Motility of IL-2-stimulated lymphocytes in neutral and acidified extracellular matrix.

The migration of lymphocytes through extracellular matrix (ECM) is an essential feature of the infiltration process. In the course of their extravasation into poorly perfused neoplastic lesions, lymphocytes often encounter regions of acidified ECM. This study was designed to determine whether lymphocyte adherence and motility in ECM are influenced by ambient pH. Murine splenic lymphocytes, activated by culture with high-titer IL-2, were allowed to migrate into three-dimensional gels of Type I collagen, a major component of interstitial stroma, or into Matrigel, a basement membrane model. After 18 hr at pH 7.1, the leading cell front traveled a mean distance of approx 475 microM into Type I collagen gel. Approx 50% of the cells remained nonadherent, 25% adhered to the gel surface, and 25% were motile (penetrated beneath the surface). At pH 6.7, the leading-front distance increased significantly, by a factor of 1.4X, but there was little change in the proportion of cells exhibiting nonadherence, surface adherence, or motility. The relative motilities of CD3+ and AsGM1+ subsets were also unaltered. It therefore appears that acidification of collagen matrix increases the locomotory activity of motile lymphocytes, but causes little recruitment of nonmotile lymphocytes into the motile pool. Similar results were obtained in experiments with Matrigel. The increased motility observed at pH 6.7 did not reflect breakdown or relaxation of matrix lattices, as measured by the passive diffusion of latex beads of defined diameter. Preincubation of lymphocytes at pH 6.7 did not alter their subsequent motility in pH 7.1 gels. The findings establish ambient pH as a microenvironmental variable which can influence lymphocyte migration through ECM. The weak acidity characteristic of certain tumor microenvironments may be a factor which encourages lymphocyte infiltration through tissue matrix. Treatments which alter intratumor pH could potentially be used to manipulate the infiltration process for immunotherapeutic benefit.     

Lymphocyte-fibroblast adhesion induced by interferon-gamma

Adhesion of lymphocytes to vascular endothelium is thought to be of importance in regulating the passage of lymphocytes from the circulation to areas of inflammation. Evidence suggests the presence of site-specific lymphocyte receptor molecules on the endothelial cell surface which can be modulated by soluble immune factors. The factors responsible for maintaining lymphocyte infiltration at tissue sites are unknown. We have examined the adherence of human peripheral blood T lymphocytes to human fibroblast monolayers in vitro and the role of interferon-gamma in enhancing adherence. Treatment of fibroblasts with interferon-gamma resulted in an increase in the number of adherent T cells in a dose- and time-dependent manner. Enhanced adhesion was noted as early as 4 hr after interferon stimulation (291 +/- 7 T cells/field vs 51 +/- 10 without IFN stimulation) and binding was further increased by lengthening the exposure time of fibroblasts to interferon up to 72 hr (475 +/- 86 T cells/field). Kinetic and inhibition experiments using monoclonal antibody to HLA-DR demonstrated that adhesion of T lymphocytes to interferon-stimulated fibroblasts proceeds by a mechanism independent of DR induction. In addition, adherence was not histocompatibility antigen-restricted, as adherence to autologous and allogeneic fibroblast monolayers was not significantly different. Nonadherent T cells, collected at the end of adhesion assays, were deficient in their capacity to bind to a second interferon-treated monolayer, suggesting the depletion of a subpopulation of T cells responsible for adhesion. Alterations of fibroblasts in vivo by immune cell-derived cytokines may be an important mechanism for the localization of lymphocytes at sites of connective tissue inflammation.     

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture

Summary Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of ³H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members.MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; TIL, tumour infiltrating lymphocytes; MLTC, mixed lymphocyte tumour culture; IL-2, interleukin-2; MLC, mixed lymphocyte culture; LSM, lymphocyte separation medium; BSS, balanced salt solution; HuSe, human serum; PBS, phosphate-buffered saline; CTC, cultured T cells; PHA, phytohaemagglutinin; CM, cultured medium; NK, natural killer; FcR, receptor for the Fc portion of IgG     

Contact-activated migration of melanoma B16 and sarcoma XC cells.

