Antigen reactive memory T cells are defined by Ta1

Ta1 is a 105,000 dalton protein that is weakly expressed on a small fraction of resting human peripheral blood T cells but strongly expressed in vitro on T cell clones and a substantial proportion of activated T cells. Unlike receptors for growth factors such as IL 2, the Ta1 antigen is present on T cell lines and clones irrespective of cell cycle. The function of Ta1 was investigated after separation of T lymphocytes into Ta1-enriched and Ta1-depleted subpopulations that were obtained from normal human subjects. Although Ta1-enriched T cells constitute only 10 to 15% of the E rosette-positive lymphocyte population, most, if not all, of the anamnestic response to the recall antigens tetanus toxoid and mumps reside in the Ta1+ population. Both Ta1-enriched and -depleted cells responded equally well to the mitogen PHA. The autologous mixed lymphocyte response was also greater in the Ta1-enriched subpopulation but not to the degree seen with soluble antigen. Increased proliferation was not due simple to increased inducer cell function within the Ta1+ subpopulations because both Ta1- and Ta1+ cells induced similar amounts of Ig synthesis in the presence of PWM. Additionally, increasing numbers of Ta1- cells did not suppress the enhanced proliferative responses of Ta1+ cells, and thus Ta1- cells do not appear to be functioning as suppressor cells. The Ta1 antigen appears to be a marker for previously activated T cells in peripheral blood, and this subpopulation appears to include T memory cells.     

Immortalized myeloid suppressor cells trigger apoptosis in antigen-activated T lymphocytes

We described a generalized suppression of CTL anamnestic responses that occurred in mice bearing large tumor nodules or immunized with powerful recombinant viral immunogens. Immune suppression entirely depended on GM-CSF-driven accumulation of CD11b(+)/Gr-1(+) myeloid suppressor cells (MSC) in secondary lymphoid organs. To further investigate the nature and properties of MSC, we immortalized CD11b(+)/Gr-1(+) cells isolated from the spleens of immunosuppressed mice, using a retrovirus encoding the v-myc and v-raf oncogenes. Immortalized cells expressed monocyte/macrophage markers (CD11b, F4/80, CD86, CD11c), but they differed from previously characterized macrophage lines in their capacities to inhibit T lymphocyte activation. Two MSC lines, MSC-1 and MSC-2, were selected based upon their abilities to inhibit Ag-specific proliferative and functional CTL responses. MSC-1 line was constitutively inhibitory, while suppressive functions of MSC-2 line were stimulated by exposure to the cytokine IL-4. Both MSC lines triggered the apoptotic cascade in Ag-activated T lymphocytes by a mechanism requiring cell-cell contact. Some well-known membrane molecules involved in the activation of apoptotic pathways (e.g., TNF-related apoptosis-inducing ligand, Fas ligand, TNF-alpha) were ruled out as candidate effectors for the suppression mechanism. The immortalized myeloid lines represent a novel, useful tool to shed light on the molecules involved in the differentiation of myeloid-related suppressors as well as in the inhibitory pathway they use to control T lymphocyte activation.     

Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-l-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1 ^a . Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1^b- and Qa-1^c-encoded determinants. Qa-1 ^d target cells are unique in that they express determinants recognized by anti-Qa-1^a CTL and by anti-Qa-1^b CTL. Killing of Qa-1 ^d targets by anti-Qa-1^a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1 ^d cells by one anti-Qa-1^b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1^a CTL and Qa-1.1-specific antibodies did not have identical specificities.Abbreviations used in this paper B6 C57BL/6J - CAB concanavalin A stimulated lymphoblasts - CML cell-mediated lympholysis - CTL cytotoxic T lymphocyte - NMS normal mouse serum - MHC major histocompatibility complex - MLC mixed leukocyte culture - MR maximum release - SMDM supplemented Mishell-Dutton medium - SR spontaneous release     

New HLA-A2 variants defined by monoclonal antibodies and cytotoxic T lymphocytes

Three HLA-A2 variants, A2-DW, A2-KC, and A2-Lee, were identified in three Chinese donors using a panel of monoclonal antibodies. A2-DW was negative with two of the ten HLA-A2 monoclonal antibodies tested, whereas A2-KC was negative with five of the ten and A-2 Lee was negative with one.Epstein-Barr virus-specific cytotoxic T cells generated from the A2-DW donor recognized and killed target cells prepared from the A2-KC donor, but did not recognize target cells from HLA-A2.1, –A2.2, or –A2.4 donors. In isoelectric focusing studies, A2-DW and A2-KC focus in identical positions more acidic than the other HLA-A2 antigens tested.     

