and different culture periods. B cells, however, were found to bind as much ³H labeled Robinia lectin as unfractionated lymphocytes. In addition, treatment of cells by antihuman T-lymphocyte antigen (HTLA) serum and complement before addition of Robinia lectin completely abolished their response, whereas similar treatment by antihuman B lymphocyte and monocyte antigen (HBLMA) serum did not prevent the T cells from incorporating thymidine. The Robinia lectin, like the other phytomitogens, thus appears to be a specific T-cell activator.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. I. Activation by tuberculin and by Staphylococcus filtrate

Normal lymphocytes activated by mitogens such as phytohemagglutinin (PHA), by Staphylococcus filtrate (SF), or lymphocytes from sensitized individuals stimulated by antigen (PPD, etc.) are cytotoxic to tissue culture cells of different origins. In this and the following paper, the results of a detailed quantitative analysis of the specificity of this cytotoxic reaction are presented. Effector cells were human or mouse lymphocytes, activated by PHA, SF, PPD, or serum factors in the culture medium. Cells from established cell lines of human, mouse, hamster, or rabbit origin, or primary human or rat embryonic fibroblasts were used as target cells. Lysis was quantitated by release of ⁵¹Cr from labeled target cells.Purified human blood lymphocytes, activated by PPD, SF, or otherwise, preferentially damaged allogeneic target cells. Lysis of xenogeneic target cells was weak or did not occur. A close correlation was noted between target cell destruction and blastoid transformation of the lymphocytes, but the slope of the regression lines of xenogeneic cytotoxicity was much smaller than that of allogeneic cytotoxicity when plotted as a function of blastoid transformation.Lymph node or spleen cells from CBA mice were stimulated by PPD to transformation and DNA synthesis. CBA lymphocytes also showed an increased degree of blast transformation in medium containing fetal calf serum or certain batches of fresh human serum. Mouse lymphocytes activated in these ways damaged allogeneic L cells but had no effects on xenogeneic Chang cells.These results indicate that lymphocytes activated by various means preferentially damage target cells from their own species. The recognition mechanisms which determine the specificity of the reactions are not known.     

Control of proliferation in mitogen responsive human peripheral blood lymphocytes: restimulation of cells stimulated by sodium periodate.

Human peripheral blood lymphocytes are stimulated to a greater extent by sodium periodate when cells are incubated in medium containing human serum than when incubated in medium with fetal calf serum. NaIO4 STIMULATION CAN BE REVERSED BY TREATMENT WITH SODIUM BOROHYDRIDE BUT CELLS ALREADY COMMITTED TO DIVISION ARE NOT AFFECTED BY BOROHYDRATE TREATMENT. Maximal commitment to DNA synthesis of a NaIO4 oxidized cell suspens-on occurs after about 28 hr of incubation in medium. The committal time after periodate stimulation is identical to that after stimulation with concanavalin A. Cells treated with periodate and then reduced with borohydride immediately after oxidation are refractory to further per-odate stimulation. Cells stimulated with periodate and then incubated for 6 hr before treatment with borohydride can be restimulated with periodate, indicating a turnover of membrane sites in the 6 hr period. Periodate-stimulated cells divide only once in response to the stimulation. The progeny of cells which were stimulated with periodate can be restimulated by treatment with either periodate or concanavalin A.     

Human monocyte activation by supernatants from concanavalin A (con A) stimulated lymphocytes

Human peripheral lymphocytes were stimulated with Concanavalin A (Con A) in the absence of serum. Supernatants were collected from control and mitogen stimulated lymphocyte cultures and fractions pooled according to the elution before, together with or after human serum albumin which was added as a marker. Only one fraction derived from Con A stimulated lymphocyte culture Supernatants which eluted immediately after human serum albumin had a significant effect on the metabolism and structure of human monocytes in vitro. Monocytes separated by human serum albumin and incubated with this fraction for 20 hr had an increase in nuclear RNA synthesis. Monocytes attached to cover slips in Leighton tubes showed an increase in the percentage of phagocytizing cells and phagocytic activity. Electron microscopy demonstrated highly phagocytic cells containing numerous Golgi associated granules and strands of nondilated rough surfaced endoplasmic reticulum in presence of the active fraction.     

