CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8+ T cells: ignorance and tolerance. When ignorance is operative, naïve autoreactive CD8+ T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8+ T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8+ T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8+ T cells. When cross-presentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8+ T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.

The CXC chemokine stromal cell-derived factor 1 is not responsible for CD8+ T cell suppression of syncytia-inducing strains of HIV-1

Primary CD8+ T cells from HIV+ asymptomatics can suppress virus production from CD4+ T cells acutely infected with either non-syncytia-inducing (NSI) or syncytia-inducing (SI) HIV-1 isolates. NSI strains of HIV-1 predominantly use the CCR5 chemokine receptor as a fusion cofactor, whereas fusion of T cell line-adapted SI isolates is mediated by another chemokine receptor, CXCR4. The CCR5 ligands RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein 1α (MIP-1α), and MIP-1β are HIV-1 suppressive factors secreted by CD8+ cells that inhibit NSI viruses. Recently, the CXC chemokine stromal cell-derived factor 1 (SDF-1) was identified as a ligand for CXCR4 and shown to inhibit SI strains. We speculated that SDF-1 might be an effector molecule for CD8+ suppression of SI isolates and assessed several SDF-1 preparations for inhibition of HIV-1LAI-mediated cell–cell fusion, and examined levels of SDF-1 transcripts in CD8+ T cells. SDF-1 fusion inhibitory activity correlated with the N terminus, and the α and β forms of SDF-1 exhibited equivalent fusion blocking activity. SDF-1 preparations having the N terminus described by Bleul et al. (Bleul, C.C., Fuhlbrigge, R.C., Casasnovas, J.M., Aiuti, A. & Springer, T.A. (1996) J. Exp. Med. 184, 1101–1109) readily blocked HIV-1LAI-mediated fusion, whereas forms containing two or three additional N-terminal amino acids lacked this activity despite their ability to bind and/or signal through CXCR4. Though SDF-1 is constitutively expressed in most tissues, CD8 T cells contained extremely low levels of SDF-1 mRNA transcripts (<1 transcript/5,000 cells), and these levels did not correlate with virus suppressive activity. We conclude that suppression of SI strains of HIV-1 by CD8+ T cells is unlikely to involve SDF-1.

A transgenic mouse model to analyze CD8+ effector T cell differentiation in vivo

Antigen-specific effector T cells are prerequisite to immune protection, but because of the lack of effector cell-specific markers, their generation and differentiation has been difficult to study. We report that effector cells are highly enriched in a T cell subset that can be specifically identified in transgenic (T-GFP) mice expressing green fluorescent protein (GFP) under control of the murine CD4 promoter and proximal enhancer. Consistent with previous studies of these transcriptional control elements, GFP was strongly and specifically expressed in nearly all resting and short-term activated CD4+ and CD8+ T cells. However, when T-GFP mice were challenged with vaccinia virus, allogeneic tumor cells, or staphylococcal enterotoxin A, the cytotoxic and IFN-γ-producing T cells lost GFP expression. Upon T cell receptor (TCR) ligation by αCD3, sorted GFP+ cells fluxed calcium and proliferated vigorously. In contrast, GFP− effector cells showed a diminished calcium flux and did not proliferate. Instead, they underwent apoptosis unless supplied with exogenous IL-2. By reverse transcription–PCR analysis, the GFP− cells up-regulated the pro-apoptotic molecule, Fas-L, and down-regulated gene expression of the proximal TCR signaling molecule, CD3ζ, and c-jun, a component of the AP-1 transcription factor. Thus, differential regulation of TCR signaling may explain the divergent responses of naïve and effector T cells to antigen stimulation.

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8+ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4+ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8+ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8+ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8+ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8+ T-cell supernatants; and (iii) the CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8+ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8+ T-cell-derived HIV-suppressor factors.

A mouse CD8 T cell-mediated acute autoimmune diabetes independent of the perforin and Fas cytotoxic pathways: Possible role of membrane TNF

Double transgenic mice [rat insulin promoter (RIP)-tumor necrosis factor (TNF) and RIP-CD80] whose pancreatic β cells release TNF and bear CD80 all develop an acute early (6 wk) and lethal diabetes mediated by CD8 T cells. The first ultrastructural changes observed in β cells, so far unreported, are focal lesions of endoplasmic reticulum swelling at the points of contact with islet-infiltrating lymphoblasts, followed by cytoplasmic, but not nuclear, apoptosis. Such double transgenic mice were made defective in either the perforin, Fas, or TNF pathways. Remarkably, diabetes was found to be totally independent of perforin and Fas. Mice lacking TNF receptor (TNFR) II had no or late diabetes, but only a minority had severe insulitis. Mice lacking the TNF-lymphotoxin (LTα) locus (whose sole source of TNF are the β cells) all had insulitis comparable to that of nondefective mice, but no diabetes or a retarded and milder form, with lesions suggesting different mechanisms of injury. Because both TNFR II and TNF-LTα mutations have complex effects on the immune system, these data do not formally incriminate membrane TNF as the major T cell mediator of this acute autoimmune diabetes; nevertheless, in the absence of involvement of the perforin or Fas cytotoxic pathways, membrane TNF appears to be the likeliest candidate.

