In vitro studies on the cell-mediated immune response to human brain tumors. II. Leukocyte-induced coats of glycosaminoglycan increase the resistance of glioma cells to cellular immune attack

We have examined the ability of cultured human glioma cells to elicit allogeneic cytolytic lymphocyte responses in vitro in order to delineate properties of glioma cells that may contribute to their ability to escape cellular immune attack. When glioma cells were cultured together with allogeneic peripheral blood mononuclear cells (PBMC) in mixed lymphocyte-tumor cultures (MLTC), it was observed that cells from eight of 12 glioma lines were surrounded by clear pericellular "halos," which appeared to impede contact between PBMC and the glioma cells. Enzymatic, histochemical, and immunochemical studies indicated that these halos represented glycosaminoglycan (GAG) coats that contained hyaluronic acid (HA) as a major constituent. Electron microscopic studies demonstrated the presence of many thin microvillous processes spanning the width of the halos. The presence of GAG coats around glioma cells in MLTC reduced the generation of cytolytic T lymphocytes specific for antigens on the glioma cells. Likewise, these cell coats decreased the lysis of glioma cells by cytolytic lymphocytes, once generated. The production of thick coats of GAG by glioma cells was induced by interaction of glioma cells with a nondialyzable factor produced by PBMC in culture. This factor did not cause glioma cells to release increased amounts of HA into the medium, but rather increased the production of HA that remained associated with the glioma cell surface. The formation of thick, protective GAG coats by glioma cells as a result of their interaction with the PBMC-derived factor constitutes a nonspecific suppressor mechanism that may contribute to the ability of this class of human solid tumors to evade cellular immune attack.     

Human myelin basic protein-specific cytolytic T lymphocyte clones are functionally restricted by HLA class II gene products

Cellular immune reactions against the autoantigen myelin basic protein (MBP) are strongly implicated in the occurrence of postinfectious and postvaccination encephalomyelitis. Clinical autoimmune encephalomyelitis in experimental animals can be transferred with cloned MBP-specific cytolytic major histocompatibility complex Class II-restricted T lymphocytes. The HLA restriction pattern of specific proliferative and cytolytic functions of two human MBP-specific cytotoxic T lymphocyte clones, derived from two different multiple sclerosis patients, was analyzed in detail. Using monoclonal antibodies against various HLA gene products and allogeneic Epstein-Barr virus-transformed B cells as antigen-presenting cells and as targets for cytolysis, it was found that MBP-specific functions of the T cell clones was restricted by HLA class II antigens, and, more specifically, by molecules encoded for by DR locus genes.     

Virus-specific T-cell sensitization

Syngeneic, semiallogeneic, or allogeneic spleen lymphocytes were transferred intonu/nu BALB/c mice, which were infected with vaccinia virus. Specific Sensitization of transferred thymus-derived cells was determined in vivo by mean survival time and virus titer in the spleen six days after infection, and in vitro by cell-mediated cytolysis of vaccinia virus-infected syngeneic target cells. Virus-specific Sensitization took place only after transfer of syngeneic or semiallogeneic spleen lymphocytes; allogeneic lymphocytes had no influence on mean survival time or virus titer and showed no virus-specific cytolytic activity in vitro. Infection of mice with vaccinia virus-strain WR, Elstree, DIs, or DIs-infected syngeneic fibroblasts resulted in the generation of virus-specific effector cells, while injection of a high amount of inactivated virus particles caused no Sensitization. These results suggest H-2 homology for production of virus-specific effector cells. Propagation of virus is not necessary, since early surface antigens, combined with syngeneic H-2 antigens, suffice for Sensitization of cytolytic T lymphocytes.Abbreviations used in this paper are as follows CMC cell-mediated cytolysis - CTL cytolytic T lymphocyte - LCM lymphocytic choriomeningitis - MHC major histocompatibility complex - MST mean survival time - T cell thymus-derived cell - TCID₅₀ 50 percent tissue culture infective dose     

