Mycobacteria induce CD4+ T cells that are cytotoxic and display Th1-like cytokine secretion profile: Heterogeneity in cytotoxic activity and cytokine secretion levels

Protective immunity against mycobacteria is dependent on antigen-specific T cells. Current evidence suggests that not only helper T cells that activate infected macrophages but also cytotoxic T cells (CTL) that lyse infected macrophages are involved in protection. Mycobacterium-specific CD4⁺ CTL are readily detectable among primary peripheral T cells but what proportion of CD4⁺ T cells display cytotoxic activity is not known. Whether the cytotoxic CD4⁺ T cells are identical to or distinct from those that produce interferon (IFN)-γ is also unknown. In addition, studies on CTL in mycobacterial infections have focused primarily on selected antigens like hsp65 but have not analyzed systematically whether other mycobacterial antigens can activate CTL as well. These issues are relevant not only to a further understanding of protective immunity and immunopathology but also may have implications for the design of effective vaccines. To start addressing these issues, we have studied a large panel of CD4⁺ T cell clones specific for a broad range of mycobacterial antigens, and analyzed their ability to lyse mycobacterium-pulsed target cells and to release IFN-γ and interleukin (IL)-4. Our results show that the vast majority of CD4⁺ T cell clones are able to lyse mycobacterial antigen-pulsed target cells, and that those CTL can be triggered by a wide variety of mycobacterial antigens. CD4+ CTL released high levels of IFN-γ, but low or nondetectable levels of IL-4. In contrast, control tetanus toxoid-specific T cell clones or lines displayed poor or weak cytotoxic activity and released high levels of IL-4. The antimycobacterial clones appeared to be heterogeneous in their levels of cytotoxic activity and IFN-γ release. Interestingly one T cell clone was able to lyse only mycobacterium-pulsed macrophages but not B cells suggesting possible selectivity in target cell recognition for some CTL. These in vitro data have to be interpreted with some caution. Nevertheless they confirm and significantly extend previous observations and suggest that mycobacteria preferentially induce CD4⁺ T helper type 1 (Th1)-like cells that display cytotoxic activity, and release high levels of IFN-γ but no or little IL-4. The induction of such Th1 like cells is specific for mycobacteria since tetanus toxoid induced T cells that were poorly or not cytolytic and secreted high levels of IL-4.     

Distinct binding of T lymphocytes to ICAM-1, -2 or -3 upon activation of LFA-1

LFA-1 (CD11a/CD18) mediates leukocyte adhesion by binding to one of its ligands: ICAM-1, ICAM-2 or ICAM-3. Here, we investigated whether stimuli known to induce adhesion to ICAM-1 were also capable of inducing LFA-1-mediated adhesion of T lymphocytes to ICAM-2 and -3 transfectants. We observed that phorbol 12-myristate 13-acetate, Mn²⁺, cross-linking of CD3 or activating antibodies against LFA-1 enhanced LFA-1-mediated T cell adhesion to ICAM-2 and -3, although to a lesser extent than to ICAM-1. These results indicate that, similar to what has been reported for adhesion to ICAM-1, activation of LFA-1 is also required for adhesion to ICAM-2 and -3. Furthermore, the results suggest that ICAM-1 is the major ligand for LFA-1 on activated T lymphocytes. Interestingly, we observed that in contrast to activating antibodies against CD18, activating antibodies against CD11a were incapable of inducing adhesion of LFA-1 to all three ligands. The antibody MEM-83 stimulated binding to ICAM-1, while at the same time inhibiting the interaction of LFA-1 with ICAM-2 and -3. The antibody NKI-L16 selectively induced adhesion to ICAM-1 and -2, but not to ICAM-3. Our results suggest that different conformations of LFA-1 are required to support adhesion to ICAM-1, -2 or -3, and that ligands may bind on different sites of the LFA-1 molecule.     

