Tim-1 promotes cisplatin nephrotoxicity

Nephrotoxicity is a frequent complication of cisplatin-based chemotherapy, in which T cells are known to promote acute kidney injury. In this study, we examined the role of T cell immunoglobulin mucin 1 (Tim-1) in cisplatin-induced acute kidney injury using an inhibitory anti-Tim-1 antibody. Tim-1 acts to modulate T cell responses, but it is also expressed by damaged proximal tubules in the kidney, where it is known as kidney injury molecule-1 (Kim-1). Anti-Tim-1 antibodies attenuated cisplatin nephrotocity, with less histologic damage, improved renal function, and fewer leukocytes infiltrating the kidney compared with control antibody-treated mice. Renal NF-κB activation and apoptosis were reduced, and proinflammatory renal cytokine and chemokine mRNA expression was decreased. Renal Kim-1 expression was reduced, consistent with the diminished kidney injury after anti-Tim-1 antibody treatment. Furthermore, anti-Tim-1 antibodies reduced early systemic CD4+ and CD8+ T cell activation, apoptosis, and cytokine production. To determine whether the protective actions of anti-Tim-1 antibodies were due to effects on renal tubular cells, cisplatin nephrotoxicity was studied in Rag1(-/-) mice. Anti-Tim-1 antibodies did not affect renal dysfunction or histologic damage in Rag1(-/-) mice, showing that the benefits of inhibiting Tim-1 come from T cell effects. As Tim-1 plays an important role in promoting cisplatin nephrotoxicity, inhibiting Tim-1 may be a therapeutic strategy to prevent cisplatin-induced acute kidney injury.     

Effects of interferon-gamma on the activation of human T lymphocytes

The role of interferon (IFN)-gamma in the activation of human T cells was investigated. Addition of IFN-gamma to mixed-lymphocyte cultures (MLC) augmented both the proliferation and the development of T-cell-mediated cytotoxicity. IFN-gamma also augmented the early expression on CD8+ but not CD4+ lymphocytes of IL-2 receptor alpha chain (Tac antigen) and Class II major histocompatibility antigen (HLA-DR). This effect synergized with that caused by interleukin 2 and was not observed with IFN-alpha. The addition of neutralizing antibody against IFN-gamma to MLC suppressed the development of cytotoxicity and proliferation and the expression of activation antigens on CD8+ cells. In experiments in which highly purified CD8+ T cells were activated with cell-free stimuli, IFN-gamma slightly but significantly augmented proliferation, antibody to IFN-gamma suppressed proliferation, and excess IFN-gamma reversed this suppression. It is concluded that (i) IFN-gamma augmented activation of T cells in human MLC, (ii) IFN-gamma exerted effects directly on T cells, and (iii) IFN-gamma preferentially augmented CD8+ cell activation.     

The Tim-3 ligand galectin-9 negatively regulates CD8+ alloreactive T cell and prolongs survival of skin graft

CD8+ alloreactive T cells are the key mediators of accelerated rejection. Vigorous CD8+ alloreactive T cells responses against alloantigens, which is the main effector mechanism in acute allograft rejection, has been well described. But the molecular mechanisms to dampen activated CD8+ T cells are largely unknown. On the other hand, Tim-3 is a molecule expressed on terminally differentiated CD4+ Th1 cells. Engaging Tim-3 with its ligand galectin-9 causes an inhibitory signal, resulting in apoptosis of Th1 cells and negatively regulates Th1 type immunity. However, the question whether CD8+ T cells express surface molecular Tim-3 has not been fully elucidated. In this study, we have investigated which CD8+ subset express molecular Tim-3 by flow cytometric assay. In addition, cytotoxic assay was applied to analyze whether CD8+ alloreactive T cells were sensitive to galectin-9 induced apoptosis. Here, our results demonstrated that Tim-3 was expressed on activated CD8+ alloreactive T cells (CD8+CD44highCD62Llow), but not expressed on na?ve CD8+ T cells. Furthermore, alloreactive CD8+ cytotoxic T cells were sensitive to galectin-9 induced apoptosis both in vitro and vivo, resulting in attenuation of CD8+ alloreactive T cells mediated cytotoxicity and prolonged survival of skin graft.     

