CD4-negative cytotoxic T cells with a T cell receptor α/βintermediate expression in CD8-deficient mice

Targeted disruption of the CD8 gene results in a profound block in cytotoxic T cell (CTL) development. Since CTL are major histocompatibility complex (MHC) class I restricted, we addressed the question of whether CD8–/– mice can reject MHC class I-disparate allografts. Studies have previously shown that skin allografts are rejected exclusively by T cells. We therefore used the skin allograft model to answer our question and grafted CD8–/– mice with skins from allogeneic mice deficient in MHC class II or in MHC class I (MHC-I or MHC-II-disparate, respectively). CD8–/– mice rejected MHC-I-disparate skin rapidly even if they were depleted of CD4⁺ cells in vivo (and were thus deficient in CD4⁺ and CD8⁺ T cells). By contrast, CD8+/+ controls depleted of CD4⁺ and CD8⁺ T cells in vivo accepted the MHC-I-disparate skin. Following MHC-I, but not MHC-II stimulation, allograft-specific cytotoxic activity was detected in CD8–/– mice even after CD4 depletion. A population expanded in both the lymph nodes and the thymus of grafted CD8–/– animals which displayed a CD4^?8^?3^intermediateTCRα/β^intermediate phenotype. Indeed its T cell receptor (TCR) density was lower than that of CD4⁺ cells in CD8–/– mice or of CD8⁺ cells in CD8+/+ mice. Our data suggest that this CD4^?8^?T cell population is responsible for the CTL function we have observed. Therefore, MHC class I-restricted CTL can be generated in CD8–/– mice following priming with MHC class I antigens in vivo. The data also suggest that CD8 is needed to up-regulate TCR density during thymic maturation. Thus, although CD8 plays a major role in the generation of CTL, it is not absolutely required.     

CD5− CD8αβ intestinal intraepithelial lymphocytes (IEL) are induced to express CD5 upon antigen-specific activation: CD5− and CD5+ CD8αβ IEL do not represent separate T cell lineages

We followed αβ T cell receptor (TCR) usage in subsets of gut intraepithelial lymphocytes (IEL) in major histocompatibility complex class I-restricted αβ TCR-transgenic (tg) mice. The proportion of tg αβ TCR⁺ CD8αβ IEL is reduced compared with CD8⁺ splenocytes of the same animal, particularly under conventional conditions of maintenance. Further fractionation of CD8αβ IEL according to the expression level of surface CD5 revealed that in conventionally housed animals tg TCR⁺ CD5^? CD8αβ IEL are as frequent as in specific pathogen-free (SPF) mice, whereas tg TCR⁺ CD5^int or, even more pronounced, tg TCR⁺ CD5^hi CD8αβ IEL are greatly diminished when compared with mice kept under SPF conditions. Upon antigen-specific stimulation of CD5^? CD8αβ IEL in vitro, CD5 surface expression is up-regulated on a large fraction of cells within 48 h. Up-regulation of CD5 surface expression is further enhanced by the presence of the anti-αIEL monoclonal antibody 2E7. This clearly demonstrates that CD5^?, and CD5⁺ CD8αβ IEL cannot be considered as separate T cell lineages.     

CD8α+ dendritic cells prime TCR‐peptide‐reactive regulatory CD4+FOXP3− T cells

CD4⁺ T cells with immune regulatory function can be either FOXP3⁺ or FOXP3^?. We have previously shown that priming of naturally occurring TCR‐peptide‐reactive CD4⁺FOXP3^? Treg specifically controls Vβ8.2⁺CD4⁺ T cells mediating EAE. However, the mechanism by which these Treg are primed to recognize their cognate antigenic determinant, which is derived from the TCRVβ8.2‐chain, is not known. In this study we show that APC derived from splenocytes of naïve mice are able to stimulate cloned CD4⁺ Treg in the absence of exogenous antigen, and their stimulation capacity is augmented during EAE. Among the APC populations, DC were the most efficient in stimulating the Treg. Stimulation of CD4⁺ Treg was dependent upon processing and presentation of TCR peptides from ingested Vβ8.2TCR⁺CD4⁺ T cells. Additionally, DC pulsed with TCR peptide or apoptotic Vβ8.2⁺ T cells were able to prime Treg in vivo and mediate protection from disease in a CD8‐dependent fashion. These data highlight a novel mechanism for the priming of CD4⁺ Treg by CD8α⁺ DC and suggest a pathway that can be exploited to prime antigen‐specific regulation of T‐cell‐mediated inflammatory disease.     

