Radiosensitivity of T and B lymphocytes. II. Effect of irradiation on response of T cells to alloantigens

The radiosensitivity of T cells was investigated by studying the effect of irradiation in vitro in suppressing the capacity of parental strain thoracic duct lymphocytes (a) to induce splenomegaly in newborn F₁ mice, and (b) to proliferate in adult irradiated F₁ mice as measured by incorporation of tritiated thymidine ([³H]dThd) 4 days after transfer. By these parameters, small T lymphocytes were found to be highly radiosensitive. It was calculated that, of cells with the reactivity to the alloantigens studied, 0.3 % were capable of a proliferative response after exposure to 500 r. Radiosensitivity was considered to be a reflection of lymphocyte death in interphase. The radiosensitivity of H-2-activated T cells (T. TDL) differed from that of small T cells. Thus, [³H]dThd incorporation by T. TDL measured 1 day after transfer to irradiated F₁ hosts was not abolished, although lowered, by exposure to doses as high as 5000 r; [³H]dThd incorporation measured at 2 days, however, was greatly reduced by much smaller doses of irradiation. In view of evidence obtained elsewhere that the response of T. TDL to alloantigens involves DNA synthesis but not cell division, the present studies were interpreted in terms of irradiation causing death of T. TDL in interphase before entry into DNA synthesis. It was concluded that T. TDL were far more resistant to irradiation-induced interphase death than were small T cells. The small numbers of lymphocytes obtained from thoracic duct lymph of mice exposed to whole body irradiation 4 days before consisted almost entirely of T cells; these cells, although viable, were found incapable of mounting a proliferate response when exposed to alloantigens on transfer.     

Targeting cytotoxic T cells to antigen-specific B lymphocytes

A recent development in immunomanipulation involves the targeting of cytotoxic T lymphocytes (CTL) to cell-bound antigens using bispecific antibodies. These antibodies have been engineered such that specificity is directed against the T cell receptor (TCR) or TCR-associated T3 molecules, as well as against the chosen antigen. The present study was aimed to force interactions between T and B cells by bridging their receptors. F23.1 antibodies, which are specific for gene products of the TCR V beta 8 gene family, were conjugated with TNP (2,4,6-trinitrophenyl) and this construct was used to bridge the receptors of V beta 8+ T cells with the receptors of TNP-specific B cells. The bridging was demonstrated by direct killing of both a TNP-specific B hybridoma and of blast cells from mice transgenic for mu, kappa of the TNP-specific antibody Sp6. Further, F23.1-TNP constructs in conjunction with V beta 8+ CTL were shown to specifically deplete Ig-secreting B cells from Sp6 transgenic mice. Conjugates of TCR-specific antibodies and antigen are theoretically useful in vivo to either deplete or expand B cells of a given specificity by coupling their receptors to the TCR of CTL or T helper cells, respectively.     

Intra-islet proliferation of cytotoxic T lymphocytes contributes to insulitis progression

Infiltration of pancreatic islets by immune cells, termed insulitis, increases progressively once it begins and leads to clinical type 1 diabetes. But even after diagnosis some islets remain unaffected and infiltration is patchy rather than uniform. Traffic of autoreactive T cells into the pancreas is likely to contribute to insulitis progression but it could also depend on T-cell proliferation within islets. This study utilizes transgenic NOD mice to assess the relative contributions of these two mechanisms. Progression of insulitis in NOD8.3 TCR transgenic mice was mildly reduced by inhibition of T-cell migration with the drug FTY720. In FTY720-treated mice, reduced beta cell MHC class I expression prevented progression of insulitis both within affected islets and to previously unaffected islets. CTL proliferation was significantly reduced in islets with reduced or absent beta cell expression of MHC class I protein. This indicates that intra-islet proliferation, apparently dependent on beta cell antigen presentation, in addition to recruitment, is a significant factor in progression of insulitis.     

