Mature Cytotoxic T Lymphocytes Research Articles

Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

Ferroptosis and Pyroptosis Co‐Activated Nanomodulator for “Cold” Tumor Immunotherapy and Lung Metastasis Inhibition

AbstractImmune checkpoint blockade (ICB) therapy is an emerging strategy for cancer immunotherapy; however, the actual effects of ICB therapy are greatly limited by the immunosuppressive tumor microenvironment (TME, i.e., “cold” tumors). Although engineered nanomaterials display significant importance to regulate TME in cancer treatment, most of them focus on “immunosilent” apoptotic processes that cannot elicit sufficient immune responses for further immunotherapy. Herein, a GSH‐responsive nanomodulator is reported that can reverse the immunosuppressive TME for “cold” tumor immunotherapy and lung metastasis inhibition through simultaneous ferroptosis and pyroptosis induction. The nanomodulator is constructed by loading FDA‐approved sulfasalazine (SAS) and doxorubicin (DOX) on disulfide‐doped organosilica hybrid micelles, where SAS and DOX are released through the GSH‐stimulated rupture of micelles to induce ferroptosis and pyroptosis, respectively, promoting dendritic cells (DCs) maturation and cytotoxic T lymphocytes (CTLs) elevation through massive tumor‐associated antigen release. In vivo experimental results verify that desirable tumor destruction of the nanomodulator at low concentrations is achieved. More importantly, combination of this nanomodulator and programed death ligand‐1 antibodies significantly inhibits primary tumors and distant lung metastases as a result of elevated mature DCs and CTLs. This strategy to modulate immunosuppressive TME by nanomodulator‐induced non‐apoptotic death provides a new promising paradigm for ICB therapy.

Rejuvenated BCMA-Specific CD8 + Cytotoxic T Lymphocytes Derived from Antigen-Specific Induced Pluripotent Stem Cells : Immunotherapeutic Application in Multiple Myeloma

T cell regenerative medicine represents an emerging immunotherapeutic approach using antigen-specific Induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8 + cytotoxic T lymphocytes (CTL). Here we report on an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) against multiple myeloma (MM) via establishment of antigen-specific iPSC, followed by differentiation into highly functional BCMA-specific CD8 + CTL. The reprogrammed BCMA-specific iPSC displayed normal karyotypes and pluripotency potential as evidenced by expression of stem cell markers (SSEA-4, TRA1-60) and alkaline phosphatase, along with differentiation into three germ layers (Ectoderm, Mesoderm, Endoderm). During embryoid body formation, BCMA-specific iPSC further polarized into the mesoderm germ layer, evidenced by the activation of SNAI2, TBX3, PLVAP, HAND1 and CDX2 transcriptional regulators. Next, the BCMA-specific iPSC clones committed to CD8 + T cell differentiation were characterized by analyzing their hematopoietic progenitor cells (HPC; CD34 + CD43 +/CD14 - CD235a -) for specific transcriptional regulation. RNAseq analyses indicated a low variability and similar profiles of gene transcription within the iPSC clones committed to CD8 + CTL compared to increased transcriptional variability within iPSC clones committed to different cell types. The unique transcriptional profiles of the iPSC committed to CD8 + T cells included upregulation of transcriptional regulators controlling CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, and cytokine transporter/cytotoxic mediator development, as well as downregulation of regulators controlling B and T cell interactions, CD4 + Th cells, and inhibitory receptor development. Specifically, a major regulatory shift, indicated by upregulation of specific genes involved in immune function, was detected in HPC from the iPSC committed to CD8 + T cells. BCMA-specific T cells differentiated from the iPSC were characterized as displaying mature CTL phenotypes including high expression of CD3, CD8a, CD8b, TCRab, CD7 along with no CD4 expression (Fig. 1). In addition, the final BCMA iPSC-T cells were predominantly CD45RO + memory cells (central memory and effector memory cells) expressing high level of T cell activation (CD38, CD69) and costimulatory (CD28) molecules. Importantly, these BCMA iPSC-T cells lacked immune checkpoints (CTLA4, PD1, LAG3, Tim3) expression and regulatory T cells induction, distinct from other antigen-stimulated T cells. The rejuvenated BCMA iPSC-T cells demonstrated a high proliferative (1,000 folds increase) during the differentiation process as well as poly-functional anti-tumor activities and Th1 cytokine (IFN-g, IL-2, TNF-a) production triggered in response to MM patients' cells in HLA-A2-restricted manner (Fig. 2). Furthermore, the immune responses induced by these BCMA iPSC-T cells were specific to the parent heteroclitic BCMA 72-80 (YLMFLLRKI) peptide used to reprogram and establish the antigen-specific iPSC. Evaluation of 88 single cell Tetramer + CTL from the BCMA iPSC-T cells revealed a clonotype of unique T cell receptor (TCRa, TCRb) sequence. The BCMA-specific iPSC clones maintained their specific differentiation potential into the antigen-specific CD8 + memory T cells, following multiple subcloning in long-term cultures under feeder-free conditions or post-thaw after long-term (18 months) cryopreservation at -140 oC, which provides additional benefits to treat patients in a continuous manner. Taken together, rejuvenated CD8 + CTL differentiated from BCMA-specific iPSC were highly functional with significant (*p < 0.05) levels of anti-MM activities including proliferation, cytotoxic activity and Th-1 cytokine production. Therefore, the antigen-specific iPSC reprogramming and T cells rejuvenation process can provide an effective and long-term source of antigen-specific memory CTL lacking immune checkpoints and suppressors for clinical application in adoptive immunotherapy to improve patient outcome in MM. [Display omitted] DisclosuresMunshi: Amgen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Legend: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