Fluorescence-activated cell sorting of human T and B lymphocytes. II. Identification of the cell type responsible for interferon production and cell proliferation in response to mitogens

We have employed the fluorescence-activated cell sorter to separate pure viable preparations of human T and enriched B lymphocytes. Using such preparations, we have demonstrated that both human T and B cells can respond to PHA and PWM in vitro in the presence of macrophages with proliferation and the production of interferon, a mediator of cellular immunity. However, selective T cell interferon production and proliferative response can be assessed at 3 days in culture; B cell interferon production and proliferative response is delayed to 5 and 7 days. T cells or T cell products are ineffective in inducing or accelerating B cell interferon or proliferative response at 3 days. The use of 3-day T cell interferon production as a new technique for the assessment of T cell effector function and competence is suggested.     

A ground-based model to study the effects of weightlessness on lymphocytes

The mitogenic response of human lymphocytes was found to be markedly reduced in weightlessness conditions as compared to normal gravity. One possible explanation is that due to the non-existent sedimentation in space the lymphocytes could not adhere and spread on a substratum. Thus, we investigated the effect of substratum adhesiveness on lymphocyte responsiveness by reducing and blocking cell adhesion with poly-HEMA in a simple on-ground system. Lymphocyte adhesiveness was assessed by measuring the proportion of non-adhesive, slightly, and strongly adhesive 51Cr-radiolabelled cells on uncoated and poly-HEMA coated plastic. The amount of cell spreading on surfaces with varying adhesiveness was determined by measuring the area of cells. Cells grown on medium and thick poly-HEMA films were rounded in shape. By contrast, on tissue culture plastic, they showed clear signs of spreading. The mitogenic response of lymphocytes grown on thick poly-HEMA films was reduced by up to 68% of the control (tissue culture plastic). Interferon-gamma production was virtually nil when the cells were grown on the least adhesive substratum. These results show that activated lymphocytes need to anchor and spread prior to achieving an optimal proliferation response. We conclude that decreased lymphocyte adhesion could contribute to the depressed in vitro lymphocyte responsiveness found in the microgravity conditions of space flight.     

Activation of human B lymphocytes: XVI. Cellular requirements,interactions, and immunoregulation of pokeweed mitogen-induced total-immunoglobulin producing plaque-forming cells in peripheral blood

The optimal culture and assay conditions for the detection of spontaneously occurring and pokeweed mitogen (PWM)-induced polyvalent Ig (IgG + IgM + IgA) and individual Ig class-specific plaque-forming cells (PFC) in human peripheral blood have been described in detail. Culture conditions are critical, particularly with regard to cell density and batches of supplemental serum. Fetal calf serum is a much more supportive serum supplement for PWM-induced PFC than is human serum. The assay system is a modified reverse hemolytic PFC assay using staphylococcal protein A coupled to sheep red blood cells by the chromic chloride method. PFC are developed by rabbit anti-human polyvalent Ig or anti-human individual Ig class antisera. Human peripheral blood contains 468 (±78) spontaneously occurring Ig secreting PFC per 10⁶ lymphocytes at Day 0 and 20,500(± 1971) PWM-induced Ig secreting PFC after 6 days in culture. The response is T-cell dependent; however, T cells can be replaced by a soluble T-cell factor prepared from a 48-hr allogeneic mixed lymphocyte reaction supernatant. The relative dependence on monocytes is a reflection of the culture conditions employed. Under the conditions of round-bottom tubes which promote cell-to-cell contact, depletion of monocytes to 0 to 2% does not result in a diminution of PFC responses. In fact, under such conditions, in certain individuals monocytes are markedly suppressive such that removal of monocytes results in a substantial enhancement of PFC responses. This system is simple and reproducible and should prove extremely useful in the delineation of the mechanisms of B-cell triggering and immunoregulation in normals and in disease states.     