During migration, tumour cells interact with neighbouring neoplastic and normal host cells, and such interaction may influence their motile activity. We investigated the effect of homotypic collisions on the motile activity of two tumour cell lines, mouse melanoma B16 and rat sarcoma XC, and nontransformed human skin fibroblasts. It was found that the tumour cells show only limited motile activity when moving as single cells without contact with neighbours. At a higher density of the culture (and also at a greater number of cell to cell contacts) the activation of motility of investigated tumour cells was observed. On the other hand, the normal human skin fibroblasts showed a typical reaction of density-dependent inhibition of motility. The motile activity of tumour cells was not affected by conditioned media and was visibly dependent on a direct physical contact among colliding cells. The activation of cell movement was observed about 40-50 min after the initial contact between tumour cells. Contact-activated migration of neoplastic cells was inhibited by 50 microM verapamil (a selective voltage-gated calcium channel inhibitor) and 10 microM gadolinium chloride (a nonspecific blocker of mechanosensitive ion channels) but not by pertussis toxin. The observation that homotypic collisions among tumour cells strongly increase their motile activity suggests that contact-activated migration may play a significant role in tumour invasion and metastasis.     

A labile antigen complex at the lymphocyte surface: the effect of the substratum on its expression

Human T lymphocytes carry a surface antigen, detectable by a monoclonal antifibronectin antibody, which appears to consist of 150 and 55 kd components as revealed by SDS-PAGE. After in vitro culture of the lymphocytes on a plastic substratum for 48 hr comparatively few cells (40 +/- 18% in separate individuals) express the antigen. In contrast, the vast majority of lymphocytes cultured on a collagen matrix for the same time period maintains surface expression of the antigen (76 +/- 14% in separate individuals). Conditioned media from lymphocytes on plastic contain substantial amounts of antigenicity detectable by the same antibody, whereas conditioned media from lymphocytes on collagen are devoid of such antigenicity. The expression at the cell surface of other T lymphocyte antigens (Leu-4, Leu-3, and OKT8) is identical during culture on plastic and collagen for 48 hr. Collagen does not activate the cells to DNA synthesis or expression of IL 2 receptors, and consequently the potentiation of antigen expression by this substratum cannot be attributed to a mitogenic effect. The composition of subsets of T lymphocytes and the viability of the cells are the same on plastic and collagen, which excludes that the substratum-dependent variations in antigenicity reflect selection or loss of antigen-bearing cells. Thus, substratum-dependent regulation of the expression at the cell surface appears to be a unique property of the 150/55 kd T cell surface antigen. Culture on collagen substrata augments the number of lymphocytes showing motile behavior two to four times compared with culture on plastic.     

Lymphocyte development of adherence and motility in extracellular matrix during IL-2 stimulation.

To extravasate into normal and neoplastic tissue, lymphocytes must migrate through the subendothelial basement membrane and underlying interstitium, structures rich in extracellular matrix (ECM). We have performed a time-course study of the development of motility in ECM by murine lymphocytes during in vitro exposure to high titers of IL-2 (1000 Cetus units/ml). This protocol generates immunotherapeutic lymphocyte populations expressing lymphokine-activated killer activity. Spontaneous motility was measured in three-dimensional gels of type I (interstitial) collagen or Matrigel, a model basement membrane. A newly developed assay permitted not only the measurement of distance traveled by the leading cell front, but also the separation of lymphocytes on the basis of three types of behavior. The motile fraction consisted of lymphocytes that penetrated beneath the ECM gel surface during an 18-h migration period. There were also two nonmotile fractions: the nonadherent fraction, which failed to bind to the gel surface; and the adherent fraction, which bound but did not penetrate during the assay period. During a 3- to 5-day exposure to high titer IL-2, both adherence and motility increased significantly. In type I collagen, cells of the NK lineage developed greater surface adherence and less motility than cells of the T lineage. The surface-adherent fraction expressed higher lymphokine-activated killer and NK activity than did the nonadherent or motile fractions. Under prolonged IL-2 stimulation (7 to 12 days), there was a decline in the percentage of cells exhibiting motility in both types of ECM, and an increase in the percentage of surface-adherent cells. The findings indicate that the behavior of an IL-2-stimulated lymphocyte population in ECM is profoundly influenced by the duration of IL-2 exposure. Furthermore, lack of lymphocyte motility may reflect two different behaviors, nonadherence and adherence without motility. The nonadherent and surface-adherent populations may differ in phenotypic distribution and function. The motility system described in this report will be useful in separating and studying the mechanisms that produce lymphocyte adherence and motility, and in understanding the in vivo implications of these behaviors.     

