Cutting edge: positive selection induced by a self-peptide with TCR antagonist activity.

Antagonist-like engagement of the TCR has been proposed to induce T cell selection in the thymus. However, no natural TCR ligand with TCR antagonist activity is presently known. Using a combination of bioinformatics and functional testing we identified the first self-peptide that can both deliver antagonist-like signals and promote T cell selection in the thymus. The peptide is presented by appropriate MHC class I molecules in vivo. Thus, endogenous antagonist peptides exist and may be involved in TCR repertoire selection.

CD8(+) tumor-infiltrating T cells are deficient in perforin-mediated cytolytic activity due to defective microtubule-organizing center mobilization and lytic granule exocytosis.

Tumor-infiltrating lymphocytes (TIL) are well known to be functionally impaired typified by the inability to lyse cognate tumor cells in vitro. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. CD8(+) TIL are nonlytic immediately on isolation even though they express surface activation markers, contain effector phase cytokine mRNAs, and contain perforin and granzyme B proteins which are packaged into lytic granules. Ag-specific lytic capability is rapidly recovered if purified TIL are briefly cultured in vitro and tumor lysis is perforin-, but not Fas ligand mediated. In response to TCR ligation of nonlytic TIL in vitro, proximal and distal signaling events are normal; calcium flux is rapid; mitogen-activated protein/extracellular signal-related kinase kinase, extracellular regulatory kinase 2, phosphoinositide-3 kinase, and protein kinase C are activated; and IL-2 and IFN-gamma is secreted. However, on conjugate formation between nonlytic TIL and cognate tumor cells in vitro, the microtubule-organizing center (MTOC) does not localize to the immunological synapse, thereby precluding exocytosis of preformed lytic granules and accounting for defective TIL lytic function. Recovery of TCR-mediated, activation-dependent MTOC mobilization and lytic activity requires proteasome function, implying the existence of an inhibitor of MTOC mobilization. Our findings show that the regulated release of TIL cytolytic granules is defective despite functional TCR-mediated signal transduction.

Modulation of CD8+ T cell response to antigen by the levels of self MHC class I.

The response of H-Y-specific TCR-transgenic CD8(+) T cells to Ag is characterized by poor proliferation, cytolytic activity, and IFN-gamma secretion. IFN-gamma secretion, but not cytotoxic function, can be rescued by the B7.1 molecule, suggesting that costimulation can selectively enhance some, but not all, effector CD8(+) T cell responses. Although the H-Y epitope binds H-2D(b) relatively less well than some other epitopes, it can induce potent CTL responses in nontransgenic mice, suggesting that the observed poor responsiveness of transgenic CD8(+) T cells cannot be ascribed to the epitope itself. Previously reported reactivity of this TCR to H-2A(b) is also not the cause of the poor responsiveness of the H-Y-specific CD8(+) T cells, as H-Y-specific CD8(+) T cells obtained from genetic backgrounds lacking H-2A(b) also responded poorly. Rather, reducing the levels of H-2(b) class I molecules by breeding the mice to (C57BL/6 x B10.D2)F(1) or TAP1(+/-) backgrounds partially restored cytotoxic activity and enhanced proliferative responses. These findings demonstrate that the self MHC class I gene dosage may regulate the extent of CD8(+) T cell responsiveness to Ag.

Alpha beta TCR+ cells are a minimal fraction of peripheral CD8+ pool in MHC class I-deficient mice.