Soluble form of T cell Ig mucin 3 is an inhibitory molecule in T cell-mediated immune response

T cell Ig mucin 3 (Tim-3) has been found to play an important role in Th1-mediated auto- and alloimmune responses, but the function of soluble form of Tim-3 (sTim-3) remains to be elucidated. In this study, we report the inhibitory effect of sTim-3 on T cell-mediated immune response. In this study, sTim-3 mRNA was found, among different tissues and organs, only in splenic cells, and the activation of splenocytes resulted in up-regulated production of both sTim-3 mRNA and protein. We constructed a eukaryotic expression plasmid, psTim-3, which expresses functional murine sTim-3. In C57BL/6 mice inoculated with B16F1 melanoma cells, the growth of tumor was facilitated by the expression of this plasmid in vivo. Furthermore, sTim-3 inhibited the responses of T cells to Ag-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation and the production of cytokines IL-2 and IFN-gamma in vitro. In tumor rejection model, sTim-3 significantly impaired T cell antitumor immunity, evidenced by decreased antitumor CTL activity and reduced amount of tumor-infiltrating lymphocytes in tumor. Real-time PCR analysis of gene expression in tumor microenvironment revealed the decreased expression of Th1 cytokine genes and the unchanged profile of the genes related to T regulatory cell function, suggesting that the inhibitory effect of sTim-3 on the generation of Ag-specific T cells in vivo is dominated by T effector cells rather than T regulatory cells. Our studies thus define sTim-3 as an immunoregulatory molecule that may be involved in the negative regulation of T cell-mediated immune response.     

Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor

This study focuses on the specific CD4+ T cell requirement for optimal induction of cytotoxicity against MHC class II negative autologous tumors (AuTu) collected from patients with various types of cancer at advanced stages. CD4+ T cells were induced in cultures of cancer patients' malignant effusion-associated mononuclear cells with irradiated AuTu (mixed lymphocyte tumor cultures (MLTC)) in the presence of recombinant IL-2 and recombinant IL-7. Tumor-specific CD4+ T cells did not directly recognize the AuTu cells, but there was an MHC class II-restricted cross-priming by autologous dendritic cells (DCs), used as APC. CD8+ CTL, also induced during the MLTC, lysed specifically AuTu cells or DCs pulsed with AuTu peptide extracts (acid wash extracts (AWE)) in an MHC class I-restricted manner. Removal of CD4+ T cells or DCs from the MLTC drastically reduced the CD8+ CTL-mediated cytotoxic response against the AuTu. AWE-pulsed DCs preincubated with autologous CD4+ T cells were able, in the absence of CD4+ T cells, to stimulate CD8+ T cells to lyse autologous tumor targets. Such activated CD8+ T cells produced IL-2, IFN-gamma, TNF-alpha, and GM-CSF. The process of the activation of AWE-pulsed DCs by CD4+ T cells could be inhibited with anti-CD40 ligand mAb. Moreover, the role of CD4+ T cells in activating AWE-pulsed DCs was undertaken by anti-CD40 mAb. Our data demonstrate for the first time in patients with metastatic cancer the essential role of CD4+ Th cell-activated DCs for optimal CD8+ T cell-mediated killing of autologous tumors and provide the basis for the design of novel protocols in cellular adoptive immunotherapy of cancer, utilizing synthetic peptides capable of inducing T cell help in vivo.     

Tim-3-Expressing CD4+ and CD8+ T Cells in Human Tuberculosis (TB) Exhibit Polarized Effector Memory Phenotypes and Stronger Anti-TB Effector Functions

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4⁺ and CD8⁺ T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3⁻ counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4⁺ and CD8⁺ T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.     

Splenic dendritic cell subsets prime and boost CD8 T cells and are involved in the generation of effector CD8 T cells

The ability of the dendritic cell (DC) subsets, CD8alpha+ and CD8alpha- DCs, to initiate a CD8 T cell response or to activate memory CD8 T cells and generate effector CD8 T cells has been controversial. In this study, we analyse the capacity of splenic DC subsets to induce CD8 T cell responses to a CD8 T cell epitope (pb9) of a malaria antigen. The administration of peptide-pulsed CD8alpha- or CD8alpha+ DCs primes and boosts a primed CD8 T cell response against the malaria epitope. In vitro, depletion of CD11c(+) DCs from mouse splenocytes, immunised with recombinant vaccinia virus Ankara (MVA) expressing pb9 epitope, significantly reduced the generation of pb9-specific IFNgamma producing effector CD8 T cells, indicating that splenic DCs are involved in the development of pb9-specific IFNgamma producing effector cells. Taken together, this result shows that both DC subsets have the ability to prime and boost CD8 T cell responses and are involved in the activation of memory CD8 T cells.     