Hypergravity speeds up the development of T-lymphocyte motility

The effect of altered gravity on single cells has been reported in a number of studies. From the investigation of the immune system response to spaceflight conditions, interest has focused on the influence of gravity on single lymphocytes. Microgravity has been shown to decrease lymphocyte activation and to influence motility. On the other hand, the effect of hypergravity on lymphocyte motility has not been explored. We studied the migration of human peripheral blood T lymphocytes cultured in vitro in a hypergravity environment (10g). After hypergravity culture for 1–11 days, T cells were seeded on a fibronectin-coated glass surface, observed by time-lapse bright-field microscopy, and tracked by a computer program. We found that T cells, activated and then cultured in hypergravity, become motile earlier than cells cultured at normal gravity. These results suggest that hypergravity stimulates T cell migration.     

HLA-DR-restricted cytotoxicity of cytomegalovirus-infected monocytes mediated by Leu-3-positive T cells

Mononuclear leukocytes from 14 cytomegalovirus (CMV)-seropositive and six CMV-seronegative normal healthy donors were treated with soluble CMV antigen for 5 days to generate cytotoxic T lymphocyte (CTL) activity. CMV-antigen-stimulated lymphocytes from CMV-seropositive but not CMV-seronegative donors lysed autologous peripheral blood monocyte targets infected with CMV in 13 of 14 donors (mean percentage of virus-specific lysis = 19.0 +/- 4.5%, effector to target ratio of 50:1). Freshly donated, unstimulated lymphocytes displayed little or no lysis of CMV-infected monocytes. Lysis was virus specific in that CMV-stimulated CTL did not kill herpes simplex virus-infected monocytes. The mean level of lysis of CMV-infected autologous targets was equivalent to that of HLA-DR-matched targets (20.0 +/- 8.0%), and was significantly greater than that of HLA-A/B-matched targets (6.3 +/- 2.5%, p less than 0.035) and HLA-mismatched targets (3.3 +/- 2.5%, p less than 0.01). Enrichment for T cell subsets with the use of selective depletion methods with monoclonal antibodies showed that CTL activity against autologous and HLA-DR-matched allogeneic targets was present predominantly in Leu-3-positive T lymphocytes. These results show for the first time that short term stimulation of heterogeneous lymphocytes from CMV-seropositive donors with CMV antigen can generate CMV-specific, Leu-3-positive CTL that are primarily restricted in their activity to autologous and class II, HLA-DR-matched targets. Our findings suggest a role for Leu-3-phenotypic CTL in immunity to CMV, and provide a model for analysis of this antiviral effector function during immunodeficient states.     

Herpes simplex virus-infected human fibroblasts are resistant to and inhibit cytotoxic T-lymphocyte activity.

