Ontogeny of lymphoid organs and development of IgM-bearing cells in Atlantic halibut (Hippoglossus hippoglossus L.)

In teleost fish, the head kidney, thymus, and spleen are generally regarded as important immune organs. In this study, the ontogeny of these organs was studied in Atlantic halibut (Hippoglossus hippoglossus), larvae at various stages of development. We observed that the kidney was present at hatching, the thymus at 33 days post hatch (dph), while the spleen was the last organ to be detected at 49 dph. All three lymphoid organs were morphologically well developed during late metamorphic stages. Real time RT-PCR analysis showed that IgM mRNA expression could be observed at 66 dph and later, which correlates well with in situ hybridisation data showing that a few IgM positive cells could be detected in the anterior kidney and spleen from 66 dph. Our data also showed that the highest levels of IgM mRNA could be detected in halibut spleen. Immunostaining using a monoclonal antibody against halibut IgM detected IgM positive cells at 94 dph in both the head kidney and the spleen, which is much later than the IgM mRNA. Numerous cells expressing both IgM mRNA and protein could be detected in the spleen and anterior kidney and also to some extent in thymus specimens from adult halibut.     

Quantitative investigation of antigen and immune response in nervous and lymphoid tissues of Atlantic halibut (Hippoglossus hippoglossus) challenged with nodavirus

The present study reports the quantitative analysis of the spatio-temporal development of nodavirus infection and corresponding immune response in juvenile Atlantic halibut (Hippoglossus hippoglossus) challenged by intramuscular injection of nodavirus. Novel quantitative real-time RT-PCR protocols were applied to evaluate the absolute copy numbers of nodavirus RNA2 (RNA2) and secretory-IgM mRNA (sec-igmicro) in the eye, brain, mid/posterior kidney and spleen sampled over a period of 81 days. In the eye and brain, levels of both RNA2 and sec-igmicro increased significantly early in the infection. In the spleen and mid/posterior kidney, both RNA2 and sec-igmicro were detected but the levels remained unchanged during the experimental period. The levels of RNA2 and sec-igmicro in the eye and brain were strongly correlated (P<0.0001). Nodavirus antigen was demonstrated by immunohistochemistry (IHC) in the retina of eyes from a relatively few fish from day 34 post challenge (brain not examined), but not at any time in the spleen and anterior kidney. By IHC, IgM+ cells were observed in conjunction with nodavirus positive IHC labelling in the retina. In both the spleen and anterior kidney, the number of IgM+ cells increased from day 3 post challenge. By conventional real-time RT-PCR, RNA2 was only sporadically demonstrated in the posterior intestine, heart and gills. ELISA analysis revealed a nodavirus specific antibody response in serum that was significant from day 18 post challenge. No clinical signs or mortality related to nodavirus infection were observed in the challenged halibut. The results suggest that the nodavirus infection induced a significant antibody response through activation of B-cells in the kidney and spleen, and involved a substantial migration of antibody-secreting cells to infected peripheral tissues.     

Intestinal CD8 alpha alpha and CD8 alpha beta intraepithelial lymphocytes are thymus derived and exhibit subtle differences in TCR beta repertoires

Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the first line of cellular defense against enteric pathogens. Therefore, determining the origin of these cells has important implications for the mechanisms of T cell maturation and repertoire selection. Recent evidence suggests that murine CD8 alpha alpha intestinal IELs (iIELs) can mature and undergo selection in the absence of a thymus. We analyzed IEL origin by cell transfer, using two congenic chicken strains. Embryonic day 14 and adult thymocytes did not contain any detectable CD8 alpha alpha T cells. However, when TCR(+) thymocytes were injected into congenic animals, they migrated to the gut and developed into CD8alphaalpha iIELs, while TCR(-) T cell progenitors did not. The TCR V beta 1 repertoire of CD8 alpha alpha(+) TCR V beta 1(+) iIELs contained only part of the TCR V beta 1 repertoire of total iIELs, and it exhibited no new members compared with CD8(+) T cells in the thymus. This indicated that these T cells emigrated from the thymus at an early stage in their developmental process. In conclusion, we show that while CD8 alpha alpha iIELs originate in the thymus, T cells acquire the expression of CD8 alpha alpha homodimers in the gut microenvironment.     

