Expression of CD30 ligand (CD153) on murine activated T cells

CD30, a member of the TNF receptor family, has been implicated in the activation of T cells and B cells. In the present study, we characterized the expression and function of murine CD30 ligand (mCD153) by utilizing mCD153 transfectants and a novel mAb against mCD153 (RM153), which can inhibit the binding of murine CD30 to mCD153. The mCD153 transfectants did not co-stimulate the proliferation of anti-CD3-stimulated naive T cells but enhanced the proliferation of anti-CD28-co-stimulated T cells. The mCD153 transfectants exhibited a potent co-stimulatory activity for proliferation of pre-activated T cells that expressed CD30 after anti-CD3 and anti-CD28 stimulation. In contrast to the CD30 expression on naive T cells that required anti-CD28 co-stimulation, mCD153 expression was observed on anti-CD3-stimulated T cells without the anti-CD28 co-stimulation, predominantly on CD4(+) T cells with a transient kinetics which peaked at 24 h but disappeared at 48 h. In contrast to the preferential expression of CD30 on Th2 cells, mCD153 was expressed on both Th1 and Th2 cells after anti-CD3 stimulation. These results indicated a differential regulation of CD30 and CD153 expression in T cells, which may be relevant to immuno-regulatory role of the CD30-CD153 interaction.     

Cloning, recombinant expression and biochemical characterization of the murine CD83 molecule which is specifically upregulated during dendritic cell maturation

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC) and represents the best marker for mature DC. Here, we report the cloning of the cDNA encoding mouse CD83 (mCD83) from a murine bone marrow-derived DC (BM-DC) cDNA library. DNA sequence analysis revealed a 196 amino acid protein including a signal peptide of 21 amino acids which shares 63% amino acid identity with hCD83. Using Northern blot analyses, mCD83 mRNA was found to be strongly expressed in mouse BM-DC and its expression was upregulated following stimulation with LPS or TNF-alpha. Transfection experiments using COS-7 cells revealed that mCD83 is glycosylated. Furthermore, the extracellular CD83 domain was recombinantly expressed in Escherichia coli and one-dimensional NMR data strongly support that the protein is structurally folded.     

Automatic generation of lymphocyte heterogeneity: Division-dependent changes in the expression of CD27, CCR7 and CD45 by activated human naive CD4+ T cells are independently regulated

Lymphocyte differentiation is a complex process regulated by the integration of signals received through a variety of cell surface receptors that results in populations of differentiated cells that have acquired novel characteristics and effector functions. Differentiation of T and B lymphocytes into effector cells, such as cytokine-secreting CD4+ T cells, cytotoxic CD8+ T cells and Ig-secreting B cells, as well as alterations in cell surface phenotype, have been reported to be associated with cell division. Nevertheless, the genesis of heterogeneity in effector cell type is unknown. A strictly deterministic view holds that heterogeneity arises from distinct signalling histories for each functionally or phenotypically different cell type. In contrast, a probabilistic interpretation proposes that internal stochastic regulation of gene expression gives rise to lymphocytes of mixed phenotypes. To help distinguish between these explanations, we examined the expression of CD27, CCR7, CD45RA and CD45RO by human naive CD4+ T cells in the context of the division history of the lymphocyte. Our results show that each marker independently changes with progressive divisions, strongly supporting the proposal that phenotypic heterogeneity in lymphocytes can arise as the result of independent stochastic processes controlling the expression of individual molecules.     

CD27-mediated activation of murine NK cells

CD27, a member of the TNF receptor superfamily, has been implicated in T cell activation, T cell development, and T cell-dependent Ab production by B cells. In the present study we examined the expression and function of CD27 on murine NK cells. Murine NK cells constitutively expressed CD27 on their surface. Stimulation with immobilized anti-CD27 mAb or murine CD27 ligand (CD70) transfectans solely could induce proliferation and IFN-gamma production of freshly isolated NK cells and enhanced the proliferation and IFN-gamma production of anti-NK1.1-sutimulated NK cells. Although NK cell cytotoxicity was not triggered by anti-CD27 mAb or against CD70 transfectants, prestimulation via CD27 enhanced the cytotoxic activity of NK cells in an IFN-gamma-dependent manner. These results suggest that CD27-mediated activation may be involved in the NK cell-mediated innate immunity against virus-infected or transformed cells expressing CD70.     