ICAM-3, the third LFA-1 counterreceptor,is a co-stimulatory molecule for both resting and activated T lymphocytes

Optimal activation of human T cells mediated by ligation of CD3/T cell receptor (TcR) complex requires co-stimulatory signals. These can be provided by the adhesive interaction between receptor molecules on T cells and their counter-receptors on antigen-presenting cells. Soluble ICAM-3, anti-ICAM-3 and anti-CD3 mAb were utilized to address the role of the ICAM-3/LFA-1 pathway in TcR/CD3-dependent or -independent T cell activation. Immunoaffinity-purified ICAM-3 co-immobilized with suboptimal concentrations of anti-CD3 monoclonal antibody (mAb) stimulated T lymphocytes as monitored by the expression of the lymphocyte activation antigens CD25 and CD69. The mechanism underlaying this activation appear to involve the interaction of ICAM-3 with a β2 integrin, likely to be LFA-1, since mAb to the CD18 chain completely inhibited T cell activation. Similar experiments demonstrated that anti-ICAM-3 mAb were able to co-stimulate both resting (cord blood) and activated (T cell clones) T lymphocytes. On the contrary, anti-ICAM-1 mAb were only co-stimulatory for CD25 expression on activated but not on resting T cells. In addition, we have found that some γδ T cell clones bearing the Vδ1 segment were activated by direct mAb engagement of ICAM-3 in the absence of TcR/CD3 occupancy. Furthermore, immobilized anti-ICAM-3 mAb also induced development of dentritic processes. In conclusion, our data suggest that ICAM-3 on the surface of both T cells and antigen-presenting cells plays an essential role in the initiation of the immune response.     

Reversible HLA multimers (Streptamers) for the isolation of human cytotoxic T lymphocytes functionally active against tumor- and virus-derived antigens

The development of MHC/peptide multimers has facilitated the visualization and purification of antigen-specific T cells. However, the persistence of multimers leads to prolonged T cell receptor signaling and subsequently to altered T-cell function. We have recently developed a new type of MHC/peptide multimers, which can be dissociated from the T cell. Herein, we have generated and tested for the first time reversible HLA/peptide multimers, termed Streptamers, for the isolation of human T cells. The Streptamer technique demonstrates the specificity and sensitivity of conventional HLA/peptide tetramers with regards to the sorting of human T lymphocytes. This is shown for T cells directed against immunogenic peptides derived from viral and tumor-associated antigens. We show that antigen-specific cytotoxic T cells remain functionally active following Streptamer dissociation, whereas lytic function and proliferation of the T cells is impaired in the presence of conventional tetramers. These novel HLA/peptide Streptamer reagents allow the isolation of antigen-specific T cells with preserved function and, therefore, facilitate the development of adoptive T cell transfer regimens for the treatment of patients with cancer or infectious diseases.     

Human recombinant interferon-β influences T helper subset differentiation by regulating cytokine secretion pattern and expression of homing receptors

Type I interferons (IFN) are important regulators of both innate and acquired immunity. We have used an in vitro system of human CD4⁺ T cell differentiation to determine how IFN-β influences development of T helper (Th) subsets and homing receptor expression. IFN-β promoted differentiation of CD4⁺ T cells that produce low levels of both IFN-γ and lymphotoxin compared to interleukin (IL)-12-derived Th1 CD4⁺ T cells. IFN-β inhibited production of Th2 cytokines (IL-5 and IL-13) and augmented IL-12-mediated IL-10 secretion. In addition, IFN-β significantly enhanced L-selectin expression on CD4⁺ T cells and synergized with IL-12 to induce expression of cutaneous lymphocyte-associated antigen (CLA). This Th1 L-selectin⁺, CLA⁺ phenotype is characteristic of T cells found in normal human skin and suggests a role for type I IFN in the regulation of Th subset differentiation and tissue-specific homing receptors.     

Identification of a shared epitope recognized by melanoma-specific, HLA-A3-restricted cytotoxic T lymphocytes

We previously established a melanoma-reactive cytotoxic T lymphocyte (CTL) line that recognizes multiple epitopes in the context of HLA-A3. To increase the number of peptides available for use in a vaccine for the treatment of melanoma, we identified one of these epitopes, SQNFPGSQK, through a combination of epitope reconstitution experiments and mass spectrometry. The SQNFPGSQK peptide was also found to be associated with HLA-A3 on an additional melanoma tumor line, thus indicating that the peptide is not unique to the melanoma tumor line from which it was isolated and thus, unlikely to arise through a mutational event. Although the protein origin of SQNFPGSQK has yet to be established, the shared nature of this epitope and the fact that it elicits a natural immune response indicates that it warrants further study to determine its usefulness as a vaccine component for the treatment of melanoma. The peptide may also be useful as a research tool for evaluating spontaneous anti-tumor immune responses in patients with melanoma.     

