The peripheral lymphoid compartment is disrupted in flaky skin mice

Flaky skin (fsn) is an autosomal recessive mutation on mouse chromosome 17 that causes severe anaemia, forestomach papillomatosis and a papulosquamous skin disease that resembles psoriasis in humans. In the present paper, it is reported that fsn causes peripheral lymphadenopathy, CD4/CD8 imbalance and hyperresponsiveness to T cell growth factors. Peripheral lymph nodes (PLN) of adult mutant (fsn/fsn) mice were found to contain almost 10-fold more leucocytes than PLN from phenotypically normal littermates (+/fsn or +/+, hereafter referred to as +/?). Analysis of PLN cells using mAbs and flow cytometry revealed that this predominantly lymphoid hyperplasia was characterized by approximately equivalent increases in the numbers of CD3+ T cells and CD19+ B cells. However, expansion within the T cell compartment was non-random, because fsn/fsn PLN had a considerably reduced ratio of CD4+ to CD8+ T cells (1.08 +/- 0.37) compared to +/? PLN (2.47 +/- 0.44, P < 0.0001). In vitro assays of cellular proliferation in response to T and B cell growth factors showed that fsn/fsn PLN cells were hyperresponsive to IL-2, IL-4 and IL-7 when compared with PLN cells from +/? mice. Studies using mesenteric lymph node and peripheral blood cells showed that hyperresponsive cells are widely distributed in fsn/fsn mice. Experiments in newborn mice showed that the lymphoid disturbances caused by fsn are established at least as early as 2 weeks of age, a time that precedes the onset of the earliest clinical skin lesions. These data implicate a role for the fsn gene product in regulating the size and content of the peripheral lymphoid compartment.     

Expansion of CD22lo B cells in the spleen of autoimmune-prone flaky skin mice

Similar to murine models with compromised CD22/SHP-1 function, flaky skin (fsn) mutant mice exhibit lymphocyte hyperactivation and an autoimmune phenotype characterized by circulating autoantibodies to dsDNA and glomerulonephritis. Immunophenotyping of fsn/fsn splenic B cells was performed to determine if abnormalities in CD22 expression contributed to the phenotype. We identified an expansion of an IgM(bright) CD22lo population consistent with immature B-lymphocytes. While normal B-lymphocytes require IL-4 to achieve down-modulation of CD22 expression in response to BCR cross-linking, culture with anti-IgM alone led to reduced CD22 expression in fsn/fsn mice. Furthermore, when IL-4 was added to fsn/fsn cultures, no further reduction in CD22 expression was observed. This suggested that fsn/fsn B cells were pre-activated in vivo by chronic IL-4 exposure. A portion of these CD22lo cells expressed the B-1 surface marker CD11b. We contend that decreased activation thresholds among CD22lo B-lymphocytes contributes to the expansion of immature and B-1 B cell populations and to the development of autoimmune pathology in fsn/fsn mice.     

Expression of intracellular IFN-gamma in HSV-1-specific CD8+ T cells identifies distinct responding subpopulations during the primary response to infection

Cutaneous infection in the footpads of C57BL/6 mice with HSV-1 results in an accumulation of activated (CD44high CD25+) CD8+ T cells within the draining popliteal lymph node (PLN). These studies were undertaken to evaluate the frequency and phenotype of the CD8+ T cell population within the PLN, recognizing the single immunodominant HSV-1 epitope derived from the viral envelope glycoprotein, glycoprotein B (gB), using an intracellular IFN-gamma-staining assay. It revealed that approximately 6% of the CD8+ T cells were specific for the gB epitope. Phenotypic analysis of the IFN-gamma-producing gB-specific CD8+ T cells generated in the PLN during the course of the acute infection expressed the CD44high CD25+ phenotype on days 3-5 postinfection. Surprisingly, IFN-gamma-producing CD8+ T cells expressed the CD44high CD25- phenotype on days 5-8 postinfection, in contrast to expectations for a CD8+ effector T cell. IFN-gamma-producing CD25- CD8+ T cells were detected in the PLN on day 21 postinfection, long after infectious virus had been cleared. Throughout the response, the spleen was found to be the major reservoir of gB-specific CD8+ T cells, even during the peak of the response. In contrast to the gB-specific CD8+ T cell population within the PLN, the entire gB-specific CD8+ T cell population within the spleen was CD25-. Collectively, these results suggest the generation of subpopulations of virus-specific CD8+ T cells, distinguished by the expression of CD25, during the acute phase of the primary response to a localized viral infection.     

Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells

We recently identified CD8+CD122+ regulatory T cells that directly control CD8+ and CD4+ cells without intervention of APCs. In this study, we investigated the effector mechanism of CD8+CD122+ regulatory T cells by using an in vitro regulation system. The profile of cytokine expression revealed that IL-10 was predominantly produced by CD8+CD122+ cells, whereas other cytokines were similarly expressed in CD8+CD122+ cells and CD8+CD122- cells. Suppression of both proliferation and IFN-gamma production by CD8+CD122- cells by CD8+CD122+ cells was blocked by adding anti-IL-10 Ab to the culture but not by adding anti-TGF-beta Ab. When IL-10 was removed from the conditioned medium from CD8+CD122+ cells, the conditioned medium no longer showed regulatory activity. Finally, CD8+CD122+ cells from IL-10-deficient mice had no regulatory activity in vitro and reduced regulatory activity in vivo. Our results clearly indicate that IL-10 is produced by CD8+CD122+ cells and mediates the regulatory activity of these cells.     

IFN-gamma controls the generation/activation of CD4+ CD25+ regulatory T cells in antitumor immune response

Immunization with serological identification of Ags by recombinant expression cloning (SEREX)-defined self-Ags leads to generation/activation of CD4+ CD25+ regulatory T cells with suppressive activities and enhanced expression of Foxp3. This is associated with increased susceptibility to pulmonary metastasis following challenge with syngeneic tumor cells and enhanced development of 3-methylcholanthrene-induced primary tumors. In contrast, coimmunization with the same SEREX-defined self-Ags mixed with a CTL epitope results in augmented CTL activity and heightened resistance to pulmonary metastasis, both of which depend on CD4+ Th cells. These active regulatory T cells and Th cells were derived from two distinct CD4+ T cell subsets, CD4+ CD25+ T cells and CD4+ CD25- T cells, respectively. In the present study, IFN-gamma was found to abrogate the generation/activation of CD4+ CD25+ regulatory T cells by immunization with SEREX-defined self-Ag. CD4+ CD25+ T cells from these IFN-gamma-treated mice failed to exhibit immunosuppressive activity as measured by 1) increased number of pulmonary metastasis, 2) enhanced development of 3-methylcholanthrene-induced primary tumors, 3) suppression of peptide-specific T cell proliferation, and 4) enhanced expression of Foxp3. The important role of IFN-gamma produced by CD8+ T cells was shown in experiments demonstrating that CD4+ CD25+ T cells cotransferred with CD8+ T cells from IFN-gamma(-/-) mice, but not from wild-type BALB/c mice, became immunosuppressive and enhanced pulmonary metastasis when recipient animals were subsequently immunized with a SEREX-defined self-Ag and a CTL epitope. These findings support the idea that IFN-gamma regulates the generation/activation of CD4+ CD25+ regulatory T cells.     

CD4+ T-Cell Help Is Required for Effective CD8+ T Cell-Mediated Resolution of Acute Viral Hepatitis in Mice

Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA−/− mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the anti-viral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-γ production and degranulation capacity were impaired in CIITA−/− mice. The impaired CD8+ T-cell function in CIITA−/− mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.     

