T cell clones from an X-linked hyper-immunoglobulin (IgM) patient induce IgE synthesis in vitro despite expression of nonfunctional CD40 ligand.

The induction of immunoglobulin E (IgE) switching in B cells requires at least two signals. The first is given by either of the soluble lymphokines interleukin 4 (IL-4) or IL-13, whereas the second is contact dependent. It has been widely reported that a second signal can be provided by the CD40 ligand (CD40L) expressed on the surface of T cells, mast cells, and basophils. A defect in the CD40L has been shown recently to be responsible for the lack of IgE, IgA, and IgG, characteristic of the childhood X-linked immunodeficiency, hyper IgM syndrome (HIGM1). IgE can however be detected in the serum of some HIGM1 patients. In this study, we isolated T cell clones and lines using phytohemagglutinin (PHA) and allergen, respectively, from the peripheral blood of one such patient who expressed a truncated form of CD40L, and investigated their ability to induce IgE switching in highly purified, normal tonsillar B cells in vitro. Unexpectedly, 4 of 12 PHA clones tested induced contact-dependent IgE synthesis in the presence of exogenous IL-4. These clones were also shown to strongly upregulated IL-4-induced germline epsilon RNA and formed dense aggregates with B cells. Of the four helper clones, three were CD8+, of which two were characteristic of the T helper cell 2 (Th2) subtype. Two allergen-specific HIGM1 T cell lines, both of the Th0 subtype, could also drive IgE synthesis when prestimulated using specific allergen. All clones and lines were negative for surface expression of CD40L, and the mutated form of CD40L was confirmed for a representative clone by RNase protection assay and sequencing. The IgE helper activity could not be attributed to membrane tumor necrosis factor alpha (TNF-alpha) although it was strongly expressed on activated clones, and the addition of neutralizing anti-TNF-alpha antibody did not abrogate IgE synthesis. These results therefore suggest the involvement of T cell surface molecules other than CD40L in the induction of IgE synthesis, and that these molecules may also be implicated in other aspects of T-B cell interactions.

Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain?

Astrocytomas are among the most common brain tumors that are usually fatal in their malignant form. They appear to progress without significant impedance from the immune system, despite the presence of intratumoral T cell infiltration. To date, this has been thought to be the result of T cell immunosuppression induced by astrocytoma-derived cytokines. Here, we propose that cell contact-mediated events also play a role, since we demonstrate the in vivo expression of Fas ligand (FasL/CD95L) by human astrocytoma and the efficient killing of Fas-bearing cells by astrocytoma lines in vitro and by tumor cells ex vivo. Functional FasL is expressed by human, mouse, and rat astrocytoma and hence may be a general feature of this nonlymphoid tumor. In the brain, astrocytoma cells can potentially deliver a death signal to Fas+ cells which include infiltrating leukocytes and, paradoxically, astrocytoma cells themselves. The expression of FasL by astrocytoma cells may extend the processes that are postulated to occur in normal brain to maintain immune privilege, since we also show FasL expression by neurons. Overall, our findings suggest that FasL-induced apoptosis by astrocytoma cells may play a significant role in both immunosuppression and the regulation of tumor growth within the central nervous system.

Heterogeneous T-cell response to MAGE-A10(254-262): high avidity-specific cytolytic T lymphocytes show superior antitumor activity.

MAGE-encoded antigens, which are expressed by tumors of many histological types but not in normal tissues, are suitable candidates for vaccine-based immunotherapy of cancers. Thus far, however, T-cell responses to MAGE antigens have been detected only occasionally in cancer patients. In contrast, by using HLA/peptide fluorescent tetramers, we have observed recently that CD8(+) T cells specific for peptide MAGE-A10(254-262) can be detected frequently in peptide-stimulated peripheral blood mononuclear cells from HLA-A2-expressing melanoma patients and healthy donors. On the basis of these results, antitumoral vaccination trials using peptide MAGE-A10(254-262) have been implemented recently. In the present study, we have characterized MAGE-A10(254-262)-specific CD8(+) T cells in polyclonal cultures and at the clonal level. The results indicate that the repertoire of MAGE-A10(254-262)-specific CD8(+) T cells is diverse both in terms of clonal composition, efficiency of peptide recognition, and tumor-specific lytic activity. Importantly, only CD8(+) T cells able to recognize the antigenic peptide with high efficiency are able to lyse MAGE-A10-expressing tumor cells. Under defined experimental conditions, the tetramer staining intensity exhibited by MAGE-A10(254-262)-specific CD8(+) T cells correlates with efficiency of peptide recognition so that "high" and "low" avidity cells can be separated by FACS. Altogether, the data reported here provide evidence for functional diversity of MAGE-A10(254-262)-specific T cells and will be instrumental for the monitoring of peptide MAGE-A10(254-262)-based clinical trials.

