Human thymus exports naive CD8 T cells that can home to nonlymphoid tissues

Functionally naive CD8 T cells in peripheral blood from adult humans can be fully described by their CD45RA(bright)CCR7(+)CD62L(+) cell surface phenotype. Cord blood lymphocytes, from healthy newborns, are homogenously functionally naive. Accordingly, the majority of cord blood CD8 T cells express the same pattern of cell surface molecules. Unexpectedly, however, a significant fraction of cord blood CD8 T cells express neither CCR7 nor CD62L. Yet these cells remain functionally naive as they contain high levels of TCR excision circles, have long telomeres, display highly polyclonal TCRs, and do not exhibit immediate effector functions. In addition, these CD8 T cells already represent a significant fraction of the mature naive CD8 single-positive thymocyte repertoire and may selectively express the cutaneous lymphocyte Ag. We suggest that CD8 single-positive thymocytes comprise two pools of naive precursors that exhibit distinct homing properties. Once seeded in the periphery, naive CCR7(+)CD62L(+) CD8 T cells patrol secondary lymphoid organs, whereas naive CCR7(-)CD62L(-) CD8 T cells selectively migrate to peripheral tissues such as skin.     

On how monospecific memory-like autoregulatory CD8+ T cells can blunt diabetogenic autoimmunity: a computational approach

We have recently shown that during progression to autoimmune diabetes in NOD mice, memory autoreactive regulatory CD8(+) T cells arising from low-avidity precursors can be expanded to therapeutic levels using nanoparticles coated with disease-relevant peptide-major histocompatibility complexes (pMHCs). Here we examine the dynamics of memory autoregulatory CD8(+) T cells specific for islet-specific glucose-6-phosphatase catalytic subunit-related protein(206-214), a prevalent β cell autoantigen; their high-avidity counterparts (dominant effectors); and all other autoreactive non-islet-specific glucose-6-phosphatase catalytic subunit-related protein(206-214)-specific CD8(+) T cell specificities (subdominant effectors) in response to pMHC-coated nanoparticle (pMHC-nanoparticle) therapy. We combine experimental data with mathematical modeling to investigate the clonal competition dynamics of these T cell pools. To mimic the response diversity observed in NOD mice, we simulated many individual mice, using a wide range of parameters, and averaged the results as done experimentally. We find that under certain circumstances, pMHC-nanoparticle-induced expansion of autoregulatory CD8(+) T cells can effectively suppress the expansion of dominant and subdominant effectors simultaneously but, in some few cases, can lead to the substitution (or switching) of one effector population by another. The model supports the idea that disease suppression is based on the elimination of autoantigen-loaded APCs by the expanded autoregulatory CD8(+) T cells. The model also predicts that treatment strategies that operate by selectively inhibiting autoantigen-loaded APCs, such as the pMHC-nanoparticle approach, have the highest promise to blunt polyclonal, multiantigen-specific autoimmune responses in vivo without impairing systemic immunity.     

Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of multipotent haemopoietic stem cells

When highly enriched multipotential spleen colony forming cells (CFU-S) obtained following fluorescence activated cell sorting (FACS-CFU-S) are cultured on marrow stromal cells, they undergo proliferation and development to produce mature haemopoietic cells (Spooncer et al., Nature, 316:62-64, 1985). We now show that FACS-CFU-S behave in a similar way when cultured on monolayers of 3T3 cells, indicating that the 3T3 cells can supply at least part of the environment which is representative of marrow stromal cells and provide, therefore, a system for studying stromal cell: haemopoietic cell interactions. We also demonstrate that IL-3-dependent multipotential stem cell lines (FDCP-Mix), but not a variety of other "committed" IL-3-dependent cell lines, resemble FACS-CFU-S in terms of their ability to proliferate and differentiate when cultured on 3T3 cells in the absence of IL-3. In this system, attachment of the FDCP-Mix to the 3T3 cells is critical for the subsequent maintenance of viability and stimulation of development of the cells. When the FDCP-Mix cells are physically separated from the 3T3 cells, they die and their death cannot be prevented by using 3T3-cell-conditioned medium. The extracellular matrix generated by 3T3 cells is not sufficient for promoting attachment or viability of the FDCP-Mix cells, indicating the importance of integral membrane components. However, attachment and development of FDCP-Mix cells occurs on 3T3 cells that have been lightly fixed with glutaraldehyde indicating that active metabolism is not essential for the effects promoted by the 3T3 cells. We suggest that the ability of FACS-CFU-S and FDCP-Mix cells to respond to 3T3 cells involves specific ligand/receptor interactions.     