CD7 and CD28 are required for murine CD4+CD25+ regulatory T cell homeostasis and prevention of thyroiditis

CD7 and CD28 are T cell Ig superfamily molecules that share common signaling mechanisms. To determine roles CD7 and CD28 might play in peripheral lymphocyte development and function, we have generated CD7/CD28-double-deficient mice. CD7- and CD28-single-deficient and CD7/CD28-double-deficient mice had normal levels of CD4 and CD8-single-positive T cells in thymus and spleen. However, CD28-deficient mice had decreased CD4+CD25+ T cells in spleen compared with wild-type mice, and CD7/CD28-double-deficient mice had decreased numbers of CD4+CD25+ T cells in both thymus and spleen compared with both wild-type and CD28-deficient mice. Functional studies demonstrated that CD4+CD25+ T cells from CD28-deficient and CD7/CD28-double-deficient mice could mediate suppression of CD3 mAb activation of CD4+CD25- wild-type T cells, but were less potent than wild-type CD4+CD25+ T regulatory cells. Thyroiditis developed in aged CD7/CD28-double-deficient mice (>1 year) that was not seen in age-matched control mice or single CD7- or CD28-deficient mice, thus suggesting in vivo loss of T regulatory cells allowed for the development of spontaneous thyroiditis. Taken together, these data demonstrated collaborative roles for both CD7 and CD28 in determination of number and function of CD4+CD25+ T regulatory cells in the thymus and peripheral immune sites and in the development of spontaneous thyroiditis.     

CD25+CD4+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses.

Specific and selective immunological unresponsiveness to donor alloantigens can be induced in vivo. We have shown previously that CD25+CD4+ T cells from mice exhibiting long-term operational tolerance to donor alloantigens can regulate rejection of allogeneic skin grafts mediated by CD45RB(high)CD4+ T cells. In this study, we wished to determine whether donor-specific regulatory cells can be generated during the induction phase of unresponsiveness, i.e., before transplantation. We provide evidence that pretreatment with anti-CD4 Ab plus a donor-specific transfusion generates donor-specific regulatory CD25+CD4+ T cells that can suppress rejection of skin grafts mediated by naive CD45RB(high)CD4+ T cells. Regulatory cells were contained only in the CD25+ fraction, as equivalent numbers of CD25-CD4+ T cells were unable to regulate rejection. This pretreatment strategy led to increased expression of CD122 by the CD25+CD4+ T cells. Blockade of both the IL-10 and CTLA-4 pathways abrogated immunoregulation mediated by CD25+ T cells, suggesting that IL-10 and CTLA-4 are required for the functional activity of this population of immunoregulatory T cells. In clinical transplantation, the generation of regulatory T cells that could provide dynamic control of rejection responses is a possible route to permanent graft survival without the need for long-term immunosuppression.     

Mycobacterial trehalose-containing glycolipid with immunomodulatory activity on human CD4+ and CD8+ T-cells

Protection against Mycobacterium tuberculosis is based on cell-mediated immunity, most importantly involving CD4+ and CD8+ T-cell subsets. One of the key features of the tubercle bacillus is its cell envelope, characterized by extremely abundant and specific lipids. The cell-surface glycolipid 2,3-di-O-acyl-trehalose (DAT) has been consistently found in M. tuberculosis strains. In this study, analysis of proliferation, activation markers and cytokine release was performed in human peripheral blood mononuclear cells (PBMC) activated in the presence and absence of DAT. We present evidence that mycobacterial DAT is able to reduce antigen-induced proliferation of human CD4+ and CD8+ T-cell subsets. We show that the effect is associated with a decrease of cells expressing the T-cell surface activation markers CD25 and CD69, and down-modulation of IL-2, IL-12, TNF-alpha and IL-10 cytokines. Data indicating that fine acyl chain structural variations in the trehalose-containing lipid may be involved in the degree of immune modulation are also presented.     

Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice

RasGRP1 and Sos are two Ras-guanyl-nucleotide exchange factors that link TCR signal transduction to Ras and MAPK activation. Recent studies demonstrate positive selection of developing thymocytes is crucially dependent on RasGRP1, whereas negative selection of autoreactive thymocytes appears to be RasGRP1 independent. However, the role of RasGRP1 in T regulatory (Treg) cell development and function is unknown. In this study, we characterized the development and function of CD4(+)CD25(+)Foxp3(+) and CD8(+)CD44(high)CD122(+) Treg lineages in RasGRP1(-/-) mice. Despite impaired CD4 Treg cell development in the thymus, the periphery of RasGRP1(-/-) mice contained significantly increased frequencies of CD4(+)Foxp3(+) Treg cells that possessed a more activated cell surface phenotype. Furthermore, on a per cell basis, CD4(+)Foxp3(+) Treg cells from mutant mice are more suppressive than their wild-type counterparts. Our data also suggest that the lymphopenic environment in the mutant mice plays a dominant role of favored peripheral development of CD4 Treg cells. These studies suggest that whereas RasGRP1 is crucial for the intrathymic development of CD4 Treg cells, it is not required for their peripheral expansion and function. By contrast to CD4(+)CD25(+)Foxp3(+) T cells, intrathymic development of CD8(+)CD44(high)CD122(+) Treg cells is unaffected by the RasGRP1(-/-) mutation. Moreover, RasGRP1(-/-) mice contained greater numbers of CD8(+)CD44(high)CD122(+) T cells in the spleen, relative to wild-type mice. Activated CD8 Treg cells from RasGRP1(-/-) mice retained their ability to synthesize IL-10 and suppress the proliferation of wild-type CD8(+)CD122(-) T cells, albeit at a much lower efficiency than wild-type CD8 Treg cells.     

A mutant mouse with severe anemia and skin abnormalities controlled by a new allele of the flaky skin (fsn) locus.

We found a novel recessive mutation in an inbred strain, INT, that was derived from an ICR closed colony. Mice homozygous for this mutation are identified by severe anemia, dysgenesis and neonatal death. This mutation was tentatively named int. Intercrosses of int heterozygotes (+/int) and the flaky skin heterozygotes (+/fsn) resulted in abnormal mice (int/fsn heterozygotes) showing anemia and flaky skin with the expected frequency for autosomal recessive mutation. The int gene was therefore named fsn(Jic) as an allele of the fsn locus on chromosome 17. We carried out phenotype analyses using B6.INT- fsn(Jic) mice to observe phenotypes of blood and skin in the embryonic and neonatal stages. Discrimination of fsn(Jic) embryos from normal embryos was performed by an indirect diagnosis of the fsn(Jic) gene using the D17Mit130 microsatellite marker tightly linked to the fsn locus. The number of fetal nucleated RBC of normal embryos decreased gradually to 17.5 dpc, but that of the abnormal embryos decreased to 14.5 dpc followed by a gradual increase to 17.5 dpc. Skin of fsn(Jic) embryos did not show any abnormalities and expressed cytokeratins normally as skin epithelial cell markers at each embryonic stage (15.5 dpc to 18.5 dpc). Time differences in the appearance of the different phenotypes observed in various tissue and organs of fsn homozygotes suggest they are caused by expression of the fsn gene at different developmental stages.     

T cell activation via Leu-23 (CD69)