Description of an ectothermic TCR coreceptor, CD8 alpha, in rainbow trout

We have cloned the first CD8 alpha gene from an ectothermic source using a degenerate primer for Ig superfamily V domains. Similar to homologues in higher vertebrates, the rainbow trout CD8 alpha gene encodes a 204-aa mature protein composed of two extracellular domains including an Ig superfamily V domain and hinge region. Differing from mammalian CD8 alpha V domains, lower vertebrate (trout and chicken) sequences do not contain the extra cysteine residue (C strand) involved in the abnormal intrachain disulfide bridging within the CD8 alpha V domain of mice and rats. The trout membrane proximal hinge region contains the two essential cysteine residues involved in CD8 dimerization (alpha alpha or alpha beta) and threonine, serine, and proline residues which may be involved in multiple O-linked glycosylation events. Although the transmembrane region is well conserved in all CD8 alpha sequences analyzed to date, the putative trout cytoplasmic region differs and, in fact, lacks the consensus p56lck motif common to other CD8 alpha sequences. We then determined that the trout CD8 alpha genomic structure is similar to that of humans (six exons) but differs from that of mice (five exons). Additionally, Northern blotting and RT-PCR demonstrate that trout CD8 alpha is expressed at high levels within the thymus and at weaker levels in the spleen, kidney, intestine, and peripheral blood leukocytes. Finally, we show that trout CD8 alpha can be expressed on the surface of cells via transfection. Together, our results demonstrate that the basic structure and expression of CD8 alpha has been maintained for more than 400 million years of evolution.     

Studies of CD4- CD8- alpha beta bone marrow T cells with suppressor activity.

The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mice expressed the alpha beta+ TCR CD4- CD8- surface phenotype. Purified C57BL/Ka alpha beta+ TCR CD4- CD8- marrow cells obtained by cell sorting suppressed the MLR of C57BL/Ka responder and BALB/c stimulator spleen cells. Although the percentage of typical T cells in the spleen was markedly reduced in adult nude mice or normal neonatal mice as compared to the normal adult, the percentage of alpha beta+ TCR CD4- CD8- cells in the spleen and marrow was not. The percentage of "self-reactive" V beta 5+ T cells in the BALB/c spleen was markedly reduced as compared to that in the C57BL/Ka spleen. However, the percentages in the bone marrow were similar. The results indicate that the predominant subset of marrow T cells in these pathogen-free mice differ with regard to surface marker phenotype, function, dependence on the adult thymus, and deletion of certain self-reactive V beta receptors as compared to typical spleen T cells. The marrow T cells appear to develop directly from marrow precursors without rearranged beta chain genes during a 48 hour in vitro culture.     

Differential expression of CD8 alpha and CD8 beta associated with MHC-restricted and non-MHC-restricted cytolytic effector cells

The differential expression of the alpha and beta chains of the CD8 glycoprotein was examined in three functionally distinct cytolytic effector cell populations: (i) T cells (CD3+ CD56-), (ii) NK cells (CD56+ CD3-), and (iii) non-MHC-restricted T cells (CD56+ CD3+). Twenty-four percent of T cells were CD8+, and they consistently coexpressed both CD8 alpha and CD8 beta. Moreover, CD8+ T cells uniformly expressed high-density CD8 alpha. Forty percent of NK cells were CD8+ but the vast majority (approximately 75%) expressed only CD8 alpha without CD8 beta. In addition, CD8+ NK cells uniformly expressed low-density CD8 alpha. In comparison, 75% of non-MHC-restricted T lymphocytes were CD8+ but they displayed an intermediate phenotype: 60% coexpressed CD8 alpha and CD8 beta while 40% expressed only CD8 alpha. Within this population, CD8 alpha was expressed at high density, similar to that of T cells. Following IL-2 activation, enhancement of non-MHC-restricted cytotoxicity was not associated with any changes in either the quantitative or qualitative pattern of expression of CD8 alpha or CD8 beta by these cells. Addition of either anti-CD8 alpha or anti-CD8 beta mAb did not alter non-MHC-restricted cytotoxicity of either CD56+ CD3- or CD56+ CD3+ effector cells. However, within the CD56+ cell population, non-MHC-restricted cytotoxicity was almost entirely found within the CD8- and CD8 alpha + beta- populations, and both subsets displayed a similar level of killing. In contrast, CD8 alpha+ beta+ cells exhibited very little non-MHC-restricted cytotoxicity. Thus, the coexpression of CD8 alpha and CD8 beta in conjunction with the TCR/CD3 complex appears to characterize MHC restricted cells while the expression of CD8 alpha alone is associated with non-MHC-restricted cytotoxicity. Taken together, these findings suggest that neither CD8 alpha nor CD8 beta is involved in the initial phases of target cell binding or recognition during NK cell-mediated lysis. However, the selective expression of CD8 alpha by a large fraction of non-MHC-restricted effector cells suggests that this antigen may play a different functional role in this unique subset of cytolytic lymphocytes.     