Transformed and nontransformed human T lymphocytes migrate to skin in a chimeric human skin/SCID mouse model.

To study human T cell migration to human skin in vivo, we grafted severe combined immunodeficient mice with 500-microm thick human skin. Two weeks after grafting, epidermal and dermal structures in the grafts were of human origin. When we intraperitoneally injected grafted mice with clones of the human HUT-78 T cell line derived from a patient with cutaneous T cell lymphoma and Sézary syndrome, we detected in the grafts the rare Vbeta23-Jbeta1.2 T cell receptor transcripts characteristic for the HUT-78 clones. These signals were found 2-6 d after cell injection in about 40% of the grafted and HUT-78 cell injected mice but not in grafts from mice that received no exogenous T cells. In contrast to HUT-78 cells, which only accumulate in low number, grafts topically challenged with nickel sufate in vaseline from mice that were injected with autologous nickel-reactive T cell lines led to massive accumulation of T cells within 3 d. Only scattered T cells accumulated in the skin when grafted mice received vaseline plus T cells, nickel sulfate alone, T cells alone, or nickel sulfate plus an allogeneic nickel-nonreactive T cell clone. When the T cell lines were labeled with the fluorochrome PKH-26 before cell injection, spots of fluorescent label in the size and shape of cells were found in the grafts challenged with nickel. Together, these results clearly demonstrate that human T cells can migrate to human skin in this chimeric human/mouse model.

The neuropeptide calcitonin gene-related peptide differently modulates proliferation and differentiation of smooth muscle cells in culture depending on the cell type.

Calcitonin gene-related peptide (CGRP) is a neuropeptide present around vasculature very early during development, when smooth muscle cells (SMC) are still proliferating and not yet totally differentiated. We investigated the effects of CGRP on proliferation and differentiation of SMC in culture; 10(-7) M CGRP added in the medium of cultured smooth muscle cells every 2 days did not significantly changed cells growth rate in 1% FCS. At the opposite, this treatment modulated proliferation of cells grown in 10% FCS medium. Two distinct populations of SMC with different growth rates were obtained from our primary cultures. SMC which proliferated slowly in the presence of 10% fetal calf serum (FCS) had growth rates positively influenced by CGRP. The quantity of alpha-smooth actin expressed by these cells was not influenced by the peptide. On the contrary, SMC which proliferated more rapidly in 10% FCS medium had growth rate inhibited by CGRP. In these cells, CGRP significantly reduced the amount of expressed alpha-smooth actin, an index of SMC differentiation. In both cases, the peptide significantly increased the level of mRNA for all the actin genes. In the light of this dual role of CGRP, it can be presumed that this peptide controls smooth muscle cells proliferation and differentiation in vivo and could thus regulate the homeostasis of the vessel wall.

Loss of Fas (CD95/APO-1) expression by antigen-specific cytotoxic T cells is reversed by inhibiting DNA methylation.

Elimination of clonally expanded peripheral CD8 T cells was thought to involve apoptosis induction mediated principally by TNF, but recently Fas (CD95/APO-1) has been shown to play a role in certain responses. Here we study Fas expression and sensitivity to its ligation on murine CD8 cells specific for the CW3 antigen expressed by transfected P815 cells. Fas was progressively downregulated after successive in vitro restimulations of antigen-specific CD8 cells, until clones became Fas negative and totally resistant to the effects of recombinant Fas ligand. In contrast, Fas expression by in vivo restimulated antigen-specific cells did not diminish. Loss of Fas expression in vitro was not totally irreversible, since it could be reinduced by inhibition of DNA methylation. Understanding how Fas may be differentially regulated in vivo and in vitro is an important issue for the optimal manipulation of T cells for adoptive immunotherapy protocols.

T-cell receptor beta chain variability in bone marrow and peripheral blood in severe acquired aplastic anemia.

