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Infiltration of CD8(+)TCRalphabeta(+) T-effector populations (CD8 effectors) into graft epithelial compartments has long been recognized as a key lesion in progression of clinical renal allograft rejection. While the afferent phase of allograft immunity is increasingly well-defined, the efferent pathways by which donor-reactive CD8-effector populations access and ultimately destroy the graft renal tubules (rejection per se) have received remarkably little attention. This is an important gap in our knowledge of transplantation immunology, because epithelial compartments comprise the functional elements of most commonly transplanted organs including not only kidney, but also liver, lung, pancreas, and intestine. Furthermore, there is increasing evidence that attack of graft epithelial elements by CD8-effector populations not only causes short-term graft dysfunction but is also a major contributor to development of chronic allograft nephropathy and late graft loss, which now represent the salient clinical problems. Recent studies of the T-cell integrin, alpha(E)beta(7) (CD103), have provided insight into the mechanisms that promote interaction of CD8 effectors with graft epithelial compartments. The purpose of this communication is to review the known properties of the CD103 molecule and its postulated role in the efferent phase of renal allograft rejection.

Iron accumulation in the liver in hereditary hemochromatosis (HH) has been shown to be highly variable. Some studies point to the importance of major histocompatibility complex (MHC) class I (MHC-I) and CD8(+) cells as modifiers of iron overload. In this report, using mice knockout for H2K(b-/-) and H2D(b-/-) genes, it is demonstrated that lack of classical MHC-I molecules results in a spontaneous increase of nonheme iron content in the liver (mainly located in the hepatocytes) when compared to wild-type mice. In CD8(-/-) and Rag2(-/-) mice, no spontaneous hepatic iron accumulation was observed. These results demonstrate for the first time that classical MHC-I molecules could be involved in the regulation of iron metabolism and contribute to the established genotype/phenotype discrepancies seen in HH.

Molecular mechanisms by which the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) negatively regulates phagocytosis in macrophages are unclear. We addressed the issue using bone marrow-derived macrophages from FcgammaR- or SHIP-deficient mice. Phagocytic activities of macrophages from FcgammaRII(b)(-/-) and SHIP(-/-) mice were enhanced to a similar extent, relative to those from wild type. However, calcium influx was only marginally affected in FcgammaRII(b)(-/-), but greatly enhanced in SHIP(-/-) macrophages. Furthermore, SHIP was phosphorylated on tyrosine residues upon FcgammaR aggregation even in macrophages from FcgammaRII(b)(-/-) mice or upon clustering of a chimeric receptor containing CD8 and the immunoreceptor tyrosine-based activation motif (ITAM)-bearing gamma-chain or human-restricted FcgammaRIIa. These findings indicate that, unlike B cells, SHIP is efficiently phosphorylated in the absence of an immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing receptor. We further demonstrate that SHIP directly bound to phosphorylated peptides derived from FcgammaRIIa with a high affinity, comparable to that of FcgammaRII(b). Lastly, FcgammaRIIa-mediated phagocytosis was significantly enhanced in THP-1 cells overexpressing dominant-negative form of SHIP in the absence of FcgammaRII(b). These results indicate that SHIP negatively regulates FcgammaR-mediated phagocytosis through all ITAM-containing IgG receptors using a molecular mechanism distinct from that in B cells.

CD8alphabeta(+) and CD4(+) intraepithelial lymphocytes, the progeny of double-positive thymocytes, are oligoclonal T-cell populations that have accumulated in the gut wall as the result of repeated antigenic stimulations, which lead to rounds of traffic through the lymph/blood circuit ending in an alpha4beta7-integrin-driven homing all along the gut mucosa. In contrast, CD8alphaalpha(+) intraepithelial lymphocytes, which may be TCRgammadelta(+) or alphabeta(+), result in part from local differentiation in the gut, but studies comparing euthymic and athymic mice suggest a thymic double-negative origin for many of them.

