IL-12 Reverses Established Tolerance Mediated by Tcrαlβ+ but not by Tcrγδ+ Suppressor T Cells

Topical cutaneous painting with chemically reactive haptens induces the ability to subsequently elicit contact sensitivity (CS) responses in the skin. These CS responses are in vivo examples of acquired, antigen (Ag)-specific T cell immunity, and are a form of delayed-type hypersensitivity (DTH). In contrast, high dose i.v. administration of the hapten can induce Ag-specific tolerance. In some instances this specific immune hyporeactivity is due to suppressor T cells. We investigated the effect of IL-12 on reversal of tolerance due to suppressor T cells that were induced by i.v. administration of hapten in either normal TCRα^+/+, or in immunodeficient TCRα^?/- mice. In the TCRα^+/+ mice, tolerance is mediated by TCRαβ⁺ suppressor T cells, while in the TCRα^?/- mice the tolerance is due to suppressive TCRγδ⁺ cells. Treatment with IL-12 reversed suppressor mediated by the TCRαβ⁺ cells, but did not affect tolerance due to TCRγδ⁺ suppressor cells. Another difference was that the αβTCR⁺ suppressor cells produced a soluble suppressor factor that could replace the surppressor cells, while γδTCR⁺ suppressor cells did not. We hypothesized that IL-12 may strengthen responses of target CS-effector T cells influenced by the hapten-MHC-specificity of αβ suppresssor cells, or suppressor factor. On the other hand, γδTCR⁺ suppressive cells likely have specificity for the hapten alone, and are not MHC-restricted, and therefore probably do not operate via peptide-MHC interactions, that could be strengthened by IL-12. The ability of IL-12 to strengthen the resistance of CS-effector T cells to αβ TCR suppressor cells, may be due to the ability of IL-12 to increase T cell costimulation mediated by signaling mechanisms acting via B7.1 and B7.2. In contrast, αβTCR⁺ suppressor cells, that are largely hapten-specific, probably do not interact with peptide/MHC complexes on APC, and thus are not affected by IL-12 strengthening of co-stimulation.     

Human intraepithelial lymphocytes

The location of intraepithelial lymphocytes (IEL) between epithelial cells, their effector memory, cytolytic and inflammatory phenotype positions them to kill infected epithelial cells and protect the intestine against pathogens. Human TCRαβ⁺CD8αβ⁺ IEL have the dual capacity to recognize modified self via natural killer (NK) receptors (autoreactivity) as well as foreign antigen via the T cell receptor (TCR), which is accomplished in mouse by two cell subsets, the naturally occurring TCRαβ⁺CD8αα⁺ and adaptively induced TCRαβ⁺CD8αβ⁺ IEL subsets, respectively. The private/oligoclonal nature of the TCR repertoire of both human and mouse IEL suggests local environmental factors dictate the specificity of IEL responses. The line between sensing of foreign antigens and autoreactivity is blurred for IEL in celiac disease, where recognition of stress ligands by induced activating NK receptors in conjunction with inflammatory signals such as IL-15 can result in low-affinity TCR/non-cognate antigen and NK receptor/stress ligand interactions triggering destruction of intestinal epithelial cells.     

Changes in the Intestinal Lymphoid Compartment Throughout Life: Implications for the Local Generation of Intestinal T Cells

The intestinal lymphoid compartment has a rather stable composition throughout life. However, both during early neonatal development and at high age unique cell populations can be recognized at distinct sites in the intestinal tissue. Directly after birth all intestinal CD3⁺ cells are found in the lamina propria. At this time the epithelium does not contain T cells. These CD3⁺ lamina propria lymphocytes co-express both TCRβ and TCRδ chains, probably reflecting the expression of a TCRβδ heterodimer on the cell surface. Cells with this particular phenotype are present in comparable numbers in the lamina propria of both neonatal euthymic and athymic mice, indicating the thymus-independent nature of these cells. During aging the frequency of TCRαβ⁺ CD8αα⁺ intestinal T cells increases. These cells are also considered to be thymus-independent. The appearance of high numbers of CD4⁺ CD8αα⁺ intestinal T cells in aged mice is even more striking. It is postulated that the neonatal TCRβδ⁺ cells, and probably also the CD4⁺ CD8αα⁺ cells as found in old mice, are intermediates in the extrathymic differentiation pathway of TCRαβ⁺ CD8αα⁺ intestinal T cells.     