Allogeneic stem cell transplantation for multiple myeloma

The sensitivity of myeloma cells to high dose chemotherapy has led to the use of allogeneic hematopoietic stem cell transplantation (HSCT) as a therapeutic modality in this disease. In addition to providing more effective chemotherapy, the transplantation of allogeneic stem cells also initiates the development of an allogeneic immune response directed against residual myeloma cells. Direct evidence for a graft vs. myeloma (GVM) effect is provided by the ability of donor lymphocyte infusion (DLI) to induce significant responses in 30-50% of patients with myeloma who have relapsed after allogeneic HSCT. Nevertheless, allogeneic stem cell transplantation is also associated with a high incidence of transplant related toxicities, including regimen-related toxicities, graft vs. host disease (GVHD) and opportunistic infections. DLI has been shown to enhance immune reconstitution after allogeneic HSCT in addition to inducing a GVM response. Current efforts are directed at reducing the toxicities associated with allogeneic HSCT, identification of the target antigens of GVM and the development of new strategies to selectively enhance the immune response to myeloma cells.     

Clonal and frequency analyses of tumor-infiltrating T lymphocytes from human solid tumors

A limiting dilution analysis (LDA) was used to assess the functional profiles of tumor-infiltrating lymphocytes (TIL) recovered from 15 human solid tumors. The microculture system applied in this study has been shown to allow virtually all normal peripheral blood T lymphocytes (PBL-T) to undergo clonal proliferation and was applied to obtain estimates of the frequency of both proliferating and cytolytic cells among the TIL population. A total of 624 microcultures proliferating in the presence of irradiated allogeneic spleen cells and interleukin 2 (IL 2) were expanded for clonal analysis. These TIL microcultures were assessed for surface antigen phenotype, IL 2 production (helper function) and for their cytolytic capabilities against the human erythroleukemic line K562 (natural killer (NK)-like activity) and P815, a mouse mastocytoma line, in the presence of phytohemagglutinin (PHA), i.e., lectin-dependent cell cytotoxicity (LDCC) which allows the detection of cytolytic activity irrespective of the antigenic specificity of the effector cells. Whenever feasible, cytolytic activity against autologous and allogeneic tumor cells was tested. LDA first demonstrated that the proliferative potential was decreased in T lymphocytes infiltrating human solid tumors (approximately 1 in 50 to 1 in 2 proliferating T lymphocyte precursors (PTL-P) in this series) as compared to normal PBL-T (1 in 2 to 1 in 1 PTL-P). The growth pattern in the titration cultures showed a remarkable agreement with the single-hit Poisson model implying that third party cells are unlikely to be involved in the reduced proliferative potential. Quantitative estimates of functional precursors showed that, in spite of reduced proliferative potential, cytolytic T lymphocyte precursors (CTL-P) against unknown antigens (LDCC-reactive) accounted for a considerable part of the microcultures in many cases. The precursor frequency of T lymphocytes with NK-like activity was usually low in situ (with the exception of glioma), whereas it was in the normal range in the patient's autologous PBL-T. In four evaluable cases, quantitative assessment showed that 1 in 200 to 1 in 1000 T lymphocytes from TIL was cytolytic against allogeneic tumor cells, which is in the range of alloreactive cytolytic T lymphocytes (CTL) generated in the mixed lymphocyte culture from normal PBL. Cytolytic activity against autologous target cells could not be quantitatively estimated but out of 88 clones from 4 patients, 3 clones originating from 2 glioma patients showed high lytic values against autologous tumor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proliferation and cytotoxicity of thymus lymphocytes in vitro]

Proliferative and cytollytical activity of lymphocytes was compared in lymphocyte alloimmunization of the spleen and intact thymus. The count of live cells and DNA-synthesizing cells in the thymocyte monoculture was 10--15-fold, and in mixed thymus cell culture--about 5-fold lower than the corresponding amounts of spleen cells. The index of immune thymocyte stimulation was several times greater than that of the immune cells of the spleen. The cytotoxicity peak was observed on the 4th--5th day of stimulation when the cytolytic activity of the immune thymocytes approached the action of the immune cells of the spleen. Low DNA synthesis and a marked cytotoxic activity of immune thymocytes signified that stimulation of the thymus cells in vitro permitted to obtain cell population with a high content of cytolytic T-lymphocytes.     