Human CD8 beta, but not mouse CD8 beta, can be expressed in the absence of CD8 alpha as a beta beta homodimer

The T cell coreceptor CD8 exists on mature T cells as disulfide-linked homodimers of CD8 alpha polypeptide chains and heterodimers of CD8 alpha- and CD8 beta-chains. The function of the CD8 alpha-chain for binding to MHC class I and associating with the tyrosine kinase p56lck was demonstrated with CD8 alpha alpha homodimers. CD8 alpha beta functions as a better coreceptor, but the actual function of CD8 beta is less clear. Addressing this issue has been hampered by the apparent inability of CD8 beta to be expressed without CD8 alpha. This study demonstrates that human, but not mouse, CD8 beta can be expressed on the cell surface without CD8 alpha in both transfected COS-7 cells and murine lymphocytes. By creating chimeric proteins, we show that the murine Ig domain of CD8 beta is responsible for the lack of expression of murine CD8 beta beta dimers. In contrast to CD8 alpha alpha, CD8 beta beta is unable to bind MHC class I in a cell-cell adhesion assay. Detection of this form of CD8 should facilitate studies on the function of the CD8 beta-chain and indicates that caution should be used when interpreting studies on CD8 function using chimeric protein with the murine CD8 beta beta Ig domain. In addition, we demonstrate that the Ig domains of CD8 alpha are also involved in controlling the ability of CD8 to be expressed. Mutation of B- and F-strand cysteine residues in CD8 alpha reduced the ability of the protein to fold properly and, therefore, to be expressed.     

Immunomodulatory effects of transforming growth factor-beta on T lymphocytes. Induction of CD8 expression in the CTLL-2 cell line and in normal thymocytes.

We investigated the role of transforming growth factor-beta 1 (TGF-beta) in regulation of T cell growth and differentiation. Treatment of CTLL-2 cells with TGF-beta inhibited IL-2-dependent proliferation and caused morphologic changes as well as increased adherence. A major change of phenotype in TGF-beta-treated cells was the de novo expression of CD8 alpha chain in 35% of cells, which required the continuous presence of TGF-beta. Of the CD8 alpha+ cells, 20 to 30% co-expressed CD8 beta chain. Increased CD8 expression occurred even in the total absence of cell growth, was not a consequence of growth inhibition, and was not a result of selective growth or survival of CD8+ cells. New RNA synthesis was required for TGF beta-induced CD8 alpha surface expression, inasmuch as this was prevented by treatment with actinomycin D. Northern blot analysis demonstrated that cells treated with IL-2 + TGF-beta rapidly accumulated mRNA encoding both chains of the CD8 dimer, to a level fourfold greater than control by 6 to 12 h. In contrast, the IL-2-dependent increases in IL-2R alpha, IL-2R beta, and Granzyme B mRNA levels in these cultures were profoundly inhibited by TGF-beta. When unfractionated murine thymocytes were stimulated with phorbol dibutyrate plus ionomycin and cultured with IL-2 + TGF-beta, an increase in CD8 alpha mRNA was seen and greater numbers of CD8+ cells with higher levels of CD8 alpha and CD8 beta surface expression resulted, as compared to controls treated with IL-2 alone. Furthermore, similar treatment of CD4-CD8-(double negative) thymocytes with TGF-beta induced de novo CD8 alpha expression by a substantial number of cells, and the majority of these CD8+ cells lacked TCR/CD3. These data suggest that TGF-beta has both positive and negative regulatory effects on the expression of gene products important for T lymphocyte differentiation and function.     

Development of the CD4 and CD8 lineage of T cells: instruction versus selection

T cells bearing the alpha beta T cell receptor (TCR) can be divided into CD4+8- and CD4-8+ subsets which develop in the thymus from CD4+8+ precursors. The commitment to the CD4 and CD8 lineage depends on the binding of the alpha beta TCR to thymic major histocompatibility complex (MHC) coded class II and class I molecules, respectively. In an instructive model of lineage commitment, the binding of the alpha beta TCR, for instance to class I MHC molecules, would generate a specific signal instructing the CD4+8+ precursors to switch off the expression of the CD4 gene. In a selective model, the initial commitment, i.e. switching off the expression of either the CD4 or the CD8 gene would be a stochastic event which is then followed by a selective step rescuing only CD4+ class II and CD8+ class I specific T cells while CD4+ class I and CD8+ class II specific cells would have a very short lifespan. The selective model predicts that a CD8 transgene which is expressed in all immature and mature T cells should rescue CD4+ class I MHC specific T cells from cell death. We have performed experiments in CD8 transgenic mice which fail to support a selective model and we present data which show that the binding of the alpha beta TCR to thymic class I MHC molecules results in up-regulation of the TCR in the CD4+8+ population. Therefore, these experiments are consistent with an instructive model of lineage commitment.     