Localization of two cytotoxic T lymphocyte epitopes and three anchoring residues on a single nonameric peptide that binds to H-2Ld and is recognized by cytotoxic T lymphocytes against mouse tumor P815

Mouse mastocytoma P815 expresses tumor antigens P815A and P815B encoded by a single gene called P1A and carried by a single peptide named P1A 35–43 (NH₂-Leu-Pro-Tyr-Leu-Gly-Trp-Leu-Val-Phe-COOH). P1A 35–43 is presented to anti-P815A and anti-P815B cytotoxic T lymphocytes (CTL) by major histo-compatibility complex (MHC) H-2L^d molecules. In order to determine the individual role played by each amino acid residue of P1A 35-43 in binding to H-2L^d and in recognition by anti-A and anti-B T cell receptors (TcR), a series of P1A 35-43 peptides substituted by alanine at single positions was synthesized and tested for binding to H-2L^d and for CTL recognition. Binding to H-2L^d was estimated by measuring the ability of the peptide to up-regulate cell surface expression of H-2L^d. We found that three residues were important for interaction of P1A 35-43 with H-2L^d. Two of them, Pro at position 2 and Phe at position 9 were consistent with the described H-2L^d binding motif. A third residue, Trp at position 6, was also required for effective MHC binding of the tumor antigen. CTL sensitization assays showed that alanine substitution at position 7 (Leu) or at position 8 (Val) dramatically affected peptide recognition by anti-A CTL while positions 3 (Tyr) and 4 (Leu) were critical for recognition by anti-B CTL. We conclude that Pro², Trp⁶ and Phe⁹ constitute the anchor residues of P1A 35-43 to H-2L^d, whereas the dipeptidyl sequences Tyr³-Leu⁴ and Leu⁷-Val⁸ form the core epitopes recognized by the TcR of anti-P815B and anti-P815A CTL, respectively.     

Tumor necrosis factor receptor p55 mediates deletion of peripheral cytotoxic T lymphocytes in vivo

Cellular death of activated lymphocytes down-regulates immune responses and is involved in maintaining self tolerance. Signals associated with ligation of the membrane molecule Fas lead to lymphocyte apoptosis, but additional, Fasindependent mechanisms have been postulated. Here, we show a marked expansion and prolonged persistence of functional activated cytotoxic T cells in mice lacking the tumor necrosis factor (TNF) receptor p55. In the absence of this receptor, peripheral lymphocyte apoptosis was significantly reduced in vivo. The prolonged thymocyte survival was associated with functional anergy, since the T cells no longer proliferated in vitro when stimulated with peptide antigen. However, specific cytotoxic effector function was easily detected in vitro. We conclude that the TNF receptor p55 is involved in peripheral T cell deletion in vivo.     

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.     

Differential modulation of T helper type 1 (Th1) and T helper type 2 (Th2) cytokine secretion by prostaglandin E2 critically depends on interleukin-2

Prostaglandin E₂ (PGE₂) favors T helper type 2 (Th2)-like cytokine secretion profiles in murine and human CD4⁺ T cells by inhibiting the production of the Th1-associated cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) and up-regulating the production of the Th2-associated cytokines IL-4 and IL-5 in a dose-dependent way. However, the potent inhibition of IL-2 production by PGE₂ seems to be in contrast with the simultaneous up-regulation of IL-4 and IL-5 production, because the induction of these cytokines requires IL-2. We, therefore, investigated to which extent the net modulatory effect of PGE₂ is determined by the availability of IL-2. To this aim, we examined the effects of PGE₂ on the cytokine secretion profiles of a panel of human Th0 clones upon stimulation via different activation pathways, resulting either in high or low IL-2 production. The differential modulation of Th1 and Th2 cytokines by PGE₂ was observed only upon modes of stimulation resulting in high IL-2 production. When IL-2 production was low, PGE₂ inhibited the secretion of all four cytokines. These different modulation patterns were directly related to the IL-2 availability, because (i) neutralizing antibody to IL-2 abrogated the up-regulatory effect of PGE₂ on IL-4 and IL-5 secretion in experiments with high endogenous IL-2 levels, (ii) lack of differential cytokine modulation by PGE₂ in conditions with low levels of endogenous IL-2 could be restored with exogenous IL-2, and (iii) cell viability was comparable in all conditions. These results demonstrate that the net modulatory effect of PGE₂ on the cytokine secretion profile of T cells critically depends on the availability of IL-2. Since this parameter varies with the experimental conditions and the T cell population studied, this finding may explain why certain immune responses may be either up- or down-regulated by PGE₂ under different conditions.     