Accessory signals in murine cytolytic T cell responses. Dual requirement for IL-1 and IL-6

We have examined the nature of the accessory factors required for the development of primary murine CTL in accessory cell-depleted allogeneic mixed lymphocyte cultures. None of the known macrophage-derived factors was active by itself but the combination of IL-1 and IL-6 induced cytolytic responses equivalent to those of unfractionated cultures. Experiments with purified T cell subsets and antibodies blocking the action of IL-2 and IL-4 demonstrated that IL-1 and IL-6 acted directly on CD8+ cells capable of growth factor production. Analysis of the respective roles of IL-1 and IL-6 indicated that IL-6 acts before IL-1 and is capable apparently by itself to induce responsiveness to IL-2. By contrast, production of IL-2 required the additional presence of IL-1.     

Suppression of T-lymphocyte cytotoxicity following exposure to sinusoidally amplitude-modulated fields

Significant inhibition of allogeneic cytotoxicity of the target cell MPC-11 by the murine cytotoxic T-lymphocyte line CTLL-1 was observed when the 4-h cytotoxicity assay was conducted in the presence of a 450-MHz field sinusoidally amplitude-modulated at 60 Hz. Exposure of the effector cells to the field prior to adding them to the target cells in the cytolytic assay resulted in a similar inhibition, suggesting a direct interaction of the field with the cytolytic T lymphocyte. The inhibition was preferentially expressed during the early allogeneic recognition phase. Field-exposed cytolytic cells recovered their full cytolytic capacity in 12.5 h. A differential susceptibility was observed with modulation frequencies from 0 to 100 Hz. Peak suppression occurred at 60 Hz modulation, with progressively smaller effects at 40, 16, and 3 Hz. The unmodulated carrier wave did not affect the cytotoxicity. Effects with 80- and 100-Hz modulation were smaller than at 60 Hz. These results demonstrate an inhibitory but recoverable effect by certain amplitude modulations of weak nonionizing radiation upon the cell-mediated cytolytic immune response.     

The "public" determinants HLA-Bw4/Bw6 do not generate cytotoxic T lymphocytes (CTLs)

The human leucocyte antigens (HLA)-Bw4/Bw6 antigens detected serologically are "public" determinants located in the HLA-B molecule. They do not generate cytotoxic T lymphocytes (CTLs) in primary allogeneic cultures (mixed lymphocyte antigens) and secondary (primed lymphocyte typing) cultures indicate that they do not behave like normal HLA "private" cell-mediated lympholysis determinants. Therefore, the contribution of the 79-83 (alpha 1) residues in the generation of the epitopes Bw4/Bw6 does not seem to be critical for the examination by T cell receptor in allogeneic CML. The different overlapping patterns of the serological and CTL examinations are discussed, based on the structure of HLA class I antigens.     

Stimulation with allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines generates HLA class I-specific CTLs with different target cell avidity.

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) are potent inducers of cytotoxic T-lymphocytes (CTL) in allogeneic mixed lymphocyte cultures (MLC). The contribution of EBV antigens to the induction of cytotoxic responses was investigated by comparing CTL clones derived from allogeneic MLCs of lymphocytes from one EBV seropositive and one seronegative donor for their capacity to lyse paired EBV positive and negative targets. The majority of the clones showed a conventional "HLA-specific" cytotoxicity and lysed equally well HLA-matched LCLs and mitogen-induced T- or B-blasts. A minority of the clones from both donors exhibited an "LCL-selective" killing potential as they lysed poorly T- and B-blasts. The LCL-selective clones did not recognize EBV antigens because they could not discriminate between EBV negative Burkitt lymphoma (BL) lines and their in vitro EBV-converted sublines. MAbs to CD3, CD8, and MHC class I antigens blocked the lysis of LCLs by HLA-specific and LCL-selective CTLs with comparable efficiency suggesting that the two effector types express T-cell receptors of similar affinity. T-blasts were unable to inhibit the lysis of LCLs in cross competition assays. This correlated with a significantly lower expression of the cell adhesion molecules ICAM-1 and LFA-3. The results suggest that stimulation with allogeneic LCLs activates HLA class I-specific CTLs with variable target cell avidity. Only CTLs that act independently of the enhancing effect of cell adhesion molecules are able to lyse mitogen-induced T- and B-blasts.     