MHC class I molecules play a role in the maintenance of the naive peripheral CD8+ T cell pool. The mechanisms of the peripheral maintenance and the life span of residual CD8+ cells present in the periphery of beta 2-microglobulin-deficient (beta 2m-/-) mice are unknown. We here show that very few CD8+ cells in beta 2m-/- mice coexpress CD8 beta, a marker of the thymus-derived CD8+ T cells. Most of the CD8 alpha+ cells express CD11c and can be found in beta 2m/RAG-2 double-deficient mice, demonstrating that these cells do not require rearranged Ag receptors for differentiation and survival and may be of dendritic cell lineage. Rare CD8 alpha+CD8 beta+ cells can be detected following in vivo alloantigenic stimulation 2 wk after the adult thymectomy. Selective MHC class I expression by bone marrow-derived cells does not lead to an accumulation of CD8 beta+ cells in beta 2m-/- mice. These findings demonstrate that 1) thymic export of CD8+ T cells in beta 2m-/- mice is reduced more severely than previously thought; 2) non-T cells expressing CD8 alpha become prominent when CD8+ T cells are virtually absent; 3) at least some beta 2m-/- CD8+ T cells have a life span in the periphery comparable to wild-type CD8+ cells; and 4) similar ligands induce positive selection in the thymus and survival of CD8+ T cells in the periphery.

Type I IFN modulates innate and specific antiviral immunity.

IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-gamma was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication.

CD8(+) T cell cytolytic activity independent of mitogen-activated protein kinase / extracellular regulatory kinase signaling (MAP kinase / ERK).

Cytotoxicity is a major effector function of CD8(+) T cells. Although mitogen-activated protein kinase (MAP kinase) / extracellular regulatory kinase (ERK) activity is indispensable for cytotoxic activity of most CD8(+) T cells a portion of CD8(+) T cells appears resistant to MEK inhibition as cytotoxicity of bulk cultures was partially preserved in the presence of a MEK inhibitor. We have also identified a long-term CD8(+) T cell line with unaltered cytolytic activity after prevention of ERK activation. Antigen-induced microtubule organizing center (MTOC) reorientation was not prevented in this CD8(+) cell line by MEK inhibition, in sharp contrast to the MTOC reorientation prevention in a CD8(+) T cell clone with MEK inhibition-sensitive cytolytic activity. These findings suggest that resistance of lysis to MEK inhibition may be due to a lack of ERK control over MTOC reorientation in some CD8(+) T cells. Thus, there appears to be a heterogeneity of ERK-regulated cytolytic activity in CD8(+) T cells, most likely resulting from a differential control of ERK over MTOC motility.

Prevention of antigen-induced microtubule organizing center reorientation in cytotoxic T cells by modulation of protein kinase C activity.

Lysis of target cells (TC) by cytotoxic T lymphocytes (CTL) is achieved by directional exocytosis of cytolytic molecules-perforin and granzymes. They are stored within lytic granules which can be readily released following antigenic stimulation. Secretion of lytic molecules appears to be controlled by protein kinase C (PKC) activity, since specific modulators of PKC activity abolish the lysis of TC. We have examined the effect of PKC modulation on some of the earliest events in the perforin/granzyme-mediated cytotoxicity. De novo synthesis of perforin mRNA, required for the refilling of granules and sustained cytotoxicity, seems to be unaltered in the presence of PKC modulators. Immunofluorescent studies of CTL-TC conjugates revealed that PKC modulation impairs reorientation of the microtubule organizing center toward the contact point with the TC, which accounts for the specific direction of lytic granules exocytosis. Thus, it appears that PKC regulates exocytosis of lytic granules by governing microtubule reorganization, one of the initial steps in perforin/granzyme-mediated cytotoxicity.

Altered effector responses of H-Y transgenic CD8+ cells.

The primary role of CD8+ T cells is to destroy virus-infected or tumor cells expressing cognate antigens in the form of peptide-MHC class I complexes. This destruction is primarily achieved by the actions of lytic mediators and/or lymphokines. In this report, we show that mature, H-Y/Db-specific CD8+ T cells from H-Y TCR transgenic mice were unable to efficiently release lytic mediators after antigenic stimulation. However, anti-TCR antibody induced granule exocytosis and target cell lysis, arguing against signaling and/or cytolytic machinery defects in CD8+ cells, and demonstrating that male antigen induced differentiation of 'naive' into effector CD8+ cells. Stimulation of H-Y-specific effector CD8+ T cells with male stimulators, although insufficient to induce lytic granule release, was sufficient for H-Y-specific IFN-gamma production. Unexpectedly, this effector-phase IFN-gamma production was dependent on B7-2 engagement. We hypothesize that altered effector functions in H-Y-specific CD8+ cells are due to the low affinity of TCR-antigen-MHC interaction and/or the elevated threshold of CD8+ T cell activation.