Lymphocyte subpopulations in man: induction of cytotoxic T lymphocytes in vitro by allogeneic B and T cells

Testing B and T cells as allogeneic stimulators of cytotoxic T lymphocytes in primary as well as secondary in vitro cultures, reveals that fresh, nonactivated B cells isolated from peripheral blood have an enhanced cytotoxic T cell stimulating capacity compared to T cells, although target determinants are present both on B and T cell blasts. Similarly, the capacity of T and B cells to stimulate proliferation in MLC is also quantitatively different. These results are in accordance with the hypothesis concerning the in vitro generation of alloreactive cytotoxic T lymphocytes, which postulates concurrent stimulation by strong lymphocyte activating determinants and target determinants for the generation of cytotoxic effector T lymphocytes, as both determinants are simultaneously found on B lymphocytes. Three cell experiments performed by coculturing allogeneic stimulating B and T cells with responding T cells show that strong lymphocyte activating determinants found on B cells enhance the cytotoxicity against target determinants on cocultured B cells but not on cocultured T cells, indicating qualitative differences between target determinants on B and T cells with respect to specific CTL stimulating capacity. Furthermore, primed resting CTLs in secondary cultures could unspecifically be restimulated by third party B cells or pokeweed mitogen. These results are the basis for a hypothesis concerning activation of CTLs, postulating nonspecific triggering of cytotoxic precursor cells by lymphocyte activating properties intrinsic to target determinants (TD) on B cells, preferentially activating clones of cytotoxic cells. The clonal proliferation is further unspecifically amplified by products of the T cell recognition of strong lymphocyte activating determinants (LAD).     

Suppression of the cytotoxic T cell response to minor alloantigens in vivo. Linked recognition by suppressor T cells

This report describes the activity of transferable suppressor T cells (Ts) generated in vivo in response to minor alloantigens. These Ts cells are antigen specific in both primary and secondary in vivo cytotoxic T lymphocyte responses to minor alloantigens and are the result of a host response rather than of a graft-vs.-host reaction. The Ts cells are produced soon after immunization and their activity is transient. They act via "linked recognition", since they can suppress the cytotoxic T lymphocyte response to noncross-reactive minor antigens, but only if these are presented on the same antigenic cell. A model for dominant low responsiveness in (high X low responder)F1 animals is proposed, whereby Ts cells, activated via the low responder allele, work by linked recognition to suppress helper cells activated via the high responder allele.     

The induction of murine cytotoxic T lymphocytes by bluetongue virus

Summary After inoculation with live bluetongue virus, mice produced cytotoxic T lymphocytes (CTL) which showed virus and H-2 restriction. Inactivated preparations failed to induce CTLs. On secondaryin vitro stimulation, specifically sensitised memory cells also produced high numbers of CTLs. The need for replicating virus to induce primary CTLs, evidence for partial type specificity and the role which cell-mediated immunity might play in the early stages of a bluetongue virus infection are discussed.With 1 Figure     

Effect of pre-existing cytotoxic T lymphocytes on therapeutic vaccines

Therapeutic vaccines which aim to induce CD8(+) cytotoxic T lymphocyte (CTL) responses will often be required to perform in the presence of pre-existing CTL which recognize epitopes within the vaccine. Here we explore the ability of a viral vaccine vector presenting several co-dominant CTL epitopes to prime CTL responses in animals that have a pre-existing CTL response to one of the epitopes in the vaccine. The vaccine was usually capable of inducing multiple new responses, suggesting that immunodomination effects of pre-existing CTL may generally be minimal following vaccination. However, when large numbers of pre-existing CTL were present, a novel type of immune modulation was observed whereby (1) the vaccine failed to prime efficiently new CTL responses that were restricted by the same MHC gene as the pre-existing responses, and (2) vaccine-induced CTL responses restricted by other MHC genes were enhanced. These results may have implications for therapeutic multi-epitope vaccines for diseases like HIV and melanoma, which aim to broaden CTL responses.     

In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies

To study the efficiency of RNA-based vaccines, RNA coding for the model antigen beta-galactosidase (beta-gal) was transcribed in vitro from a lacZ gene flanked by stabilizing Xenopus laevis beta-globin 5' and 3' sequences and was protected from RNase degradation by condensation with the polycationic peptide protamine. The liposome-encapsulated condensed RNA-peptide complex, the condensed RNA-peptide complex without liposome or naked, unprotected RNA, was injected into BALB/c (H-2(d)) mice. All preparations led to protein expression in the local tissue, activation of L(d)-restricted specific cytotoxic T lymphocytes (CTL) and production of IgG antibodies reactive against beta-gal. RNA-triggered CTL were as efficient in the lysis of lacZ-transfected target cells as CTL triggered by a lacZ-DNA eukaryotic expression vector. Immunization with RNA transcribed from a cDNA library from the beta-gal-expressing cell line P13.1 again led to beta-gal-specific CTL and IgG induction. Thus, both naked and protected RNA can be used to elicit a specific immune response in vivo, whereby the protected RNA is stable in vitro for a longer period of time. RNA vaccines can be produced in high amounts and have the same major advantages as DNA vaccines but lack the potentially harmful effect of DNA integration into the genome.     