Human macrophages and dendritic cells can equally present MART-1 antigen to CD8(+) T cells after phagocytosis of gamma-irradiated melanoma cells

Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine. Other clinical trials have used IFN-gamma activated macrophage killer cells (MAK) to treat cancer patients. However, the clinical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Thus, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by clinical grade DC or MAK as well as the ability of these cells to cross present MART-1 to CD8⁺ T cells. Using a high affinity antibody against MART-1, 2A9, which specifically stains melanoma tumors, melanoma cell lines and normal melanocytes, the expression level of MART-1 in melanoma cell lines could be related to their ability to stimulate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8⁺ T cell clone. Confocal microscopy with Alexa Fluor®⁶⁴⁷-labelled 2A9 also showed that MART-1 could be detected in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and DC co-cultured with melanoma gamma-irradiated cells for different time-points. Thus, naturally occurring MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8⁺ T cell cross-presentation thereafter.     

Cycloheximide or puromycin can substitute for PDGF in inducing cellular DNA synthesis in quiescent 3T3 cells

A brief exposure of quiescent (Go) Swiss 3T3 mouse fibroblasts to inhibitors of protein synthesis can replace platelet-derived growth factor in the stimulation of cellular DNA synthesis. When 3T3 cells, after a 6 hr exposure to either cycloheximide or puromycin, are incubated with platelet-poor plasma, a significant percentage of cells enters DNA synthesis. Either inhibition of protein synthesis, or platelet poor plasma by themselves are totally ineffective. A possible mechanism by which inhibitors of protein synthesis may initiate cell cycle progression is through the activation of the c-myc gene.     

A transfected L-myc gene can substitute for c-myc in blocking murine erythroleukemia differentiation.

We investigated the ability of the proto-oncogene L-myc to substitute for c-myc in blocking murine erythroleukemia differentiation. Murine erythroleukemia cells (line C19) were transfected with recombinant plasmids containing genomic and cDNA fragments of the L-myc gene driven by a Moloney murine leukemia virus long terminal repeat. Clones expressing constitutive high levels of L-myc failed to differentiate in response to the chemical inducer N,N'-hexamethylene bisacetamide (HMBA). The block to differentiation correlated with the level of L-myc expression. Furthermore, transfected clones grown in the presence of inducer for an extended period of time showed an increased level of L-myc expression. These results suggest that functional domains of the c-myc gene involved in differentiation are located in the discrete regions of homology between the c- and L-myc genes.     

Truncated forms of the polyomavirus middle T antigen can substitute for the small T antigen in lytic infection.

Cloned polyomavirus genomes encoding the small T antigen or truncated forms of the middle T antigen facilitated the growth of genomes encoding only the large T antigen in mouse 3T6 cells. We conclude that an N-terminal domain of the middle T antigen, in the appropriate cellular location, can substitute for the small T antigen during lytic infection.     

B cells participate in thymic negative selection of murine auto-reactive CD4+ T cells

It is well documented that thymic epithelial cells participate in the process of negative selection in the thymus. In recent years it was reported that also dendritic cells enter the thymus and contribute to this process, thus allowing for the depletion of thymocytes that are specific to peripherally expressed self-antigens. Here we report that also B cells may take part in the elimination of auto-reactive thymocytes. Using a unique mouse model we show that B cells induce negative selection of self-reactive thymocytes in a process that leads to the deletion of these cells whereas regulatory T cells are spared. These findings have direct implication in autoimmunity, as expression of a myelin antigen by B cells in the thymus renders the mice resistant to autoimmune inflammation of the CNS.     

