Expression of the CD28 costimulatory molecule on subsets of murine intestinal intraepithelial lymphocytes correlates with lineage and responsiveness

The CD28 antigen has been recently demonstrated to be a costimulatory molecule and is expressed by almost all thymic and peripheral T cell receptor (TcR) α^δ+ and γ^λ+ cells in the mouse system. We show here that expression of CD28 is heterogeneous among murine intestinal intraepithelial lymphocytes (IEL). Whereas some TcR αβ-expressing IEL subsets such as CD4⁺8^? and CD4^?8α⁺β⁺ cells express CD28 at the same levels as their phenotypic counterparts in lymph node, other subsets of TcR αβ cells (including CD4^?8α⁺β^? and CD4⁺8αβ⁺β cells) as well as TcR γ^λ+ IEL fail to express CD28. Parallel experiments using aged BALB/c-nu/nu mice indicated that CD28 expression patterns among IEL are quite similar to those of normal BALB/c mice. Furthermore, forward light scatter analysis showed that CD28^? cells are considerably larger than CD28⁺ cells in the gut, although cycling cells were rare in both subsets. Finally CD28^? cells in the gut did not proliferate or produce IL-2 upon stimulation by anti-CD3 monoclonal antibodies (mAb) and phorbol 12-myristate 13-acetate, whereas CD28⁺ cells in the gut and lymph nodes responded to these stimuli. The response of the CD28⁺ cells was enhanced by anti-CD28 mAb. These results suggest that CD28^? IEL (CD4-8α⁺β^? cells, and some CD4⁺8α⁺β^?cells) may follow a different developmental pathway from that of CD28⁺ IEL in a thymus-independent environment, and that expression of CD28 correlates with responsiveness of the cells to triggering via the TcR-CD3 complex.     

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.     

Demonstration of identical expanded clones within both CD8+ CD28+ and CD8+ CD28− T cell subsets in HIV type 1-infected individuals

In HIV-1-infected individuals, the CD8⁺ CD28⁻ T cell subset is considerably expanded and is frequently the largest subset of T cells found in peripheral blood. It has been assumed, but not proven, that CD8⁺ CD28⁻ T cells derive from CD8⁺ CD28⁺ T cells in vivo. To further study the ontogeny of CD8⁺ CD28⁻ T cells, we have performed analyses of the complementarity determining region 3 (CDR3) of the TCRB of CD8⁺ CD28⁺ and CD8⁺ CD28⁻ T cells from the peripheral blood of HIV-1-infected individuals. When cells from the same individual were compared, expanded peaks in CDR3 length analysis within a given BV family were frequently observed at the same location in both CD8⁺ subsets ( p < 0.001). Sequencing of cDNA corresponding to dominant peaks revealed the presence of identical expanded CD8⁺ T cell clones within both the CD28⁺ and CD28⁻ subsets on eight of nine attempts. Our results show that CD8⁺ CD28⁺ and CD8⁺ CD28⁻ T cells are phenotypic variants of the same lineage, most likely evolving from CD8⁺ CD28⁺ to end-stage CD8⁺ CD28⁻ T cells.     

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.     

Characteristics of Expanded CD4+CD28null T Cells in Patients with Chronic Hepatitis B

Expanded CD4⁺CD28^null T cells have not been described in the circulation of patients with chronic hepatitis B (CHB). The aim of the present was to detect and characterize the surface phenotype and functional capacity of these cells in CHB patients. Expanded CD4⁺CD28^null T cells were detected in the circulation of CHB patients with high viral load and elevated aminotransferase levels. Most CD4⁺CD28^null T cells showed a CD27?CD45RA^?CD45RO⁺ surface phenotype. The markers CD56, CD57 and killer immunoglobulin-like receptor (KIR) were detected on CD4⁺CD28^null T cells, but the majority were positive for CD57. Functionally, CD4⁺CD28^null T cells were found to be potent cytotoxic T lymphocytes with perforin and granzyme B secretion profiles. These findings indicate that the expanded CD4⁺CD28^null T cells are cytotoxic memory T cells and display a distinct functional phenotype in comparison with CD4⁺CD28⁺ T cells. The presence of these cells appears to be associated with inflammatory conditions, suggesting that these elevated CD4⁺CD28^null T cells might be involved in the pathogenesis of CHB.     