A novel peripheral CD4+CD8+ T cell population: Inheritance of CD8α expression on CD4+ T cells

In this study we show the inheritance of a CD4⁺CD8⁺ peripheral T cell population in the H.B15 chicken strain. A large proportion of αβ T cells in peripheral blood (20–40%), spleen (10–20%) and intestinal epithelium (5–10%) co-express CD4 and CD8α, but not CD8β. CD4⁺ CD8αα cells are functionally normal T cells, since they proliferate in response to mitogens and signals delivered via the αβT cell receptor as well as via the CD28 co-receptor. These cells induce in vivo a graft versus host-reaction, providing further evidence for their function as CD4⁺ T cells. The CD4⁺CD8αα T cell population was found in 75% of the first progeny and in 100% of further progenies, demonstrating that co-expression of CD4 and CD8 on peripheral T cells is an inherited phenomenon. In addition, cross-breeding data suggest a dominant Mendelian form of inheritance. The hereditary expression of CD8α on peripheral CD4⁺ T cells in chicken provides a unique model in which to study the regulation of CD4 and CD8 expression.     

CD8α+ DC are not the sole subset cross‐presenting cell‐associated tumor antigens from a solid tumor

One of the clear paradoxes in tumor immunology is the fact that cross‐presentation of cell‐associated tumor antigens to CD8⁺ T cells is efficient, yet CTL generation is weak, and tumors continue to grow. We examined, for the first time whether this may be due to alterations in the phenotype or function of cross‐presenting DC using a solid tumor model expressing a membrane bound neo‐antigen (hemagglutinin, HA). Tumor antigen was constitutively cross‐presented in the tumor‐draining LN throughout tumor progression by CD11c⁺ DC. Further analysis revealed that both CD8α⁺ and CD8α^? DC subsets, but not plasmacytoid DC, were effective at cross‐presenting HA tumor antigen. The proportions of DC subsets in the tumor‐draining LN were equivalent to those seen in the LN of naïve mice; however, a significant increase in the expression of the potential inhibitory B7 molecule, B7‐DC, was noted and appeared to be restricted to the CD8α^– DC subset. Therefore LN resident CD8α⁺ DC are not the sole DC subset capable of cross‐presenting cell‐associated tumor antigens. Migratory tumor DC subsets with altered co‐stimulatory receptor expression may contribute to induction and regulation of tumor‐specific responses.     

Neoplastic thymic epithelial cells of human thymoma support T cell development from CD4−CD8− cells to CD4+CD8+ cells in vitro

Human thymoma is a thymic epithelial cell tumour which often contains a large number of immature T cells and is frequently associated with autoimmune diseases. Since thymic epithelial cells play key roles in the development and selection of T cells in the normal thymus, we hypothesized that the neoplastic thymic epithelial cells of thymoma may support T cell differentiation in the tumour. We characterized CD4^?CD8^? cells in thymoma and applied an in vitro reconstitution culture system using the CD4^?CD8^? cells and the neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on 29.9 ± 12.2% of CD4^?CD8^? cells in thymoma. TCRγδ was expressed on 27.4 ± 15.1% of CD4^?CD8^? cells and CD19, a B cell marker, was expressed on 14.1 ± 23.1% of CD4^?CD8^? cells. CD4^?CD8^? cells expressed both IL-7R α-chain and common γ-chain. Purified CD4^?CD8^? cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4⁺CD8⁺ cells via CD4 single-positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. Furthermore, we examined the reconstitution culture using CD34⁺CD4^?CD8^? cells purified from normal infant thymus. The CD34⁺CD4^?CD8^? cells in normal thymus also differentiated to CD4⁺CD8⁺ cells in the allogeneic co-culture with the neoplastic epithelial cells of thymoma. These results indicate that the tumour cells of thymoma retain the function of thymic epithelial cells and can induce differentiation of T cells in thymoma.     

Co-expression of CD8α in CD4+ T cell receptor αβ+ T cells migrating into the murine small intestine epithelial layer