Cell-mediated immunity against herpes simplex induction of cytotoxic T lymphocytes.

The conditions required for the induction of both primary cytotoxic T lymphocytes (CTL) in vivo and secondary CTL in vitro against herpes simplex virus type 1 (HSV-1)-infected cells were defined. Primary CTL responses occurred only in mice exposed to infectious HSV-1. These responses, which were shown to be mediated by T lymphocytes, peaked at 1 week and had disappeared by 2 weeks after infection. The level of primary cytotoxicity was enhanced by treatment of mice with cyclophosphamide before infection. Secondary in vitro CTL responses were more pronounced and were induced by some forms of inactivated virus as well as by infectious HSV-1. Thus, both ultraviolet light- and glutaraldehyde-inactivated preparations of HSV-1 induced CTL, but heat-inactivated and detergent-extracted antigens failed to do so. The reasons for the differing efficiency of infectious and noninfectious HSV-1 for induction of CTL are discussed.     

The lysis of cytotoxic T lymphocytes and their blasts by cytotoxic T lymphocytes.

After binding to their targets, cytotoxic T lymphocytes (CTL) deliver a lethal hit signal, ultimately leading to target cell lysis, and then can recycle to lyse additional targets, without themselves being destroyed. If non-specific secreted lytic mediators are involved in such lysis. CTL survival would not be expected unless the effectors are immune to CTL-mediated lysis. Therefore the lytic susceptibilities of alloimmune peritoneal exudate lymphocytes (PEL), containing up to 50% CTL, and of the cytolytic PEL blasts (PEB), obtained by culturing with interleukin-2 (IL-2), were examined. 51Cr-labelled BALB/c (H-2d) anti-EL4 (H-2b) (d alpha b) PEL were lysed 88%, 78%, and 48% by C3H/eb (H-2k) anti-P815 (H-2d) (k alpha d) PEL, C57BL/6 (H-2b) anti-P815 (b alpha d) PEL and b alpha d PEB, respectively. Similarly, b alpha d PEL were lysed 82% and 21% by d alpha b PEL and PEB, respectively. b alpha d PEB were lysed 82%, 28-39% and 39-51% by k alpha d PEL, b alpha d PEL and b alpha d PEB, respectively, b alpha d PEB were lysed 29-55% by d alpha b PEL. Furthermore, the CTL-containing populations were no less susceptible to lysis than normal lymphocytes. Since the majority (80-90%) of cells in these two types of CTL-containing populations can be directly and specifically lysed by appropriately immunized PEL CTL, we conclude that both the lytic granule and perforin lacking (PEL) and containing (PEB) CTL are not a priori immune to CTL-mediated lysis. These findings are in accord with theories proposing lysis to be induced by receptor-mediated contact between effector CTL and target cells, and challenge those suggesting the involvement of secreted lytic mediators.     

Interactions of human T cell subsets during the induction of cytotoxic T lymphocytes: the role of interleukins.

In this work we study the role of subsets of human T cells, detectable by the OKT series of monoclonal antibodies, in the production of and the response to the lymphokine interleukin-2 (Il-2) during the course of an allogeneic cytotoxic T lymphocyte response in vitro. The results obtained establish that the Il-2 producer cells reside within the OKT4 positive T cell subset. Once produced, Il-2 mediates the clonal expansion of alloantigen-activated cytotoxic T killer cells which reside in the OKT8 positive T cell subset. Il-2 appears to have no mitogenic activity on the activated OKT4 positive T cells which produce the lymphokine. In order to release Il-2, the OKT4 positive T cell requires a stimulus, such as allogeneic cells or the lectin phytohaemagglutinin A (PHA). Macrophages are also required for Il-2 production, but the macrophage requirement can be bypassed by a soluble macrophage product as found in supernatants of lymphocyte cultures stimulated with lipopolysaccharide (LPS), the biological activity presumably representing Interleukin-1 (Il-1).     