Stimulation of CD8+ T cell responses to MAGE-3 and Melan A/MART-1 by immunization to a polyvalent melanoma vaccine.

A critical requirement for cancer vaccines is that they stimulate CD8+ T cell responses. In this study, we tested the ability of a polyvalent melanoma vaccine to induce CD8+ T cell responses to the melanoma associated antigens MAGE-3 and Melan A/MART-1. Fifteen HLA-A2+ patients with resected malignant melanoma were immunized with the vaccine s.c. every 2-3 weeks. CD8+ T cells in peripheral blood reacting to HLA-A2 restricted epitopes on MAGE-3 (FLWGPRALV) and Melan A/MART-1/(AAGIGILTV) were quantitated using a filter spot assay at baseline and following 4 immunizations. Vaccine immunization induced CD8+ T cells reacting to one or both of these peptides in 9 of the 15 (60%) patients. These cells were CD8+ and HLA-A2 restricted, as reactivity was abrogated by monoclonal antibodies (MAbs) to CD8 and class I HLA, but not by anti-CD4. All responding patients remained recurrence-free for at least 12 months (median 15 months, range 12 to >21 months), whereas melanoma recurred within 3-5 months in non-responders. The differences in outcome were unrelated to differences in disease severity or overall immunological competence between responders and non-responders. Our results demonstrate directly that MAGE-3 and Melan A/MART-1 can stimulate CD8+ T cell responses in humans, and suggest that these responses are protective and surrogate markers of vaccine efficacy.

The role of protein kinase C in CD8+ T lymphocyte effector responses.

Rare CD8+ T cells present in beta2microglobulin-deficient (beta2m-/-) mice reject allogeneic tumors but not syngeneic wild-type tumors. The lack of syngeneic tumor rejection in vivo is correlated with a partial response of beta2m-/- CD8+ cell lines to syngeneic tumor cells in vitro. This partial response is characterized by perforin/granzyme-mediated cytolytic activity in the absence of cytokine secretion or proliferation. Allogeneic tumors induce cytolysis, cytokine secretion, and proliferation. Cytokine secretion may therefore be an important effector mechanism for tumor rejection by CD8+ T cells. To determine the missing signaling events needed for cytokine secretion as well as the events inducing the isolated cytotoxic response, we attempted to restore cytokine secretion of beta2m-/- CD8+ cells to syngeneic MHC class I. Phorbol ester and syngeneic tumor cells acted synergistically to induce full responsiveness of beta2m-/- CD8+ cells. However, this synergistic induction of cytokine secretion used a different pathway than that induced by alloantigen. Protein kinase C (PKC) inhibitor prevented the syngeneic class I plus PMA-induced cytokine secretion, but not allo-class I-induced cytokine secretion. In contrast to the PKC independent alloantigen-induced cytokine secretion, cytolysis of both allogeneic and syngeneic targets was PKC dependent. The differential dependence of effector functions on PKC activation was also found in beta2m+/+ CD8+ T cells. Thus, two distinct signaling pathways (PKC dependent and PKC independent) may ultimately converge to induce cytokine secretion in CD8+ cells. The TCR engagement-initiated pathway is PKC independent, whereas the phorbol ester-activated pathway is PKC dependent.