We investigated the surface phenotype of CD3⁺CD4⁺ T cell receptor (TCR) αβ⁺ T cells repopulating the intestinal lymphoid tissues of C.B-17 scidlscid (severe-combined immunodeficient; scid) (H-2^d, L^d+) mice. CD4⁺ CD8^? T cells were cell sorter-purified from various secondary and tertiary lymphoid organs of congenic C.B-17 +/+ (H-2^d, L^d+) or semi-syngeneic dm2 (H-2^d, L^d?) immunocompetent donor mice. After transfer of 10⁵ cells into young scid mice, a mucosa-homing, memory CD44^hi CD45RB^lo CD4⁺ T cell population was selectively engrafted. Large numbers of single-positive (SP) CD3⁺ CD2⁺ CD28⁺ CD4⁺ CD^8? T cells that expressed the α₄ integrin chain CD49d were found in the spleen, the mesenteric lymph nodes, the peritoneal cavity and the gut lamina propria of transplanted scid mice. Unexpectedly, large populations of donor-type doublepositive (DP) CD4⁺ CD8α⁺ CD8β^? T cells with high expression of the CD3/TCR complex appeared in the epithelial layer of the small intestine of transplanted scid mice. In contrast to SP CD4⁺ T cells, the intraepithelial DP T cells showed low expression of the CD2 and the CD28 co-stimulator molecules, and of the α₄ integrin chain CD49d, but expressed high levels of the α_IEL integrin chain CD103. The TCR-Vβ repertoire of DP but not SP intraepithelial CD4⁺ T cells was biased towards usage of the Vβ6 and Vβ8 viable domains. Highly purified populations of SP and DP CD4⁺ T cell populations from the small intestine epithelial layer of transplanted scid mice had different abilities to repopulate secondary scid recipient mice: SP CD4⁺ T cells repopulated various lymphoid tissues of the immunodeficient host, while intraepithelial DP CD4⁺ T cells did not. Hence, a subset of CD3⁺ CD4⁺ TCRαβ⁺ T cells apparently undergoes striking phenotypic changes when it enters the microenvironment of the small intestine epithelial layer.     

Characterization of CD4−CD8− T cell receptor αβ+ T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis

Inflammation of the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) starts in the subarachnoid space (SAS) and spreads later to the adjacent CNS parenchyma. To characterize the nature of lesion-forming T cells in situ in more detail, T cells were isolated from the SAS and their surface phenotype and the nucleotide sequence of the junctional region of the T cell receptor (TCR) was determined and compared with those of the lymph node (LN) and spinal cord (SC) T cells. Characteristically, more than 70% of SAS TCR αβ⁺ T cells isolated at the early stage of EAE lacked both CD4 and CD8 molecules, whereas those from LN and SC were either CD4⁺ or CD8⁺. Analysis of nucleotide sequences of the junctional region of TCR revealed that T cells bearing a sequence identical to that for encephalitogenic T cell clones were found in both SAS and SC. Furthermore, purified CD4^?CD8^? T cells expressed CD4 molecules after culture. At the same time, these T cells acquired reactivity to myelin basic protein and induced passive EAE in naive animals after adoptive transfer. Our results suggest that CD4^?CD8^? T cells in the SAS are precursors of lesion-forming T cells in the SC and that phenotype switching takes place during the process of T cell infiltration into the CNS parenchyma. The double-negative nature of these T cells may explain an escape of encephalitogenic T cells from negative selection in T cell differentiation.     

Helper activity of CD4+ αβ T cells is required for the avian γδ T cell response

We have studied the in vitro activation of chicken γδ T cells. Both splenic αβ and γδ T cells obtained from complete Freund's adjuvant-primed chickens proliferated in vitro when stimulated with mycobacterial sonicate or purified protein derivative of Mycobacterium tuberculosis. When CD4⁺ cells or αβ T cell receptor (TcR)-positive cells were removed, both the proliferation and the blast formation of γδ T cells in response to mycobacterial antigens were abrogated. The response was restored if supernatant from concanavalin A (Con A)-activated lymphocyte cultures (CAS) as a source of helper factors was added together with the specific antigen purified protein derivative. The CD4- or αβ TcR-depleted cells still proliferated in response to Con A, although a decrease of the response was observed. To analyze the γδ T cell response more specifically we stimulated peripheral blood cells with immobilized monoclonal antibodies against T cell receptor. Anti-γδ TcR antibody alone did not induce significant proliferation. When CAS was added together with the anti-γδ TcR monoclonal antibody, a strong proliferation of γδ T cells was observed. In contrast, both Vβ1- and Vβ2-expressing αβ T cells proliferated in vitro in response to stimulation with the relevant anti-TcR monoclonal antibody alone. Depletion of either Vβ1⁺ or Vβ2⁺ T cell subset alone had no negative effect on the proliferation or blast formation of γδ T cells stimulated with mycobacterial antigens. Taken together our results suggest that CD4⁺ αβ T cells (both Vβl- and Vβ2-expressing) play a role in the activation and response of chicken γδ T cells.     