Recruitment of functional subpopulations of T lymphocytes. Initiator T lymphocytes recruit helper T cells for cytotoxic response

Initiator T (Ti) lymphocytes are defined by their ability to recruit other T cell populations in vivo. In this study the function of T cells recruited into draining lymph nodes following injection of Ti cells primed to alloantigens in mixed lymphocyte culture was examined. The results demonstrate that alloantigen-specific helper T cells that interact with cytotoxic T (Tc) lymphocyte precursors are recruited, as shown by the significantly higher frequencies of helper cells in draining lymph nodes compared with controls. However, neither Tc cells nor their precursors are recruited. Recruitment by Ti lymphocytes is therefore selective for certain T cell subsets. Proposals to explain the mechanism, specificity, and selectivity of recruitment are discussed. We suggest that Ti cells have a central role in both the initiation of T cell-dependent immune responses and in the maintenance of immunologic memory. Their function is the rapid mobilization of T cell subclasses to a regional lymphoid organ where the immune response subsequently develops.     

Alloantigen pretreatment induces a down-regulation of the in vivo subsequent development of cytotoxic T lymphocytes directed against linked alloantigens

In the present study, we describe a new regulatory system that influences the in vivo development of cytotoxic T lymphocytes (CTL) and that could be related to epitopic suppression. Epitopic suppression has been previously shown to occur when carrier-primed mice are subsequently immunized with a "new" epitope coupled to the priming carrier. The suppression specifically inhibited the antibody response to the "new" epitope without affecting the secondary antibody response to the carrier. In this report, using a carrier/hapten-carrier type of immunization protocol, we have demonstrated that a similar regulatory system could also affect the induction of CTL directed against allogeneic cells. Priming mice with an alloantigen 1 (carrier) inhibits the induction of alloantigen 2 (hapten)-specific cytotoxic responses when the alloantigen 2 is presented in association with the alloantigen 1 on an F1 stimulator cell (hapten-carrier conjugate). This has been demonstrated by the specific decrease of anti-H-2b or anti-H-2d CTL responses generated in C3H/He mice (H-2k) previously primed with, respectively, H-2d or H-2b spleen cells before immunization with F1 (H-2d x b) spleen cells. This suppression of the CTL responses against the second immunizing alloantigen is associated with a strong CTL response against the first priming alloantigen. The induction of the suppression is dependent on the dose of H-2d spleen cells administered before immunization with F1 spleen cells and is not related to antigen elimination since a strong suppression of the CTL response against H-2b antigens is shown following immunization with a mixture of F1 cells and H-2b-bearing cells of H-2d-primed animals.     

Primary T-cell responses to minor alloantigens. II. Analysis of accessory cell requirements for the development of cytotoxic T lymphocytes.

The accessory cell requirements of cytotoxic T-lymphocyte (CTL) responses directed at multiple minor alloantigens are examined using a short-term, combined in-vivo and in-vitro protocol. The development of cytotoxic activity in vitro from T cells sensitized in vivo requires low-density accessory cells derived from the immunizing strain which have to be H-2 compatible with the responder population. The accessory activity of these cells can be by-passed by interleukin-2 (IL-2)-containing supernatant. Since IL-2 is a product of helper T (Th) cells secreted upon activation by antigen recognized in context of self H-2 and is known to stimulate antigen-activated cytotoxic T-lymphocyte precursors (CTL-P) non-specifically to effector CTL function, the results indicate that accessory cells interact in an H-2 restricted fashion with helper rather than cytotoxic T-lymphocyte precursors. The low-density accessory cells active in this system do not express Fc receptors (FcR) and are present in both the adherent and non-adherent fractions of spleen or lymph nodes.     

Comparison of numerous delivery systems for the induction of cytotoxic T lymphocytes by immunization

A variety of vaccine delivery systems including peptides with various adjuvants, recombinant particles, live recombinant viruses and bacteria and plasmid DNA were tested for their ability to induce CD8⁺ cytotoxic T lymphocytes (CTL) against a well-defined epitope (amino acids 252–260) from the circumsporozoite (CS) protein of Plasmodium berghei. We compared routes of immunization that would be applicable for the administration of a malaria vaccine in humans. The majority of these vaccines did not induce high CTL responses in the spleens of immunized mice. However, both a yeast-derived Ty virus-like particle expressing the optimal nine-amino acid epitope SYIPSAEKI from the CS protein (CSP-VLP) and a lipid-tailed peptide of this same sequence induced high levels of the major histocompatibility complex (MHC) class I-restricted CTL with one and three subcutaneous immunizations, respectively. Moreover, these CTL were able to recognize naturally processed antigen expressed by a recombinant vaccinia virus. The levels of CTL induced by CSP-VLP could be augmented by co-immunization with certain cytokines. Target cells pulsed with CSP-VLP were recognized and lysed, showing that the particles were effectively processed and presented through MHC class I presentation pathway. The levels of CTL induced using CSP-VLP and lipopeptides are comparable to those observed after immunization with multiple doses of irradiated sporozoites.     

