Secondary cytotoxic allograft responses in vitro. II. Differentiation of memory T cells into cytotoxic T lymphocytes in the absence of cell proliferation.

Murine cytotoxic T lymphocytes (CTL) were induced in "one-way" mixed lymphocyte cultures and their physical characteristics investigated by velocity sedimentation at 1 X g. The in vitro differentiation of progenitors of CTL into primary and secondary CTL was paralleled by characteristic changes in the size of the responder cells. Fractionated cells enriched for primary blast CTL reverted into clonally restricted "nonlytic" secondary T lymphocytes. Upon antigenic reexposure, these lymphocytes differentiated into secondary CTL within 18-32 h. This took place in the absence of cell proliferation and could be triggered by UV light irradiated allogeneic stimulator cells. It is suggested the different characteristics for the induction of either a primary or secondary cytotoxic T cell response reflect qualitative differences between unprimed T cells and memory T lymphocytes.     

Immunoregulation by mouse T cell clones. III. Cloned H-Y-specific cytotoxic T cells secrete a soluble mediator(s) that inhibits cytotoxic responses by acting on both Lyt-2- and L3T4- lymphocytes

In this study we report that cloned Thy-1+, L3T4-, Lyt-1-, Lyt-2+, H-Y-specific and H-2Db-restricted cytotoxic T cell lines (CTLL) when induced by lectin or antigen secrete a soluble mediator(s) (SF) that inhibits proliferation and generation of cytotoxic lymphocytes (CTL) in mixed lymphocyte cultures (MLC). The biological activity was separable by gel filtration and appeared as a broad peak in the molecular mass range between 10 000 and 50 000 kDa. It was found that the suppressive activity released by CTLL neither strictly correlates with their cytotoxic potential nor with their ability to produce immune interferon or lymphotoxin. SF was shown to elicit its activity in an antigen-nonspecific manner in that it suppressed the maturation of T lymphocytes responding to both, the appropriate H-Y antigen as well as to unrelated H-2d alloantigens or to the hapten 2,4,6-trinitrophenyl (TNP). The effect of SF on CTL responses was most pronounced in early phases of primary or secondary MLC. When analyzed for its inhibitory activity on precursor cells in populations selected for either Lyt-2- or L3T4- lymphocytes, it was found that SF interfered with the maturation of both subsets. The inhibition of CTL responses elicited by SF could not be reversed by the addition of exogenous interleukin 2. The finding that SF also inhibited the proliferation of some but not all antigen-dependent cloned T cells with helper or cytotoxic potential provides evidence that the factor also may regulate effector lymphocytes. In addition, the results support the assumption that SF exerts its effect directly on the responder rather than the stimulator population, and demonstrate that the development of CTL from their precursor cells is controlled at least in part by the cytotoxic effector cells themselves via a soluble factor(s) that interferes with distinct stages of T cell maturation. These findings again emphasize the expression of multiple functions by CTL and indicate their possible role during the course of an immune response by their capability to eliminate target cells and to secrete a soluble product(s) that mediates feedback control.     

I. Characterization of cytotoxic effector cells generated from regional lymph nodes after immunization in the footpad.

A system is described which involves an in-vivo and an in-vitro step for the generation of strong primary cytotoxic T lymphocyte (CTL) responses to multiple minor alloantigens. The protocol consists of immunizing mice in the footpad with irradiated, minor alloantigen-different spleen cells. Four days later, cells from the lymph nodes draining the site of injection are cultured for 3 days in the absence of antigen to allow the development of cytotoxic activity. The analysis of the effector phase of the response indicates that the killer cells are H-2 restricted cytotoxic cells specific for minor alloantigens. The in-vitro phase of these cytotoxic responses is T cell-dependent and the responder cells proliferate strongly during the culture period. Both cytotoxic and proliferative responses are abolished by passing responder cells over Sephadex G-10 prior to culture. The results suggest that the maturation process of CTL-precursor (CTL-P) cells into effector CTL in vitro depends upon proliferating T cells and accessory cells.     