We examined the ability of human anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL) to lyse autologous human fibroblasts infected with HSV. In contrast to HSV-infected human Epstein-Barr virus-transformed B cells (LCL), which were lysed by HLA-restricted anti-HSV CTL, autologous fibroblasts infected with HSV were resistant to lysis. This resistance was not due to a lack of infectivity or production of HSV proteins since greater than 90% of the cells were infected and expressed abundant levels of viral proteins. HSV-infected human fibroblasts were also tested for susceptibility to lysis by alloantigen-specific CTL. Although allogeneic LCL and uninfected allogeneic fibroblasts were killed, human fibroblasts infected with HSV demonstrated a time-dependent resistance to lysis by alloantigen-specific CTL. HSV-infected human fibroblasts were not resistant to all forms of cell-mediated cytotoxicity since they were sensitive to antibody-dependent cellular cytotoxicity. Although one may suspect that the resistance of HSV-infected human fibroblasts to anti-HSV CTL and alloantigen-specific CTL-mediated lysis was due to a lack of major histocompatibility complex expression, Confer et al. (Proc. Natl. Acad. Sci. USA 87:3609-3613, 1990) previously demonstrated that incubation of human natural killer and lymphokine-activated killer cells with monolayers of human fibroblasts infected with HSV "disarmed" the killers in that they were unable to lyse sensitive target cells. We extend their results and show that incubation of anti-HSV CTL or alloantigen-specific CTL with uninfected fibroblasts did not affect their lytic activity, whereas CTL incubated with HSV-infected fibroblasts for 2 to 6 h rendered the CTL incapable of lysing their normally sensitive target cells. Indeed, human fibroblasts infected for merely 2 h with HSV were able to profoundly inhibit the cytotoxic activity of alloantigen-specific CTL. Thus, HSV-infected human fibroblasts are not inherently resistant to lysis by anti-HSV CTL or alloantigen-specific CTL, but rather contact of CTL with HSV-infected fibroblasts resulted in inactivation of the CTL. The inactivation of CTL appears to be HSV specific since incubation of alloantigen-specific CTL in sandwich assays with fibroblasts infected with HSV type 1 (HSV-1) or HSV-2 resulted in inactivation, whereas incubation of CTL with fibroblasts infected with adenovirus or vaccinia virus had no effect. Further, although incubation of alloantigen-specific CTL in sandwich assays with HSV-infected fibroblasts resulted in inhibition of CTL activity, exposure of CTL in Transwell cultures to cell-free supernatant from HSV-infected fibroblasts did not mediate this inhibitory effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin-13 secretion by normal and posttransplant T lymphocytes; in vitro studies of cellular immune responses in the presence of acute leukaemia blast cells

T lymphocyte secretion of interleukin-13 (IL-13) in response to different activation signals was characterized in vitro. IL-13 release was investigated when virus transformed B lymphocytes or acute myelogenous leukaemia (AML) blasts were used as accessory cells during T cell activation. First, a majority of both CD4⁺ and CD8⁺ TCRαβ⁺ T lymphocyte clones, derived from normal individuals and bone marrow transplant recipients, secreted IL-13 in response to a standardized mitogenic activation signal (phytohaemagglutinin+IL-2+ B lymphocyte accessory cells). The CD4⁺ cells showed significantly higher IL-13 levels than the CD8⁺ subsets. Second, when leukaemic accessory cells (more than 95% AML blasts) were used during T cell activation, IL-13 was released both during alloactivation of normal T lymphocytes and during mitogen activation of posttransplant T cells. Third, when normal T lymphocytes were stimulated with allogeneic AML blasts, addition of IL-13-neutralizing monoclonal antibodies decreased interferon γ levels. Although addition of IL-13-neutralizing antibodies did not alter granulocyte-colony-stimulating factor secretion by allostimulating AML blasts, altered blast proliferation was detected for certain patients. Thus, most T cell clones can release IL-13, and IL-13 can modulate cytokine responses during T cell recognition of allogeneic AML cells. Received: 24 April 1997 / Accepted: 24 July 1997     

Auto-tumor lysis by blood lymphocytes in vitro

Summary Selectivity of the lysis of the tumor cells by autologous blood lymphocytes and its various subsets was investigated by means of the cold target competition assay. The effectors were autologous lymphocytes passed through a nylon-wool column (unfractionated: U) and their low-and high-density subsets, either without or after activation. The lymphocytes were activated (a) in autologous mixed lymphocyte tumor cell culture in autologous (MLTC), (b) in mixed lymphocyte culture (MLC), without and with interleukin-2, for 6 days, or (c) by phytohaemagglutinin for 3 days. Autologous-lymphocyte-mediated cytotoxicity (auto-tumor lysis: ALC) by the unfractionated, unmanipulated blood lymphocyte (U) population, its high-density fraction and those induced for auto-tumor lysis in the MLTC is regularly weak and affects only the autologous tumor cells. Their ALC function was inhibited only by the target identical unlabelled cells while the effect of separated low-density lymphocytes was inhibited also by allogeneic tumor cells.The cold-target competition assay indicated that several subsets with different specificities exist simultaneously in the effector populations activated in MLC, because the various targets did not cross-compete or did so only partially. Whenever interleukin-2 was added, at the start of the mixed cultures (MLTC or MLC), the lytic effects were no longer selective. Phytohaemagglutinin-activated effectors lysed several targets. These targets were inhibitory in a criss-cross fashion. Generally, populations showing auto-tumor selectivity had weak lytic effects, while the strongly activated effectors, with strong cytotoxic function, were not selective.     