Impaired clonal expansion in athymic nude CD8+CD4- T cells

A comparative study of the phenotype and immune functions of highly purified CD8+CD4- T cells obtained from the spleen and thymus of normal mice and from the spleen of athymic nude mice was conducted. Of seven individual normal and nude mice examined, the range of V beta 8+ cells among CD8+ T cells was a heterogeneous 4.3 to 30.5% for athymic nude mice and a much more uniform spread from 14.7 to 18.5% for normal mice. In six of the seven nude mice examined, the fraction of V beta 8+ cells was below the lower limit of the V beta 8 distribution in normal mice. However, one of the seven nude mice contained nearly twice the percentage of normal V beta 8+ cells. A reduction in the density of V beta 8 as well as CD3 Ag expression was also observed in athymic CD8+CD4- cells although an Ly-6-linked Ag, B4B2 displayed a highly increased expression. Considering the battery of Ag analyzed in entirety, athymic CD8+CD4- T cells were clearly distinct from their "counterpart" CD8+CD4- T cells isolated from either thymus or spleen of normal (euthymic) mice. Anti-CD3-mediated triggering of the TCR:CD3 complex caused extensive clonal proliferation in cultures to which single responding CD8+ T cells had been deposited. Under identical conditions, however, anti-CD3 caused little, if any clonal expansion in CD8+ cells from athymic nude mice. Highly purified athymic CD8+CD4- cells produced readily detectable IL-2R expression and IL-2 synthesis and secretion upon stimulation by anti-CD3 and by Con A. Production of IL-2 by purified athymic CD8+CD4- cells was due to CD8+CD4- cells and not due to a minor population of contaminating CD8- cells as anti-CD8 + C treatment completely abrogated the ability of athymic CD8+CD4- cells to produce IL-2. Despite IL-2 production and IL-2R expression by athymic nude CD8+CD4- T cells in response to anti-CD3 and to Con A, an impaired proliferative response followed.     

Alpha beta TCR+ cells are a minimal fraction of peripheral CD8+ pool in MHC class I-deficient mice

MHC class I molecules play a role in the maintenance of the naive peripheral CD8+ T cell pool. The mechanisms of the peripheral maintenance and the life span of residual CD8+ cells present in the periphery of beta 2-microglobulin-deficient (beta 2m-/-) mice are unknown. We here show that very few CD8+ cells in beta 2m-/- mice coexpress CD8 beta, a marker of the thymus-derived CD8+ T cells. Most of the CD8 alpha+ cells express CD11c and can be found in beta 2m/RAG-2 double-deficient mice, demonstrating that these cells do not require rearranged Ag receptors for differentiation and survival and may be of dendritic cell lineage. Rare CD8 alpha+CD8 beta+ cells can be detected following in vivo alloantigenic stimulation 2 wk after the adult thymectomy. Selective MHC class I expression by bone marrow-derived cells does not lead to an accumulation of CD8 beta+ cells in beta 2m-/- mice. These findings demonstrate that 1) thymic export of CD8+ T cells in beta 2m-/- mice is reduced more severely than previously thought; 2) non-T cells expressing CD8 alpha become prominent when CD8+ T cells are virtually absent; 3) at least some beta 2m-/- CD8+ T cells have a life span in the periphery comparable to wild-type CD8+ cells; and 4) similar ligands induce positive selection in the thymus and survival of CD8+ T cells in the periphery.     