Prolonged exposure of dendritic cells to maturation stimuli favors the induction of type-2 cytotoxic T lymphocytes

Dendritic cell (DC) maturation influences the priming and polarization of T lymphocytes. We recently found that early activated DC (i.e. DC exposed to pro-maturation stimuli for 8 h) were more prone to prime in vivo a type-1 cytotoxic T cell (Tc1) response than DC exposed to pro-maturation stimuli for 48 h (48h-DC). We investigated whether 48h-DC, conversely, allowed the induction of Tc2 cells. Antigen-pulsed mouse bone-marrow-derived DC at any maturation stage, in the presence of exogenous IL-12, skewed in vitro naive CD8(+) T cells towards Tc1 cells, but 48h-DC most potently, in the presence of exogenous IL-4, favored the induction of Tc2 cells. In vivo, full maturation of DC promoted expansion of Tc2 and fall of Tc1 cells. Tc2 cells maintained a high cytolytic activity and produced significant amounts of IL-4, IL-5, IL-10 and TGF-beta. Our results indicate that polarization of naive CD8(+) T cells to Tc2 cells is dependent on the amount of time DC have been exposed to maturation stimuli, and might be favored in late and/or chronic phases of an immune response.     

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.     

Blastogenic factors: production by T and B lymphocytes. Effect on T and B cells.

The aim of the present report is to identify the cells responsible for the in vitro production of blastogenic factor and to evaluate the effect of blastogenic factor on T and B lymphocytes. Human T lymphocytes were purified by a nylon wool column filtration method and B-cell-enriched populations were purified by sedimentation of E rosettes on Ficoll-Hypaque. The results were summarized as follows: Both T cells and B cells can produce blastogenic factor. However, blastogenic factor produced by T cells differed in some respects from blastogenic factor produced by B cells. (a) Blastogenic factor produced in unmixed B-cell cultures stimulated both B cells and T cells to proliferate, whereas blastogenic factor produced in unmixed T-cell cultures stimulated only B cells. (b) The production of T-cell blastogenic factor was accelerated when stimulated by allogeneic cells. In contrast, the blastogenic activity of supernatants from mixed cultures of B cells was not different from that of supernatants from unmixed cultures. The significance of these findings for mixed leucocyte reaction is discussed.     

Regulatory T-cell response and tumor vaccine-induced cytotoxic T lymphocytes in human melanoma

A role of CD4(+) cells in the regulation of immune responses has steadily gained renewed recognition. The understanding of these T-regulatory (T-reg) cells in the generation of antitumor cytolytic T lymphocyte (CTL) response is therefore important. It has been shown that immunization with specific peptides, DNA, or tumor lysate-based vaccines can induce CTL responses in vivo. We have immunized melanoma patients with major histocompatibility complex (MHC) class I restricted peptide- or melanoma tumor lysate-loaded antigen-presenting cell (APC)-based vaccines and have monitored the generation of CTL responses and T-reg cell responses, if any. Using tetramer staining and limiting dilution analyses as monitors of CTL responses, we found significant increases in the number of antigen-specific CTL in circulation after vaccination with the MART-1(27-35) peptide (AAGIGILTV)-pulsed autologous APC, the MAGE-1(161-169) peptide (EADPTGHSY)-pulsed APC, or with autologous tumor lysate-pulsed APC. The antigen-specific CTL reached the peak expansion by day 7 and then declined to the prevaccine levels by day 28. The decline in the CTL response was associated by a concomitant expansion of CD4(+) CD25(+)T cells. Analysis of postvaccine peripheral blood lymphocytes (PBL) from patients showed an increased amount of interleukin (IL)-10 secretion on in vitro stimulation with IL-2 after successive vaccination. Triple color flow cytometric analyses revealed cytoplasmic IL-10 in the CD4(+)CD25(+) T-cell fraction and the number of CD4(+)CD25(+) IL-10(+) T cells were found to increase significantly in postvaccine PBL. These observations have implications in tumor antigen and APC/dendritic cell (DC)-based cancer vaccine strategies.