Apparent split tolerance of CD8+ T cells from beta 2-microglobulin-deficient (beta 2m-/-) mice to syngeneic beta 2m+/+ cells.

beta 2-microglobulin-deficient (beta 2m-/-) mice express reduced levels of MHC class I molecules and, consequently, have impaired positive selection of CD8+ T lymphocytes in the thymus. However, small numbers of CD8+ CTLs can be found in beta 2m-/- mice after immunization with allogeneic as well as syngeneic beta 2m+/+ tumor or spleen cells. It has been proposed, therefore, that because of the low ligand density in beta 2m-/- mice, negative selection does not remove cells capable of recognizing syngeneic MHC class I expressed at normal levels. We report here that beta 2m-/- CD8+ T cells are partially tolerant to syngeneic beta 2m+/+ cells. Despite the ability of beta 2m-/- mice to raise CD8+ CTLs against syngeneic beta 2m+/+ cells, these CD8+ cells do not proliferate and do not secrete IFN-gamma or IL-3/granulocyte-macrophage-CSF upon in vitro stimulation with syngeneic beta 2m+/+ cells. In contrast, all of these cellular responses are displayed by the beta 2m-/- CD8+ cells upon recognition of the allogeneic MHC class I. These in vitro findings of partial responsiveness to syngeneic and of full responsiveness to allogeneic MHC class I correlate well with the ability of beta 2m-/- mice to reject allogeneic, but not syngeneic, tumors in vivo. It appears, thus, that the significantly reduced levels of MHC class I molecules found in beta 2m-/- mice, although not capable of inducing deletion of all reactive clones, can induce deletion of high affinity clones and, therefore, maintain tolerance to self-MHC class I, even when expressed at much higher (beta 2m+/+) levels.

A positively selecting thymic epithelial cell line lacks costimulatory activity.

The participation of costimulatory molecule interactions in positive selection of T lymphocytes was addressed by assessing the ability of a positively selecting thymic epithelial cell (TEC) line, 427.1, to stimulate allospecific CTL responses. Stimulation of H-2s spleen cells with the H-2b expressing 427.1 line does not result in the generation of cells capable of lysing H-2b target cell lines. The level of expression of MHC class I molecules by 427.1 is lower than that found in other stimulatory TEC lines. However, this finding does not account for the nonstimulatory phenotype. Up-regulation of MHC class I did not result in stimulation, and fusion of 427.1 cells with stimulatory TEC resulted in a line with low MHC class I molecule expression and stimulatory phenotype. The TEC line 427.1 does not express the costimulatory molecule B7/BB1, and transfection of the B7/BB1-encoding DNA results in expression of the molecule and conversion into a stimulatory phenotype, demonstrating directly that the non-stimulatory phenotype is a result of lack of costimulation. However, B7/BB1 expression does not improve the ability of 427.1 TECs to induce positive selection. Intrathymic injection of the B7/BB1 transfected, compared with mock transfected 427.1 cells, rescued fewer CD8+ mature thymocytes in beta 2-microglobulin negative mice. Therefore, unlike the peripheral T cell responses to Ag, positive selection of T cells in the thymus may not depend on the costimulation provided by the presenting cell.

A thymic epithelial cell line induces both positive and negative selection in the thymus.

TCR engagement in the thymus results in both survival and elimination signals for developing thymocytes. To examine whether both signals can be provided by the same cell type, we investigated the ability of a thymic epithelial cell (TEC) line 427.1, previously shown to allow positive selection in the thymus, to induce clonal deletion of immature thymocytes. [H-2b/s-->H-2s] bone marrow chimeras are non-responsive to antigens in the context of H-2b. However, chimeras that underwent intrathymic injection of H-2b/s 427.1 cells expressing vesicular stomatitis virus (VSV) nucleocapsid antigen acquired the ability to raise influenza, but not VSV specific H-2b restricted cytotoxic T lymphocyte (CTL) responses. The ability of 427.1 cells to delete CD4+CD8+ thymocytes was determined using mice transgenic for the TCR specific for ovalbumin (OVA) in the context of H-2Kb. OVA transfected, but not mock transfected 427.1 TECs, induced in vitro deletion of CD4+CD8+ TCR transgenic thymocytes manifested as a down-modulation of CD4 and CD8 molecules, a shift in the side versus forward scatter characteristics of thymocytes, and appearance of thymocytes with subdiploid content of DNA indicated the ongoing process of DNA fragmentation. The finding that the same TEC line is capable of inducing both positive and negative selection in the thymus suggests that thymocytes bearing TCRs specific for self peptides expressed by positively selecting thymic epithelium can be deleted. Therefore the expression of a unique set of MHC associated peptides by TECs does not appear to be the basis for the positive outcome of the TCR ligation on immature thymocytes.