急性移植物抗宿主病中Ly49A+T淋巴细胞的分布及其调节

目的 :探讨急性GVHD模型中小鼠杀伤性细胞抑制性受体Ly4 9A在造血系统T淋巴细胞亚群的表达及CsA、IL 4、IL 15对Ly4 9A表达的调节 ;方法 :BALB c、C5 7BL 6、CB6F1间脾细胞和骨髓细胞混合移植 ,建立急性GVHD模型 ,利用FCM双色免疫荧光检测 ,动态观察植入后不同时间造血器官T淋巴细胞亚群中Ly4 9A表达情况 ;体外T淋巴细胞培养观察IL 4、IL 15对Ly4 9A表达的调节 ;结果 :①Ly4 9AB6 在B6、F1小鼠的外周血、脾脏和骨髓细胞中均有表达 ,但在胸腺中基本无表达 ;②aGVHD中CD3 Ly4 9A 及CD8 Ly4 9A T细胞在移植早期增加 ,1w后迅速减低 ,至移植后 5w开始升高 ,并可超过移植前 ;CsA明显下调Ly4 9A变化幅度 ;③IL 4、IL 15均有上调脾CD3 Ly4 9A 细胞群的作用 (P =0 0 0 1)。结论 :Ly4 9A T细胞主要出现在外周淋巴器官 ;供者Ly4 9A T细胞在改变的MHC背景环境下 ,趋向于对宿主的免疫耐受 ,抑制性受体表达增加 ;骨髓的造血微环境可影响骨髓Ly4 9A T细胞比例的变化 ;一些细胞因子可以调节抑制性受体表达率     

T cell receptor and CD8-dependent tyrosine phosphorylation events in cytotoxic T lymphocytes: activation of p56lck by CD8 binding to class I protein

Tyrosine phosphorylation of proteins plays a central role in T cell activation. Mitogens or anti-receptor antibodies have been employed to study these signaling events, but the extent to which these mimic receptor interactions with native ligands is unclear. Cytotoxic T lymphocytes can be activated for functional responses using purified, native class I ligands presented on a surface. Previous work showed that stimulation with fluid-phase anti-T cell receptor (TCR) monoclonal antibody (mAb) activates CD8 to mediate adhesion to class I proteins and that activated CD8 generates a co-stimulatory signal upon binding to class I. Changes in tyrosine phosphorylation of substrates and activity of the p56^lck kinase have now been examined in this two-step process. The observed changes are small in comparison to those found using more potent nonphysiological stimuli, but may more accurately reflect the events required for activation of functional responses. Fluid-phase anti-TCR mAb caused increased tyrosine phosphorylation of a discrete subset of cellular substrates. Increased phosphorylation of additional substrates occurred upon CD8 binding to class I, resulting in a phosphorylation pattern comparable to that found in cells stimulated with class I alloantigen. Anti-TCR mAb alone caused increased tyrosine phosphorylation of p56^lck. When CD8 bound to class I, phosphorylation of p56^lck decreased to below the basal level found in unstimulated cells, accompanied by a substantial increase in kinase activity. These results are consistent with the two-step model for TCR activation of CD8/class I interactions and directly demonstrate that CD8 binding to class I leads to up-regulation of p56^lck activity.     

Tubeimoside-1 inhibits the proliferation and activation of mouse T lymphocytes through signal transduction pathways

Abstract

Tubeimoside-1 (TBMS1) is one of the important components in Bolbostemma paniculatum (Maxim.) Franque. In the study, its immunosuppressive effects on murine T lymphocyte responses were evaluated in vitro and in vivo. The data showed that TBMS1 inhibited ConA-induced T lymphocyte proliferation, decreased the ratio of CD4⁺/CD8⁺, suppressed IL-2, IFN-γ, IL-4 and IL-6 production and mRNA expression, down-regulate activation of NF-κB, NFAT2 and AP-1 signal transduction pathways in vitro. In addition, administration of TBMS1 significantly inhibited T cell-mediated DTH response in vivo. These findings indicated that TBMS1 inhibits the proliferation and activation of T lymphocytes in mice.     