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.     

Conversion of alloantigen-specific CD8+ T cell anergy to CD8+ T cell priming through in vivo ligation of glucocorticoid-induced TNF receptor

In this study, we investigated the effect of an agonistic mAb (DTA-1) against glucocorticoid-induced TNF receptor (GITR) in a murine model of systemic lupus erythematosus-like chronic graft-vs-host disease (cGVHD). A single dose of DTA-1 inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis, typical symptoms of cGVHD. DTA-1-treated mice showed clinical and pathological signs of acute GVHD (aGVHD), such as lymphopenia, loss of body weight, increase of donor cell engraftment, and intestinal damage, indicating that DTA-1 shifted cGVHD toward aGVHD. The conversion of cGVHD to aGVHD occurred because DTA-1 prevented donor CD8+ T cell anergy. Functionally active donor CD8+ T cells produced high levels of IFN-gamma and had an elevated CTL activity against host Ags. In in vitro MLR, anergic responder CD8+ T cells were generated, and DTA-1 stimulated the activation of these anergic CD8+ T cells. We further confirmed in vivo that donor CD8+ T cells, but not donor CD4+ T cells, were responsible for the DTA-1-mediated conversion of cGVHD to aGVHD. These results indicate that donor CD8+ T cell anergy is a restriction factor in the development of aGVHD and that in vivo ligation of GITR prevents CD8+ T cell anergy by activating donor CD8+ T cells that otherwise become anergic. In sum, our data suggest GITR as an important costimulatory molecule regulating cGVHD vs aGVHD and as a target for therapeutic intervention in a variety of related diseases.     

Differential expression of CD8 alpha and CD8 beta associated with MHC-restricted and non-MHC-restricted cytolytic effector cells

The differential expression of the alpha and beta chains of the CD8 glycoprotein was examined in three functionally distinct cytolytic effector cell populations: (i) T cells (CD3+ CD56-), (ii) NK cells (CD56+ CD3-), and (iii) non-MHC-restricted T cells (CD56+ CD3+). Twenty-four percent of T cells were CD8+, and they consistently coexpressed both CD8 alpha and CD8 beta. Moreover, CD8+ T cells uniformly expressed high-density CD8 alpha. Forty percent of NK cells were CD8+ but the vast majority (approximately 75%) expressed only CD8 alpha without CD8 beta. In addition, CD8+ NK cells uniformly expressed low-density CD8 alpha. In comparison, 75% of non-MHC-restricted T lymphocytes were CD8+ but they displayed an intermediate phenotype: 60% coexpressed CD8 alpha and CD8 beta while 40% expressed only CD8 alpha. Within this population, CD8 alpha was expressed at high density, similar to that of T cells. Following IL-2 activation, enhancement of non-MHC-restricted cytotoxicity was not associated with any changes in either the quantitative or qualitative pattern of expression of CD8 alpha or CD8 beta by these cells. Addition of either anti-CD8 alpha or anti-CD8 beta mAb did not alter non-MHC-restricted cytotoxicity of either CD56+ CD3- or CD56+ CD3+ effector cells. However, within the CD56+ cell population, non-MHC-restricted cytotoxicity was almost entirely found within the CD8- and CD8 alpha + beta- populations, and both subsets displayed a similar level of killing. In contrast, CD8 alpha+ beta+ cells exhibited very little non-MHC-restricted cytotoxicity. Thus, the coexpression of CD8 alpha and CD8 beta in conjunction with the TCR/CD3 complex appears to characterize MHC restricted cells while the expression of CD8 alpha alone is associated with non-MHC-restricted cytotoxicity. Taken together, these findings suggest that neither CD8 alpha nor CD8 beta is involved in the initial phases of target cell binding or recognition during NK cell-mediated lysis. However, the selective expression of CD8 alpha by a large fraction of non-MHC-restricted effector cells suggests that this antigen may play a different functional role in this unique subset of cytolytic lymphocytes.     

Expression of programmed death 1 ligands by murine T cells and APC

Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-gamma, GM-CSF, or IL-4, and on DCs by IFN-gamma, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-gamma, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.     