Aplastic anemia (AA) is characterized by multilineage bone marrow failure of unknown etiology. In order to assess the role of immune-mediated mechanisms in hematopoietic suppression, we examined the diversity of T lymphocyte repertoire in terms of variable (V) gene segment usage of the T cell receptor (TCR) beta chain in bone marrow and peripheral blood of six patients with severe untreated AA. Expression of transcripts encoding Vbeta1-Vbeta24 subfamilies was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that T lymphocytes in AA utilize highly diverse segments of the beta chain loci. Over the heterogenous Vbeta expression background, transcripts encoding Vbeta3, Vbeta20, Vbeta21, and Vbeta22 subfamilies were enhanced by at least threefold in 5 of 6 patients as compared to normal samples, but a different transcript species was over expressed in each patient. To evaluate clonality of T cells, size diversity within the complementarity determining region 3 (CDR3) and usage of TCRbeta joining (J) gene segments were analyzed in PCR products specific for each of the 24 Vbeta subfamilies. We found that the majority of transcripts display normal CDR3 size patterns, as is characteristic of polyclonal populations. Nevertheless, one or two predominating junctional rearrangements were observed in each patient. They were identified in Vbeta5, Vbeta7, Vbeta8, Vbeta13, Vbeta15, Vbeta16, and Vbeta23 transcripts, which differed from patient to patient and did not correspond to transcripts with an abnormally high expression level. Our results demonstrate that T cell repertoire in AA is random with respect to the TCR beta chain. Unique rearrangements detected in the CDR3 region are suggestive of a limited process of an antigen-driven (oligo)clonal T cell expansion which may take place over the overwhelmingly polyclonal repertoire of T lymphocytes at the onset of severe AA.

CD28 functions as an adhesion molecule and is involved in the regulation of human IgE synthesis.

Activated T cells induce IgE switching in B cells via a combination of lymphokines and direct T:B cell contact. As CD28-deficient mice have reduced basal levels of IgG1 and IgG2a and diminished Ig class switching, we investigated whether the CD28/B7.1 (CD80) ligand pairing might also be involved in human IgE regulation. Co-incubation of an allergen-specific, human T cell clone with tonsillar B cells caused a marked up-regulation of CD28 expression, whereas, in contrast, CD45 RB expression was unaffected. To test whether blocking the CD28: B7.1 interaction affected IgE synthesis, a dialyzed anti-CD28 monoclonal antibody (mAb) was added to cultures containing tonsillar B cells, pre-activated T cell clones and interleukin-4. Anti-CD28 treatment caused a reproducible, dose-dependent inhibition of IgE, but not IgG synthesis that was accompanied by a visible decrease in cell aggregate formation. Conversely, an anti-B7.1 mAb had no effect in this system. The effect of blocking CD28-ligand interactions on lymphocyte adhesion was formally assessed on human T cell clones and B cell lines using dual intracellular staining and flow cytometry. Co-incubation with an anti-CD28 mAb, but not control IgG or anti-B7.1 mAb, resulted in a marked impairment of conjugate formation that correlated well with T cell surface expression of CD28. Using this system we found that an anti-CTLA-4 mAb but not an anti-B7.2 mAb inhibited T:B cell conjugate formation. Lastly, in addition to a direct effect of anti-CD28 mAb on conjugate formation, 14-day culture of T and B cells in the presence of anti-CD28 caused a marked decrease of ICAM-1 (CD54) expression on aggregated lymphocytes. In contrast, LFA-1 (CD18) expression was unaffected. We, therefore, conclude that the T cell co-stimulatory molecule CD28 is involved in the regulation of IgE synthesis in vitro. CD28 may act to a limited extent as an adhesion molecule, though apparently not by pairing with B7.1 or B7.2. It is more likely that ligation of CD28 under certain conditions modulates the expression of other T and B cell surface molecules.

The brain parenchyma is permissive for full antitumor CTL effector function, even in the absence of CD4 T cells.

Effective antitumor immune responses against cerebral malignancies have been demonstrated in several models, but precise cellular function of specific effector cells is poorly understood. We have explored this topic by analyzing the MHC class I-restricted T cell response elicited after implantation of HLA-CW3-transfected P815 mastocytoma cells (P815-CW3) in syngeneic mice. In this model, tumor-specific CTLs use a distinctive repertoire of TCRs that allows ex vivo assessment of the response by immunophenotyping and TCR spectratyping. Thus, for the first time in a brain tumor model, we are able to directly visualize ex vivo CTLs specific for a tumor-expressed Ag. Tumor-specific CTLs are detected in the CNS after intracerebral implantation of P815-CW3, together with other inflammatory cells. Moreover, despite observations in other models suggesting that CTLs infiltrating the brain may be functionally compromised and highly dependent upon CD4 T cells, in this syngeneic P815-CW3 model, intracerebral tumors were efficiently rejected, whether or not CD4 T cells were present. This observation correlated with potent ex vivo cytotoxicity of brain-infiltrating CTLs, specific for the immunodominant epitope CW3170-179 expressed on P815-CW3 tumor cells.