Interleukin 5 is a differentiation factor for cytotoxic T lymphocytes

The role of IL-5 on the generation of cytotoxic T lymphocytes (CTL) was analysed using a culture system in which production of T helper cell factors was abrogated by exposure of the stimulator cells to ultraviolet irradiation. Supernatants from a T helper cell line (2.19 sup), recombinant (r) IL-2, rIL-4 and rIL-5 were then tested for the capacity to replace T cell help, on the generation of CTL. The results showed that the specific CTL response, in unseparated spleen cells, could be reconstituted by either 2.19 sup, IL-2 or IL-4. However, if the responder cells were purified in nylon wool, only 2.19 sup or rIL-4 plus rIL-5, but not each lymphokine per se, reconstituted the CTL response. Because IL-5 does not support T cell proliferation, it is suggested that IL-5 induces differentiation of immature precursors into CTL. Based on these findings and in an attempt to conciliate the conflicting views that have emerged from different reports, as to whether IL-2 by itself could support generation of CTL in purified T cells, a hypothesis is formulated, suggesting that T cell at different stages of differentiation require distinct lymphokines to acquire CTL effector function.     

The effect of cyclophosphamide on cytotoxic T-lymphocyte responses: inhibition of helper T-cell induction in vitro.

The effects of cyclophosphamide (Cy) on the different cell populations participating in the cytotoxic T lymphocyte (CTL) response against haptenated (trinitrophenyl, TNP) syngeneic cells were studied. Pretreatment of responder cell donor mice with 150 mg/kg Cy decreased the cytotoxicity against TNP-modified syngeneic target cells almost to the background level. When TH cells were added to the culture the cytotoxicity increased significantly. Helper T cells were generated in vivo by priming the mice with TNP-modified syngeneic spleen cells or sensitizing the mice with a reactive hapten (TNCB). However, if the TH cell donor mice were treated with Cy before in vivo priming, the cytotoxicity reached the normal level, which indicated that TH precursors were not destroyed by Cy treatment and TH induction was even more effective after Cy. These data indicate that the decrease of the response by this Cy dose is not due to the sensitivity of CTL or TH precursors. Mice could be primed with male-specific (HY) antigen in spite of Cy pretreatment. However, Cy pretreatment caused a latent period of 2 weeks when effective CTL could not be generated in vitro, but after that the capacity for CTL generation was restored. These experiments confirm that pretreatment of responder cell donor mice with Cy does not destroy CTL or TH precursors, but rather affects their in vitro restimulation probably by destroying a short lived 'inducer' cell that is needed.     

Trophoblast Does Not Cause the Cytotoxic T Lymphocyte Generation Due to the Lack of Ability to Stimulate lnterleukin-2 Production

ABSTRACT: Trophoblast was demonstrated to be unable to cause the production of interleukin-2 (IL-2) by allogeneic splenocytes in vitro in two ways: 1) The addition of lymphocyte-trophoblast culture-supernatant (LTC-SN) did not stimulate the proliferation of IL-2 dependent cytotoxic T lymphocyte (CTL-L cells; 2) When responder cells were cultured with heat-treated splenocytes (usually no CTL generation) an increase of CTL formation could be seen in the presence of mixed lymphocyte culture-supernatant (MLC-SN) but not of SN from the cultures in which trophoblast cells served as stimulators. In parallel, the trophoblast cells were found to be very poor stimulators of alloreactive CTL. The addition of interleukin-2-enriched media resulted in a significant amplification of trophoblast-induced CTL generation. The resulting killing lymphocytes were capable of destroying the only specific targets, did not lyse syngeneic target cells, and could not be generated in the absence of allogeneic trophoblast. The incubation of these lymphocytes with anti-Thy 1.2 monoclonal antibody in the presence of complement eliminated their killing effect. Lack of class II antigenic determinants on the surface of trophoblast and/or local immunosuppression of IL-2 production by trophoblast cells seem to be responsible for nondelivery of helper factor, which is essential for CTL production.     

Secondary cytotoxic allograft response in vitro. I. Antigenic requirements

The antigenic requirementsfor in vitro induction of secondary murine cytotoxic allograft responses were tested. The proliferative responses were assayed by the [³H]thymidine uptake technique; the generation of cytotoxic T lymphocytes (CTL) was tested in a ⁵¹Cr-cytotoxicity assay. Spleen cells from normal or alloantigen preimmunized CBA mice (H-2^k) were used as responder cells. Allogeneic x-irradiated splenic lymphocytes (normal stimulator cells) were UV light treated, heat treated or glutaradehyde fixed and subsequently tested for their capacity to induce CTL in a primary or secondary mixed lymphocyte culture (MLC). In addition allogeneic fibroblasts were tested as stimulator cells. The results obtained suggest that although they fail to trigger significant proliferative and cytotoxic T cell responses, in a primary MLC certain allogeneic stimulator cells, are able to induce strong cytotoxic T cell activity in a secondary MLC. The generation of these secondary CTL is preceded by only marginal cell proliferation.     