Melan-A/MART-151–73 represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4+ T cells

The human Melan-A/MART-1 gene encodes an HLA-A2-restricted peptide epitope recognized by melanoma-reactive CD8+ cytotoxic T lymphocytes. Here we report that this gene also encodes at least one HLA-DR4-presented peptide recognized by CD4+ T cells. The Melan-A/MART-151–73 peptide was able to induce the in vitro expansion of specific CD4+ T cells derived from normal DR4+ donors or from DR4+ patients with melanoma when pulsed onto autologous dendritic cells. CD4+ responder T cells specifically produced IFN-γ in response to, and also lysed, T2.DR4 cells pulsed with the Melan-A/MART-151–73 peptide and DR4+ melanoma target cells naturally expressing the Melan-A/MART-1 gene product. Interestingly, CD4+ T cell immunoreactivity against the Melan-A/MART-151–73 peptide typically coexisted with a high frequency of anti-Melan-A/MART-127–35 reactive CD8+ T cells in freshly isolated blood harvested from HLA-A2+/DR4+ patients with melanoma. Taken together, these data support the use of this Melan-A/MART-1 DR4-restricted melanoma epitope in future immunotherapeutic trials designed to generate, augment, and quantitate specific CD4+ T cell responses against melanoma in vivo.

Suppressor T cells regulate the nonanergic cell population that remains after peripheral tolerance is induced to the Mls-1 antigen in T cell receptor Vβ8.1 transgenic mice

We have found suppressor T cells that inhibit the proliferative response of naive CD4+ T cells in T cell receptor (TCR) Vβ8.1 transgenic mice rendered tolerant in vivo by inoculation of Mls-1a-positive cells. This suppression was mediated by CD4+ T cells but not by CD8+ T cells or double-negative (DN) cells, and splenic CD4+ T cells from tolerant mice displayed a greater suppression than lymph node CD4+ T cells. Cell contact was required for efficient suppression, and known inhibitory cytokines such as IL-4, IL-10, and transforming growth factor β were not involved. Suppressor T cells inhibited IL-2 production by naive CD4+ T cells, and the addition of exogenous IL-2 diminished the suppressed activity while having little activity on tolerant T cells. Suppression was abolished by the elimination of CD25+ T cells in the tolerant CD4+ T cell subset. CD25+CD4+ T cells suppressed the proliferative response of the residual fraction of the nonanergic population, namely, 6C10+CD4+ T cells still present in the tolerant mice. However, 6C10−CD4+ T cells still had reduced reactivity to Mls-1a even after CD25+CD4+ T cells were removed and exogenous IL-2 was added. Suppressor cells appear to affect only residual nonanergic cells in situ, thereby facilitating the maintenance of the unresponsive state in vivo. These data provide a framework for understanding suppressor T cells and explain the difficulties and variables in defining their activity in other systems, because suppressor T cells apparently control only a small population of nonanergic cells in the periphery and may be viewed as a homeostatic mechanism.

Increased rates of CD4+ and CD8+ T lymphocyte turnover in simian immunodeficiency virus-infected macaques

Defining the rate at which T cells turn over has important implications for our understanding of T lymphocyte homeostasis and AIDS pathogenesis, yet little information on T cell turnover is available. We used the nucleoside analogue bromodeoxyuridine (BrdUrd) in combination with five-color flow cytometric analysis to evaluate T lymphocyte turnover rates in normal and simian immunodeficiency virus (SIV)-infected rhesus macaques. T cells in normal animals turned over at relatively rapid rates, with memory cells turning over more quickly than naive cells. In SIV-infected animals, the labeling and elimination rates of both CD4+ and CD8+ BrdUrd-labeled cells were increased by 2- to 3-fold as compared with normal controls. In normal and SIV-infected animals, the rates of CD4+ T cell BrdUrd-labeling and decay were closely correlated with those of CD8+ T cells. The elimination rate of BrdUrd-labeled cells was accelerated in both naive and memory T lymphocytes in SIV-infected animals. Our results provide direct evidence for increased rates of both CD4+ and CD8+ T cell turnover in AIDS virus infection and have important implications for our understanding of T cell homeostasis and the mechanisms responsible for CD4+ T cell depletion in AIDS.