Interactions between lymphocytes and dermal fibroblasts: an in vitro model of cutaneous lymphocyte trafficking.

Cultures of dermal fibroblasts were established from skin biopsies of CBA mice and used to study the interactions with murine T-lymphocytes. Electron microscopy showed that zones of contact developed between the fibroblasts and the T-cells, particularly after mitogenic activation. The adhesion of the lymphocytes was temperature-dependent, and many more lymphoblasts than resting cells attached to the fibroblast monolayers. Flow cytometry analysis of the adherent population showed that the most prominent type of resting lymphocyte was of the CD4 phenotype, which was also observed using a T-helper lymphoid cell line. However, neither the CD4 nor the CD8 (T-cytotoxic) antigens were involved in the binding process, and while the fibroblasts expressed Class I MHC molecules (but not Class II), these also had no role in mediating lymphocyte adhesion. Although the fibroblasts did not express the ligand Mala-2, the murine homologue of human ICAM-1, a monoclonal antibody against LFA-1, its cognate receptor on the lymphocytes, nevertheless effectively inhibited binding. T-cell attachment was also partially prevented by antibody against the lymphocyte CD2 antigen and by RGDS, a protein epitope known to mediate a number of receptor-integrin interactions. Moreover, this peptide also rapidly and preferentially detached T-lymphocytes which had previously adhered to the fibroblast monolayers. Lymphocyte binding was substantially elevated following treatment of the fibroblasts with cytokines such as tumor necrosis factor-alpha and interferon-gamma, but not interleukin-1 alpha. This increase in adhesiveness was, however, almost completely abolished by monoclonal antibodies specific for LFA-1 or for Mala-2. The results of this study show that while lymphocytes recognize fibroblasts normally via a number of constitutively expressed receptor-integrin interactions, their adhesion can also be modulated by cytokine-induced changes in the expression of other surface ligands.     

Human cytotoxic lymphocytes. V. Frequency and specificity of gamma delta+ cytotoxic lymphocyte precursors activated by allogeneic or autologous stimulator cells

We have investigated the frequency and specificity of gamma delta+ cytotoxic lymphocyte precursors (CLP) under limiting dilution culture conditions. E rosette separated total T cells and CD3+CD4-CD8-TCR alpha beta- double-negative (DN) T cells were cocultured with allogeneic or autologous PBMC stimulator cells, and frequencies of alloreactive and autoreactive CLP were determined after 12 to 14 days against Con A blast target cells. Freshly isolated DN cells consisting of 82.3 +/- 8.2% gamma delta+ T cells did not exert cytolytic activity against K562 or anti-TCR gamma delta mAb-producing hybridoma cells. In striking contrast to E+ cells, the vast majority of alloantigen-stimulated clonally developing DN CLP did not show specificity for stimulator-derived target cells. Thus, frequencies of alloreactive and autoreactive CLP after alloantigenic stimulation were in the range of 1/100 to 1/4800 and 1/450 to 1/5000, respectively. After coculture with autologous stimulator cells, frequencies of autoreactive and alloreactive DN CLP were 1/700 to 1/2700 and 1/1360 to 1/4500, respectively. Split culture analysis revealed that most proliferating DN colonies selected for high probability of clonality simultaneously killed both autologous and HLA-mismatched allogeneic targets. The majority of the DN cells expressed the CD3+/TCR gamma delta+ phenotype after culture, and thus were not CD2+CD3- NK cells. Taken together, our results show that 1) freshly isolated peripheral blood gamma delta+ T cells lack cytotoxic activity, and 2) most cytotoxic gamma delta+ T cells activated by autologous or allogeneic stimulator cells under limiting dilution conditions do not discriminate between autologous and allogeneic targets.     

Lymphocyte transformation induced by autologous cells. III. Lymphoblast-induced lymphocyte to stimulation does not correlate with EB viral antigen expression or immunity.