The CD69 (Leu-23) activation Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer that is rapidly induced after lymphocyte activation. CD69 is not present on the surface of peripheral blood resting T cells, but is constitutively expressed by CD3bright thymocytes. Activation of protein kinase C (PKC) by stimulation of the TCR/CD3 or by phorbol esters directly induces CD69 expression on T cells. In the attempt to elucidate the function of CD69 we investigated the ability of the CD69 glycoprotein to transmit an activation signal. Cross-linking of CD69 by mAb induced a prolonged elevation of intracellular [Ca2+], mostly due to an influx of extracellular Ca2+. This signal alone was unable to effectively activate PKC. When PKC was simultaneously activated by PMA, stimulation of CD69 induced IL-2 and IFN-gamma gene expression, enhancement of CD25 expression, and ultimately IL-2-dependent T cell proliferation. Both CD4+ and CD8+ peripheral T cells responded to CD69-mediated activation. Stimulation of CD69 induced proliferation of thymocytes as well as peripheral T cells, but both required independent PKC activation by PMA. Cyclosporin A, which does not prevent PKC-induced CD69 expression, completely suppressed CD69-induced IL-2 and IFN-gamma gene expression. Although the signal delivered by the CD69 initiates T cell proliferation, it is unable to trigger cytotoxicity programs in CD69+-activated T cells or T cell clones.     

Lymphotoxin controls alphaEbeta7-integrin expression by peripheral CD8+ T cells

Lymphotoxin (LT)-alpha, a member of the TNF family, is recognized as an important mediator in different aspects of lymphoid organ development. Targeted disruption of this molecule resulted in a substantial reduction in the proportion of alphaEbeta7-integrin(high) CD8+ T cells detectable in peripheral lymphoid organs. This defect, however, was not observed on mature CD4-CD8+ thymocytes. To determine whether this was due to downregulation of beta7-integrin expression by peripheral CD8+ T cells or a failure of thymic emigration of CD8+ beta7-integrin(high) T cells, beta7-integrin was examined on recent thymic emigrants (RTE). When analysed within 16 h after leaving the thymus CD4-CD8+ RTE in both LT-alpha-/- and wild type (wt) mice remained beta7-integrin(high) and were indistinguishable. However, within 3-5 days, emigration loss of beta7-integrin became evident in LT-alpha-/- mice. Despite this loss, the proportion of thymically derived alphabetaTCR+ T-cell populations in the intestinal epithelium, an important target tissue of CD8+ alphaEbeta7-integrin(high) T cells, was increased in the absence of LT-alpha. In contrast, B cells were detectable only rarely in the intestinal tissue of LT-alpha-/- mice. The expression of E-Cadherin remained unchanged. These results indicate that a LT-alpha-dependent process maintains a high level of alphaEbeta7-integrin expression by peripheral CD8+ T cells, and with this control mechanism LT-alpha may help to regulate CD8+ T-cell numbers in the tissues.     

Differential role of lipid rafts in the functions of CD4+ and CD8+ human T lymphocytes with aging

Lipid rafts are critical to the assembly of the T-cell receptor (TCR) signaling machinery. It is not known whether lipid raft properties differ in CD4+ and CD8+ T cells and whether there are age-related differences that may account in part for immune senescence. Data presented here showed that time-dependent interleukin-2 (IL-2) production was different between CD4+ and CD8+ T cells. The defect in IL-2 production by CD4+ T cells was not due to lower levels of expression of the TCR or CD28. There was a direct correlation between the activation of p56(Lck) and LAT and their association/recruitment with the lipid raft fractions of CD4+ and CD8+ T cells. p56Lck, LAT and Akt/PKB were weakly phosphorylated in lipid rafts of stimulated CD4+ T cells of elderly as compared to young donors. Lipid rafts undergo changes in their lipid composition (ganglioside M1, cholesterol) in CD4+ and CD8+ T cells of elderly individuals. This study emphasizes the differential role of lipid rafts in CD4+ and CD8+ T-cell activation in aging and suggests that the differential localization of CD28 may explain disparities in response to stimulation in human aging.     