The lack of CD8α cytoplasmic domain resulted in a dramatic decrease in efficiency in thymic maturation but only a moderate reduction in cytotoxic function of CD8+ T lymphocytes

The glycoprotein CD8 is believed to play an important role in the maturation and function of MHC class I-restricted T lymphocytes. CD8 has been proposed to function as a co-receptor of the TcR to participate in signal transduction, possibly through its cytoplasmic domain that binds to protein tyrosine kinase p56^lck. A T cell-specific transgene encoding CD8α truncated at the cytoplasmic domain (“tailless CD8α”), was introduced into CD8α-deficient mice. This animal model was used to study the role of the CD8 cytoplasmic domain in T cell ontogeny and function. “Tailless CD8α” was expressed on the cell surface of thymocytes and peripheral T cells. A small population of peripheral CD4^? T cells (6% of T lymphocytes) was found to have cell surface expression of “tailless CD8α” and endogenous CD8β indicating that these cells may belong to the CD8⁺ T cell lineage. A consistent result was obtained from CD8α-deficient mice bearing the “tailless CD8α” and the MHC class I-restricted 2C TcR transgenes. A small population of CD4 T cells expressing CD8β the “tailless CD8α” and the 2C TcR transgenes was present in the periphery of these mice in a selecting background, but was absent in a deleting background. When “tailless CD8α” mice were infected with lymphocytic choriomeningitis virus (LCMV), the peripheral CD8⁺ CD4^? T cell subset expanded dramatically and a significant LCMV-specific cytolytic activity was detected. The results suggest that the cytoplasmic portion of CD8α is not absolutely required but dramatically enhances the eficiency of thymic maturation of CD8⁺ T cells. The lack of CD8α cytoplasmic domain in peripheral CD8⁺ T cells does not abolish the generation of cytotoxicity in response to an in vivo LCMV infection, although the cytolytic activity is slightly reduced compared to that in control mice.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

A human CD4 transgene rescues CD4−CD8+ cells in β2-microglobulin-deficient mice

The specificity of the αβ T cell receptor for class I or class II major histocompatibility complex (MHC) molecules determines whether a mature T cell will be of the CD4^?CD8⁺ or CD4⁺CD8^? phenotype, respectively. We show here that a human CD4 transgene can rescue a significant fraction of CD4^?CD8⁺ T cells in β2-microglobulin-deficient mice. Cells with this phenotype could be induced to become potent killers of targets expressing allogeneic MHC antigens, indicating that lineage commitment can precede the rescue of developing cells by the T cell receptor for antigen and the CD4 coreceptor.     

Progenies of fetal thymocytes are the major source of CD4−CD8+ αα intestinal intraepithelial lymphocytes early in ontogeny

Present literature supports the view of an extrathymic origin for the subset of intestinal intraepithelial lymphocytes (IEL) that express the CD4^?CD8⁺ αα phenotype. This subset would include virtually all T cell receptor (TCR) γδ IEL and a portion of TCR αβ IEL. However, these reports do not exclude the possibility that some CD4^?CD8⁺ αα IEL are actually thymically derived. To clarify this issue, we examined the IEL day 3 neonatally thymectomized (NTX) mice. NTX resulted in as much as 80 % reduction in total TCR γδ IEL and in a nearly complete elimination of TCR αβ CD4^?CD8⁺ αα IEL early in ontogeny (3-to 5-week-old mice). The thymus dependency of TCR γδ IEL and TCR αβ CD4^?CD8⁺ IEL was less prominent in older mice (7- to 10-week-old mice), as the total number of these IEL increased in NTX mice, but still remained severalfold less than that in euthymic mice. Furthermore, we demonstrate, by grafting the fetal thymus of CBF1 (H-2^b/d) mice under the kidney capsule of congenitally nude athymic mice of BALB/c background (H-2^d), that a substantial number of TCR γδ IEL and TCR αβ CD4^?CD8⁺ αα IEL can be thymically derived (H-2^b+). In contrast, but consistent with our NTX data, grafting of adult thymi into nude mice generated virtually no TCR γδ IEL and relatively less TCR αβ CD4^?CD8⁺ αα IEL than did the grafting of fetal thymi. These results suggest that the thymus is the major source of TCR γδ and TCR αβ CD4^?CD8⁺ αα IEL early in ontogeny, but that the extrathymic pathway is probably the major source of these IEL later in ontogeny. A reassessment of the theory that most CD4^?CD8⁺ IEL are extrathymically derived is needed.     