Reversing tumor immune suppression with intratumoral IL-12: activation of tumor-associated T effector/memory cells, induction of T suppressor apoptosis, and infiltration of CD8+ T effectors

A single intratumoral injection of IL-12 and GM-CSF-loaded slow-release microspheres induces T cell-dependent eradication of established primary and metastatic tumors in a murine lung tumor model. To determine how the delivery of cytokines directly to the microenvironment of a tumor nodule induces local and systemic antitumor T cell activity, we characterized therapy-induced phenotypic and functional changes in tumor-infiltrating T cell populations. Analysis of pretherapy tumors demonstrated that advanced primary tumors were infiltrated by CD4+ and CD8+ T cells with an effector/memory phenotype and CD4+CD25+Foxp3+ T suppressor cells. Tumor-associated effector memory CD8+ T cells displayed impaired cytotoxic function, whereas CD4+CD25+Foxp3+ cells effectively inhibited T cell proliferation demonstrating functional integrity. IL-12/GM-CSF treatment promoted a rapid up-regulation of CD43 and CD69 on CD8+ effector/memory T cells, augmented their ability to produce IFN-gamma, and restored granzyme B expression. Importantly, treatment also induced a concomitant and progressive loss of T suppressors from the tumor. Further analysis established that activation of pre-existing effector memory T cells was short-lived and that both the effector/memory and the suppressor T cells became apoptotic within 4 days of treatment. Apoptotic death of pre-existing effector/memory and suppressor T cells was followed by infiltration of the tumor with activated, nonapoptotic CD8+ effector T lymphocytes on day 7 posttherapy. Both CD8+ T cell activation and T suppressor cell purge were mediated primarily by IL-12 and required IFN-gamma. This study provides important insight into how local IL-12 therapy alters the immunosuppressive tumor milieu to one that is immunologically active, ultimately resulting in tumor regression.     

Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12

We investigated the effects of IL-18 on the development of CD8+ effector T cells in DBA/2 anti-BDF1 whole spleen cell MLC and compared the results with those of IL-12. Addition of IL-18 to the MLC resulted in a twofold increase in CD8/CD4 ratios compared with the control cultures when cells were expanded in IL-2-containing medium following MLC. Purified CD8+ T cells recovered from the IL-18-stimulated MLC produced 20- to 30-fold more IFN-gamma after secondary stimulation with C57BL/6 spleen cells or anti-CD3 mAb, and exhibited strong allospecific CTL activity. Neither IL-18 nor IL-18-supplemented culture supernatants from DBA/2 anti-BDF1 MLC induced type I CD8+ effector T cells when purified CD8+ T cells were used as responder cells in primary MLC. Furthermore, CD4+ T cell depletion from the responder cells abrogated the IL-18-induced increase in secondary IFN-gamma production by CD8+ T cells, suggesting that IL-18-induced type I effector CD8+ T cell development was CD4+ T cell dependent. In marked contrast, adding IL-12 to primary MLC decreased CD8/CD4 ratios by 50% and suppressed secondary IFN-gamma production and CTL activity by CD8+ T cells regardless of concentration, whereas Th1 development was promoted by IL-12. Moreover, both IL-12 and IL-18 efficiently induced type I CD8+ effector T cells in C57BL/6 anti-BDF1 MLC. These findings show that IL-18 plays an important role in the generation of type I CD8+ effector T cells, and further suggest that functional maturation of CD8+ T cells is differentially regulated by IL-18 and IL-12.     

Preferential blockade of CD8(+) T cell responses by administration of anti-CD137 ligand monoclonal antibody results in differential effect on development of murine acute and chronic graft-versus-host diseases.