Cellular immunity in Q fever: modulation of responsiveness by a suppressor T cell-monocyte circuit

Human infection with the rickettsia Coxiella burnetii presents as an acute flulike primary Q fever, as a subacute granulomatous hepatitis, or, rarely, as chronic endocarditis. We have previously described lymphocyte unresponsiveness to Coxiella antigen in patients with Q fever endocarditis. This unresponsiveness was antigen specific and was mediated in part by adherent suppressor cells. In this report we show that the adherent suppressor cells work via prostaglandin E2 (PGE2)4 production. Addition of the cyclooxygenase inhibitor indomethacin to cultures of PBMC from patients with endocarditis or chronic laboratory exposure resulted in consistent increases in Coxiella-specific lymphocyte proliferation. The degree of increase in proliferation induced by indomethacin correlated strongly with the amount of PGE2 produced in a 4-hr culture stimulated by Coxiella antigen, but it also correlated with the sensitivity to inhibition of mitogenesis by PGE2. The suppressor mechanism was antigen nonspecific, because induction of suppression in vitro by Coxiella antigen also suppressed Candida-induced proliferation when both antigens were present in the same culture. Addition of indomethacin to these antigen cocultures totally reversed the Coxiella-induced suppression, confirming the evidence above that the nonspecific effector mechanism of suppression was prostaglandin (PG)-mediated. Elicitation of suppression, however, was antigen specific and involved a T cell-monocyte suppressor circuit. Supernatants from Coxiella-stimulated immune T cells and from the suppressor subset (OKT8+-enriched) of those T cells, but not unstimulated immune cells, induced augmented PGE2 production by unrelated nonimmune PBMC. We conclude that the lymphocyte unresponsiveness characterizing patients with Q fever endocarditis is modulated in part by an antigen-specific T suppressor cell which secretes a lymphokine to stimulate PGE2 production by adherent cells.     

Recognition of EBV plasma membrane protein expressed on murine cells after gene transfer

The immune response to B lymphocytes infected with Epstein-Barr virus (EBV) prevents their overgrowth in normal humans. A murine model is now described for analyzing the T cell immune response to Epstein-Barr virus genes expressed in murine lymphoblasts by gene transfer. In mice, a 60,000 dalton virus-encoded protein characteristically found in the plasma membrane of latently infected human lymphocytes readily induces both proliferative and cytolytic T lymphocytes specific for both the EBV protein and murine major histocompatibility proteins. Longterm cultures of L3T4+ cells, some of which were cytolytic, were found to be restricted by H-2I-Ed and the latent membrane protein. Similarly, Lyt-2+ cells were cytolytic and were restricted by H-2Ld and the lymphocyte membrane protein gene product. The similarity in murine and human effector cell responses suggests that this is a useful experimental model, and the EBV latent infection membrane protein may be an important antigen in the immune restriction of growth transformed latently infected lymphocytes.     

Cloned cytolytic T cells can suppress primary cytotoxic responses directed against them