树突状细胞疫苗与泌尿系统肿瘤的生物治疗

肿瘤生物治疗的基本策略之一是激活肿瘤抗原特异性的细胞毒T细胞,诱导抗肿瘤免疫反应,这一过程必须要有抗原提呈细胞的参与.树突状细胞的抗原提呈功能极强,它能激活幼稚T细胞分化增殖,并建立初级免疫反应.近年来研究应用多种形式的抗原(肿瘤细胞裂解物、肿瘤蛋白、RNA等)负载树突状细胞,再将经过修饰的树突状细胞回输入动物模型或人体内,取得了较好的效果.本文综述了近年树突状细胞的研究进展及其在泌尿系统肿瘤的中研究和应用情况.     

Suppression of inducible CD4 regulatory cells by MHC class I-restricted human tumor epitope specific TCR engineered multifunctional CD4 T cells

Regulatory T cells (Treg) can interfere with the generation and function of anti-tumor immune effectors. Accordingly, ways that could block Treg function would be useful in cancer immunotherapy. We have previously shown that incorporation of CD4+CD25-ve T cells in an in vitro cytolytic T lymphocyte (CTL) generation assay leads to generation of induced regulatory T cells (iTregs), and that these iTreg block the generation of productive CTL response (Chattopadhyay et al., 2006). We here show that human CD4 T cells engineered to express MHC class I-restricted human melanoma associated epitope, MART-1_27–35, specific T cell receptor (TCR), that can simultaneously exhibit helper as well as cytolytic effector functions (Chhabra et al., 2008, Ray et al., 2010), can interfere with the generation of inducible Treg, block iTreg-mediated suppression, and allow the activation and expansion of MART-1_27–35 specific CTL responses, in vitro. We also show that mitigation of Treg generation by TCR engineered CD4 T cells is not mediated by a soluble factor and may involve “licensing/conditioning” of the dendritic cells (DC). Our data offer novel insights on the biology of MHC class I restricted TCReng CD4 T cells and have translational implications.     

Mutual tolerization of histoincompatible lymphocytes

T lymphocytes react strongly against foreign major histocompatibility complex encoded class I antigens by destroying incompatible tissue in vivo, and by generating cytotoxic T lymphocytes (CTL) in vitro. The absence of reactivity against self antigens may be due to clonal deletion of self-reactive T cells during their ontogeny in the thymus. The functional clonal deletion of mature T cells in the periphery was described recently: CTL recognizing antigen on other CTL are eliminated (Rammensee et al., Immunol. Today 1985. 6: 41). Hence, in a normal immune system only autoreactive cells would be eliminated. Here we show that injection of lymphocytes into class I-incompatible mice leads to abrogation of host anti-donor as well as donor anti-host reactivity, leaving a mixed population of host and donor T cells reactive against third-party antigens. The results demonstrate the existence of a peripheral failsafe mechanism for the elimination of autoaggressive CTL. Whether this failsafe mechanism is actually used under physiological conditions is a different question.     

Three-dimensional collagen matrices as cell culture substrata affect the generation of alloreactive cytotoxic T lymphocytes

Primary one-way mixed lymphocyte cultures (MLC) of C3H/He responder and DBA/2 stimulator were performed in three-dimensional (3-D) collagen matrices and the generation of alloreactive cytotoxic T cell (CTL) responses was compared to those in MLC which were done on usual plastic surfaces or on collagen-coated plastic surfaces. MLC in the 3-D collagen matrices were found to generate strong CTL responses. Flow cytometric analysis of Lyt-2 and L3T4 antigen expressions on the effector cells showed that the Lyt-2/L3T4 ratios were substantially higher in the 3-D collagen matrices, and that a larger proportion of the cells in the 3-D collagen matrices were Lyt-2+ lymphoblasts. These results indicate that the milieu of the 3-D collagen matrices favors the proliferation of Lyt-2+ lymphocytes, and suggests that cell-to-matrix interactions in 3-D collagen matrices may play a regulatory role in the maturation process of alloreactive CTLs.     