Ia-positive T lymphocytes are the producer cells of interferon gamma

The production of γ-interferon (IFNγ) in human peripheral blood T lymphocytes was induced by stimulation with PHA. For identification of the producer cell of IFNγ, double fluorescence studies were undertaken and titers of interferon were determined in preparatively separated T-cell subpopulations reactive with one of the monoclonal antibodies OKT3, OKT4, OKT8, and OKIa1. Production of IFNγ was found in OKT3⁺, OKT4⁺, and OKT8⁺ cells. However, IFNγ production occurred only in T cells also reactive with the monoclonal antibody OKIa1. Addition of macrophages had no substantial effect on interferon titers in these subpopulations. It is suggested that the T cell subset producing IFNγ is characterized by its reactivity with the monoclonal antibodies OKT3, OKT4 or OKT8, and OKIa1.     

Stepwise differentiation of CD4 memory T cells defined by expression of CCR7 and CD27

To study the steps in the differentiation of human memory CD4 T cells, we characterized the functional and lineage relationships of three distinct memory CD4 subpopulations distinguished by their expression of the cysteine chemokine receptor CCR7 and the TNFR family member CD27. Using the combination of these phenotypic markers, three populations were defined: the CCR7+CD27+, the CCR7-CD27+, and the CCR7-CD27- population. In vitro stimulation led to a stepwise differentiation from naive to CCR7+CD27+ to CCR7-CD27+ to CCR7-CD27-. Telomere length in these subsets differed significantly (CCR7+CD27+ > CCR7-CD27+ > CCR7-CD27-), suggesting that these subsets constituted a differentiative pathway with progressive telomere shortening reflecting antecedent in vivo proliferation. The in vitro proliferative response of these populations declined, and their susceptibility to apoptosis increased progressively along this differentiation pathway. Cytokine secretion showed a differential functional capacity of these subsets. High production of IL-10 was only observed in CCR7+CD27+, whereas IFN-gamma was produced by CCR7-CD27+ and to a slightly lesser extent by CCR7-CD27- T cells. IL-4 secretion was predominantly conducted by CCR7-CD27- memory CD4 T cells. Thus, by using both CCR7 and CD27, distinct maturational stages of CD4 memory T cells with different functional activities were defined.     

CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes

The success of immunization with irradiated sporozoites is unparalleled among the current vaccination approaches against malaria, but its mechanistic underpinnings have yet to be fully elucidated. Using a model mimicking natural infection by Plasmodium yoelii, we delineated early events governing the development of protective CD8(+) T-cell responses to the circumsporozoite protein. We demonstrate that dendritic cells in cutaneous lymph nodes prime the first cohort of CD8(+) T cells after an infectious mosquito bite. Ablation of these lymphoid sites greatly impairs subsequent development of protective immunity. Activated CD8(+) T cells then travel to systemic sites, including the liver, in a sphingosine-1-phosphate (S1P)-dependent fashion. These effector cells, however, no longer require bone marrow-derived antigen-presenting cells for protection; instead, they recognize antigen on parenchymal cells-presumably parasitized hepatocytes. Therefore, we report an unexpected dichotomy in the tissue restriction of host responses during the development and execution of protective immunity to Plasmodium.     

IFN regulation and functions in myeloid dendritic cells

A central issue in dendritic cells (DC) biology is to understand how type I IFNs modulate the immuno-regulatory properties of DC. In this review I will address this issue in light of the recent experimental evidence on the expression and function of these cytokines in myeloid DC. This knowledge may have important therapeutic implications in infectious and neoplastic diseases and open new perspectives in the use of IFNs as vaccine adjuvants and in the development of DC-based vaccines.     

Cerebral vascular endothelial cells are effective targets for in vitro lysis by encephalitogenic T lymphocytes.

Lysis of cerebral vascular endothelial cells (EC) by CD4-positive, myelin basic protein-specific encephalitogenic T cell lines was investigated. Unstimulated EC were not lysed, but culture in the presence of murine rIFN-gamma resulted in the expression of class II MHC (Ia) molecules and the concomitant ability to function as effective target cells for lysis. The possible requirement for Ia molecules was further demonstrated by antibody-blocking experiments. Lysis of EC targets also required the presence of specific Ag (myelin basic protein); PPD-specific T cell lines also lysed the PPD-pulsed EC. In all cases, lysis was directly proportional to E:T ratios. In addition, continuous passage of T cell lines resulted in the concomitant loss of encephalitogenicity and ability to affect EC lysis, indicating a possible relationship between these two factors. These results demonstrate that CD4+ T cells interact with cerebral vascular EC. It is suggested that such interactions may be important in the pathogenesis of diseases involving migrations of these cells across the blood-brain barrier.     

Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression

Fractalkine/CX3C ligand 1 and its receptor CX3CR1 are known to mediate both cell adhesion and cell migration. Here we show that CX3CR1 defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B, which include NK cells, gammadelta T cells, and terminally differentiated CD8(+) T cells. In addition, soluble fractalkine preferentially induced migration of cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic effector lymphocytes with membrane-bound fractalkine promoted subsequent migration to the secondary chemokines, such as macrophage inflammatory protein-1beta/CC ligand 4 or IL-8/CXC ligand 8. Thus, fractalkine expressed on inflamed endothelium may function as a vascular regulator for cytotoxic effector lymphocytes, regardless of their lineage and mode of target cell recognition, through its ability to capture them from blood flow and to promote their emigration in response to other chemokines.     

Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.     

Molecular analysis of an HLA-A2 functional variant CLA defined by cytolytic T lymphocytes

By using cytolytic T lymphocytes (CTL), the HLA-A2 serologic specificity may be divided into at least four subtypes designated as A2.1 to A2.4. The HLA-A2.4 antigen expressed by donor CLA is not recognized by allogeneic CTL specific for either A2.1, A2.2, or A2.3, but is indistinguishable from HLA-A2.1 by H-Y-specific, HLA-A2-restricted CTL and by isoelectric focusing. The structure of this HLA-A2.4 antigen was compared with the known structure of the main A2.1 subtype expressed on JY cells to establish the molecular basis for the immunologic differences between the two antigens. Comparative peptide mapping and radiochemical sequence analysis were used to establish that they differed by a single amino acid change: Phe at position 9 in HLA-A2.1 was replaced by Tyr in HLA-A2.4 from donor CLA. This position displays the highest variability score among all polymorphic residues of the class I HLA antigens. But its participation in the specific determinants recognized by CTL has not been previously established, because no other known HLA variant or H-2 mutant has been found to vary at this position. In addition, HLA-A2.4 from CLA is the only HLA-A2 subtype antigen that is identical to A2.1 in the segment spanning residues 147 to 157, a region in which all three A2.1, A2.2, and A2.3 antigens are different.     

Recognition and lysis of target cells by cytotoxic T lymphocytes

A single cytotoxic T lymphocyte (CTL) is capable of performing the two most fundamental functions of an immune response, recognition and elimination of foreign antigens. It is now clear that in a CTL these two functions are linked via the antigen-specific, heterodimeric receptor. We review here some experimental approaches that justify this conclusion and provide the means for further examination of the mechanisms by which CTLs lyse their target cells. When antireceptor antibodies serving as antigen substitutes are attached to various cells, they trigger the lytic activity of particular CTLs, which results in lysis of the antibody-modified cell. In the process, a novel serine esterase, which is located within cytolytic granules of the CTL, is released. The presence of this enzyme and a complement-like protein, perforin, in granules of a CTL has led to the suggestion that CTLs and complement have similar cytolytic mechanisms. However, the resistance of some CTLs to lysis by other CTLs, but not to lysis by antibody-activated complement, suggests fundamental differences between cytolytic mechanisms of CTLs and complement.     

Glucocorticoid-induced TNF receptor-triggered T cells are key modulators for survival/death of neural stem/progenitor cells induced by ischemic stroke

Increasing evidences show that immune response affects the reparative mechanisms in injured brain. Recently, we have demonstrated that CD4(+)T cells serve as negative modulators in neurogenesis after stroke, but the mechanistic detail remains unclear. Glucocorticoid-induced tumor necrosis factor (TNF) receptor (GITR), a multifaceted regulator of immunity belonging to the TNF receptor superfamily, is expressed on activated CD4(+)T cells. Herein, we show, by using a murine model of cortical infarction, that GITR triggering on CD4(+)T cells increases poststroke inflammation and decreases the number of neural stem/progenitor cells induced by ischemia (iNSPCs). CD4(+)GITR(+)T cells were preferentially accumulated at the postischemic cortex, and mice treated with GITR-stimulating antibody augmented poststroke inflammatory responses with enhanced apoptosis of iNSPCs. In contrast, blocking the GITR-GITR ligand (GITRL) interaction by GITR-Fc fusion protein abrogated inflammation and suppressed apoptosis of iNSPCs. Moreover, GITR-stimulated T cells caused apoptosis of the iNSPCs, and administration of GITR-stimulated T cells to poststroke severe combined immunodeficient mice significantly reduced iNSPC number compared with that of non-stimulated T cells. These observations indicate that among the CD4(+)T cells, GITR(+)CD4(+)T cells are major deteriorating modulators of poststroke neurogenesis. This suggests that blockade of the GITR-GITRL interaction may be a novel immune-based therapy in stroke.     

Kinetics of activation antigen expression by in vitro-stimulated human T lymphocytes

In this study a panel of monoclonal antibodies was used to investigate the kinetics of the appearance of activation-linked surface determinants as well as cytoplasmic and nuclear determinants in human T cells following lectin stimulation. Well known activation markers, such as Ia/DR, transferrin receptor, IL-2 receptor, T10, and gp24, were compared and investigated together with the T13 structure, recently found in this laboratory. T13, not demonstrable on resting T cells, could be seen within 24 hr after lectin stimulation. Kinetics of the appearance were similar to IL-2 receptor and transferrin receptor expression. Ia/DR synthesis was investigated separately for each polypeptide and the cytoplasmic invariant gamma-chain expression could be demonstrated for the first time with a gamma-chain-specific monoclonal antibody VIC-Y1. Moreover, gamma-chain synthesis seems to precede alpha- and beta-chain occurrence in human T cells. In addition, data from quantitative studies on antigenic densities are presented.