Fate of Lymphocytes after Withdrawal of Tofacitinib Treatment

Tofacitinib (Tofa) is an inhibitor of Janus Kinase 3, developed for the treatment of autoimmune diseases and for the prevention of transplant rejection. Due to its selective action on proliferating cells, Tofa can offer a way to block T cell activation, without toxic effects on resting cells. However, few studies have investigated the effects of Tofa on lymphocyte activation in vitro. Our aim was to study the action of Tofa on different lymphocyte subsets after in vitro stimulation and to track the behaviour of treated cells after interruption of the treatment. Peripheral blood lymphocytes were stimulated in vitro with mitogen and treated with two concentrations of Tofa. After a first period in culture, cells were washed and further incubated for an additional time. Lymphocyte subsets, activation phenotype and proliferation were assessed at the different time frames. As expected, Tofa was able to reduce the activation and proliferation of lymphocytes in the first four days of treatment. In addition the drug led to a relative decrease of Natural Killer, B cells and CD8 T cells compared to CD4 T cells. However, treated cells were still viable after the first period in culture and begun to proliferate, strikingly, in a dose dependent manner when the drug was removed from the environment by replacing the culture medium. This novel data does not necessarily predict a similar behaviour in vivo, but can warn about the clinical use of this drug when a discontinuation of treatment with Tofa is considered for any reason.     

In vitro allogeneic response of human lymphocytes dependent upon dialyzable plasma components and a thymic hormone, THF.

The in vitro response to allogeneic stimulation of human lymphocytes obtained from umbilical cord blood and from adult peripheral blood is dependent upon the presence of dialyzable plasma components. We observed here a reduced allogeneic response of human lymphocytes in the presence of a dialyzed human plasma as compared to their reactivity in the presence of whole human plasma. This impaired mixed lymphocyte culture response was restored by the addition of thymus humoral factor (THF), a calf thymus extract. It is suggested that dialysis depletes the plasma of its natural thymic hormone(s) content, this being replaced by the addition of THF. Restoration of the in vitro allogeneic response of human lymphocytes by THF was shown to be specific, since such effect was not observed when calf spleen extract or bovine serum albumin were tested.     

In vitro response of subpopulations of human lymphocytes. II. DNA synthesis induced by anti-immunoglobulin antibodies.

A significant and constant increase in DNA synthesis was observed in human lymphocytes cultured in the presence of purified anti-immunoglobulin antibodies specific for human IgG, IgA, and IgM. This has been found in cultures of lymphocytes isolated from blood, tonsils, spleen, and lymph nodes. The optimal culture conditions for blood and tonsil lymphocytes were determined. As a rule 6-day cultures containing 2 x 10(6) cells/ml and 100 mug/ml of antibody yielded the highest 3H-thymidine uptake. Purified T cell cultures could not be stimulated, whereas a low response could be observed in most of the purified B cell cultures. Optimal culture conditions were the same for the B and total tonsil lymphocytes. However, when the purified B cells were totally depleted of T cells, no response was observed. A T and B cell synergy has been demonstrated by supplementing B cell cultures with purified T cells, whether treated or not with mitomycin. These experiments indicated a permissive and potentiating effect of T cells on the B cell response. Cultures containing mitomycin-treated B cells and purified T cells (mB + T) could be stimulated by a-Ig, thus indicating a T cell proliferation. In keeping with this finding was the observation of an increased response of total lymphocytes supplemented with T cells but not with B cells. Adherent cells are necessary for an optimal response to a-Ig; they enhanced the B cell proliferation observed in (Tm + B) cultures and suppressed the response of T cells in (T + Bm) cultures.