Primary cytotoxic T-cell response in vitro against Mls antigens in NZB-mice.

NZB-mice have a T cell hyperreactivity based on a primary response to minor histocompatibility antigens (MIH) on target cells identical to NZB on the H-2 complex (MHC). We tested the idea that a single MIH difference on MHC identical target cells is sufficient to elicit such a primary response in vitro. Cytotoxic T lymphocyte (CTL) response and activated T-helper cell (THC) frequencies were evaluated. NZB x CBA/J (Mls a/d) and NZB x CBA/Ca (Mls a/b) hybrids, which differ only at the M-locus, were raised. A primary cytotoxic response in the direction Mls b anti Mls d, but not vice versa was observed in vitro. In the assay used no unusual THC frequencies against Mls d could be demonstrated. The results favour cellular hyperreactivity in NZB which can be elicited by a single MIH antigen alone.     

Lymphocyte subpopulations in man: enhancement and inhibition in generation of cytotoxic T lymphocytes in vitro

The requirements for generation of allospecific CTLs in vitro have been studied by "three cell" experiments, with two allogeneic T cell suspensions as cocultured responders, stimulated by irradiated B cells. This study describes enhancement as well as inhibition of the response, functionally defining T amplifier and T depressor cells regulating the differentiation of CTLs. Enhancement is the result of an amplifying effect of cytotoxic precursor T cells. Amplification is due to HLA-D region incompatibility between the responding T cell donor and the stimulating donor resulting in strong proliferation. Inhibition is the result of a depressing effect on cytotoxic precursor T cells mediated by cocultured T cells. The depression seems to be due to HLA-A, -B, -D identity between one of the responder T cell donors and the stimulator cells. The induction of depression is radiosensitive, accompanied by strong proliferation and CTL generation with cytotoxic specificity against the cocultured and depressed donor target cells. It is suggested that the functionally defined T depressor cells are cytotoxic precursors mediating cytostatic functions before strong cytotoxicity is detectable.     

Cellular cytotoxicity generated in a canine mixed kidney lymphocyte cell culture

Canine cultured kidney epithelial cells were stimulatory to allogeneic lymphocytes in mixed kidney cell - lymphocyte cultures (MKLC). Generation of cytotoxic cells, cytotoxic for both kidney cells and PHA stimulated lymphoblasts of the stimulator have been observed. Lymphocyte stimulation and generation of CTL occurred in the MKLC at lower stimulator: responder cell ratios as in the mixed lymphocyte culture (MLC).     

Modulation on immunogenicity by HLA-B27 subtype polymorphism

Cells from the same HLA-B27- individual, PA, were stimulated in vitro in primary mixed lymphocyte culture, with either B*2705+ or B*2704+ lymphoblastoid cell lines, in independent experiments. Cytolytic T lymphocytes (CTL) were cloned at limiting dilution and the clones obtained were screened for anti-B27 alloreactivity. Most of the CTL clones generated against the B*2705+ stimulator cells were directed against the B*2705 antigen. In contrast, no anti-B27 CTL clones were found among those derived against the B*2704+ stimulator cells. This was not due to a poor cytotoxic response against these cells because a large proportion of the T cell clones derived from this stimulation were cytotoxic. B2704 differs from B*2705 by only two amino acid changes at positions 77 and 152. Previous studies (Aparacio, P. et al., Eur. J. Immunol. 1988.18: 203) have shown that none of the anti-B*2705 CTL clones derived from donor PA and amenable to detailed characterization cross-reacted with B*2704, suggesting that most of this cytotoxic response was directed against an immunodominant determinant contributed for by residues 77 and/or 152 from B*2705. The present results further suggest that the changes at these positions in B*2704 alter this determinant in such a way that B*2704 becomes less immunogenic for the particular individual PA. Furthermore, a similar poor anti-B*2704 CTL response was obtained from a second B27- responder individual, AE, stimulated with another B2704+ cell line. The single anti-B*2704 CTL clone, 64.8P, isolated from this second individual, displayed an unusual reaction pattern in that it cross-reacted with all B27 subtypes with changes only at or close to positions 77 and 152, including B*2705. Significantly, the only HLA-B27 subtype that was not recognized by CTL 64.8P was B*2703, which differs from B*2705 only at residue 59. This residue is located in the three-dimensional structure at the opposite end from residues 77 and 152 at the surface of the antigen-binding groove of the class I molecule. Thus, the area around residues 77 and 152 is not an essential part of the epitope recognized by CTL 64.8P.     