High Frequency Production of T Cell-Derived iPSC Clones Capable of Generating Potent Cytotoxic T Cells

Current adoptive T cell therapies conducted in an autologous setting are costly, time-consuming, and depend on the quality of the patient’s T cells, and thus it would be highly beneficial to develop an allogeneic strategy. To this aim, we have developed a method by which cytotoxic T lymphocytes (CTLs) are regenerated from induced pluripotent stem cells that are originally derived from T cells (T-iPSCs). In order to assess the feasibility of this strategy, we investigated the frequency of usable T-iPSC clones in terms of their T cell-generating capability and T cell receptor (TCR) affinity. We first established eight clones of T-iPSCs bearing different MART-1-specific TCRs from a healthy volunteer. Whereas all clones were able to give rise to mature CTLs, cell yield varied greatly, and five clones were considered to be usable. TCR affinity in the regenerated CTLs showed a large variance among the eight clones, but functional avidities measured by cytotoxic activity were almost equivalent among three selected clones representing high, medium, and low TCR affinity. In a total of 50 alloreactivity tests using five CTL clones versus ten target cells, alloreactivity was seen in only three cases. These findings collectively support the feasibility of this T-iPSC strategy.

IL-21 regulates SOCS1 expression in autoreactive CD8+ T cells but is not required for acquisition of CTL activity in the islets of non-obese diabetic mice

In type 1 diabetes, maturation of activated autoreactive CD8+ T cells to fully armed effector cytotoxic T lymphocytes (CTL) occurs within the islet. At present the signals required for the maturation process are poorly defined. Cytokines could potentially provide the necessary “third signal” required to generate fully mature CTL capable of killing insulin-producing β-cells. To determine whether autoreactive CTL within islets respond to cytokines we generated non-obese diabetic (NOD) mice with a reporter for cytokine signalling. These mice express a reporter gene, hCD4, under the control of the endogenous regulatory elements for suppressor of cytokine signalling (SOCS)1, which is itself regulated by pro-inflammatory cytokines. In NOD mice, the hCD4 reporter was expressed in infiltrated islets and the expression level was positively correlated with the frequency of infiltrating CD45+ cells. SOCS1 reporter expression was induced in transferred β-cell-specific CD8+ 8.3T cells upon migration from pancreatic draining lymph nodes into islets. To determine which cytokines induced SOCS1 promoter activity in islets, we examined hCD4 reporter expression and CTL maturation in the absence of the cytokine receptors IFNAR1 or IL-21R. We show that IFNAR1 deficiency does not confer protection from diabetes in 8.3 TCR transgenic mice, nor is IFNAR1 signalling required for SOCS1 reporter upregulation or CTL maturation in islets. In contrast, IL-21R-deficient 8.3 mice have reduced diabetes incidence and reduced SOCS1 reporter activity in islet CTLs. However IL-21R deficiency did not affect islet CD8+ T cell proliferation or expression of granzyme B or IFNγ. Together these data indicate that autoreactive CD8+ T cells respond to IL-21 and not type I IFNs in the islets of NOD mice, but neither IFNAR1 nor IL-21R are required for islet intrinsic CTL maturation.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human β2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