Encephalitogenic,myelin basic protein-specific T cells from naive rat thymus: preferential use of the T cell receptor gene Vβ8.2 and expression of the CD4− CD8− phenotype

Using a primary limiting dilution approach to generate T cell lines, we compared myelin basic protein (MBP)-specific T cell clones from naive unprimed Lewis rat thymuses with the corresponding T cell repertoire of primed rats. We found that in the naive thymus repertoire MBP-specific, encephalitogenic T cell clones preferentially use T cell receptor Vβ8.2 genes, along with CDR3 sequences typical for the primed Lewis anti-MBP response. In contrast to T cells from primed immune organs, which all display the CD4⁺ CD8^? phenotype, the majority of naive thymus-derived T cell clones expressed reduced levels of the CD4 co-receptor. Some clones were completely CD4^?CD8^?, while others included CD4^? CD8^? subpopulations along with CD4⁺CD8^? T cells. In the one mixed population examined in detail, the CD4^?CD8^? and CD4⁺CD8^? T cell subpopulations used a T cell receptor with identical β chain sequence. The data suggest that in the Lewis rat the biased T cell receptor gene usage by encephalitogenic T cells is a property of the natural thymic T cell repertoire, possibly as a consequence of positive selection. The unusually low expression of CD4 in the major histocompatibility complex class II-restricted autoreactive T cells could be related to their escape from negative selection within the thymus.     

TGF‐β downregulates KLRG1 expression in mouse and human CD8+ T cells

The inhibitory receptor killer cell lectin‐like receptor G1 (KLRG1) and the integrin α_E (CD103) are expressed by CD8⁺ T cells and both are specific for E‐cadherin. However, KLRG1 ligation by E‐cadherin inhibits effector T‐cell function, whereas binding of CD103 to E‐cadherin enhances cell–cell interaction and promotes target cell lysis. Here, we demonstrate that KLRG1 and CD103 expression in CD8⁺ T cells from untreated and virus‐infected mice are mutually exclusive. Inverse correlation of KLRG1 and CD103 expression was also found in human CD8⁺ T cells‐infiltrating hepatocellular carcinomas. As TGF‐β is known to induce CD103 expression in CD8⁺ T cells, we examined whether this cytokine also regulates KLRG1 expression. Indeed, our data further reveal that TGF‐β signaling in mouse as well as in human CD8⁺ T cells downregulates KLRG1 expression. This finding provides a rationale for the reciprocal expression of KLRG1 and CD103 in different CD8⁺ T‐cell subsets. In addition, it points to the limitation of KLRG1 as a marker for terminally differentiated CD8⁺ T cells if lymphocytes from tissues expressing high levels of TGF‐β are analyzed.     

Blood and beyond: Properties of circulating and tissue‐resident human virus‐specific αβ CD8+ T cells

CD8⁺ αβ T‐cell responses form an essential line of defence against viral infections. An important part of the mechanisms that control the generation and maintenance of these responses have been elucidated in experimental mouse models. In recent years it has become clear that CD8⁺ T‐cell responses in humans not only show similarities, but also display differences to those occurring in mice. Furthermore, while several viral infections occur primarily in specialised organ systems, for obvious reasons, most human CD8⁺ T‐cell investigations were performed on cells deriving from the circulation. Indeed, several lines of evidence now point to essential functional differences between virus‐specific CD8⁺ memory T cells found in the circulation and those providing protection in organ systems, such as the lungs. In this review, we will focus on summarising recent insights into human CD8⁺ T‐cell differentiation in response to several viruses and emphasise that for a complete understanding of anti‐viral immunity, it is pivotal to scrutinise such responses in both blood and tissue.     

Carbon monoxide‐treated dendritic cells decrease β1‐integrin induction on CD8+ T cells and protect from type 1 diabetes

Carbon monoxide (CO) treatment improves pathogenic outcome of autoimmune diseases by promoting tolerance. However, the mechanism behind this protective tolerance is not yet defined. Here, we show in a transgenic mouse model for autoimmune diabetes that ex vivo gaseous CO (gCO)‐treated DCs loaded with pancreatic β‐cell peptides protect mice from disease. This protection is peptide‐restricted, independent of IL‐10 secretion by DCs and of CD4⁺ T cells. Although no differences were observed in autoreactive CD8⁺ T‐cell function from gCO‐treated versus untreated DC‐immunized groups, gCO‐treated DCs strongly inhibited accumulation of autoreactive CD8⁺ T cells in the pancreas. Interestingly, induction of β1‐integrin was curtailed when CD8⁺ T cells were primed with gCO‐treated DCs, and the capacity of these CD8⁺ T cells to lyse isolated islet was dramatically impaired. Thus, immunotherapy using CO‐treated DCs appears to be an original strategy to control autoimmune disease.