We investigated the effect of CD137 costimulatory blockade in the development of murine acute and chronic graft-vs-host diseases (GVHD). The administration of anti-CD137 ligand (anti-CD137L) mAb at the time of GVHD induction ameliorated the lethality of acute GVHD, but enhanced IgE and anti-dsDNA IgG autoantibody production in chronic GVHD. The anti-CD137L mAb treatment efficiently inhibited donor CD8(+) T cell expansion and IFN-gamma expression by CD8(+) T cells in both GVHD models and CD8(+) T cell-mediated cytotoxicity against host-alloantigen in acute GVHD. However, a clear inhibition of donor CD4(+) T cell expansion and activation has not been observed. On the contrary, in chronic GVHD, the number of CD4(+) T cells producing IL-4 was enhanced by anti-CD137L mAb treatment. This suggests that the reduction of CD8(+) T cells producing IFN-gamma promotes Th2 cell differentiation and may result in exacerbation of chronic GVHD. Our results highlight the effective inactivation of CD8(+) T cells and the lesser effect on CD4(+) T cell inactivation by CD137 blockade. Intervention of the CD137 costimulatory pathway may be beneficial for some selected diseases in which CD8(+) T cells are major effector or pathogenic cells. Otherwise, a combinatorial approach will be required for intervention of CD4(+) T cell function.     

IL-7 regulation of cytotoxic lymphocytes: pore-forming protein gene expression, interferon-gamma production, and cytotoxicity of human peripheral blood lymphocytes subsets.

The effects of IL-7 on the generation of cytolytic human peripheral blood lymphocytes (PBL) were investigated. Induction of T-cell pore-forming protein (PFP) mRNA and cytotoxic potential by IL-7 was both slow and minor compared with that observed in IL-2-cultured T cells. IL-7 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in T cells. Clearly, however, both IL-7 and IL-2/IL-7 induced the PFP gene expression and cytotoxic potential of CD8+ T cells and not CD4+ T cells. In addition, neither monoclonal antibodies (mAb) to the p55 or p75 IL-2-receptor subunits had any effect upon IL-7 induction of CD8+ T-cell cytotoxicity, indicating that IL-7 induction of cytotoxic CD8+ T cells was IL-2 independent. IL-7 induction of CD3- large granular lymphocyte (LGL) and PB gamma delta T-cell cytotoxicity was also delayed and reduced compared with that effected by IL-2. IL-7 (10 or 1000 U/ml, 72 hr) enhanced the NK and LAK cytotoxic of LGL and PB gamma delta T cells. By contrast IL-7 or IL-2 augmented the redirected cytotoxic potential of PB gamma delta T cells, but not that of LGL, and neither lymphokine had any effect on constitutive PFP mRNA expression in either lymphocyte subset. In addition, IL-7 induction of LGL IFN-gamma production was weak and delayed compared with that effected by IL-2 and neither IL-2 nor IL-7 stimulated IFN-gamma production in PB gamma delta T cells. Therefore, overall the effects of IL-2 and IL-7 on various cytotoxic human PBL were qualitatively similar, but quantitatively and kinetically different.     

Cbl-b regulates antigen-induced TCR down-regulation and IFN-gamma production by effector CD8 T cells without affecting functional avidity

The E3 ubiquitin ligase Cbl-b is a negative regulator of TCR signaling that: 1) sets the activation threshold for T cells; 2) is induced in anergic T cells; and 3) protects against autoimmunity. However, the role of Cbl-b in regulating CD8 T cell activation and functions during physiological T cell responses has not been systematically examined. Using the lymphocytic choriomeningitis virus infection model, we show that Cbl-b deficiency did not significantly affect the clonal expansion of virus-specific CD8 T cells. However, Cbl-b deficiency not only increased the steady-state cell surface expression levels of TCR and CD8 but also reduced Ag-induced down-modulation of cell surface TCR expression by effector CD8 T cells. Diminished Ag-stimulated TCR down-modulation and sustained Ag receptor signaling induced by Cbl-b deficiency markedly augmented IFN-gamma production, which is known to require substantial TCR occupancy. By contrast, Cbl-b deficiency minimally affected cell-mediated cytotoxicity, which requires limited engagement of TCRs. Surprisingly, despite elevated expression of CD8 and reduced Ag-induced TCR down-modulation, the functional avidity of Cbl-b-deficient effector CD8 T cells was comparable to that of wild-type effectors. Collectively, these data not only show that Cbl-b-imposed constraint on TCR signaling has differential effects on various facets of CD8 T cell response but also suggest that Cbl-b might mitigate tissue injury induced by the overproduction of IFN-gamma by CD8 T cells. These findings have implications in the development of therapies to bolster CD8 T cell function during viral infections or suppress T cell-mediated immunopathology.     

Regulation of lymphokine-activated killer activity and pore-forming protein gene expression in human peripheral blood CD8+ T lymphocytes. Inhibition by transforming growth factor-beta.