TGF-beta requires CTLA-4 early after T cell activation to induce FoxP3 and generate adaptive CD4+CD25+ regulatory cells

Although positive CD28 costimulation is needed for the generation of natural CD4+CD25+ regulatory T cells, we report that negative CTLA-4 costimulation is necessary for generating phenotypically and functionally similar adaptive CD4+CD25+ suppressor cells. TGF-beta could not induce CD4+CD25- cells from CTLA-4(-/-) mice to express normal levels of FoxP3 or to develop suppressor activity. Moreover, blockade of CTLA-4 following activation of wild-type CD4+ cells abolished the ability of TGF-beta to induce FoxP3-expressing mouse suppressor cells. TGF-beta accelerated expression of CTLA-4, and time course studies suggested that CTLA-4 ligation of CD80 shortly after T cell activation enables TGF-beta to induce CD4+CD25- cells to express FoxP3 and develop suppressor activity. TGF-beta also enhanced CD4+ cell expression of CD80. Thus, CTLA-4 has an essential role in the generation of acquired CD4+CD25+ suppressor cells in addition to its other inhibitory effects. Although natural CD4+CD25+ cells develop normally in CTLA-4(-/-) mice, the lack of TGF-beta-induced, peripheral CD4+CD25+ suppressor cells in these mice may contribute to their rapid demise.     

Soluble factor-mediated differentiation of CD4+CD8+ thymocytes to single positives in vitro

Although the thymic microenvironment provides the necessary elements for T-cell differentiation, the precise role of individual components remains to be determined. In this paper, attempts were made to address the possibility that CD4 or CD8 single-positive (SP) thymocytes could be developed from immature CD4+CD8+ (double-positive; DP) thymocytes in a suspension culture in the presence of soluble factors. We observed that IL-4 and IFN-gamma weakly induced DP cells to differentiate to CD4 cells, but not to CD8. In contrast, IL-2 weakly induced differentiation to CD8. Interestingly, Con A sup strongly induced differentiation to CD8 SP from the purified DP thymocytes prepared from C57BL/6 or LCMV TCRtg mice. In particular, it was found that thymocyte culture with Con A sup generated CD69+DP cells, and the CD69+DP differentiated to CD8 SP under the suspension culture with soluble factors. Thus, Con A sup or combinations of IL-2, IL-4 and IL-7 strongly induced differentiation of CD69+DP to CD8 SP, whereas individual cytokines did not. These results suggest that soluble factors like cytokines play an important role in the generation of SP thymocytes in the absence of thymic stromal cells, at least from a distinctive subpopulation like CD69+DP thymocytes, and perhaps from those of broader range when in conjunction with TCR/MHC interaction.     

Prognostic value of the immunological phenomena and relationship with clinicopathological characteristics of the tumor--the expression of the early CD69+, CD71+ and the late CD25+, CD26+, HLA/DR+ activation markers on T CD4+ and CD8+ lymphocytes in squamous cell laryngeal carcinoma. Part II

One of the most important challenges in contemporary oncology is to find objective biomarkers of tumor aggressiveness, which help to identify more invasive phenotypes of the carcinoma. The purpose of this study was to investigate the relationships between the early and the late activation markers expression on T CD4(+) and CD8(+) cells subpopulations and certain clinicopathological characteristics of the neoplastic infiltration in order to determine their role as biomarkers for tumor behavior in squamous cell laryngeal carcinoma. Analysis of the early (CD69(+), CD71(+)) and the late activation antigens (CD25(+) (high), CD26(+), HLA/DR(+)) expression on T CD4+ and CD8(+) lymphocytes by cytofluorymetry in 55 patients treated for squamous cell laryngeal carcinoma was performed. Clinicomorphological analysis on the basis of TNM criteria and tumor front grading, which included tumor-related features and adjacent stroma-related characteristics of the peripheral edge of infiltration was carried out. The relationships between the activation markers expression and parameters of tumor aggressiveness were investigated. Our work revealed statistically significant differences in the expression of the studied activation markers on T cells with regard to certain clinicomorphological features. The expressions of CD69(+) and CD71(+) antigens on T CD3(+)CD4(+) and CD3(+)CD8(+) cells as well as CD4(+)HLA/DR(+) markers were higher for pT3 and pT4 tumors, in comparison with pT2 carcinomas. Moreover, tumors with the smallest number of TFG points were characterized by significantly lower values of the average expression of CD3(+)CD69(+) and CD3(+)CD71(+) as well as CD4(+)HLA/DR(+) markers on T lymphocytes. In addition, more aggressive and deeply infiltrating laryngeal carcinomas were most often characterized by significantly higher values of the average expression of CD69(+) and CD71(+) antigens on CD8(+) as well as HLA/DR(+) markers on CD4(+). Our study confirmed the implication of the early and the late activation antigens expression on CD4(+) and CD8(+) T lymphocytes in clinicomorphological parameters of the tumor, especially TFG total score and depth of invasion, and their importance as indicators of the invasive phenotype of laryngeal carcinoma.     