Immune effect and safety evaluation of vaccine prepared by dendritic cells modified by rAAV-carrying BCSG1 gene.

The immune effect and safety evaluation of rAAV (recombinant adeno-associated virus)-containing Bcsg1 (breast cancer-specific gene 1) (rAAV/Bcsg1)-transfected DC (dendritic cell) (rAAV/Bcsg1-DC) vaccine in immunotherapy for (BCSG1) (+) BC was assessed. Immune effect of cytotoxic T lymphocytes (CTLs) on Bcsg1 (+) BC cells, and rAAV gene residuals in mature CTL cells and culture medium were determined. Nude mouse xenograft tumor model was established to assess the inhibition effects of DC-activated CTLs on tumor growth. DC cell surface markers were highly expressed in rAAV/Bcsg1 group and lysate-DC group, and rAAV/Bcsg1-DC-CTL showed stronger cytotoxic activity targeting Bcsg1 (+) BC cells. The rAAV/Bcsg1-DC vaccine-treated groups showed lower mean tumor weight, higher tumor inhibition rates and slower tumor growth. rAAV/Bcsg1-DC can induce highly efficient CTL-targeting Bcsg1 antigen without rAAV gene residuals.

In Vivo Suppression of HIV by Antigen Specific T Cells Derived from Engineered Hematopoietic Stem Cells

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.

What’s New in Shock, March 2012?

What’s New in Shock, March 2012?

Intrinsic and Estrinsic Mechanism Contributes to Maintain the JAK/STAT Pathway Aberrantly Activated in T-Type Large Granular Lymphocyte Leukemia