Differential expression of the human CD8beta splice variants and regulation of the M-2 isoform by ubiquitination

The CD8alphabeta heterodimer functions as a coreceptor with the TCR, influencing the outcome of CD8(+) T cell responses to pathogen-infected and tumor cells. In contrast to the murine CD8B gene, the human gene encodes alternatively spliced variants with different cytoplasmic tails (M-1, M-2, M-3, and M-4). At present, little is known about the expression patterns and functional significance of such variants. We used quantitative RT-PCR to demonstrate differential mRNA expression patterns of these splice variants in thymocytes and in resting, memory, and activated primary human CD8(+) T cells. In total CD8(+) T cells, mRNA levels of the M-1 variant were the most predominant and levels of M-3 were the least detected. The M-4 isoform was predominant in effector memory CD8(+) T cells. Upon stimulation of CD8(+) T cells, the M-2 variant mRNA levels were elevated 10-20-fold relative to resting cells in contrast to the other isoforms. Curiously, the M-2 isoform was not expressed on the cell surface in transfected cell lines. Using fluorescent chimeras of the extracellular domain of mouse CD8beta fused to the cytoplasmic tails of each isoform, the M-2 isoform was localized in a lysosomal compartment regulated by ubiquitination of a lysine residue (K215) in its cytoplasmic tail. In contrast, upon short-term stimulation, the M-2 protein localized to the cell surface with the TCR complex. The relatively recent evolution of CD8B gene splice variants in the chimpanzee/human lineage is most likely important for fine-tuning the CD8(+) T cell responses.     

Regulation of lymphocyte clustering by CD30-mediated ICAM-1 up-regulation

CD30 is expressed transiently on activated B and T lymphocytes and constitutively on several B- and T cell lymphomas. CD30 functions include participation in negative selection of thymocytes, costimulation of activated T cells, isotype switching of B cells, and regulation of the effector activity of cytotoxic lymphocytes. Although CD30 is not a marker for T helper 2 (TH2) cells, it may participate in the polarization of TH1 and TH2 cells. The pleiotropic functions of CD30 are initiated by interaction of CD30-expressing cells with other immune competent cells expressing CD30-L and providing the signals for modulation of effector cell activity. Here, we report that CD30 signals generated by anti-CD30 on activated, normal murine T cells strongly up-regulate the expression of intercellular adhesion molecule 1 (ICAM-1, CD54), and to a lesser extent, ICAM-2 (CD102). CD30 signals moreover delay the subsequent decline of ICAM expression. CD30 cross-linking did not alter the expression of CD11a/CD18 (LFA-1), the counter receptor for ICAM abundant on T cells. CD30-mediated ICAM-1 up-regulation is independent of cytokine secretion and appears to be transmitted directly through NF-kappaB activation. CD30-mediated up-regulation of ICAM-1 expression led to a significant increase in cluster formation of lymph node cells. Increased lymphocyte self-aggregation mediated by CD30 may set the stage for fraternal signaling to modulate lymphocyte function.     

Differentiation and functional maturation of bone marrow-derived intestinal epithelial T cells expressing membrane T cell receptor in athymic radiation chimeras

The thymus dependency of murine intestinal intraepithelial lymphocytes (IEL) was studied in an athymic F1----parent radiation chimera model. IEL, although not splenic or lymph node lymphocytes, from athymic chimeras displayed normal levels of cells bearing the class-specific T cell Ag, CD4 and CD8; the TCR-associated molecule, CD3; and the Thy-1 Ag. Moreover, two-color flow cytometric analyses of IEL from athymic mice demonstrated regulated expression of T cell Ag characteristic of IEL subset populations from thymus-bearing mice. In immunoprecipitation experiments, surface TCR-alpha beta or TCR-gamma delta were expressed on IEL, although not on splenic lymphocytes, from athymic chimeras. That IEL from athymic chimeras constituted a population of functionally mature effector cells activated in situ, similar to IEL from thymus-bearing mice, was demonstrated by the presence of CD3-mediated lytic activity of athymic lethally irradiated bone marrow reconstituted IEL. These data provide compelling evidence that intestinal T cells do not require thymic influence for maturation and development, and demonstrate that the microenvironment of the intestinal epithelium is uniquely adapted to regulate IEL differentiation.     