T-cell precursors that undergo productive rearrangements at the T-cell receptor (TCR) beta locus are selected for proliferation and further maturation, before TCRalpha expression, by signaling through a pre-TCR composed of the TCRbeta chain paired with a pre-TCRalpha (pTalpha) chain. Such a critical developmental checkpoint, known as beta-selection, results in progression from CD4(-) CD8(-) double negative (DN) to CD4(+) CD8(+) double positive (DP) TCRalphabeta(-) thymocytes. In contrast to mice, progression to the DP compartment occurs in humans via a CD4(+) CD8(-) intermediate stage. Here we show that the CD4(+) CD8(-) to CD4(+) CD8(+) transition involves the sequential acquisition of the alpha and beta chains of CD8 at distinct maturation stages. Our results indicate that CD8alpha, but not CD8beta, is expressed in vivo in a minor subset of DP TCRalphabeta(-) thymocytes, referred to as CD4(+) CD8alphaalpha(+) pre-T cells, mostly composed of resting cells lacking cytoplasmic TCRbeta chain (TCRbeta(ic)). In contrast, expression of CD8alphabeta heterodimers was selectively found on DP TCRalphabeta(-) thymocytes that express TCRbeta(ic) and are enriched for cycling cells. Interestingly, CD4(+) CD8alphaalpha(+) pre-T cells are shown to be functional intermediates between CD4(+) CD8(-) TCRbeta(ic)(-) and CD4(+) CD8alphabeta(+) TCRbeta(ic)(+) thymocytes. More importantly, evidence is provided that onset of CD8beta and TCRbeta(ic) expression are coincident developmental events associated with acquisition of CD3 and pTalpha chain on the cell surface. Therefore, we propose that the CD4(+) CD8alphaalpha(+) to CD4(+) CD8alphabeta(+) transition marks the key control point of pre-TCR-mediated beta-selection in human T-cell development.

Mice expressing a Kb-restricted transgenic T cell receptor (TCR) and a naturally occurring MHC class I variant molecule, Kbm8, were used to study thymic selection. The transgenic TCR was specific for the major peptide determinant from ovalbumin (OVA(257-264)), while Kbm8 has a mutation that alters the position 2 binding pocket of the Kb molecule, abolishing antigenic peptide presentation and positive selection of transgenic T cells. Peptide presentation was restored by identifying a position 2 analog peptide with Kbm8-binding capacity. In combination with Kbm8, the E2 peptide variant was capable of deleting immature double-positive thymocytes in suspension culture. Similarly, addition of exogenous E2 peptide to fetal thymic organ culture resulted in efficient deletion of double-positive thymocytes. However, there remained a population of CD8 single-positive T cells that exhibited impaired responsiveness to the antigenic peptide and lacked expression of the CD8 beta-chain. These results suggest a mechanism whereby developing thymocytes bearing an alphabetaTCR can modify their expression of the CD8 coreceptor to escape thymic deletion and achieve self-tolerance.

Introduction of MHC class I Kb cDNA via recombinant retrovirus into B10.AKM (Kk) bone marrow cells (BMC) has been shown to confer specific hyporesponsiveness to B10.MBR (Kb) allogeneic skin grafts in lethally irradiated B10.AKM recipients of the transduced syngeneic BMC. We have recently developed a nonmyeloablative conditioning regimen that allows engraftment of fully MHC-mismatched allogeneic bone marrow and the induction of donor-specific tolerance. We ultimately plan to adapt this nonmyeloablative regimen for the use of retroviral transfer of the Kb gene to syngeneic marrow. As a step toward this goal, we have assessed the effects of our current BMC transduction protocol on engraftment of class I mismatched marrow in mice prepared using the nonmyeloablative regimen. BMC from B10.MBR (KbIkDq) mice treated 2 days earlier with 5-fluorouracil (5-FU) were cultured for 4 days with rIL-3 and rIL-6, and then injected into B10.AKM (KkIkDq) recipients conditioned with anti-CD4 and anti-CD8 mAbs, 7 Gy of thymic irradiation and 3 Gy of whole body irradiation. Engraftment was comparable to that of freshly prepared normal B10.MBR marrow. All recipients of 10(6) precultured BMC developed long-term multilineage mixed WBC (white blood cells) chimerism, and six of seven of these animals showed long-term specific tolerance to B10.MBR skin grafts. Four of seven recipients of 2 x 10(5) precultured BMC showed long-term repopulation by the donor of > 1% of multiple WBC lineages and four of five recipients showed specific tolerance to B10.MBR tail skin. These data suggest that our previously described nonmyeloablative conditioning regimen could be applicable to the gene therapy approach for the induction of donor-specific transplantation tolerance.