Primary defect in CD8+ lymphocytes in the antibody deficiency disease (common variable immunodeficiency): abnormalities in intracellular production of interferon-gamma (IFN-γ) in CD28+ (‘cytotoxic’) and CD28− (‘suppressor’) CD8+ subsets

We have measured by flow cytometry the ability of subsets of CD8⁺ CD3⁺ lymphocytes within mononuclear cell preparations to make intracellular cytokines (IL-2, tumour necrosis factor-alpha (TNF-α) and IFN-γ) on stimulation in vitro with phorbol myristate acetate (PMA) and ionomycin for 16 h. These CD8⁺ subsets were defined by the presence or absence of CD28 or HLA-DR. Subsets of normal CD8⁺ cells were compared with cells from the antibody deficiency disease common variable immunodeficiency (CVID). In CVID there was a significant increase in the production of IFN-γ in the CD8⁺ CD28⁺ subset (‘cytotoxic’). This reflects a shift in this disease towards an excessive Th1 response away from B cell help. Paradoxically, some CVID patients also showed a reduction in IFN-γ production in the CD8⁺ CD28^? subset (‘suppressor’) which was associated with a failure of these cells to maintain a state of activation after a stimulus in vitro. The B cell problem in this disease is known to be related to a failure of T cell help shown by an inability to produce the antigen-specific CD4⁺ memory T cells needed for successful B cell maturation. The two pathological CD28 subsets of CD8⁺ cells we have found in CVID may both be detrimental to a normal CD4-dependent immune response. The CD28^? suppressor subset expands and is unable to maintain activation and cytokine secretion, and the CD28⁺ cytotoxic subset is over-producing the Th1 cytokine IFN-γ.     

Expression in vivo of CD45RA,CD45RB and CD44 on T cell receptor-transgenic CD8+ T cells following immunization

We used mice transgenic for a major histocompatibility complex class I-restricted T cell receptor to study the changes of phenotype in vivo which follow priming by antigen of CD8 T cells. We show that following priming with peptide, CD44 on CD8 T cells is up-regulated. The change of phenotype was relatively stable, as primed CD8 cells isolated from thymectomized mice 6 weeks after priming still expressed increased levels of CD44. CD8 T cells in these mice are still responsive to peptide and could represent long-lived primed cells. No downregulation in vivo of the CD45RA or CD45RB isoforms was found, indicating that there is a differential regulation of the expression of CD44 and CD45RB by activated CD8 transgenic T cells. These results contradict earlier studies in vitro which showed that CD8 T cells which have been primed earlier belong to the CD45RA^? or CD45RB^? subset.     

Murine lupus in MRL/lpr mice lacking CD4 or CD8 T cells

MRL/lpr mice develop a systemic autoimmune disease similar to systemic lupus erythematosus in humans. The mice show progressive lymphadenopathy due to the accumulation of an unusual population of CD4^?8^?(DN) B220⁺ αβ⁺ T cells. We bred MRL/lpr mice with mice lacking CD4⁺ or CD8⁺ T cells by gene targeting via homologous recombination in embryonal stem cells to determine the roles of these cells in the autoimmune disease. No difference in survival or autoantibody levels was noted between CD8-/- lpr and littermate controls. Interestingly, these CD8-/- lpr mice have a reduced level of B220⁺ DN T cells despite the fact that the degree of lymphadenopathy was unaltered. CD4-/- lpr mice had a diminished autoimmune disease with a reduction in autoantibody production and skin vasculitits, and increased survival compared to littermate controls. However, CD4-/- lpr mice had an enhanced splenomegaly that developed massively by 16–20 weeks of age (5 to 8 greater than lpr control mice) due to the accumulation of DN B220⁺ T cells. In addition, there were no differences in peripheral lymph node enlargement, although the proportion of DN B220⁺ T cells was about twofold higher in the CD4-/- lpr mice. These cells were phenotypically identical to the DN population in control lpr mice, indicating that the accumulating DN T cells can be dissociated from the autoimmune disease in these mice. Collectively, our results reveal that the autoimmune disease is dependent on CD4⁺, but not CD8⁺ T cells, and that many of the B220⁺ DN T cells traverse a CD8 developmental pathway.     

Lamina Propria T Cell Subsets in the Small and Large Intestine of Euthymic and Athymic Mice

We investigated lamina propria T cells from the small intestine (jejunum/ileum) and the large intestine (colon) of euthymic (BALB/c, C. B-17, C57BL/6) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/ xid) mice. CD3⁺ T cells represented about 40% of the lamina propria lymphocytes (LPL) from the small or the large intestine of euthymic mice, and 20–30% of the LPL populations from the small or large intestine of athymic mice. In the lamina propria T cell population of the small intestine, 85% were of the αβ lineage in euthymic mice, but only 40% were of the αβ lineage in athymic mice. T cells of the αβ lineage were thus more frequent than T cells of the αβ lineage in the intestinal lamina propria T cells of extrathymic origin. CD4⁺ T cells represented 40% of the lamina propria T cells in the small as well as in the large intestine of euthymic mice, and 20–30% of the T cells in the lamina propria of the nude mouse gut. In euthymic mice, 40% of the T cells in the small intestine lamina propria, and 30% of the T cells in the colonic lamina propria were CD8⁺, In intestinal lamina propria T cell populations of athymic mice, the CD8⁺ T cell population was expanded. Most (60–70%) CD8+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice expressed the homodimeric CD8α⁺β^? form of the CD8 coreceptor. A fraction of 15–20% of all CD3+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice were ‘double negative’ CD4^? CD8^?. A large fraction of the TCRαβ⁺ T cells in the colonic lamina propria (but not in the small intestine lamina propria) of euthymic mice expressed the CD2 and the CD28 costimulator molecules, the adhesion molecule LECAM-1 (CD62 L), and could be activated in vitro by CD3 ligation. These data reveal a considerable heterogeneity in the surface phenotype and the functional phenotype of murine lamina propria T cells.     