Abstract 1375T-cell large granular lymphocyte (T-LGL) leukemia is a heterogeneous disorder characterized by the chronic expansion of a terminally differentiated cytotoxic T lymphocytes (CTLs) with a CD3+/CD8+/CD57+ phenotype. Although the pathogenesis of T-LGL leukemia is still unknown, the hallmark of the disease is the abnormal clonal expansion of antigen-primed mature CTLs that successfully escape activation-induced cell death (AICD) and remain long-term competent. Similar to normal activated CTLs, leukemic T-LGLs exhibit activation of multiple survival signaling pathways. Among the intracellular signaling pathways altered in T-LGL leukemia, JAK/STAT (Janus Kinase/signal transducer and transcription factor) signaling has been associated with LGL transformation. In particular, it has been demonstrated that the STAT3, a pivotal element of this pathway, is over-expressed and constitutively activated in leukemic LGLs as compared to normal peripheral blood mononuclear cells (PBMCs). STAT3 is a transcription factor inducing a number of genes promoting cell survival and the involvement of an aberrant STAT3 expression has been suggested to play a role in the pathogenesis of this disease.In order to investigate the mechanism through which STAT3 is maintained in a state of activation in T-LGL leukemia, in patients in 27 patients we studied the expression of “suppressor of cytokine signaling 3 protein” (SOCS3) which is the specific negative regulator of STAT3 signaling, and the interleukin 6 (IL-6), which strongly induces STAT3 activation. Expression analysis was performed by Real Time-PCR and Western Blot assay in highly purified LGLs from PBMCs of patients by magnetic microbeads system.By Real Time-PCR, the expression level of IL-6 was shown to be increased in patients’ PBMCs (56.84 ± 17.01) as compared to normal PBMCs (0.53 ± 0.14, p<0.05). However, we found that SOCS3 was down-expressed, both at mRNA and protein level, in neoplastic LGLs as compared to control LGL (mRNA: 0.26 ± 0.04 vs 0.63 ± 0.17, p<0.01; protein: 0.59 ± 0.05 vs 0.85 ± 0.10, p<0.01). We also observed that in vitro SOCS3 was unresponsive to IL-6 in patients’ LGL, whereas in LGLs of healthy individuals this cytokine was definitely able to increase SOCS3 expression level.To better understand the mechanism responsible for SOCS3 low expression and unresponsiveness to IL-6 and since we found that this epigenetic mechanism is implicated in the NK type of this disease (Haematologica 2010. 95:1722), we evaluated whether an aberrant methylation of SOCS3 promoter takes place in T-LGL patients. Neoplastic LGLs were treated with the demethylating agent 5-aza-2'-deoxycytidine (DAC) and we observed that DAC re-established SOCS3 expression after IL-6 stimulation. Interestingly, as consequence of IL-6-induced SOCS3 expression we observed the inhibition of STAT3 phosphorylation and a reduction of Mcl1 protein level. These results support that in LGLs, SOCS3 tightly controls STAT3 and is able to modulate STAT3-induced gene Mcl1, that could be involved in LGL survival. Studies are now in progress to confirm the hypothesis that SOCS3 silencing is caused by the SOCS3 promoter methylation.In conclusion, our results suggest that the downregulation of SOCS3, likely related to aberrant methylation, cooperates with IL-6 in the activation of JAK/STAT pathway, leading to the constitutive phosphorylation of STAT3, and thus ultimately resulting into increased survival of leukemic LGLs. Disclosures:No relevant conflicts of interest to declare.

Cytolytic mechanisms and T‐cell receptor Vβ usage by ex vivo generated Epstein–Barr virus‐specific cytotoxic T lymphocytes

Ex-vivo-generated Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable beta-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0.0001) and ethylene glycol-bis tetraacetic acid (P < 0.0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-gamma and TNF-alpha. Granzyme B, perforin and Fas ligand were detected in CD8(+) and CD4(+) cells in all CTL; however, a greater proportion of CD8(+) than CD4(+) T cells expressed granzyme B (P < 0.0001) and more granzyme B was detected in CD8(+) T cells than in CD4(+) T cells (P = 0.001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.

Dendritic Cell–Tumor Cell Fusion and Staphylococcal Enterotoxin B Treatment in a Pancreatic Tumor Model

Background. Surgical resection of pancreatic tumors removes gross disease but not metastases. Adjuvant therapy such as chemotherapy and radiation treatment is of little value in metastatic pancreatic cancer. The hypothesis of this investigation is that specific and effective immunotherapeutic vaccine (dendritic/tumor cell fusion) will activate cytotoxic T lymphocytes (CTLs), leading to the eradication of spontaneous pancreatic cancer. Methods. We have developed a double transgenic mouse model (MET) that forms spontaneous pancreatic tumors and expresses the human MUC1 antigen. Seven-week-old MET mice ( n = 8) were treated every 3 weeks with the vaccine. In addition, these mice received 50 μg of superantigen staphylococcal enterotoxin B (SEB), a known T cell stimulant, prior to the first vaccination. A second treatment group received SEB alone ( n = 8) and controls received no treatment ( n = 9). MUC1-specific CTLs were measured by chromium release assay. At 10 weeks of age and at necropsy, MUC1 serum levels were measured using a MUC1-specific ELISA. Results. Mice were known to harbor microscopic foci of cancer at birth. Survival was enhanced in vaccine as well as SEB-treated mice (75% CI ± 0.42) compared to controls (11% CI ± 0.28) and both groups of treated mice exhibited mature CTLs without in vitro stimulation. MUC1 serum levels of the vaccine group were 50% less than that of control ( P < 0.04) at 10 weeks. MUC1 serum levels directly correlated with tumor weight at necropsy ( r = 0.86). Conclusions. This is the first evidence that MUC1-specific CTLs can be stimulated to enhance survival in a spontaneous tumor model.