Human mitogen-induced and cell line B lymphoblasts stimulate the proliferation of allogeneic and autologous lymphocytes in culture. The role in thes reaction of EB viral determinants on the stimulating cells and immunity of the lymphocyte donor to the EB virus has been studied. The stimulatory capacity of cultured cell line lymphoblasts is not inhibited by incubating lymphoblasts with antisera to EB viral determinants. Cultured cell line B lymphoblasts stimulate as much thymidine incorporation by lymphocytes from donors with or without immunity to the EB virus. Further, a B lymphoblast cell line (U-698) which lacks the EB viral genome stimulated as much lymphocyte proliferation as did B lymphoblasts with the EB genome. Cultured T lymphoblast cell lines do not stimulate allogeneic lymphocyte proliferation. These cells appear to lack the determinants which stimulate lymphocyte transformation. No evidence was found that cultured cell line T lymphoblasts suppressed allogeneic lymphocyte proliferation. Mitogeninduced lymphoblasts from EB-immune and non-immune subjects stimulated the proliferation of autologous lymphocytes comparably. It is concluded that neither immunity to the EB virus nor expression of EB viral antigens on mitogen-induced on cell line lymphoblasts is necessary for the stimulation of lymphocyte proliferation.     

Simple sugars inhibit proliferation of human T lymphocytes in autologous and allogeneic mixed lymphocyte reactions

Several oligo- and monosaccharides were studied for their capacity to modulate lymphocyte proliferation in human allogeneic and autologous mixed lymphocyte reactions (MLR). A defined subset of sugars showed a marked inhibitory effect on lymphocyte proliferative response in the majority of the allogeneic MLR combinations studied. The inhibitory effect disappeared when sugars were added to allogeneic MLR 96 hr after the beginning of culture. These sugars also showed a significant inhibitory power on autologous MLR, performed by using T- and non-T-enriched lymphocytes from the same donor. The reported data suggest that carbohydrate determinants are involved in the proliferative response of human lymphocytes in both autologous and allogeneic MLR.     

Human immune responses to hapten-conjugated cells. II. The roles of autologous and allogeneic histocompatibility determinants in proliferative responses in vitro.

Proliferative responses of human lymphocytes primed in vitro to autologous TNP-cells were found to be associated with autologous D-region determinants irrespective of HLA-B locus antigens. Family studies of secondary TNP-conjugate proliferative responses demonstrated a gene dosage effect in this phenomenon. Moreover, co-culture with allogeneic cells did not affect the net TNP-conjugate proliferative responses of primed responder cells, suggesting that HLA-D region preference was due to a requirement for representation of TNP-molecules in association or combination with autologous MHC structures. Alloantigens were found to influence the sensitization of lymphocytes to autologous hapten-conjugated cells. Co-culture of allogeneic and TNP-modified autologous stimulator cells in primary cultures enhanced the secondary TNP proliferative response. Sensitization of human lymphocytes to allogeneic cells alone did not prime responses to autologous modified cells. However, priming lymphocytes to modified autologous cells potentiated responses to allogeneic cells. The data suggest a complex relationship between responses to alloantigens and modified autologous cells.     

Immunomodulating activity of p-hydroxyphenyllactic and ascorbic acids

p-Hydroxyphenyl lactic acid (PHA) in a concentration of 5 . 10(-5) M produced a significant inhibition of cell proliferation in response to alloantigens in a one-way mixed lymphocyte culture (MLC) in colonic cancer patients and in blast transformation in response to suboptimal doses of Con A. Multiple administration of ascorbic acid in an optimal concentration to the culture increased the proliferative response of lymphocytes to alloantigens and Con A. PHA and ascorbic acid did not exhibit any immunomodulating action during the use of healthy donors' lymphocytes or lymphocytes from colonic cancer patients, transformed with optimal mitogen doses. PHA did not affect the production of cytotoxic T lymphocytes in the MLC of the spleens of allogeneic mice but inhibited lymphocyte proliferation in response to alloantigens in the MLC of the spleens obtained from B6 and vitamin A deficient animals.     