巨噬细胞型骨髓基质细胞对肿瘤抗原提呈的体外实验研究

目的　探讨骨髓基质细胞对肿瘤抗原的提呈功能。方法　小鼠骨髓贴壁细胞经 Ｇ Ｍ Ｃ Ｓ Ｆ 诱导,形成以成熟巨噬细胞为主的基质细胞,用小鼠红白血病细胞 Ｆ Ｂ Ｌ３ 肿瘤抗原刺激,然后再与 Ｆ Ｂ Ｌ３ 肿瘤抗原致敏的 Ｔ 淋巴细胞混合培养。结果　骨髓基质细胞经 Ｆ Ｂ Ｌ３ 肿瘤抗原刺激后, Ｔ Ｎ Ｆα和 Ｉ Ｌ１β的分泌水平明显升高,经抗原预激的骨髓基质细胞能特异性地刺激同种抗原致敏的 Ｔ 淋巴细胞增殖和分泌高水平的 Ｉ Ｌ２ 。单抗阻断试验发现, Ｍ Ｈ ＣⅡ类分子和 Ｂ７２ 分子的联合阻断能有效地抑制致敏 Ｔ 淋巴细胞分泌 Ｉ Ｌ２ 。结论　本实验证实骨髓基质细胞具有抗原提呈功能, Ｍ Ｈ ＣⅡ类分子和 Ｂ７２ 分子在其抗原提呈中发挥了重要作用。     

A wild-type p53 cytotoxic T cell epitope is presented by mouse hepatocarcinoma cells

The possibility to identify epitopes presented by tumor cells to cytotoxic T lymphocytes (CTL) has given rise to new fields in tumor immunology. The tumor suppressor gene product p53 is a good candidate antigen because it is involved in the tumorigenesis of many cancers. It accumulates in an inactivated form due to mutation or formation of heterodimers with an oncogene product. Epitopes from the mutant or wild-type p53 proteins are thought to be presented by tumor cells and to induce a tumor-specific CTL response. To identify such epitopes, mouse wild-type p53 peptides encompassing the H-2 D^b anchoring motif were tested for their association with the D^b molecule. Positive peptides were assayed for their ability to induce CTL in C57BL/6 mice. CTL specific for one wild-type p53 peptide, p232–240, were isolated and found to lyse hepatocarcinoma cell lines established from mice transgenic for simian virus 40 large T antigen which overexpress p53. These results show that the p232–240 epitope from wild-type p53 is naturally processed and presented in H-2^b tumor cells.     

TCR-independent cytokine stimulation induces non-MHC-restricted T cell activity and is negatively regulated by HLA class I

Recent evidence suggests that the functional status of T cells activated independently from their TCR differs substantially from classical MHC-restricted T cells. Here, we show that TCR-independent, short-term stimulation via the common gamma-chain of the IL-2/IL-15 receptor induces non-MHC-restricted cytotoxicity and sustained cytokine secretion in purified CD4+ or CD8+ T cells. NK-like cytotoxicity is directed against MHC class I-negative targets and can be inhibited by classical and non-classical HLA class I molecules. Known inhibitory receptors, such as CD85j (ILT2) and leukocyte-associated Ig-like receptor-1, are not responsible for this HLA-mediated inhibition. NK-like cytotoxicity can be costimulated by NKG2D (CD314) triggering, but 2B4 (CD244) and DNAM-1 (CD226) are not involved. NK-like T cells display an activated phenotype and secrete various cytokines, including IFN-gamma, TNF-alpha, IL-5, IL-13 and MIP-1beta. Under normal conditions, HLA class I-mediated inhibition may function as a safety mechanism to prevent unbalanced cytokine production and effector killing mechanisms by T cells that were activated independently from their TCR. Non-MHC-restricted activity represents a functional status rather than a property of distinct T cell subpopulations. Thus, cytokine-induced, non-MHC-restricted T cells may be relevant in immune responses against tumors showing aberrant MHC expression through their capacities of cytokine production and direct tumor cell eradication.     