In vivo and in vitro, murine peripheral T cells can suppress or "veto" the activation of cytotoxic T lymphocytes directed against antigens presented by those T cells. This suppression is antigen-specific and H-2-restricted. The recognition event initiating this suppression appears to be unidirectional; precursors of cytotoxic T lymphocytes recognize the antigen-bearing veto cell and are thereby inactivated--the veto cell need not recognize the CTL precursor. We show here that 3/3 cytolytic T cell clones can exert veto activity in vitro on normal spleen cells which do not bear antigens the T cell clones can recognize. This suppression results in greatly diminished cytotoxic activity generated during a primary 5-day mixed lymphocyte culture against antigens which the veto cell expresses, but not against third-party antigens present in the same culture. In this same system, a noncytolytic T cell clone will not serve as a source of veto cells. Secondary cytotoxic responses are relatively resistant to the veto cell activity of cloned cytolytic T cells. The cloned veto cells do not suppress the generation of cytotoxic activity directed against antigens they recognize (and presumably carry over via antigen-specific receptors). Cold target competition during the cytotoxic assay has been eliminated as a possible mechanism for T cell clone-induced suppression, and suppression cannot be reversed by the addition to the mixed lymphocyte cultures of supernatants from concanavalin A-activated spleen cells. It is suggested that this mechanism of inactivating primary cytotoxic T lymphocyte responses could play an important role in the maintenance of self-tolerance and in the induction and maintenance of tolerance to allografts.     

Inhibitory activity of medium-dialyzed transfer factor on lymphocyte blastogenesis.

Human transfer factor was prepared from normal donors with marked skin reactivity against common microbial antigens by dialysis of lymphocyte extracts versus tissue culture medium (TFmd). Several batches of TFmd were tested for their ability to specifically increase the DNA synthesis of unsensitized lymphocytes in vitro in the presence of the corresponding antigens (PPD, SKSD, Candida, Thistoplasmin). No transfer or immunologic reactivity by TFmd was observed. There was, however, a significant unspecific inhibition by TFmd of lymphocyte cultures stimulated by antigens, PHA or allogeneic cells.     

The role of cytostasis in antitumor immunity: comparison between syngeneic and allogeneic systems

Different aspects of the immune response differ with respect to sensitivity of neonatal thymectomy (NTx). The effect of thymectomy on Days 1 and 7 after birth (NTx-1, NTx-7) on antitumor immune responses to both a syngeneic and an allogeneic tumor graft was examined. The tumor used was the same, Meth A fibrosarcoma of BALB/c origin, but the mice inoculated differed. The results show that the immune response to a syngeneic tumor graft is primarily cytostatic rather than cytolytic and is relatively insensitive to neonatal thymectomy, while the response to a large dose of allogeneic tumor cells includes a cytolytic mechanism of high thymus dependence.     

Inhibition of T cell-mediated functions by MVM(i), a parvovirus closely related to minute virus of mice

A purified preparation of MVM(i), a murine parvovirus closely related to minute virus of mice (MVM), was found to inhibit various functions mediated by murine T cells in vitro. Addition of MVM(i) virus to secondary allogeneic mixed leukocyte cultures resulted in the inhibition of both lymphocyte proliferation (3H-thymidine incorporation) and the generation of cytolytic T lymphocyte activity but not interferon production. MVM(i) virus also inhibited the growth and cytolytic activity of several cloned, long-term Lyt-2+ cytolytic T cell lines. Furthermore, the antigen-induced proliferative responses of parasite- (Leishmania) specific Lyt-1+ T cells in vitro was abrogated by the addition of MVM(i) virus to the culture. Finally, the suppression of an in vitro antibody response to SRBC by MVM(i) virus was the result of the inhibition of T helper cells required for the B cell response. These suppressive effects were specific for MVM(i); parallel studies in which the prototype MVM parvovirus was used showed no significant inhibition in the various systems tested.     

Alloantigen-specific cytotoxic and suppressor T lymphocytes are derived from phenotypically distinct precursors