Interleukin-dependent modulation of HLA-DR expression on CD4and CD8 activated T cells

Summary Interleukins (IL) regulate different T-cell surface Ag known as activation markers that have distinct functional roles. In this paper, while studying the influence of some cytokines(IL-12, IL-2 and IL-4) on the expression of several markers [CD69,CD25, CD26, CD3, human leukocyte antigen (HLA-DR), CD45R0] in in vitro activated human T lymphocytes, we observed two groups of donors responding to phytohaemagglutinin (PHA) activation with high or low HLA-DRAg expression. We also found that CD4 and CD8 populations had different HLA-DR densities under PHA activation (particularly the high HLA-DR-expressing group). Interleukins, in a dose-dependent manner (IL-2 partially),upregulated these HLA-DR levels. In 5 day cultures, IL-12 and IL-2 enhanced the CD8/CD4 ratio of activated T cells,which was responsible, in part, for the IL-dependent HLA-DR upregulation.IL-12 and IL-2 also upregulated the HLA-DR expression at the molecular level on CD8, and IL-12 downregulated it on CD4 cells. It seems that IL-4 upregulated HLA-DR by shortening the mitogen-dependent regulation kinetics. We hypothesize that the different effect of each IL on HLA-DR expression might be related to the regulation of the dose of antigenic peptide presentation and, thus, also influence TH1/TH2 dominance.     

Human immunodeficiency virus-specific circulating CD8 T lymphocytes have down-modulated CD3zeta and CD28, key signaling molecules for T-cell activation

Although human immunodeficiency virus (HIV)-infected subjects without AIDS have a high frequency of HIV-specific CD8 T lymphocytes, cellular immunity is unable to control infection. Freshly isolated lymphocytes often do not lyse HIV-infected targets in 4-h cytotoxicity assays. A large fraction of circulating CD8 T cells from HIV-infected donors down-modulate CD3zeta, the signaling component of the T-cell receptor complex, which is reexpressed in vitro coincident with the return of cytotoxic function. To investigate further the link between CD3zeta down-modulation and possible CD8 T-cell functional defects, we used flow cytometry to characterize further the properties of the CD3zeta-down-modulated subset. HIV-specific CD8 T cells, identified by tetramer staining, are CD3zeta(-). CD8 T cells with down-modulated CD3zeta also do not express the key costimulatory receptor CD28 and have the cell surface phenotype of activated or memory T cells (HLA-DR(+) CD62L(-)). After T-cell activation, CD3zeta-down-modulated cells express the activation marker CD69 but not the high-affinity interleukin 2 (IL-2) receptor alpha-chain CD25 and produce gamma interferon but not IL-2. Therefore HIV-specific CD8 T cells have down-modulated key signaling molecules for T-cell activation and costimulation and require exogenous cytokine stimulation. The typical impairment of HIV-specific CD4 T helper cells, which would normally provide specific CD8 T-cell stimulation, means that in vivo CTL function in vivo is compromised in most HIV-infected individuals. In AIDS patients, the functional defect is more severe, since CD3zeta is not reexpressed even after IL-2 exposure.