Cytokines required for induction of histocompatibility leukocyte antigen-class I-restricted and tumor-specific cytotoxic T lymphocytes by a SART1-derived peptide.

Although there have been several reports on peptides of human tumor‐rejection antigens capable of inducing histocompatibility leukocyte antigen (HLA)‐class I‐restricted and tumor‐specific cytotoxic T lymphocytes (CTLs), it is not yet clear which cytokines are required for CTL induction. This study has investigated the cytokine combinations required for optimal induction of CTLs by SARTI690–698 peptide, which is capable of inducing HLA‐A24‐restricted and tumor‐specific CTLs in peripheral blood mononuclear cells (PBMCs). Pretreatment of PBMCs as a source of antigen‐presenting cells (APCs) with interferon (IFN)‐γ, or to some extent with IFN‐α, but not with any of the other cytokines tested, augmented the peptide‐induced CTL activity in HLA‐A24 heterozygotes, but not in HLA‐A24 homozygotes. This IFN‐γ ‐mediated augmentation was inhibited by either interleukin (IL)‐4 or IL‐10. IL‐2 alone in culture, along with weekly stimulation by peptide‐pulsed APCs, was sufficient for the differentiation and proliferation of CTLs for the initial several weeks of culture. This IL‐2‐mediated activation of CTLs was inhibited by the addition of IFN‐γ, IL‐4, or IL‐10 to the IL‐2 culture. For further expansion of the CTLs, dendritic cells (DCs) induced from PBMCs with IL‐4 and granulocyte macrophage colony‐stimulating factor (GM‐CSF) were required as APCs. These results indicate that IFN‐γ and IL‐2 are important in the activation of APCs and CTLs, respectively, while GM‐CSF and IL‐4 are needed for the induction of DCs, which in turn are required for further expansion of mature CTLs. These results are important in allowing for a better understanding of the cellular and molecular basis of tumor‐specific immunity, and also for the development of peptide‐based specific immunotherapy.

Kinetics of circulating cytotoxic T lymphocyte precursors that have a high avidity for donor antigens: correlation with the rejection status of the human cardiac allograft

Studies on graft infiltrating cells demonstrated that accumulation of cytotoxic T lymphocytes (CTL) with high avidity for donor antigens (Ag) coincided with acute cardiac rejection. In the present study, we analyse whether such high-avidity CTL are present within the peripheral blood of cardiac transplant recipients and whether their kinetics correspond with the rejection status of the allograft. Using limiting dilution analysis (LDA), donor-specific CTL were enumerated in serial blood samples of seven patients. From each patient, 7–11 samples were obtained during the first year after transplantation and up to three samples were obtained at a later date. Enumerated donor-specific CTL were divided into CTL with high or low avidity for donor Ag, depending on their sensitivity to CD8-blocking. In contrast to the situation in the graft, the donor-specific CTL present within the peripheral blood were CTL precursors (pCTL) and not fully mature CTL (cCTL). The number of donor-specific pCTL among peripheral blood cells fluctuated irrespective of the rejection grade of the allograft, indicating that the frequency of circulating donor-specific CTL does not reflect the immunological status of the allograft. During acute cardiac rejection, 66% (median) of the circulating denor-specific pCTL had a high avidity for donor Ag. This percentage significantly exceeded pre- and postrejection values obtained during the first year post-transplantation (median, 39% and 37%, respectively). The disparity in avidity increased even further more than 1 year after transplantation, when stable engraftment was achieved. Among donor-specific pCTL in peripheral blood, those with a high avidity were absent (median, 0%). Hence the avidity of circulating donor-specific CTL might inform us about the status of the cardiac allograft.

Human leukocyte antigen phenotype imposes complex constraints on the antigen-specific cytotoxic T lymphocyte repertoire.

The memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, is exceptionally restricted, being dominated by cytotoxic T lymphocytes (CTL) with a single, public T cell receptor (TCR). CTL clones that express this receptor fortuitously cross-react with the alloantigen HLA B44. However, of the two major subtypes of this HLA, B*4402 and B*4403, that differ by a single amino acid, only the former is recognized by these mature CTL clones. Individuals heterozygous for HLA B8 and B*4402 use alternative TCR for the EBV determinant since the dominant TCR is potentially self-reactive. We now demonstrate that this clonotype is also essentially absent from the repertoire of CTL directed against the viral epitope in seven from seven unrelated individuals heterozygous for HLA B8 and B*4403. Thus immune tolerance of these CTL recognizing HLA B*4402 is associated with expression of either B*4402 or B*4403. This suggests that tolerance in the human T cell compartment requires a lower threshold of recognition than for effector function, thus providing a buffer zone minimizing the risk of autoimmunity. These data also illustrate the potential for non-restricting HLA molecules to bias dramatically the T cell repertoire used for specific immune responses. Such influences may be the basis of the "protective" effects of certain HLA alleles in susceptibility to autoimmune disorders.

Cytokine patterns and cytotoxic mediators in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver with unknown etiology. Autoreactive T lymphocytes that infiltrate the liver may play a major role in the bile duct damage that accompanies the disease. We hypothesized that cytokines produced by T lymphocytes and other cells are central to the disease process. Therefore, we used reverse transcription-polymerase chain reaction (PCR) and Southern hybridization to identify cytokine message directly from liver tissue of 11 patients with PBC and 5 patients with autoimmune hepatitis (AI-CAH). Messenger RNA (mRNA) for interleukin (IL)-2, IL-5, IL-6, interferon gamma (IFN-γ), and transforming growth factor beta (TGF-β) were detected in the majority of the specimens from patients with PBC. The presence of IL-5 was associated with PBC ( P < .001, PBC vs. AI-CAH). Because IL-5 is a potent eosinophil differentiation factor, we looked for evidence of activated eosinophils within the infiltrate. We observed the deposition of the primary cytotoxic granule protein of eosinophils, major basic protein (MBP), within the portal region of livers from patients with PBC. Moreover, we detected message for a cytotoxic T-lymphocyte (CTL) granzyme in 87.5% of these livers indicating that mature CTL are present. Thus, we present evidence for two effector pathways that may contribute to the tissue damage observed in PBC and have identified message for cytokines that may regulate these pathways.

Cytokine patterns and cytotoxic mediators in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver with unknown etiology. Autoreactive T lymphocytes that infiltrate the liver may play a major role in the bile duct damage that accompanies the disease. We hypothesized that cytokines produced by T lymphocytes and other cells are central to the disease process. Therefore, we used reverse transcription-polymerase chain reaction (PCR) and Southern hybridization to identify cytokine message directly from liver tissue of 11 patients with PBC and 5 patients with autoimmune hepatitis (AI-CAH). Messenger RNA (mRNA) for interleukin (IL)-2, IL-5, IL-6, interferon gamma (IFN-gamma), and transforming growth factor beta (TGF-beta) were detected in the majority of the specimens from patients with PBC. The presence of IL-5 was associated with PBC (P < .001, PBC vs. AI-CAH). Because IL-5 is a potent eosinophil differentiation factor, we looked for evidence of activated eosinophils within the infiltrate. We observed the deposition of the primary cytotoxic granule protein of eosinophils, major basic protein (MBP), within the portal region of livers from patients with PBC. Moreover, we detected message for a cytotoxic T-lymphocyte (CTL) granzyme in 87.5% of these livers indicating that mature CTL are present. Thus, we present evidence for two effector pathways that may contribute to the tissue damage observed in PBC and have identified message for cytokines that may regulate these pathways.

Interleukin 2-independent interleukin 7 activity enhances cytotoxic immune response of HIV-1-infected individuals.