CD8+ T cells can be primed in vitro to produce IL-4.

IL-4 production by T lymphocytes from naive mice in response to stimulation by plate-bound anti-CD3 is concentrated among CD4+ T cells. In vitro stimulation of lymph node T cells with anti-CD3 plus IL-2 and IL-4 strikingly increases the frequency of cells that produce IL-4 in response to subsequent stimulation with anti-CD3 plus IL-2. Separation of these primed cell populations into CD4+ and CD8+ T cell by cell sorting reveals that the frequency of IL-4-producing cells in both population is similar. Verification that CD8+ T cells produce IL-4 is provided by the capacity of anti-IL-4 mAb to inhibit the response of the indicator cell line to the growth factor produced by the primed cells and by detection of IL-4 by an IL-4-specific ELISA. The in vitro "priming" of CD8+ T cells to produce IL-4 is not dependent on the presence of CD4+ T cells because highly purified CD8+ T cells can be stimulated to develop into cells capable of producing IL-4 by culture with plate-bound anti-CD3 plus IL-2 and IL-4.     

IL-1 as a co-factor for lymphokine-secreting CD8+ murine T cells

Immunologically important among the known biologic activities of IL-1 is its ability to function as a co-factor for responses mediated by lymphokine secreting CD4+ Th cells. In contrast to its known effects in CD4+ T cell responses, IL-1 is not known to play a role in CD8+ T cell responses. In the present study, we have assessed the ability of murine recombinant IL-1 to function as a co-factor for stimulating CD8+ T cells to secrete lymphokines such as IL-2. We found that, in conjunction with either Ag or mitogen, IL-1 is able to stimulate lymphokine-secreting CD8+ T cells. Furthermore, we found that, as a consequence of its stimulation of lymphokine-secreting CD8+ T cells, IL-1 is able to reconstitute MHC class I allospecific cytolytic T lymphocyte responses by cell populations depleted of both accessory cells and CD4+ T cells. These results demonstrate that the biologic activity of IL-1 is not restricted to CD4+ cell responses, and suggests that IL-1 can function as a co-factor for the stimulation of lymphokine-secreting Th cells regardless of their CD4/CD8 phenotype. If IL1 acts directly on lymphokine-secreting T cells or on the APC with which they interact is not yet certain.     

Efficient cellular uptake of recombinant murine Hoxc8 homeoprotein in COS-7 cells

In order to analyze the self-delivery activity of Hoxc8, recombinant Hoxc8 protein (rHoxc8) was designed to be expressed and purified in E. coli as a glutathione S-transferase and green fluorescent protein-fused form (GST-GFP-Hoxc8). After purification using glutathione sepharose beads, the 82 kDa fusion protein was separated on the SDS-PAGE gel and confirmed by detecting the fluorescence through luminescent image analyzer. When rHoxc8 was added to culture media for 30 h, most of the COS-7 cells contained the fusion proteins, showing green fluorescence under the fluorescent microscope. When the efficiency of cellular uptake was examined after Hoechst staining, almost 100% of the cells exhibited the GFP signal, revealing that rHoxc8 can traverse the cellular membrane of COS-7 cells efficiently, suggesting that the rHoxc8 could be applied in the development of efficient and useful delivery vectors for therapeutic molecules.     

In APCs, the autologous peptides selected by the diabetogenic I-Ag7 molecule are unique and determined by the amino acid changes in the P9 pocket.

We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus.     