Inhibition of tumor growth by peptide specific cytotoxic T lymphocytes in a three-dimensional collagen matrix

Tumor associated, MHC I restricted antigenic peptides have been identified in both human and mouse tumors. Cytotoxic T lymphocytes (CTL) which recognize these tumor associated antigenic peptides are potential anti-cancer effectors. The anti-tumor activity of CTL is usually measured in vitro by the ⁵¹Cr release assay and in mice by tumor growth inhibition which is the most direct assessment of anti-tumor effect. In clinical studies, an in vivo tumor growth inhibition assay is not an option and an in vitro assay which corroborates with in vivo tumor growth is needed to assess the long-term outcome of CTL activity. Here, a three-dimensional (3-D) collagen gel assay was developed to measure in vitro the inhibition of mouse mammary tumor growth by anti-tumor CTL. BALB/c mouse CTL were induced with peptide E474 SFAVATTAL which was expressed by mouse mammary tumor cells D2F2. To measure D2F2 tumor growth inhibition in vitro, a mixture of tumor cells and anti-E474 CTL in a 1 μl cell bolus was embedded in the collagen gel. Complete eradication of tumor growth was observed at E:T ratio of or greater than 1:1. rIL-2 supplementation was necessary to achieve long-term tumor growth inhibition. Even spontaneous D2 tumor explant could be grown in the collagen gel and addition of anti-E474 to this culture reduced tumor growth. This assay system provides a realistic and sensitive alternative to the in vivo tumor growth inhibition assay and allows easy adaptation to test additional therapeutic reagents.     

Impaired induction of cytotoxic T lymphocytes by antagonism of a weak agonist borne by a variant hepatitis C virus epitope

An epitope that acted as a weak agonist in the cytotoxicity assay was identified as part of the capsid protein of a hepatitis C virus (HCV) variant. In a low concentration, the variant epitope also had a weak antagonistic effect. When a minute amount of this variant epitope was added to the culture for induction, it selectively attenuated the expansion of major cytotoxic T cell populations and drastically reduced the cytotoxic responses against the wild-type epitope. Thus, antagonism to induction suppressed immune responses against both the wild type and the variant, thereby helping the persistence of not only variant itself but also the wild-type HCV. Because this variant was a weak agonist, most cytotoxic T cells induced with the wild-type epitope were cross-reactive with the variant and susceptible to the antagonism to induction. Only the T cells which were not cross-reactive with the variant and not susceptible to the antagonism survived the antagonism in induction. This implied that the specificity of the remaining immune response, if any, was directed exclusively to the wild-type epitope after the emergence of the variant. For viruses like HCV, being heterogeneous itself may contribute significantly toward persistent infection through antagonism to induction.     

Allo- and self-restricted cytotoxic T lymphocytes against a peptide library: evidence for a functionally diverse allorestricted T cell repertoire

BALB/c-derived spleen cells were depleted of cytotoxic T lymphocytes (CTL) recognizing allogeneic (H2^b) and TAP-negative cells followed by stimulation with the same cells loaded with a synthetic library binding to H2-K^b. The resulting CTL lines were found to differ widely in peptide specificity and to exhibit an avidity towards the library as that demonstrated for syngeneic CTL. These results demonstrate that positive selection in the context of a certain MHC molecule does not seem to be required for generating high-avidity TCR that are restricted by the same molecule. However, positive selection increases the frequency of such CTL. By raising T cell lines from a repertoire which did not undergo negative selection by the restriction element in question, it becomes possible to produce effective self-peptide/MHC as well as nonself-peptide/MHC-specific CTL as tools for adoptive tumor immunotherapy.     

Environmental modulation of the autonomy of cytotoxic T lymphocytes

The extent to which one compartment of the immune system depends on another for efficient function is important to establish to fully comprehend disease phenotypes arising from selective immunodeficiency. Just how much the major histocompatibility complex class I-restricted cytotoxic T cell responses depend on class II-restricted T cell help has been controversial. Using the influenza A virus system, we show that mice unable to make class II-restricted T cell responses due to an engineered defect in class II molecule expression are able to mount virtually normal cytotoxic responses when bred under specific-pathogen-free conditions. However, when exposed to the more diverse environmental challenges of a conventional breeding facility, a situation that more closely parallels immuno-deficient states in man, they show impaired cytotoxic responses.