CD70-driven costimulation induces survival or Fas-mediated apoptosis of T cells depending on antigenic load

Apoptosis plays an essential role in the removal of activated CD8 T cells that are no longer required during or postinfection. The Bim-dependent intrinsic pathway of apoptosis removes effector CD8 T cells upon clearance of viral infection, which is driven by withdrawal of growth factors. Binding of Fas ligand to Fas mediates activation-induced T cell death in vitro and cooperates with Bim to eliminate CD8 T cells during chronic infection in vivo, but it is less clear how this pathway of apoptosis is initiated. In this study, we show that the costimulatory TNFR CD27 provides a dual trigger that can enhance survival of CD8 T cells, but also removal of activated CD8 T cells through Fas-driven apoptosis. Using in vitro stimulation assays of murine T cells with cognate peptide, we show that CD27 increases T cell survival after stimulation with low doses of Ag, whereas CD27 induces Fas-driven T cell apoptosis after stimulation with high doses of Ag. In vivo, the impact of constitutive CD70-driven stimulation on the accumulation of memory and effector CD8 T cells is limited by Fas-driven apoptosis. Furthermore, introduction of CD70 signaling during acute infection with influenza virus induces Fas-dependent elimination of influenza-specific CD8 T cells. These findings suggest that CD27 suppresses its costimulatory effects on T cell survival through activation of Fas-driven T cell apoptosis to maintain T cell homeostasis during infection.     

The Pim kinase pathway contributes to survival signaling in primed CD8+ T cells upon CD27 costimulation

Stimulation of the costimulatory receptor CD27 by its ligand CD70 has proved important for the generation of primary and memory CD8(+) T cell responses in various models of antigenic challenge. CD27/CD70-mediated costimulation promotes the survival of primed T cells and thereby increases the size of effector and memory populations. In this paper, we reveal molecular mechanisms underlying the prosurvival effect of CD27. CD27 signaling upregulated expression of the antiapoptotic Bcl-2 family member Bcl-x(L). However, genetic reconstitution of Cd27(-/-) CD8(+) T cells with Bcl-x(L) alone or in combination with the related protein Mcl-1 did not compensate for CD27 deficiency in the response to influenza virus infection. This suggested that CD27 supports generation of the CD8(+) effector T cell pool not only by counteracting apoptosis via Bcl-2 family members. Genome-wide mRNA expression profiling indicated that CD27 directs expression of the Pim1 gene. Pim-1 is a serine/threonine kinase that sustains survival of rapidly proliferating cells by antiapoptotic and prometabolic effects that are independent of the mammalian target of rapamycin (mTOR) pathway. In TCR-primed CD8(+) T cells, CD27 could increment Pim-1 protein expression and promote cell survival throughout clonal expansion independent of the mTOR and IL-2R pathways. In addition, introduction of the Pim1 gene in Cd27(-/-) CD8(+) T cells partially corrected their defect in clonal expansion and formation of an effector pool. We conclude that CD27 may contribute to the survival of primed CD8(+) T cells by the upregulation of antiapoptotic Bcl-2 family members but also calls the Pim-1 kinase survival pathway into action.     

TGF-beta 1 attenuates the acquisition and expression of effector function by tumor antigen-specific human memory CD8 T cells

TGF-beta1 is a potent immunoregulatory cytokine. However, its impact on the generation and effector function of Ag-specific human effector memory CD8 T cells had not been evaluated. Using Ag-specific CD8 T cells derived from melanoma patients immunized with the gp100 melanoma Ag, we demonstrate that the addition of TGF-beta1 to the initial Ag activation cultures attenuated the gain of effector function by Ag-specific memory CD8 T cells while the phenotypic changes associated with activation and differentiation into effector memory were comparable to control cultures. These activated memory CD8 T cells consistently expressed lower mRNA levels for T-bet, suggesting a mechanism for TGF-beta1-mediated suppression of gain of effector function in memory T cells. Moreover, TGF-beta1 induced a modest expression of CCR7 on Ag-activated memory CD8 T cells. TGF-beta1 also suppressed cytokine secretion by Ag-specific effector memory CD8 T cells, as well as melanoma-reactive tumor-infiltrating lymphocytes and CD8 T cell clones. These results demonstrate that TGF-beta1 suppresses not only the acquisition but also expression of effector function on human memory CD8 T cells and tumor-infiltrating lymphocytes reactive against melanoma, suggesting that TGF-beta1-mediated suppression can hinder the therapeutic benefits of vaccination, as well as immunotherapy in cancer patients.     