Altered ex vivo balance between CD28+ and CD28− cells within HIV-specific CD8+ T cells of HIV-seropositive patients

The CD8⁺ CD28⁻ cell population in the blood of HIV-infected individuals is considerably expanded. Yet the cause of this expansion is not clear. The recent demonstration of identical TCR-rearranged genes in CD8⁺ CD28⁺ and CD8⁺ CD28⁻ expanded T cells of HIV-seropositive patients supports the hypothesis that these two subpopulations are phenotypic variants of the same lineage. To further elucidate the precise relationship between them, we measured the fraction of CD28⁺ and CD28⁻ T cell subsets in IFN-γ-producing CD8⁺ T cells by intracellular staining and cytofluorometry as a functional test for ex vivo recognition of epitopes derived from HIV-1, Epstein-Barr virus (EBV) and influenza virus. HIV-specific CD8⁺ T cells were predominantly CD28⁻ in all the eight HIV-seropositive subjects tested. In contrast, the anti-EBV and anti-influenza CD8⁺ T cells were mainly CD28⁺ in these patients as well as in HIV-seronegative individuals. This supports the notion that the CD8⁺ CD28⁻ hyperlymphocytosis observed in HIV infection is due mainly to chronic activation and differentiation of HIV-specific memory CD8⁺ CD28⁺ T cells into terminally differentiated CD8⁺ CD28⁻ lymphocytes, because of intense HIV-1 replication and without any important bystander activation. This clarification of the mechanisms underlying the CD8⁺ CD28⁻ expansion in HIV-induced pathogenesis may have important therapeutic implications.     

Progressive increase of CD7- T cells in human blood lymphocytes with ageing

CD7 is one of the major surface antigens expressed very early during T cell ontogeny. Lack of CD7 expression on mature T cells is regarded as a classical feature of malignant T cells in certain forms of cutaneous T cell lymphoma. Previously, we identified a CD7^- subset of peripheral blood T lymphocytes in normal human individuals. In this study we determined the portion of CD7^- T cells in the peripheral blood of healthy volunteers ranging in age from 8 months to 90 years (n= 85) and in cord blood of full-term infants (n= 14), Furthermore, this CD7^- subset was characterized in detail by the use of MoAbs and three-colour flow cytometry. In cord blood no CD7^- T cells could be detected. After birth, percentage and absolute number of CD7^- T cells increased with age. Independently of age, most CD7^- CD3⁺ cells belonged to the CD4⁺ subpopulation. Focusing on the latter we could demonstrate the predominance of the CD45RO⁺CD45RA^- phenotype in the CD7^- subset. Furthermore, CD7^- T cells contained a higher number of cells expressing activation markers and the CD57 antigen, but a reduced number of CD62L⁺ cells in comparison with CD7⁺ T cells.     

Importance of B cell co-stimulation in CD4(+) T cell differentiation: X-linked agammaglobulinaemia, a human model

We were interested in the question of whether the congenital lack of B cells actually had any influence on the development of the T cell compartment in patients with agammaglobulinaemia. Sixteen patients with X‐linked agammaglobulinaemia (XLA) due to mutations in Btk, nine patients affected by common variable immune deficiency (CVID) with <2% of peripheral B cells and 20 healthy volunteers were enrolled. The T cell phenotype was determined with FACSCalibur and CellQuest Pro software. Mann–Whitney two‐tailed analysis was used for statistical analysis. The CD4 T cell memory compartment was reduced in patients with XLA of all ages. This T cell subset encompasses both CD4⁺CD45RO⁺ and CD4⁺CD45RO⁺CXCR5⁺ cells and both subsets were decreased significantly when compared to healthy controls: P = 0·001 and P < 0·0001, respectively. This observation was confirmed in patients with CVID who had <2% B cells, suggesting that not the lack of Bruton's tyrosine kinase but the lack of B cells is most probably the cause of the impaired CD4 T cell maturation. We postulate that this defect is a correlate of the observed paucity of germinal centres in XLA. Our results support the importance of the interplay between B and T cells in the germinal centre for the activation of CD4 T cells in humans.     