The aim of this study was to determine the effects of the anti-CD4 monoclonal antibody (mAb) W3/25, found to be nondepleting, on the onset of rat adjuvant arthritis (AA), and, in addition, to ascertain whether depletion of CD8+ cells during the same period could interfere with those effects. Female Wistar rats in which AA had been induced were treated with W3/25 and/or OX8 (anti-rat CD8) mAb during the latency period of arthritis. W3/25 alone or in combination with OX8 prevented the inflammatory process of AA. When the protected groups were rechallenged with a second dose of Mycobacterium butyricum no arthritis was observed. Protected and nonprotected arthritic animals developed the same anti-mycobacteria antibody levels as the arthritic control group. This study indicates that a nondepleting anti-CD4 mAb can prevent AA, while CD8+ lymphocytes do not appear relevant for the development of AA and do not seem to have a regulatory role for CD4+ cells.

The immune response to a murine myeloid leukemia (cell line C1498) was studied in vitro and in vivo. Natural killer (NK) cells and CD8+ cytotoxic T lymphocytes (CTL) were shown to lyse C1498 in vitro through the binding of leukocyte function antigen-1 (LFA-1) on effectors and intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on C1498 target cells. However, the ability of nonimmunized mice to resist an in vivo challenge of a low dose (10(4)) of C1498 was NK-cell, but not T-cell dependent. The failure of T cells to participate in the immune surveillance of a low leukemia burden appeared, in part, because of a lack of expansion of leukemia reactive CTL precursors (CTLp). Leukemia reactive CTLp frequency estimations in naive and leukemia bearing mice were not significantly different (range, 1:20,600 to 1:74,000) in contrast to immunized mice (range, 1:1,400 to 1:4,400). Leukemia reactive CTLp could be expanded to a level that could apparently mediate in vivo immune surveillance of 10(5) leukemia cells by injection of irradiated leukemia cells intraperitoneally (IP) or subcutaneously (SC), but not intravenously (IV). However, IV injection of 10(5) live leukemia cells engineered to secrete interleukin-2 (IL-2) resulted in systemic immunity mediated primarily by CD8+ T cells. We conclude that NK cells can mediate immune surveillance of a low leukemia burden. CD8+ CTL-mediated immune surveillance can eliminate a higher leukemia burden than NK cells, but requires T-cell help, which can be delivered by local IL-2. Both NK and CTL-mediated immune surveillance of C1498 murine myeloid leukemia is dependent on recognition through the LFA-1:ICAM adhesion pathway.

The CD8 molecule regulates T cell activation mediated via the CD3 T cell receptor and the adhesion molecule CD2. CD8 mAbs have been found to inhibit early (Ca2+ rise) as well as late events (cytotoxicity, proliferation, and lymphokine secretion) mediated via the CD2 pathway. A panel of eight anti-human CD8 mAbs was tested for inhibition of CD2-mediated Ca2+ rise in a cytotoxic T cell clone. The inhibition ranged from 5 to 53% independently of mAb isotype and affinity measured by half saturation binding. We then characterized these mAbs for their reactivity toward three mutants of the human CD8 alpha carrying amino acid sequence changes in the surface-exposed loops homologous to the immunoglobulin CDR1, 2, and 3. The mutations included replacement of the human CD8 alpha CDR1- and CDR2-like loops by the homologous mouse sequences and the insertion of a glycine in the middle of the CDR3-like loop. Thus, five mAbs were found to be affected by the mutation in the CDR2-like loop but not by alterations in the other CDR-like loops. Conversely, the other two mAbs (8E1.7 and B9.8) were affected only by mutations in the CDR1- and CDR3-like loops, respectively. Cross-inhibition experiments were essentially in agreement with these results. Interestingly, all the mAbs directed against the CDR2-like loop were potent inhibitors of CD2-mediated Ca2+ rise, with one exception probably due to poor affinity. Thus, in addition to being a site of interaction with major histocompatibility complex Class I as recent data have indicated, this region of the CD8 alpha subunit may play a role in regulating T cell activation.