Virus-specific cytotoxic T lymphocytes (CTLs), which kill virus-infected cells, are thought to be a major host defense against viral infections. The addition of interleukin 7 (IL-7) at the onset of mitogen-stimulated cultures resulted in a marked (up to threefold) augmentation of env-specific cytotoxicity in human immunodeficiency virus type 1 (HIV-1)-infected individuals (p < 0.001). Addition of IL-7 on day 3 or 5 produced a significant but lesser augmentation of CTL response as compared to day 0. The IL-7-induced proliferative response and augmentation of cytotoxic activity was time and dose dependent, with an optimal IL-7 concentration of 1000 U/ml. Cell surface phenotypic analysis of CTL effector cells indicates that IL-7 primarily affects the proliferation of CD8+ T cells. Anti-IL-2 monoclonal antibody (MAb) substantially inhibited the proliferative effect of IL-2, but did not affect the proliferative effect of IL-7. Endogenous IL-2-induced generation of cytotoxic T cells was blocked by MAbs to IL-2 or IL-2R. The addition of IL-7 restored the process of conversion of precursor CTLs (pCTLs) to mature CTLs (mCTLs) and significantly enhanced specific cytolytic activity. It appears that IL-7 is a potent regulatory cytokine capable of acting independently of IL-2 in mitogen-specific activation of pCTLs to mCTLs. These data suggest that IL-7 should be considered as a potential therapeutic approach in AIDS and other infectious diseases in which CTL response declines.

Role of cAMP in regulating cytotoxic T lymphocyte adhesion and motility

We investigated the functional role of the cAMP pathway in human cytotoxic T lymphocyte (CTL)-target interaction. Pharmacological increase of intracellular cAMP concentration ([cAMP]i) inhibits killing, especially at low effector-to-target ratios, suggesting an inhibitory effect on CTL recycling. We show that this inhibitory effect is primarily at the level of conjugate formation. Pharmacological increase in [cAMP]i, as well as treatment with cytochalasin D, results in a "rounding up" of the CTL and inhibition of the dramatic changes in shape that occur when a CTL forms a conjugate, even with an irrelevant target. In addition, pharmacological increase in [cAMP]i affects the cytoskeleton of the CTL since it induces a decrease of filamentous actin, as detected by flow cytometry on phalloidin-stained CTL, and a stabilization of microtubules, as detected by increased resistance to the disrupting action of nocodazole. In mature CTL (but not in their immature precursors), T cell receptor triggering by specific targets results in a measurable increase in cAMP levels and strongly synergizes with adenylyl cyclase activators such as prostaglandin E2, cholera toxin and forskolin. We suggest that T cell receptor triggering may induce accumulation of cAMP that interferes with cytoskeleton function and, thus, terminates CTL secretion and adhesion. These effects of cAMP are rapidly reversible and may regulate CTL recycling.

Effects of cognate peptides on cytolytic and proliferative activities of cloned cytotoxic T lymphocytes.

When a cognate peptide is added to a culture of the corresponding clone of CD8+ cytotoxic T lymphocytes (CTLs) having the appropriate major histocompatibility complex (MHC) each cell can serve as both an antigen-presenting target cell and as a responding CTL. Under these circumstances many of the cells die. The extent of cell death is greatly diminished by Ca2+ chelation (by Mg2 EGTA) and by mAbs to CD8 and to LFA-1. Cell death is also blocked by cyclosporin A and FK-506, but not by inhibitors of protein synthesis and gene transcription (cyclohexamide and actinomycin D respectively). In contrast to the stimulatory effect on cytolytic activity, proliferation of recently stimulated CTLs is profoundly inhibited by the cognate peptide, as well as by conventional target cells that are specifically recognized and lysed by the CTLs. The inhibition of proliferation is transient and is followed by enhanced DNA synthesis of surviving CTLs. Cognate peptides also elicit fragmentation of CTL DNA, and this effect is likewise decreased by cyclosporin A and FK-506. Thus the addition of a target, either in the form of a synthetic cognate peptide that associates with MHC proteins on intact CTLs or other cells, or in the form of a conventional target cell, can simultaneously stimulate cytolytic activity and inhibit the proliferative activity of mature CTLs.