Generation of mucosal cytotoxic T cells against soluble protein by tissue-specific environmental and costimulatory signals

We compared peripheral and mucosal primary CD8 T cell responses to inflammatory and noninflammatory forms of antigen in a T cell-adoptive transfer system. Immunization with the soluble antigen, ovalbumin (ova), administered i.p. or orally without adjuvant, activated nonmucosal CD8 T cells but did not induce cytotoxic activity. However, after activation, the transferred cells entered the intestinal mucosa and became potent antigen-specific killers. Thus, exogenous intact soluble protein entered the major histocompatibility complex class I antigen presentation pathway and induced mucosal cytotoxic T lymphocytes. Moreover, distinct costimulatory requirements for activation of peripheral versus mucosal T cells were noted in that the CD28 ligand, B7-1, was critical for activated mucosal T cell generation but not for activation of peripheral CD8 T cells. The costimulator, B7-2, was required for optimum activation of both populations. Infection with a new recombinant vesicular stomatitis virus encoding ovalbumin induced lytic activity in mucosal as well as peripheral sites, demonstrating an adjuvant effect of inflammatory mediators produced during virus infection. Generation of antiviral cytotoxic T lymphocytes was also costimulation-dependent. The results indicated that induction of peripheral tolerance via antigen administration may not extend to mucosal sites because of distinct costimulatory and inflammatory signals in the mucosa.

Virus-specific CD8+ T cell numbers are maintained during γ-herpesvirus reactivation in CD4-deficient mice

The murine γ-herpesvirus 68 replicates in epithelial sites after intranasal challenge, then persists in various cell types, including B lymphocytes. Mice that lack CD4+ T cells (I-Ab−/−) control the acute infection, but suffer an ultimately lethal recrudescence of lytic viral replication in the respiratory tract. The consequences of CD4+ T cell deficiency for the generation and maintenance of murine γ-herpesvirus 68-specific CD8+ set now have been analyzed by direct staining with viral peptides bound to major histocompatibility complex class I tetramers and by a spectrum of functional assays. Both acutely and during viral reactivation, the CD8+ T cell responses in the I-Ab−/− group were no less substantial than in the I-Ab+/+ controls. Indeed, virus-specific CD8+ T cell numbers were increased in the lymphoid tissue of clinically compromised I-Ab−/− mice, although relatively few of the potential cytotoxic T lymphocyte effectors were recruited back to the site of pathology in the lung. Thus the viral reactivation that occurs in the absence of CD4+ T cells was not associated with any exhaustion of the virus-specific cytotoxic T lymphocyte response. It seems that CD8+ T cells alone are insufficient to maintain long-term control of this persistent γ-herpesvirus.

Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy

Most tumor-associated antigens represent self-proteins and as a result are poorly immunogenic due to immune tolerance. Here we show that tolerance to carcinoembryonic antigen (CEA), which is overexpressed by the majority of lethal malignancies, can be reversed by immunization with a CEA-derived peptide. This peptide was altered to make it a more potent T cell antigen and loaded onto dendritic cells (DCs) for delivery as a cellular vaccine. Although DCs are rare in the blood, we found that treatment of advanced cancer patients with Flt3 ligand, a hematopoietic growth factor, expanded DCs 20-fold in vivo. Immunization with these antigen-loaded DCs induced CD8 cytotoxic T lymphocytes that recognized tumor cells expressing endogenous CEA. Staining with peptide-MHC tetramers demonstrated the expansion of CD8 T cells that recognize both the native and altered epitopes and possess an effector cytotoxic T lymphocyte phenotype (CD45RA+CD27−CCR7−). After vaccination, two of 12 patients experienced dramatic tumor regression, one patient had a mixed response, and two had stable disease. Clinical response correlated with the expansion of CD8 tetramer+ T cells, confirming the role of CD8 T cells in this treatment strategy.

Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations.

A small percentage of human T lymphocytes, predominantly CD8+ T cells, express receptors for HLA class 1 molecules of natural killer type (NK-R) that are inhibitory for T-cell antigen receptor (TCR)-mediated functions. In the present study, it is demonstrated that the various NK-R molecules typically expressed by NK cells are also expressed on periheral blood T lymphocytes. These CD3+ NK-R+ cells have a cell surface phenotype typical of memory cells as indicated by the expression of CD45RO and CD29 and by the lack of CD28 and CD45RA. Furthermore, by the combined use of anti-TCR V beta-specific antibodies and a semiquantitative polymerase chain reaction assay, the TCR repertoire in this CD3+ NK-R+ cell subset was found to be skewed; in fact, one or two V beta families were largely represented, and most of the other V beta s were barely detected. In addition, analysis of recombinant clones of the largely represented V beta families demonstrated that these V beta s were oligoclonally or monoclonally expanded.