Human Immunodeficiency Virus Type 1 Replication,Immune Activation, and Circulating CytotoxicT Cells Against Uninfected CD4+ T Cells

Cytotoxic T lymphocytes (CTL) that kill uninfected activated CD4⁺ T cells can be induced in vitro by stimulating CD8⁺ T cells with activated autologous CD4⁺ T cells. Similar CTL have been detected in circulating T cells from human immunodeficiency virus type 1 (HIV)-infected individuals. To define the in vivo correlates of this CTL activity, we studied plasma -2 microglobulin and HIV RNA levels, T-lymphocyte subset counts, and expression of CD28 on CD8⁺ T cells concurrently with circulating CTL activity against uninfected CD4⁺ T cells in 75 HIV-infected individuals at different stages of disease progression. Mean values of each parameter were compared in subsets of this group of 75 segregated on the basis of this CTL activity. The group with CTL against uninfected activated CD4⁺ T lymphocytes had more CD8⁺ T cells, a higher percentage of CD28^– CD8⁺ T cells, and higher plasma levels of HIV RNA and -2 microglobulin. CTL against uninfected activated CD4⁺ T cells were predominantly CD28^– and in HIV-infected individuals were associated with immunological or virological evidence of progressive disease. In HIV infection, circulating CTL activity against uninfected activated CD4⁺ T lymphocytes is associated with immune activation, CD8⁺ T cell expansion, accumulation of CD28^– CD8⁺ T cells, and inadequate suppression of HIV replication.     

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.     

Human bone marrow contains a subset of quiescent early memory CD8+ T cells characterized by high CD127 expression and efflux capacity

Even today it is still not completely understood how CD8⁺ T‐cell memory is maintained long term. Since bone marrow (BM) is a niche for immunological memory, we sought to identify long‐lasting early memory CD8⁺ T cells in this compartment. To achieve this, we looked for CD8⁺ T cells that are able to efflux Rhodamine 123, a typical property of stem cells. Indeed, we identified a distinct subset of CD8⁺ T cells in BM, with the capacity to efflux and high CD127 expression. These CD127^hi effluxers are conventional CD8⁺ T cells exhibiting a broad TCR‐Vβ repertoire and are generated in response to viral peptides in vitro. CD127^hi effluxer CD8⁺ T cells have an early memory phenotype defined by preferential TNF‐α production and a Bcl‐2^hi, KLRG‐1^low profile. This population has long telomeres and shows constitutively low frequencies of Ki‐67 expression ex vivo, but has a high proliferative and differentiation capacity in vitro. However, IL‐15 downmodulates CD127 in CD127^hi effluxer CD8⁺ T cells in vitro. Consequently, the CD127^low effluxer subset may comprise cells recently exposed to IL‐15. Taken together, CD127^hi effluxer CD8⁺ T cells represent a novel population of early memory T cells resident in BM with properties required for long‐lived memory.     

Increased expression of CD80, CD86 and CD70 on T cells from HIV-infected individuals upon activation in vitro: regulation by CD4+ T cells

T cells express CD28 and CD27 which transduce co-stimulatory signals after interaction with their ligands on antigen-presenting cells (APC). These ligands, CD80, CD86 and CD70, are also expressed to some extent on activated T cells. Here, we show that in human immunodeficiency virus (HIV)-infected individuals, CD28 and CD27 expression is decreased on CD8⁺ T cells. On the other hand, T cell stimulation in vitro induced high CD80, CD86 and CD70 expression on T cells from HIV-infected individuals. It appeared that an inverted CD4:CD8 T cell ratio could explain this enhanced expression of co-stimulatory ligands. Indeed, high expression levels of CD80, CD86 and CD70 were found on activated CD8⁺ T cells from HIV⁻ individuals cultured in the absence of CD4⁺ T cells. Addition of CD4⁺ T cells prevented this up-regulation. However, in HIV-infected individuals, addition of excess autologous or healthy control CD4⁺ T cells did not completely counteract up-regulation of co-stimulatory ligand expression on CD8⁺ T cells. Thus, to some extent, CD8⁺ T cells in HIV-infected individuals appeared to be refractory to CD4⁺ T cell-mediated regulation of ligand expression in vitro. Activated T cells from HIV-infected individuals and activated CD8⁺ T cells from healthy controls were able to act as accessory cells in CD3-induced T cell proliferation, which was dependent on cell-cell contact. Thus, we showed that T cells from HIV-infected individuals express enhanced levels of co-stimulatory ligands upon activation, which provides them with accessory cell properties. Enhanced stimulatory potential of these nonprofessional APC may contribute to persistently high levels of immune activation in HIV infection related to disease progression.