The effect of transforming growth factor-beta 1 (TGF-beta) on activation-induced CD8+ T cell cytotoxicity and gene expression was investigated. TGF-beta was demonstrated to inhibit pore-forming protein (PFP) mRNA expression and total benzoyloxycarbonyl-L-lysine thiobenzyl ester esterase activity in CD8+ T cells cultured with IL-2 and OKT3 mAb for 6 to 18 days. Consistently, in the absence or presence of TGF-beta, the PFP mRNA expression and lymphokine-activated killer (LAK) activity of CD8+ T cells were closely correlated. The inhibitory effects of TGF-beta on both CD8+ T cell PFP mRNA expression and LAK activity were reversible by removal of TGF-beta from the culture. Expression of lymphokines, adhesion/recognition molecules, and activated p55 IL-2R, previously implicated in the lytic mechanism of cytotoxic lymphocytes, either was not detectable or did not correlate with TGF-beta inhibition of LAK activity. In addition, independently of effector/target cell binding, the lectin- or heteroconjugated antibody-dependent cellular cytotoxicity of IL-2/OKT3 mAb-activated CD8+ T cells was inhibited by preculture with TGF-beta. TGF-beta also inhibited the rapid activation-induced expression of PFP mRNA and cytotoxic potential in resting T cells, thereby indicating that the effect of TGF-beta was independent of T cell proliferation. TGF-beta inhibition of CD8+ T cell PFP mRNA expression and cytotoxic potential was TGF-beta dose dependent; however, a variety of activation stimuli (including IL-2, IL-6, and OKT3 mAb) were all similarly inhibited by TGF-beta. Therefore, TGF-beta may be an important general regulator of CD8+ T cell cytotoxic function, in particular by suppressing expression of PFP, a major cytolytic protein implicated in the lytic function of cytotoxic lymphocytes.     

Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially at high levels on CD25+CD4+ regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression is enhanced rapidly after activation. In this report we show that the GITR provides a potent costimulatory signal to both CD25+ and CD25- CD4+ T cells. GITR-mediated stimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of both CD25-CD4+ and CD25+CD4+ T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation was further confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokine production, such as IL-2, IFN-gamma, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-kappaB components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation.     

Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN-gamma production

Although CD8+ IL-2Rbeta (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rbeta-negative or -low (CD122-) T cells conversely decreased. The IFN-gamma production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-gamma than did CD8+ CD122- T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-gamma production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-gamma production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-gamma. The CD3-stimulated IFN-gamma production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-gamma than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-gamma production and are suggested to be involved in the immunological changes with aging.     

Expression of variable exon A-, B-, and C-specific CD45 determinants on peripheral and thymic T cell populations.

A mAb (I/24) has been generated that is specific for a determinant on mouse CD45 molecules. Reactivity of this mAb with a panel of CD45 transfected cell lines demonstrated that the determinant recognized is dependent upon expression of one or more CD45 variable exons and that exon C is sufficient for its expression. The exon C-specific epitope detected by I/24 is expressed at high density on essentially all B lymphocytes and at an intermediate density on the vast majority of CD8+ splenic T cells. Two distinct subpopulations of CD4+ splenic T cells were detected, a minor subpopulation that expresses this exon determinant at high density and a major subpopulation that expresses it at a much lower density. This first identification of a CD45RC-specific reagent allowed a comparison of the expression of exon A-, exon B-, and exon C-specific determinants on peripheral and thymic lymphoid populations. When splenic lymphocytes were analyzed for expression of CD45RA (reactive with mAb 14.8), CD45RB (reactive with mAb 23G2 or mAb 16.A), and CD45RC (reactive with mAb I/24) determinants, it was found that each of these CD45 determinants had a distinct pattern of expression on CD4+ and CD8+ T cells and B cells. CD45RB and RC epitopes were also detected at high density on a small proportion (0.7 to 4.1%) of thymocytes. Both CD45RB and RC epitopes were found predominantly on CD4-CD8- and CD4-CD8+ thymocytes but were also found on small numbers of CD4+CD8+ and CD4+CD8- cells. The population of thymocytes that expressed CD45RB and CD45RC determinants displayed a novel TCR CD3 phenotype characterized by a level of expression that was intermediate between that seen in the larger CD3 bright and CD3 dull populations of thymocytes.