Suppression of islet allogeneic immune response by indoleamine 2,3 dioxygenase-expressing fibroblasts

Success of transplantation of pancreatic islets which is a promising way for restoring efficient insulin regulation in type 1 diabetes depends on lifelong use of immunosuppressive drugs. To eliminate the use of systemic immunosuppressive drugs for islet transplantation, we examined the potential use of a local immunosuppressive factor, indoleamine 2,3-dioxygenase (IDO). Thus, the aim of this study was to determine whether local expression of IDO in bystander syngeneic fibroblasts could prevent islet allogeneic immune response in vitro. C57BL/6 (B6) mouse fibroblasts were induced to express IDO by either IFN-gamma treatment or transduction with an adenoviral vector and were co-cultured with B6 mouse lymphocytes and BALB/c mouse pancreatic islets in the presence or absence of an IDO inhibitor. Proliferation of lymphocytes were then assessed using [(3)H]-thymidine incorporation assay. IDO-expression by co-cultured syngeneic fibroblasts resulted in a five-fold decrease in lymphocyte proliferation rate upon stimulation of lymphocytes by allogeneic mouse pancreatic islets (21.9% +/- 5.3 and 22.1% +/- 4.9 in the preparations with IFN-gamma treated and genetically modified IDO-expressing fibroblasts, respectively vs. 100% in control groups, P < 0.01). Allogeneic response was restored when IDO inhibitor was added to the culture indicating that suppression was due to IDO. In conclusion, this study shows that local expression of IDO by syngeneic bystander fibroblasts can suppress in vitro proliferation of lymphocytes in response to stimulation with allogeneic pancreatic islets. This local immunosuppressive function of IDO may be employed for development of a novel alternative strategy for preventing allogeneic islet graft rejection.     

Lymphocyte transformation induced by autologous cells.

Human peripheral blood T lymphocytes are stimulated to proliferate when cultured with autologous B-lymphoblastoid cell lines, autologous mitogen-induced lymphoblasts, or autologous non-T blood lymphocytes. This reaction, the autologous mixed lymphocyte reaction, has attributes of an immune response possessing both memory and specificity. The capacity to stimulate autologous T lymphocyte proliferation depends on the lineage of the lymphoid cell and not on its establishment in continuous culture or carriage of the EB viral genome. The determinant on non-T lymphocytes which stimulates the autologous mixed lymphocyte reaction appears to be an Ia determinant. Thus, allogeneic graft rejection and the allogenic mixed lymphocyte reaction are very likely extensions of an immune response expressed within the host.     

Human cytotoxic lymphocytes. IV. Frequency and clonal specificity of CD8+CD16-(Leu2+Leu11-) and CD16+CD3-(Leu11+Leu4-) cytotoxic lymphocyte precursors activated by alloantigen or K562 stimulator cells

Cell sorter-purified CD8+CD16- (Leu2+Leu11-) cytotoxic T cell precursors and CD16+CD3-(Leu11+Leu4-) natural killer (NK) cells were cultured under limiting dilution (LD) conditions with allogeneic stimulator cells or with K562 tumor cells in the presence of exogenous interleukin 2. One out of 100-200 alloantigen-stimulated Leu2+ T cells clonally developed into an alloantigen-specific cytotoxic T cell, but only 1 out of 500-3400 of these cells lysed NK-susceptible K562 target cells. In contrast, 1 out of 2-35 alloantigen-stimulated Leu11+ precursor cells developed into an effector cell that lysed K562, but less than 1 out of 500 of these cells lysed allogeneic Con A blast targets. However, clonal activation of Leu11+ precursor cells under LD conditions did not require alloantigenic stimulator cells. Comparable high frequencies (f = 1/3 to 1/28) of anti-K562 cytotoxic lymphocyte precursors were thus measured when Leu11+ precursor cells were cultured on autologous or K562 feeder cells. As shown by a split culture approach, the vast majority of alloantigen-activated Leu2+ effector cells were highly specific for the stimulating alloantigen (i.e., they did not lyse K562), while the majority of Leu11+ microcultures lysed K562 tumor cells but neither autologous nor allogeneic Con A blast targets. On a quantitative basis, these data show that CD8+CD16- T cells and CD16+CD3-NK cells are two mutually exclusive lymphocyte populations which clonally develop into cytotoxic effector cells specific for alloantigen or K562 target cells, respectively.