Profiling the CD8low phenotype, an alternative career choice for CD8 T cells during primary differentiation

A CD8+ T cell of naive phenotype has multiple career choices during its primary differentiation into an effector cell population. One of these career options is becoming a CD8low T cell. We have previously shown by in vitro studies that CD8low T cells have lost expression of CD8 surface protein and mRNA and are poorly cytolytic. In line with poor cytolytic function, CD8low T cells express low levels of perforin and granzyme B and C, mediators of the granule-exocytosis machinery. However, CD8low T cells express IFN-gamma and substantial amounts of IL-4, the signature cytokines of type 1 and type 2 T-cell polarization, respectively. Here, we argue that the CD8low phenotype is an alternative career choice for any naive CD8+ T cell during primary activation but that the probability of choosing this option is greatly enhanced by both IL-4 and strong activation conditions. CD8low T cells have downregulated CD8 alpha/beta heterodimers and no preferential CD8 alpha/alpha homodimer expression. As shown by anti-CD8 Ab blocking experiments, surface CD8 substantially contributes to the CD8 T cell's effector function (i.e. cytokine expression and cytolytic activity). The distinct effector profile of CD8low T cells gives an example of the complexity of different CD8 T cell careers during primary effector differentiation.     

A murine thymic stromal cell line which may support the differentiation of CD4-8- thymocytes into CD4+8- alpha beta T cell receptor positive T cells.

A fibroblastoid cell line TSt-4 was established from fetal thymus tissue of C57BL/6 mice. When fetal thymus (FT) cells or CD4-8- (DN) cells of adult thymuses were cultured on the monolayer of TSt-4, a considerable proportion of lymphocytes expressed CD4 or both CD4 and CD8 within 1 day, and the CD4+CD8- cells were maintained further while the CD4+8+ cells disappeared by Day 5. A large proportion of cells generated from DN cells but not FT cells was shown to express CD3 and T cell receptor alpha beta. Addition of recombinant interleukin (IL)-7 into the cultures resulted in a marked increase of cell recovery without virtual change in differentiation process of alpha beta lineage. The present work strongly suggests that thymic fibroblasts play an important role in T cell differentiation and IL-7 contributes to supporting proliferation of differentiated cells.     

Developmentally regulated expression of the beta 4 integrin on immature mouse thymocytes.

Integrins are a superfamily of alpha beta heterodimers, most of which serve as cell surface receptors for extracellular matrix proteins. In this report, we demonstrate that the recently described alpha 6 beta 4 integrin, previously thought to be limited to epithelial cells and Schwann cells, is expressed on immature mouse thymocytes. The presence of alpha 6 beta 4 is controlled by regulation of beta 4 expression, because alpha 6 was expressed by virtually all cells examined, paired with the beta 1 integrin chain to form VLA-6. During fetal ontogeny, beta 4 was highly expressed by 35% of day-13 thymocytes, 75% of day-14 to -15 thymocytes, then rapidly declined to low levels by birth. In neonates and adults, beta 4 expression was highest on CD4- CD8- CD3- and TCR(+)-gamma delta subsets. Correlation of IL-2R, CD44 and beta 4 on CD4- CD8- thymocytes revealed maximal levels on the intermediate CD44- IL-2R+ subset. Most CD4- CD8+ TCR- thymocytes and a significant fraction of CD4+ CD8+ thymocytes were beta 4lo, whereas the most mature J11d- single positive thymocytes were beta-4. Overall, down-regulation of beta 4 was associated with up-regulation of CD4, CD8, and CD3 in the thymus. alpha 6 beta 4 was undetectable on fetal liver or bone marrow cells, lymphocytes from lymph node, spleen, or blood, and mitogen-activated splenic T cells cultured up to 10 wk with IL-2. The data suggest that alpha 6 beta 4 is up-regulated after pro-T cells enter the thymus and may have a thymus-specific function for T cells. The developmentally regulated pattern of expression and the prominence of alpha 6 beta 4 on day-13 to -16 fetal and adult CD4- CD8- CD3- thymocytes further suggest this unusual integrin may play a role in early T cell development, including stages before acquisition of the TCR.     

Expression of murine IL-2 receptor beta-chain on thymic and splenic lymphocyte subpopulations as revealed by the IL-2-induced proliferative response in human IL-2 receptor alpha-chain transgenic mice