A role for IL-5 in the induction of cytotoxic T lymphocytes in vivo

IL-5 is generally regarded as a Th2 cytokine involved in eosinophil maturation and function and in B cell growth and antibody production, but without any well-established effects on T cells. Early reports suggested that IL-5 could stimulate the production of cytotoxic T lymphocytes (CTL) in vitro, but no evidence has been obtained to date for such a role in studies with IL-5-deficient (IL-5-/-) mice. Here we demonstrate that when oxidized mannan MUC1 fusion protein (M-FP) is used as an antigen in mice, IL-5 is required for the optimal generation of the CTL response. IL-5 was as effective as IL-2 for the induction of CTL from spleen cells in vitro and both CD4+ and CD8+ T cells from M-FP-immunized animals could be shown to secrete IL-5 in culture. In IL-5-/- mice, CTLp frequency was greatly diminished resulting in the inability to reject MUC1- tumors. Clearly, IL-5 is produced by functional T cells, especially the Tc1 type, after M-FP immunization and is required for an optimal CTL response to this antigen.     

Regulation of human cytotoxic lymphocyte responses. I. Non-cytolytic suppression mediated by alloantigen-activated cells.

Alloantigen sensitized human lymphocytes obtained from a 2-3 day mixed lymphocyte culture (MLC) suppressed the in vitro generation of alloreactive cytotoxic lymphocytes (CTL). The inhibition of CTL responses was demonstrated in MLC after both 1 day and 14 days' allosensitization. The suppressor cells were nylon wool non-adherent, lacked Fc receptors and adhered to histamine columns. The MLC-activated suppressor cell population had an associated very low and transient cytotoxic response directed against the allogeneic sensitizing cell. Several procedures were used to dissociate this activity from suppressor cell function: (1) donors were preselected which showed minimal cross-killing between allogeneic stimulating cells, (2) suppressor cultures were added to the test cultures prior to the development of maximal CTL activity, (3) suppressor cultures were irradiated preventing cell proliferation associated with differentiating CTL. Lastly, by increasing stimulating antigen concentration suppressor cell activity was increased rather than being competitively diminished, which would be predicted if suppression was occurring through a cytolytic or inactivation of stimulating antigen. It was therefore concluded that alloantigen stimulation in MLC activates a non-cytolytic regulatory cell population which is capable of inhibiting CTL responses to third-party allogeneic lymphocytes.     

Antigen-independent activation of cytotoxic T cells by lymphokines.

Supernatants from phorbol myristate acetate (PMA)-stimulated EL4.IL2 cells (EL4.PMA), but not recombinant IL-2 (rIL-2), induced the production of cytotoxic T lymphocytes (CTL) in low density murine spleen cell cultures. CTL induction in these cultures was completely abrogated by treatment with anti-Thy-1 or anti-Lyt-2 antibody plus complement but not by anti-L3T4 antibody plus complement. Fractionation of EL4.PMA on a Sephadex G-150 column demonstrated that the CTL-inducing activity in EL4.PMA eluted with an apparent molecular weight of about 44,000 and was partially separated from IL-2. This 44,000 MW material was shown to contain insignificant amounts of PMA. Following a 3-day culture period with the partially purified factor, C57BL/6J thymocytes could proliferate and differentiate into cytotoxic cells in response to rIL-2, whereas there was no proliferation or generation of cytotoxic cells when the thymocytes were cultured in rIL-2 alone. The number of IL-2 receptor-positive cells in C57BL/6J thymocytes also increased from 1.1% to 22.8% after 3 days of culture in the partially purified factor. Recombinant IL-4 (BSF-1) and IL-5 (TRF), when used alone or in combination with rIL-2, were unable to induce a cytotoxic response under similar culture conditions. These findings are consistent with the interpretation that EL4.PMA contains a novel lymphokine that directly, or indirectly, induces the expression of IL-2 receptors on resting CTL precursors without intentional stimulation by specific antigen. In the presence of IL-2, these precursors may then differentiate into effector CTL.     