Although Leu-2+ (OKT8+) T cells activated in the mixed lymphocyte reaction (MLR) mediate both alloantigen-specific cytotoxicity and suppression of alloantigen-induced proliferation, it is not known whether these functions derive from a single cell type or phenotypically distinct cells. This study was undertaken to examine the alloantigen-specific cytolytic and suppressor potential of two subpopulations of Leu-2+ cells distinguishable from one another on the basis of their binding to the monoclonal antibody 9.3. Leu-2+, 9.3+ and Leu-2+, 9.3- populations were purified from peripheral blood, cultured for 7 days with autologous helper/inducer (Leu-3+) cells and allogeneic non-T cells, and reisolated before testing for cytotoxicity and suppression. All detectable alloantigen-specific cytolytic activity was confined to the Leu-2+, 9.3+ subpopulation. Killing by this subset was specific for the HLA-A and B (class I) major histocompatibility complex (MHC) antigens of the priming cell. By contrast, suppression of proliferation was mediated predominantly by the Leu-2+, 9.3- cells, and suppression by this subpopulation was specific for the HLA-DR (class II) MHC antigens of the priming cell. The development of suppression by Leu-2+, 9.3- cells was unaffected by cyclosporin A (CsA), an agent shown previously to block the development of cytolytic but not suppressor cells in MLR. Alloactivated Leu-2+, 9.3+ cells were slightly inhibitory of fresh MLR, but this effect as well as the development of cytolytic cells was completely abrogated by CsA. These results indicate that suppressor and cytolytic Leu-2+ T cells activated in MLR are derived from distinct precursors separable by antibody 9.3.     

Cytogenetic effects of the addition of leukocytes and blood sera on human fibroblasts exposed to measles virus or to streptolysin-O

Infection with measles virus and also introduction of streptolysin-O induced a significant increase in the level of cells with cytogenetic disturbances in the culture of human fibroblasts (HF). A decrease to intact condition of the number of HF with aneuploid and polyploid sets of chromosomes was observed after the introduction of non-immune autologous T-lymphocytes into the cultures. Immune homologous T-lymphocytes, unlike non-immune autologous T-lymphocytes, eliminated cells with structural disturbances of the chromosomes from the culture, but did not influence the level of aneuploid and polyploid cells. The ability of immune T-lymphocytes to exert antimutagenic effect can obviously be explained by their cytolytic action on virus-infected cells. As for non-immune autologous T-lymphocytes, two ways are equally probable: T-lymphocytes eliminate HF with virus antigens on their surface, or T-lymphocytes determine and destroy fibroblasts with changed cell surface developing as a result of cytogenetic disturbances induced by infectious factors. Specificity of the cytolytic reaction of T-lymphocytes concerning cells with some types of cytogenetic disturbances has been demonstrated.     

Rabbit antiserum to human B-cell alloantigens. II. Mechanism of inhibition of stimulation in the mixed lymphocyte response.

The process by which a rabbit antiserum to human B-cell alloantigens blocks stimulation in the mixed lymphocyte response (MLR) was investigated. A functional mammalian Fc region was necessary for the antiserum to be inhibitory, since F(ab′)₂ fragments failed to inhibit and a chicken antiserum with similar specificity to the rabbit anti-B-cell serum did not effectively block the response. Immune elimination of the stimulating cell population possibly via antibody dependent cell-mediated cytotoxicity (ADCC) or phagocytosis by macrophages was suggested by the observation that the addition of aggregated IgG to the MLR reduced the level of inhibition. It was also found that the number of immunoglobulin positive cells decreased in cultures treated with intact rabbit anti-B-cell serum, but not the corresponding F(ab′)₂ fragments, whether the cells were from a single individual or an allogeneic cell mixture. ADCC appears to be involved in the blocking process, as demonstrated by the marked reduction in MLR suppression when the MLR was initiated in the absence of ADCC effector cells. Removal or inhibition of monocytes in the MLR partially restored the response in experiments where the stimulator cells were pretreated with the antiserum, but not when the antiserum was present throughout the MLR.     

The origins of alloreactivity: differentiation of prekiller cells to viral infection results in alloreactive cytolytic T lymphocytes

Mice maintained in our animal colony become primed to Sendai virus. This "environmental" priming is reflected in a shift in prekiller activity from the Ly 123 to Ly 23 T cell set and in increased virus-specific cytolytic activity. This transition is accompanied by the development of cytolytic activity against allogeneic targets (not expressing Sendai antigens). These findings are consistent with the view that continued stimulation of Ly 123 cells by autologous MHC antigens, associated with foreign antigens such as a virus, generate Ly 23 prekiller cells that respond to alloantigens as well as autologous cells infected with the relevant virus.