Stimulation of protective CD8+ T lymphocytes by vaccination with nonliving bacteria.

Infectious diseases caused by intracellular microbes are responsible for major health problems, and satisfactory control will ultimately depend on efficient vaccination strategies. The general assumption is that activation of protective immune responses against intracellular microbes dominated by CD8+ T cells are achieved only by live vaccines. In contrast, we here demonstrate stimulation of protective immunity in mice against the intracellular pathogen Listeria monocytogenes by vaccination with heat-killed listeriae. Vaccine-induced immunity comprised cytolytic and interferon gamma-producing CD8+ T lymphocytes. CD8+ T cells from vaccinated donor mice transferred protection against listeriosis. Moreover, vaccination with heat-killed listeriae induced production in CD4+ T-cell-deficient, H2-A beta gene-disrupted mutant mice. We conclude that antigens from killed listeriae are introduced into the major histocompatibility complex class I pathway and thus are recognized by CD8+ T cells. The practicability of killed vaccines against human infectious diseases therefore should be reevaluated.

Deletion of high-avidity T cells by thymic epithelium.

Tolerance induction by thymic epithelium induces a state of so-called "split tolerance," characterized in vivo by tolerance and in vitro by reactivity to a given thymically expressed antigen. Using a model major histocompatibility complex class I antigen, H-2Kb (Kb), three mechanisms of thymic epithelium-induced tolerance were tested: induction of tolerance of tissue-specific antigens exclusively, selective inactivation of T helper cell-independent cytotoxic T lymphocytes, and deletion of high-avidity T cells. To this end, thymic anlagen from Kb-transgenic embryonic day 10 mouse embryos, taken before colonization by cells of hemopoietic origin, were grafted to nude mice. Tolerance by thymic epithelium was not tissue-specific, since Kb-bearing skin and spleen grafts were maintained indefinitely. Only strong priming in vivo could partially overcome the tolerant state and induce rejection of some skin grafts overexpressing transgenic Kb. Furthermore, the hypothesis that thymic epithelium selectively inactivates those T cells that reject skin grafts in a T helper-independent fashion could not be supported. Thus, when T-cell help was provided by a second skin graft bearing an additional major histocompatibility complex class II disparity, tolerance to the Kb skin graft was not broken. Finally, direct evidence could be obtained for the avidity model of thymic epithelium-induced negative selection, using Kb-specific T-cell receptor (TCR) transgenic mice. Thymic epithelium-grafted TCR transgenic mice showed a selective deletion of those CD8+ T cells with the highest density of the clonotypic TCR. These cells presumably represent the T cells with the highest avidity for Kb. We conclude that split tolerance induced by thymic epithelium was mediated by the deletion of those CD8+ T lymphocytes that have the highest avidity for antigen.

Priming of tumor-specific T cells in the draining lymph nodes after immunization with interleukin 2-secreting tumor cells: three consecutive stages may be required for successful tumor vaccination.

Although both CD4+ and CD8+ T cells are clearly required to generate long-lasting anti-tumor immunity induced by s.c. vaccination with interleukin 2 (IL-2)-transfected, irradiated M-3 clone murine melanoma cells, some controversy continues about the site and mode of T-cell activation in this system. Macrophages, granulocytes, and natural killer cells infiltrate the vaccination site early after injection into either syngeneic euthymic DBA/2 mice or athymic nude mice and eliminate the inoculum within 48 hr. We could not find T cells at the vaccination site, which argues against the concept that T-cell priming by the IL-2-secreting cancer cells occurs directly at that location. However, reverse transcription-PCR revealed transcripts indicative of T-cell activation and expansion in the draining lymph nodes of mice immunized with the IL-2-secreting vaccine but not in mice vaccinated with untransfected, irradiated M-3 cells. We therefore propose that the antigen-presenting cells, which invade the vaccination site, process tumor-derived antigens and, subsequently, initiate priming of tumor-specific T lymphocytes in lymphoid organs. These findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2-secreting tumor cells: (i) tumor-antigen uptake and processing at the site of injection by antigen-presenting cells, (ii) migration of antigen-presenting cells into the regional draining lymph nodes, where T-cell priming occurs, and (iii) circulation of activated T cells that either perform or initiate effector mechanisms leading to tumor cell destruction.