Modulation by IL-2 of CD70 and CD27 expression on CD8+ T cells: importance for the therapeutic effectiveness of cell transfer immunotherapy

Proper T cell function relies on the integration of signals delivered by Ag, cytokine, and costimulatory receptors. In this study, the interactions between IL-2, CD27, and its ligand CD70 and their effects on human T cell function were examined. Unstimulated CD8(+) T cells expressed relatively low levels of CD70 and high levels of CD27. Incubation in vitro with high doses of IL-2 (3,000 IU/ml) or administration of IL-2 in vivo resulted in substantial up-regulation of CD70 expression and the concomitant loss of cell surface CD27 expression on CD8(+) cells. Withdrawal of IL-2 from activated CD8(+) T cells that had been maintained in IL-2 resulted in a reversal of the expression of these two markers, whereas reciprocal changes were seen following treatment of PBMCs with IL-2. The proliferation observed in cells stimulated with IL-2 primarily occurred in a subset of the CD70(+)CD8(+) T cells that up-regulated IL-2 receptor expression but did not occur in CD70(-)CD8(+) T cells. Blocking CD70 resulted in a significant reduction of T cell proliferation induced by high-dose IL-2, indicating that the interaction of CD70 with CD27 played a direct role in T cell activation mediated by IL-2. Finally, studies conducted on tumor-infiltrating lymphocyte (TIL) samples that were administered to melanoma patients indicated that the size of the pool of CD27(+)CD8(+) T cells in bulk TILs was highly associated (p = 0.004) with the ability of these TILs to mediate tumor regression following adoptive transfer.     

Dynamic control of self-specific CD8+ T cell responses via a combination of signals mediated by dendritic cells

It is acknowledged that T cell interactions with mature dendritic cells (DC) lead to immunity, whereas interactions with immature DC lead to tolerance induction. Using a transgenic murine system, we have examined how DC expressing self-peptides control naive, self-reactive CD8+ T cell responses in vitro and in vivo. We have shown, for the first time, that immature DC can also stimulate productive activation of naive self-specific CD8+ T cells, which results in extensive proliferation, the expression of a highly activated cell surface phenotype, and differentiation into autoimmune CTL. Conversely, mature DC can induce abortive activation of naive CD8+ T cells, which is characterized by low-level proliferation, the expression of a partially activated cell surface phenotype which does not result in autoimmune CTL. Critically, both CD8+ T cell responses are determined by a combination of signals mediated by the DC, and that altering any one of these signals dramatically shifts the balance between autoimmunity and self-tolerance induction. We hypothesize that DC maintain the steady state of self-tolerance among self-specific CD8+ T cells in an active and dynamic manner, licensing productive immune responses against self-tissues only when required.     

CD70 signaling is critical for CD28-independent CD8+ T cell-mediated alloimmune responses in vivo

The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8(+) T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4(+) T cell function but prevented CD8(+) T cell-mediated rejection, inhibited the proliferation and activation of effector CD8(+) T cells, and diminished the expansion of effector and memory CD8(+) T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8(+) alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8(+) T cell depletion is currently mandatory.     

Frequency, specificity, and sites of expansion of CD8+ T cells during primary pulmonary influenza virus infection

We have used intracellular cytokine staining and MHC class I tetramer binding in conjunction with granzyme B protease expression and in vivo BrdU uptake to characterize the primary murine CD8(+) T cell response to pulmonary influenza virus infection. We have observed that the majority (>90%) of the CD8(+) T cell response to the A/Japan/305/57 virus in the lung at the peak of the response (days 9-11) is directed to four epitopes (three dominant and one subdominant). Using induction of granzyme B as a surrogate to identify specific activated CD8(+) T cells, we found that an unexpectedly large fraction ( approximately 70%) of lung-infiltrating CD8(+) T cells expressed granzyme B on day 6 of infection when estimates by MHC tetramer/intracellular cytokine staining yielded substantially lower frequencies ( approximately 30%). In addition, by using intranasal administration of BrdU during infection, we obtained evidence for proliferative expansion of activated CD8(+) T cells in the infected lung early (days 5-7) in the primary response. These results suggest that the frequency and number of specific CTL present in the lung early in infection may be underestimated by standard detection methods, and primary CD8(+) T cell expansion may occur in both secondary lymphoid organs and the infected lung.     