Cytotoxic T lymphocyte precursor cells specific for the major histocompatibility complex class I-like antigen, Qa-2, require CD4+ T cells to become primed in vivo and to differentiate into effector cells in vitro.

Experiments were performed to determine whether CD4+ T cells are required for the generation of cytotoxic T lymphocytes (CTL) specific for the nonpolymorphic major histocompatibility complex (MHC) class I-like antigen, Qa-2. Splenic T cells from BALB/cBy (Qa-2b) mice that had been immunized with irradiated BALB/cJ (Qa-2a) splenocytes generated CTL following in vitro stimulation with BALB/cJ splenocytes. These CTL lysed all Qa-2+, but not Qa-2- targets, regardless of the H-2 haplotypes of target cells or their non-MHC backgrounds. This apparent MHC class I-unrestricted recognition of Qa-2 antigen was confirmed using Qa-2-specific CTL clones. The Qa-2-primed CTL precursor cells (CTLp) and CTL were found to be CD8+ T cells. Primed splenocytes depleted of CD4+ T cells prior to culture failed to generate CTL, but addition of lymphokines to the culture restored the CTL generation. Stimulation of primed splenic T cells with irradiated Qa-2+ T blast cells, instead of splenocytes or B blast cells, led to little to no CTL generation, suggesting that MHC class II molecules are involved in the presentation of Qa-2 antigen to CD4+ T cells. This was also supported by the results of experiments using Qa-2+, class II- thymoma cells of BALB/c origin. Stimulation of the thymoma-primed splenic T cells with the mitomycin C-treated thymoma cells resulted in no generation of anti-Qa-2 CTL, despite the fact that high levels of CTL specific for minor histocompatibility (H) antigens and H-2d were generated by immunizing the corresponding allogeneic hosts with the thymoma. However, the addition of lymphokines rendered thymoma-primed T cells capable of generating anti-Qa-2 CTL. Both CD4+ and CD8+ T cell populations, isolated from the BALB/cJ splenocyte-primed responder cells, proliferated in vitro in response to the Qa-2+ splenocytes, suggesting that Qa-2-reactive CD4+ T cells were present in the immunized mice. Depletion of CD4+ T cells from thymectomized BALB/cBy mice with anti-L3T4 monoclonal antibodies markedly reduced, but did not eliminate anti-Qa-2 CTL generation. In contrast, depletion of CD8+ T cells led to a complete abrogation of the CTL response. Addition of lymphokines to the culture of responder cells depleted of either T cell subset did not restore their reactivity. It is concluded that anti-Qa-2 CTLp need "help" from CD4+ T cells to become primed in vivo. Furthermore, primed CTLp also need "help" or lymphokines provided by CD4+ T cells to differentiate into effector CTL in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

A monoclonal antibody that blocks class II histocompatibility-related immune interactions

Previous studies using conventional hetero- or isoantisera have indicated the involvement of class II (Ia) molecules in presentation of soluble by monocytes to inducer T lymphocytes, stimulation of inducer T cells in MLR, and recognition of Ia-bearing targets cells by cytotoxic T lymphocytes (CTL). The experience in using monoclonal anti-Ia reagents capable of blocking these phenomena in the human system is limited. Recently, however, we have characterized a lytic IgG2a monoclonal antibody, 9–49, that binds to functionally significant class II molecules. This antibody blocks (in the absence of complement): (1) specific binding of peripheral blood lymphocytes (PBL) to antigen-pulsed monocyte monolayers, (2) proliferation of PBL in response to soluble antigen (tetanus toxoid or mumps) or cell surface class II antigen stimulation in allogeneic or autologus MLR, (3) proliferation of cloned T4+ (inducer) lymphocyte cell lines to class II antigens, (4) generation of cytotoxic lymphocytes during allogenic MLR, and (5) recognition (and killing) of class II-bearing target cells by T4+ CTL clones. Proliferation and CTL activity of a T8+ clone is unaffected by the 9–49 antibody. These results indicate the usefulness of this monoclonal reagent in studies evaluating the functional role of Ia molecules in immune recognition phenomena.