TCR analysis reveals significant repertoire selection during in vitro lymphocyte culture.

The in vitro stimulation of T lymphocytes is frequently used as a technique to expand specific cells present at low precursor frequency in vivo. However, cells analysed after such procedures may no longer reflect those originally present in vivo because of the variable efficiency of outgrowth of different T cell subpopulations. To systematically assess this and to complement functional assays, we have analysed the TCR repertoire using a new high resolution RT-PCR method to determine TCR beta chain CDR3 transcript length. In the ex vivo analysis of tumor infiltrating lymphocytes (TIL) of renal cell carcinoma and glioblastoma patients, we observed and quantified oligoclonally expanded populations of T cells that were very susceptible to repertoire modification upon subsequent in vitro culture with autologous tumor cells. This in vitro repertoire skewing occurred preferentially with TIL rather than peripheral blood lymphocytes and we noted that tumor cells rather than normal cells of the same tissue type were the most potent inducers of the effect. It was striking that this selection was sometimes negative: certain prominent T cell populations that were highly represented in vivo disappeared after in vitro re-stimulation. This suggests that the presentation of tumor associated antigens during culture may eliminate rather than enrich for in vivo primed T cells. It is clear that in vitro functional tests cannot adequately describe all T cells with tumor specificity. Approaches that allow the assessment of potentially antigen-reactive T cell populations ex vivo are thus an important advance in the global appraisal of anti-tumor T cell immune responses.

CD95 (Fas/Apo-1) as a receptor governing astrocyte apoptotic or inflammatory responses: a key role in brain inflammation?

Astrocytes are a major cellular component of the brain that are capable of intense proliferation and metabolic activity during diverse inflammatory brain diseases (such as multiple sclerosis, Alzheimer's dementia, tumor, HIV encephalitis, or prion disease). In this biological process, called reactive gliosis, astrocyte apoptosis is frequently observed and could be an important mechanism of regulation. However, the factors responsible for apoptosis in human astrocytes are poorly defined. Here, we report that short term cultured astrocytes derived from different brain regions express significant levels of CD95 at their surface. Only late passage astrocytes are sensitive to CD95 ligation using either CD95 mAb or recombinant CD95 ligand. Blocking experiments using caspase inhibitors with different specificities (DEVD-CHO, z-VAD-fmk, and YVAD-cmk), an enzymatic activity assay, and immunoblotting show that CPP32/caspase-3 play a prominent role in CD95-induced astrocyte death. In contrast, early passage astrocytes are totally resistant to death, but a significant increase in astrocytic IL-8 secretion (p < 0.001, by Wilcoxon's test for paired samples) is observed after CD95 triggering. Production of IL-8 contributes to the resistance of astrocytes to CD95 ligation. Furthermore, in the presence of IFN-gamma, resistant astrocytes became sensitive to CD95-mediated death. These data suggest that microenvironmental factors can influence the consequences of CD95 ligation on astrocytes. Therefore, we propose that CD95 expressed by human astrocytes plays a pivotal role in the regulation of astrocyte life and death and may be a key factor in inflammatory processes in the brain, such as reactive gliosis.

Astrocytoma infiltrating lymphocytes include major T cell clonal expansions confined to the CD8 subset.

Anaplastic astrocytoma and glioblastoma are frequent and malignant brain tumors that are infiltrated by T lymphocytes. Whether these cells result from non-specific inflammation following blood-brain barrier disruption or an antigen-driven specific immune response is unknown. In this study, an in-depth characterization of TCR diversity in tumor and blood RNA biopsies was performed in a series of 16 patients with malignant astrocytoma. Whilst there was no obvious restriction of the AV and BV gene segment usage, complementarity-determining region 3 size analysis and sequencing of amplified TCR transcripts revealed multiple T cell oligoclonal expansions in all astrocytomas analyzed. Unique T cell clones were present in different adjacent areas of a given tumor, but never detected in the blood. Quantification of the number of TCR clonal transcripts per microg of tumor RNA indicated that certain T cell clonal expansions may represent at least 300 cells/10(6) tumor cells. Furthermore, we demonstrated that the in vivo expanded clones were almost exclusively confined to the CD8(+) subset. Overall, these data suggest that spontaneous antigen-driven immune responses may be elicited against human astrocytoma despite the immunosuppressive microenvironment generated by the brain and the tumor itself. However, the ultimate failure of the immune system to control tumor growth could be the consequence of a deficient CD4 T(h) component of the response. This observation could have important consequences for the development of immunotherapies for astrocytoma patients.