The soluble CD antigens sCD8, sCD23, and sCD25 are increased in untreated Graves' hyperthyroidism. These levels remain elevated when euthyroidism is established in response to propylthiouracil (PTU) therapy but decrease to control values after PTU treatment is discontinued, when euthyroidism has been established and maintained. Neither sCD8 nor sCD23 are elevated in patients with euthyroid Graves' ophthalmopathy nor in the hyperthyroid phase of subacute thyroiditis. sCD25 is increased to an intermediate degree in these disorders. Soluble CD8 > or = 450 U/ml is sensitive, specific, and predictive of PTU success as sole therapy or need for definitive therapy in untreated and PTU-treated Graves' hyperthyroidism, exceeding the predictive values of thyroid-stimulating hormone receptor antibody, thyroid peroxidase antibody, and T3 radioimmunoassay.

The past several years have seen significant progress in understanding the role of T lymphocyte coreceptors in adhesion and activation. New insights have been gained in several areas: the avidity regulation of beta 1 and beta 2 integrins and their role in signal transduction; the regulation of CD8 avidity; the role of Lck in CD4 coreceptor activity; and the novel role for CD2 adhesion in the T cell antigen response.

Thymocytes co-expressing the CD4 and CD8 co-receptors differentiate into mature T cells that express either CD4 or CD8 and have helper or cytotoxic functions, respectively. Recent studies indicate that commitment to the CD4+ or CD8+ lineages occurs stochastically, but retention of the appropriate co-receptor is required to complete development.

CD4, CD8 and CD45 regulate the coupling of the T-cell receptor complex (CD3-TCR) to tyrosine kinase activation and phosphorylation of key substrates such as phospholipase C gamma 1. CD4 and CD8 contribute to activation signals through their cytoplasmic association with p56lck. Expression of the zeta-chain is required for functional synergy of the T-cell receptor with CD4 in the activation of phospholipase C gamma 1, which probably reflects an interaction between p56lck and zeta-associated kinase ZAP-70. CD45 expression is required for CD3-TCR signaling. CD45 may positively regulate signaling by dephosphorylating the carboxyl-terminal tyrosine of p56lck and p59fyn, and negatively regulate signaling by dephosphorylation of other TCR-associated substrates directly. One ligand for CD45 receptor has been identified as the B cell CD22 molecule. The positive and negative effects of CD45 are sensitive to the composition of CD45 in receptor complexes, and may be regulated by specific associations of CD45 isoforms with other receptors such as CD3-TCR, CD2 and CD4.

Analysis of T-cell development in transgenic and gene-deficient mice suggests that the co-receptor function of CD8 is essential for positive selection. Recent data also demonstrate that the requirement for CD4 and CD8 in negative selection of T cells is not absolute and may be regulated by T-cell receptor affinity for the deleting ligand, an interpretation consistent with the affinity model of thymic selection. In addition to its association with CD4 and CD8, it appears that p56lck is also important during the early stages of thymic development.

CD8 T lymphocytes are an important component of the host immune response to human immunodeficiency virus (HIV). To characterize CD8 cell function, we have studied the in vitro phenomenon of CD8 cell-mediated inhibition of HIV replication from autologous, naturally infected CD4 cells. We describe here a reproducible assay of CD8 T cell-mediated inhibition in HIV-infected individuals. The method involves the use of a commercially available cell separation system and anti-CD3 monoclonal antibody to stimulate CD4 cells to produce HIV. Using this technique, we were able to detect HIV production from the CD4 cells of 25 of 27 HIV-infected individuals who had not progressed to AIDS. Further, in vitro CD8 cell-mediated inhibition of HIV production was noted in all of the 25 subjects whose CD4 cells produced viral p24 antigen. This assay may be useful as an in vitro correlate of protective immunity to HIV, with potential application for assessing disease progression, therapeutic efficacy, and immune mechanisms in HIV disease.

We studied the relationship of CD4+ and CD8+ depletion to initial and late opportunistic infections in 62 patients with AIDS. The mean interval between initial and late infections was 12.2 months. Geometric mean (and 95% confidence intervals) of T-cell counts at the diagnosis of each infection were: (Pneumocystis carinii pneumonia) CD4+ 0.051 (0.044-0.058) x 10(9)/l, CD8+ 0.561 (0.476-0.646) x 10(9)/l; (cytomegalovirus retinitis) CD4+ 0.025 (0.019-0.031) x 10(9)/l, CD8+ 0.333 (0.183-0.483) x 10(9)/l. Mycobacterium avium-intracellulare bacteraemia closely followed cytomegalovirus dissemination. Most patients were free from late opportunistic infections caused by disseminated cytomegalovirus and M. avium-intracellulare until CD8+ declined below 0.500 x 10(9)/l. Zidovudine improved CD4+, but less so CD8+, and similarly enhanced the survival of patients treated in 1985-1990 and 1991.