B7-1-dependent co-stimulation results in qualitatively and quantitatively different responses by CD4+ and CD8+ T cells

To characterize better the co-stimulatory activity of native B7-1 in the absence of other receptor/ligand interactions that might contribute to the response, B7-1 was purified by monoclonal antibody (mAb) affinity chromatography. Immobilization of purified B7-1 with anti-T cell receptor (TCR) mAb on cell-sized latex microspheres provided an effective stimulus for activation of both CD4⁺ and CD8⁺ T cells as measured by proliferation, development of effector function, and changes in motility and adhesion. The CD4⁺ T cell response was prolonged and resulted in efficient interleukin-2 production and clonal expansion. In contrast, CD8⁺ responses were transient. Proliferation and clonal expansion peaked on days 3 and 4, coincident with maximal expression of lytic effector function, and the cells then died. These results demonstrate that B7-1 mediated co-stimulation is sufficient for the induction of effector function in both helper and cytotoxic T cell precursors, but suggest that B7-1 co-stimulation is not sufficient to sustain helper-independent CD8⁺ CTL responses. When the dose responses of CD4⁺ and CD8⁺ T cells to B7-1 were compared, CD8⁺ T cells were found to require higher densities of B7-1 to attain an equivalent level of activation, suggesting that the level of expression of B7-1 by APC may influence the development of helper or CTL responses. Finally, in contrast to results obtained by others with B7-1 transfectants, purified B7-1 did not provide co-stimulation when presented on a surface separate from the TCR stimulus.     

Distinct involvement of CD45 in antigen receptor signalling in CD4+ and CD8+ primary T cells

We demonstrate that pretreatment of primary CD4⁺, but not CD8⁺ T cells with anti-CD45 inhibits activation signals induced through the T cell receptor for antigen (TCRαβ). Specifically, anti-TCRαβ-mediated tyrosine phosphorylation of phospholipase C-γ1 is inhibited, and this in turn correlates with the inhibition of subsequent Ca²⁺ mobilization and DNA synthesis. In marked contrast, none of these activation parameters are affected by anti-CD45 in CD8⁺ T cells. Perturbation of TCRαβ signalling in CD4⁺ cells is observed in conditions which do not detectably affect the level of CD45 expression, or its membrane distribution. Further, changes in the intrinsic phosphatase activity of CD45 are not detectable. While anti-CD45 ablates TCRαβ signalling, anti-CD3?-mediated activation is unaffected. This suggests that elements of the antigen receptor complex can be functionally uncoupled, and indicates that the requirements for CD45 in signalling through these two elements are different. The results demonstrate that the involvement of CD45 in coupling TCRαβ to second messenger-generating pathways is under distinct physical and/or functional constraints in primary CD4⁺ and CD8⁺ T cells.     

Distinct roles for CD4 and CD8 as co-receptors in T cell receptor signalling

We demonstrate that CD4 and CD8 modify signals induced through the T cell receptor for antigen (TCRαβ) in distinct fashions. Pretreatment of CD4⁺ lymph node T cells with CD4-specific monoclonal antibody results in a tenfold inhibition of DNA synthesis induced by anti-TCRαβ. In contrast, pretreatment of CD8⁺ T cells with CD8-specific mAb has no effect on DNA synthesis subsequently induced through TCRαβ. While inhibiting late activation signals, pretreatment with anti-CD4 does not detectably alter the pattern of anti-TCRαβ-induced tyrosine phosphorylation of cellular proteins, nor subsequent Ca²⁺ mobilization. The distinct biological consequences of anti-CD4 and anti-CD8 pretreatment correlate with the differential association of their respective ligands with the cellular protein tyrosine kinase, p56^lck. While both T cell lineages contain similar levels of cellular p56^lck, tenfold more is associated with CD4 than with CD8. This difference is associated with the differential effects of pretreatment with anti-CD4 and anti-CD8 on the distribution and activity of p56^lck. Further, antibody-mediated aggregation of TCRαβ on CD4⁺ T cells induces the appearance of a p56^lck species with decreased mobility in sodium dodecylsulfate-polyacrylamide gel electrophoresis. This effect is observed in CD4⁺ T cells exclusively and involves the fraction of p56^lck which is not associated with CD4. The results presented here demonstrate that the signalling elements which couple the antigen receptor to second messenger-generating systems are under distinct physical and/or functional constraints in the two T cell lineages.     

The role of the gut as a primary lymphoid organ: CD8αα intraepithelial T lymphocytes in euthymic mice derive from very immature CD44+ thymocyte precursors

Intestinal CD8αα intraepithelial T lymphocytes (T-IELs) have a key role in mucosal immunity and, unlike other T cells, were proposed to differentiate locally. In apparent contradiction, these cells were also shown to originate from a wave of thymus migrants colonizing the gut in the first 3 weeks after birth. We here identify previously uncharacterized very immature CD4⁻CD8⁻CD3⁻CD44⁺CD25^int thymocytes, which have not yet rearranged their T-cell antigen receptor (TCR), as having the capacity to leave the thymus, migrate to the blood, colonize the gut, and reconstitute CD8αα T-IEL, and show that this cell set is fully responsible for the generation of the CD8αα T-IEL pool. Thus, although the thymus may be fundamental for efficient T-cell commitment, CD8αα T-IEL' complete TCR rearrangements and TCR-αβ/γδ lineage commitment must occur in the gut. These results demonstrate a major role of the gut environment as a primary lymphoid organ.     