An unusual T-cell surface phenotype in vivo correlates with the failure to proliferate and produce IL-2 in vitro in a patient with common variable immunodeficiency.

Stimulation of T-lymphocytes derived from some patients with common variable immunodeficiency (CVID) syndrome results in defective proliferation. The underlying mechanism is related to the inability of stimulated cells to secrete IL-2 while the expression of IL-2 receptor (IL-2R) is normal. We have identified a patient whose peripheral T-cells failed to proliferate and secrete IL-2 upon stimulation. The addition of recombinant IL-2 restored proliferation. The defect did not seem to be caused by accessory cell failure since the patient's adherent cells produced IL-1 and IL-6, and addition of allogeneic irradiated cells did not induce proliferation. Stimulation of CVID T-cells with phorbol esters and Ca2+ ionophore induced both IL-2 secretion and proliferation, indicating the absence of a defect in the transcription and/or translation of the IL-2 gene. The patient's T-cells expressed high levels of CD3. The majority of T-cells expressed the CD38 molecule which is normally found on thymocytes or activated T-cells but not peripheral blood T-cells and HLA-DR, another activation marker. However, CD25 (the IL-2R) and CD1, a marker of more immature thymocytes, were not expressed. Finally, the patient's cells were sensitive to an in vitro corticosteroid treatment. The possibilities that this patient's T-cells represent anergic T-cells or not fully matured thymocytes are discussed.

Positive selection of T-lymphocytes induced by intrathymic injection of a thymic epithelial cell line.

T lymphocytes recognize antigens as peptide fragments associated with molecules encoded by the major histocompatibility complex (MHC) and expressed on the surface of antigen-presenting cells. In the thymus, T cells bearing alpha beta receptors that react with the MHC molecules expressed by radioresistant stromal elements are positively selected for maturation. In (A x B-->A) bone marrow chimaeras, T cells restricted to the MHC-A haplotype are positively selected, whereas MHC-B-reactive thymocytes are not. We investigated whether the introduction of particular thymic stromal elements bearing MHC-B molecules could alter the fate of B-reactive T cells in these (A x B-->A) chimaeras. Thymic epithelial cell (TEC) lines expressing H-2b were introduced by intrathymic injection into (H-2b/s-->H2s) bone marrow chimaeras and we measured their ability to generate H-2b-restricted cytotoxic T-lymphocytes (CTLs). We report here that one TEC line, 427.1, was able positively to select CTLs specific for influenza and vesicular stomatitis virus antigens in association with class I H-2b molecules. In addition, line 427.1 can process cytoplasmic proteins for presentation to H-2Kb- and H-2Db-restricted CTLs. Thus, a TEC line capable of normal class I MHC antigen processing and presentation in vitro can induce positive selection after intrathymic injection.

Phosphorylation of murine CD8 alpha is not essential for responses of T cell hybridomas to antigen.

CD8 T cell differentiation antigens, expressed on class I-restricted T cells, have a key role in the control of recognition and response of these cells to antigen. It has been suggested that these molecules function as co-receptors together with antigen-specific T cell receptors to regulate T cell responses. We have addressed the question of whether cytoplasmic serine phosphorylation, which occurs on CD8 molecules after activation by antigen or phorbol esters, is relevant to its co-receptor function. By mutagenesis, we show that phorbol ester-induced phosphorylation occurs exclusively on CD8 alpha serine residue 216. However, inhibition of CD8 polypeptide phosphorylation does not appear to have a detrimental effect on several responses of CD8-dependent transfectants to antigen. This is in contrast to results reported with CD4 (N.Glaichenhaus, N.Shastri, D.R. Littmann and J.M.Turner. 1991. Cell, 64:511), suggesting that CD4 and CD8 molecules may play somewhat different roles in the control of T cell activation.