Stable CD8+ suppressor T cell (Ts) clones were established by a relatively simple method. Keyhole limpet hemocyanin (KLH)-primed spleen cells from C3H mice were depleted of B cells and CD4+ T cells by panning and cytotoxic treatment, and the resulting CD8+ T cells were periodically stimulated with antigen and irradiated syngeneic spleen cells followed by manifestation in interleukin-2 (IL-2) containing medium. T cell clones with a definite suppressor function were established by limiting dilution. They were defined as classical effector type Ts of CD8+ phenotype as they had constant and definite suppressor functions in antigen-induced T cell proliferation and specific antibody response against T cell-dependent antigens without detectable cytotoxic activity against both antigen presenting cells (APC) and helper T cells (Th). They showed no helper activity for B cells and produced no detectable helper type lymphokines such as IL-2 and IL-4. CD8+ Ts clones were able to inhibit the antigen-induced IL-2 production of normal and cloned T cells. Their suppressive activity was antigen-nonspecific and major histocompatibility complex-unrestricted. CD8+ Ts clones were also able to suppress the proliferative response of Th clones induced by immobilized anti-T cell receptor (TcR) and anti-CD3 mAbs but not the response induced by concanavalin A (ConA) and IL-2. All the CD8+ T cell clones established independently utilized the TcR V beta 8 gene. Syngeneic antigen presenting cells could induce proliferation of these CD8+ clones, which was blocked by anti-CD8 and anti-I-Ak monoclonal antibody (mAb) but not by anti-class I mAbs. The stimulation of CD8+ Ts clones with immobilized anti-CD3 resulted in the release of a suppressor factor(s) that potently inhibited the antigen-induced proliferation of CD4+ Th clones and the in vitro secondary antibody formation.

In this paper, we report on our year-long experience with the magnetic cell sorter (MACS), and present a critical evaluation of its pitfalls and benefits. Satisfactory separation of lymphocytes into subsets with preservation of function can be achieved, but there are several drawbacks: in comparison with Dynal beads, MACS results in a higher cell loss due to the increased number of separation steps and requires depletion of plastic-adherent cells as these will engulf microbeads and contaminate the enriched fraction, and is more expensive. The advantage of MACS over Dynal beads is that the microbeads are biodegradable and do not interfere with proliferation assays: both the depleted and enriched fractions of cells can therefore be used in culture immediately following separation. We used MACS for the positive and negative selection of CD45RO cells: the enriched fraction was of high purity (greater than 98%), but a depleted fraction of greater than 90% purity could not be obtained even after running the same sample over three separating columns. Dynabeads, on the other hand, achieved 98% pure CD45RO-depleted fractions after three separation runs.

In vitro immunomodulatory properties of gangliosides have been well characterized such as the ganglioside-induced modulation of CD4 on T lymphocytes and inhibition of lectin-induced proliferative response of lymphocytes. These findings have led to an interesting suggestion that gangliosides play a role as in vivo immune modulators, although this possibility is not clearly defined yet. We then first confirmed in vitro effects of gangliosides on murine immunocytes and examined in vivo effects of gangliosides on immune response in mice. Murine spleen cells that were treated with a ganglioside mixture (GS) purified from bovine brain exhibited a marked decrease in CD4 expression, while CD8 expression was slightly suppressed. Transplantation of GS-untreated control immunocytes that were isolated from syngeneic mice into the immune suppressed mice by X-ray irradiation restored in vivo immune responses, while GS-treated cells could not. Immune response was assayed by the evaluation of footpad swelling which was induced by immunization with sheep erythrocytes as antigens. Moreover, intramuscular administration of gangliosides into mice suppressed both immediate (Arthus)-type and delayed-type allergic reactions. These results suggest that gangliosides would be potential in vivo immune modulators.