Positive selection by the pre-TCR yields mature CD8+ T cells

It has been of much interest whether there is functional redundancy between the constitutively signaling pre-Talpha/TCRbeta (pre-TCR) and ligated TCRalphabeta complexes, which independently operate the two distinct checkpoints during thymocyte development, i.e., the pre-TCR involved in beta-selection at the CD4(-)CD8(-) double-negative stage and the TCRalphabeta being crucial for positive/negative selection at the CD4(+)CD8(+) double-positive stage. We found that the pre-TCR expressed on double-positive cells in TCRalpha-deficient (TCRalpha(-/-)) mice produced a small number of mature CD8(+) T cells. Surprisingly, when pre-Talpha was overexpressed, resulting in augmentation of pre-TCR expression, there was a striking increase of the CD8(+) T cells. In addition, even in the absence of up-regulation of pre-TCR expression, a similar increase of CD8(+) T cells was also observed in TCRalpha(-/-) mice overexpressing Egr-1, which lowers the threshold of signal strength required for positive selection. In sharp contrast, the CD8(+) T cells drastically decreased in the absence of pre-Talpha on a TCRalpha(-/-) background. Thus, the pre-TCR appears to functionally promote positive selection of CD8(+) T cells. The biased production of CD8(+) T cells via the pre-TCR might also support the potential involvement of signal strength in CD4/CD8 lineage commitment.     

NKT cells and IFN-gamma establish the regulatory environment for the control of diabetogenic T cells in the nonobese diabetic mouse

In type 1 diabetes mellitus (T1DM), T cell-mediated destruction of insulin-producing pancreatic beta cells leads to the acute onset of hyperglycemia. The nonobese diabetic mouse model of human T1DM reveals that T cells capable of inducing diabetes can escape normal central tolerance, and can cause T1DM if left unchecked. However, several regulatory T cell subsets can temper autoaggressive T cells, although it remains undetermined when and how, and by which subset, homeostatic control of diabetogenic T cells is normally achieved in vivo. Using a cotransfer model, we find that NKT cells efficiently dampen the action of diabetogenic CD4+ T cells, and do so in an indirect manner by modifying the host environment. Moreover, the NKT cell-containing population modifies the host via production of IFN-gamma that is necessary for driving the inhibition of diabetogenic T cells in vivo.     

Immune T cells can protect or induce fatal neurological disease in murine lymphocytic choriomeningitis

Adoptively transferred immune spleen cells induce fatal neurological disease in cyclophosphamide-suppressed recipients injected intracerebrally (ic) with a large, but not small, dose of neurotropic lymphocytic choriomeningitis (LCM) virus. The elimination of virus from brain in the latter group, which survives without developing symptoms, depends upon the presence of Lyt 2+ lymphocytes. However removal of Lyt 2+ subset which is cytotoxic in vitro does not diminish the severity of the inflammatory process in vivo, though the onset of clinical disease is delayed in mice given Lyt 2-depleted populations and a larger ic dose of virus. The present findings are consistent with the idea that fatal LCM results from acute, synchronous damage to key functional cells in the central nervous system by virus-immune Lyt 2+, lymphocytes. Even so, if the number of virus-infected CNS cells is still relatively small at the time of T cell invasion, neurological symptoms are not recognized and the mice survive.     

S-100-positive T cells are largely restricted to a CD8-positive, 9.3-negative subset

The present report concerns the demonstration of the exclusive detection among peripheral blood T-lymphocytes of the S-100 protein within the CD8-positive subpopulation which lacks the antigen recognized by the 9.3 monoclonal antibody. Highly purified human peripheral blood T-cell subsets, obtained by means of panning techniques, were first stained, by an immunofluorescence method, with purified anti-S-100 protein antibodies. The vast majority of S-100 protein- (and, specifically, its beta subunit) positive cells were detected in the CD8-positive, 9.3-negative subset. This subset had previously been shown to comprise all the alloantigen-specific and histamine-inducible suppressor T-cells. Other T-subsets, even those showing either CD8-positivity (but 9.3-positivity) or 9.3-negativity (but CD8-negativity), were, as a rule, S-100 negative. Immunoelectronmicroscopy confirmed that the S-100-positive cells, showing peroxidase activity within the cytoplasm, were found exclusively within the CD8-positive, 9.3-negative subset. This finding of S-100 protein in cells of a specific T8 suppressor subset extends the range of the known distribution of this protein and may have important implications concerning its role in the modulation of immune responses.     

I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice.

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet beta cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet beta cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.