Cytokine synthesis and apoptosis by intestinal intraepithelial lymphocytes: Signaling of high density αβ T cell receptor+ and γδ T cell receptor+ T cells via T cell receptor-CD3 complex results in interferon-γ and interleukin-5 production,while low density T cells undergo DNA fragmentation

To study the biological consequences of cytokine production and apoptosis by intraepithelial lymphocytes (IEL), we have studied these characteristics in both the high and low density CD3⁺ IEL populations. Stimulation of low- or high-density CD3⁺ IEL via the T cell receptor (TCR)-CD3 complex using monoclonal anti-CD3, anti-αβ TCR or anti-γδ TCR antibodies resulted in opposing effects. In one case, a significant number of the high-density CD3⁺ T cells entered cell cycle from the resting stage (DNA replication was observed) and anti-TCR-CD3 treatment enhanced the numbers of interferon-γ and interleukin-5 spot-forming cells in this cell fraction. In contrast, when the low-density αβ TCR⁺ or γδ TCR⁺ T cells were activated via the TCR-CD3 complex, DNA fragmentation was observed. These results demonstrated that the activation signals transduced via the TCR-CD3 complex resulted in their entry into the cell cycle and subsequent interferon-γ and interleukin-5 production in the high-density IEL T cell subset. However, identical signals induced apoptosis in the majority of the low-density fraction of CD3⁺ IEL.     

TCRαβ+CD3+CD4−CD8− (double negative) T cells in autoimmunity

TCRαβ⁺CD3⁺CD4^?CD8^? “double negative” (DN) T cells comprise a small subset of mature peripheral T cells. The origin and function of DN T cells are somewhat unclear and discussed controversially. While DN T cells resemble a rare and heterogeneous T cell subpopulation in healthy individuals, numbers of TCRαβ⁺ DN T cells are expanded in several inflammatory conditions, where they also exhibit distinct effector phenotypes and infiltrate inflamed tissues. Thus, DN T cells may be involved in systemic inflammation and tissue damage in autoimmune/inflammatory conditions, including SLE, Sjögren's syndrome, and psoriasis. Here, the current understanding of the origin and phenotype of DN T cells, and their role in the instruction of immune responses, autoimmunity and inflammation will be discussed in health and disease.     

The expansion and selection of T cell receptor αβ intestinal intraepithelial T cell clones

In conventional mice, the T cell receptor (TCR)αβ⁺ CD8αα⁺ and CD8αβ⁺ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Exp. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8αα and CD8αβ⁺ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCRαβ⁺ T cells than conventional mice. The TCRβ repertoire of the CD8αα and the CD8αβ IEL populations of germ-free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show that the intestinal microflora is not responsible for the repertoire oligoclonality of TCRαβ⁺ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8αα and the CD8αβ TCRαβ IEL clones have a heterogeneous size pattern, with clones containing from 3 × 10³ cells up to 1.2 × 10⁶ cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCRαβ IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.     

Absence of cytotoxic molecules in CD8- and/or CD56-positive adult T-cell leukaemia/lymphoma

Adult T-cell leukaemia/lymphoma (ATLL) cells usually exhibit a CD4⁺ (helper/inducer) phenotype (CD4⁺/8^–/56^–), and only a minority of tumours express the CD8 (cytotoxic/suppressor) or CD56 (natural killer [NK]-associated) antigens. TIA-1 is a cytotoxic granule-associated protein expressed in NK cells and cytotoxic T lymphocytes (CTLs). Granzyme B, perforin and Fas ligand (FasL) are also expressed in activated CTLs and NK cells. To clarify the cytotoxic potential of ATLL cells, immunohistochemistry was performed in CD8⁺ and/or CD56⁺ ATLL cells, using anti-TIA-1, anti-granzyme B, anti-perforin and anti-FasL antibodies. We studied nine cases of CD8⁺ and/or CD56+ ATLL, all of which exhibited monoclonal integration of human T-cell leukaemia virus type 1 (HTLV-1) proviral DNA. Four cases exhibited a CD8⁺/CD56^– phenotype, four others had a CD8^–/CD56⁺ phenotype, and one was CD8⁺/CD56⁺. All but one case also expressed the surface antigens CD3, TCR αβ, and CD4. Expression of granzyme B and TIA-1 were demonstrated in three and two cases, respectively, but none expressed perforin or FasL. In the control study, 10 cases with typical CD3⁺/4⁺/8^–/56^– ATLL demonstrated no expression of those cytotoxic-associated proteins. Our findings suggest that CD8 and/or CD56 positivity probably confer(s) no cytotoxic function on ATLL cells, and it is possible that CD8 and CD56 may be simply aberrant surface markers in ATLL. Received: 20 January 1999 / Accepted: 13 April 1999     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.