Ly-1 (CD5), a membrane glycoprotein of mouse T lymphocytes and a subset of B cells, is a natural ligand of the B cell surface protein Lyb-2 (CD72).

CD5 is a 67-kDa glycoprotein expressed on the cell surface membrane of all T lymphocytes and on a small proportion of B lymphocytes. The physiologic role of this Ag is still unknown. Structural and functional studies of CD5 suggest that it might act as a receptor for a positive signal. CD5-specific mAb augment CD3- or mitogen-induced T cell proliferation, IL-2 secretion, and IL-2R expression and induce a rise in intracellular [Ca2+]. In this report, we describe the purification of mouse CD5 protein (mCD5) and its use as a probe to search for the ligand of CD5. We demonstrate that mCD5 specifically interacts with the mouse B cell differentiation Ag CD72/Lyb-2. Three serologically defined allelic forms of mouse CD72/Lyb-2 can all interact with mCD5. We further show that mCD5 can interact with human CD72/Lyb-2, and similarly, that human CD5 can interact with mouse CD72/Lyb-2. These studies may have major implications for the mechanisms of T-B cell communication.     

Jakmip1 is expressed upon T cell differentiation and has an inhibitory function in cytotoxic T lymphocytes

Jakmip1 belongs to a family of three related genes encoding proteins rich in coiled-coils. Jakmip1 is expressed predominantly in neuronal and lymphoid cells and colocalizes with microtubules. We have studied the expression of Jakmip1 mRNA and protein in distinct subsets of human primary lymphocytes. Jakmip1 is absent in naive CD8(+) and CD4(+) T lymphocytes from peripheral blood but is highly expressed in Ag-experienced T cells. In cord blood T lymphocytes, induction of Jakmip1 occurs upon TCR/CD28 stimulation and parallels induction of effector proteins, such as granzyme B and perforin. Further analysis of CD8(+) and CD4(+) T cell subsets showed a higher expression of Jakmip1 in the effector CCR7(-) and CD27(-) T cell subpopulations. In a gene expression follow-up of the development of CMV-specific CD8(+) response, Jakmip1 emerged as one of the most highly up-regulated genes from primary infection to latent stage. To investigate the relationship between Jakmip1 and effector function, we monitored cytotoxicity of primary CD8(+) T cells silenced for Jakmip1 or transduced with the full-length protein or the N-terminal region. Our findings point to Jakmip1 being a novel effector memory gene restraining T cell-mediated cytotoxicity.     

CD27low CD4 T lymphocytes that accumulate in the mouse lungs during mycobacterial infection differentiate from CD27high precursors in situ, produce IFN-gamma, and protect the host against tuberculosis infection

The generation of effector, IFN-gamma producing T lymphocytes and their accumulation at sites of infection are critical for host protection against various infectious diseases. The activation and differentiation of naive T lymphocytes into effector memory cells starts in lymphoid tissues, but it is not clear whether the Ag-experienced cells that leave lymph nodes (LN) are mature or if they undergo further changes in the periphery. We have previously shown that CD44(high)CD62L(low) effector CD4 T lymphocytes generated during the course of mycobacterial infection can be segregated into two subsets on the basis of CD27 receptor expression. Only the CD27(low) subset exhibited a high capacity for IFN-gamma secretion, indicating that low CD27 expression is characteristic of fully differentiated effector CD4 T lymphocytes. We demonstrate now that CD27(low) IFN-gamma-producing CD4 T lymphocytes accumulate in the lungs but are rare in LNs. Several factors contribute to their preferential accumulation. First, CD27(low) CD4 T lymphocytes present in the LN are highly susceptible to apoptosis. Second, circulating CD27(low) CD4 T cells do not enter the LN but efficiently migrate to the lungs. Third, CD27(high) effector CD4 T cells that enter the lungs down-regulate CD27 expression in situ. In genetically heterogeneous mice that exhibit varying susceptibility to tuberculosis, the accumulation of mature CD27(low) CD4 T cells in the lungs correlates with the degree of protection against infection. Thus, we propose that terminal maturation of effector CD4 T lymphocytes in the periphery provides the host with efficient local defense and avoids potentially harmful actions of inflammatory cytokines in lymphoid organs.