Lymphocytes from the human (h) IL-2R alpha chain transgenic mice (TGM) constitutively express high affinity binding sites for hIL-2, consisting of transgenic h-IL-2R alpha and endogenous murine IL-2R beta, and therefore easily proliferate in vitro in response to hIL-2. Our study was undertaken to clarify the hIL-2-responsive lymphocyte subsets in the TGM, which should most likely reflect the normal distribution of m IL-2R beta expression. In both thymus and spleen, the majority of expanded cells by hIL-2 was CD3+CD4-CD8+ TCR alpha beta+ cells. The proliferation of CD4+ cells was not observed at all from either organ despite the expression of transgenic hIL-2R alpha. Potent cellular proliferation was also observed from the thymocytes that had been depleted of CD8+ cells, the expanded cells consisting of CD3- (15-40%) and CD3+ populations (60-85%). Among CD3+ cells, approximately the half portion expressed TCR alpha beta, whereas the other half was suggested to express TCR gamma delta. A variable portion (5-20%) of the CD3+ cells expressed CD8 (Lyt-2) in the absence of Lyt-3, and the CD3+CD8+ cells were confined preferentially to the TCR alpha beta- (TCR gamma delta+) population. In the culture of splenocytes depleted of CD8+ cells, however, the proliferated cells were mostly CD3-CD4-CD8-TCR-Mac1-, whereas a minor portion (10-30%) was CD3+CD4-CD8-TCR alpha beta- (TCR gamma delta+. Analysis of TCR genes at both DNA and mRNA levels confirmed the phenotypical observations. These results strongly suggested that IL-2R beta was constitutively and selectively expressed on the primary murine thymocytes and splenic T and NK cells, except for CD4+ cells in both organs.     

The CD8alpha from sea bass (Dicentrarchus labrax L.): Cloning, expression and 3D modelling

In this paper we describe the cloning, expression and structural study by modelling techniques of the CD8alpha from sea bass (Dicentrarchus labrax L.). The sea bass CD8alpha cDNA is comprised of 1490 bp and is translated in one reading frame to give a protein of 217 amino acids, with a predicted 26 amino acids signal peptide, a 88 bp 5'-UTR and a 748 bp 3'-UTR. A multiple alignment of CD8alpha from sea bass with other known CD8alpha sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within CD8alpha's. Cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, an extra cysteine residue found in most mammals in this region is not present in sea bass. The transmembrane and cytoplasmic regions are the most conserved regions within the molecule in the alignment analysis. However, the motif (CXCP) that is thought to be responsible for binding p56lck is missing in the sea bass sequence. Phylogenetic analysis conducted using amino acid sequences showed that sea bass CD8alpha grouped with other known teleost sequences and that three different clusters were formed by the mammalian, avian and fish CD8alpha sequences. The thymus was the tissue with the highest CD8alpha expression, followed by gut, gills, peripheral blood leukocytes and spleen. Lower CD8alpha mRNA levels were found in head kidney, liver and brain. It was possible to create a partial 3D model using the human and mouse structures as template. The CD8alpha 11-120 amino acid region was taken into consideration and the best obtained 3D model shows the presence of ten beta-strands, involving about 50% of the sequence. The global structure was defined as an immunoglobulin-like beta-sandwich made of two anti-parallel sheets. Two cysteines were present in this region and they were at a suitable distance to form an S-S bond as seen in the template human and mouse structures.     

CD8 Raft localization is induced by its assembly into CD8alpha beta heterodimers, Not CD8alpha alpha homodimers

The coreceptor CD8 is expressed as a CD8alphabeta heterodimer on major histocompatibility complex class I-restricted TCRalphabeta T cells, and as a CD8alphaalpha homodimer on subsets of memory T cells, intraepithelial lymphocytes, natural killer cells, and dendritic cells. Although the role of CD8alphaalpha is not well understood, it is increasingly clear that this protein is not a functional homologue of CD8alphabeta. On major histocompatibility complex class I-restricted T cells, CD8alphabeta is a more efficient TCR coreceptor than CD8alphaalpha. This property has for the mouse protein been attributed to the recruitment of CD8alphabeta into lipid rafts, which is dependent on CD8beta palmitoylation. Here, these divergent distributions of CD8alphabeta and CD8alphaalpha are demonstrated for the human CD8 proteins as well. However, although palmitoylation of both CD8alpha and CD8beta chains was detected, this modification did not contribute to raft localization. In contrast, arginines in the cytoplasmic domain are crucial for raft localization of CD8betabeta. Most strikingly, the assembly of a non-raft localized CD8beta chain with a non-raft localized CD8alpha chain resulted in raft-localized CD8alphabeta heterodimers. Using chimeric CD8 proteins, this property of the heterodimer was found to be determined by the assembly of CD8alpha and CD8beta extracellular regions. The presence of two CD8alpha extracellular regions, on the other hand, appears to preclude raft localization. Thus, heterodimer formation and raft association are intimately linked for CD8alphabeta. These results emphasize that lipid raft localization is a key feature of human CD8alphabeta that clearly distinguishes it from CD8alphaalpha.