Thymic differentiation of TCRαβ+ CD8αα+ IELs

Summary:  Intraepithelial lymphocytes (IELs) contain several subsets, but the origin of the T-cell receptor (TCR)αβ⁺ CD8αα⁺ IELs has been particularly controversial. Here we provide a synthesis, based on recent work, that attempts to unify the divergent views. The intestine has a primordial function in lymphopoiesis, and precursors with the potential to differentiate into T cells are found both in the epithelium and underlying lamina propria. Moreover, the thymus has been reported to export cells to the intestine that are not fully differentiated. TCRαβ⁺ CD8αα⁺ IELs can differentiate in the intestine from each of these sources, but in normal euthymic mice, the thymus appears to be the major source for TCRαβ⁺ CD8αα⁺ IELs. This unique IEL subset is a self-reactive population that requires exposure to self-agonists for selection in the thymus, similar to other regulatory T-cell populations. IELs transition through a double-positive (DP) intermediate in the thymus, but they originate from a subset of the DP cells that can be identified by its expression of CD8αα homodimers. The agonist-selected cells in the thymus are TCRβ⁺ but CD4 and CD8 double negative. The evidence suggests that reacquired expression of CD8αα and downregulation of CD5 occur after thymus export, perhaps in the intestine under the influence of interleukin-15. As a result of agonist exposure, a new gene expression program is activated. Therefore, the increased understanding of the developmental origin of TCRαβ⁺ CD8αα⁺ IELs may help us to understand how they participate in immune regulation and protection in the intestine.     

Clonally Expanded CD8+ T cells in Allogeneic Bone Marrow Transplantation

Allogeneic bone marrow transplantation (BMT) is a potentially curative therapy for patients with haematologic malignancies. Several lines of evidence demonstrate that donor T cells are involved in the antitumour effects observed after BMT. Thus, patients receiving T-cell-depleted BMT have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated BMT, and patients experiencing graft-versus-host disease (GVHD) have a lower risk of disease relapse than patients who do not experience GVHD. Although the importance of donor T cells for the curative action of BMT has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. In a recently initiated project, we have conducted a longitudinal study of T-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. Peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (MNCs) were isolated and cryopreserved. CD8⁺ T cells were isolated from the MNCs by use of immunomagnetic beads or FACS and analysed for the presence of clonally expanded cells by T-cell receptor clonotype mapping based on RT-PCR and denaturing gradient gel electrophoresis (DGGE). Using this gel-based methodology, clonally expanded T cells were monitored after transplant and compared to the clinical data of the patients. The preliminary results demonstrates the presence of clonally expanded CD8⁺ T cells at all time points analysed. Furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. The appearance of newly emerged clonotypes which coincided with clinical GVHD could indicate a role for these T cells in the pathogenesis of GVHD.     

CD8+ T Cells Induce Medullary Thymic Epithelium and CD4+CD8+CD25+ TCRβ− Thymocytes in SCID Mice

During T-cell development the transition in the thymus of CD4-CD8- double negative (DN) progenitor T cells into CD4+CD8+ double positive (DP) cells is dependent on the expression of a T-cell receptor (TCR)-beta-chain protein. In this study purified peripheral CD4+ and CD8+ T lymphocytes from the C.B-17 strain of mice were adoptively transferred into syngeneic, neonatal SCID mice, where donor cells resided at constant numbers in thymus from 2 weeks until 10 weeks post cell transfer. In the recipient thymus the CD8+ donor cells outnumbered the CD4+ cells by a factor of three to five and both subsets contained a large fraction of activated cells. During the late phase of treatment, CD8+ T cells induced high numbers of DP thymocytes in the SCID mice, a process accompanied by the maturation of medullary epithelial cells. Such thymic development in the SCID mouse was inhibited by coresiding CD4+ donor T cells. These results indicate a regulatory role by mature peripheral T cells on medullary epithelial growth and thymocyte development in the treated SCID mice.     

Characterization of T-Cell Receptor Vβ Repertoire in Ovarian Tumour-Reacting CD3+ CD8+ CD4− CTL Lines

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T-cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3⁺ CD8⁺ CD4⁻ cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3⁺ CD8⁺ CD4⁻ lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8⁺ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour-reactive CD3⁺ CD8⁺ CD4⁻ CTL from different individuals of known HLA types.     

Increased Frequencies of the CD29 and CD57 Markers and Decreased Frequency of CD45RA Within CD4+ and CD8+ Subsets after Allogeneic Bone Marrow Transplantation in Man

Two monoclonal antibodies, anti-CD45RA and anti-CD29, reciprocally divide the CD4⁺ and CD8⁺ lymphocytes into CD4⁺ CD45RA⁺, CD4⁺ CD29⁺, CD8⁺ CD45RA⁺ and CD8⁺ CD29⁺ subsets. The CD4⁺ CD45RA⁺, CD4⁺ CD29⁺ and CD8⁺ CD45RA⁺ possess suppressor-inducer, helper-inducer and suppressor-effector functions respectively. Since the role of these subsets has not been established after allogeneic bone marrow transplantation we studied lymphocyte subpopulations in 12 patients 45- 227 days after the procedure. The fraction of CD4⁺ lymphocytes was significantly (P= 0.0005) decreased to 20±9% versus 43±3% in controls. Within the CD4⁺ compartment, we found an increase in the fraction of CD4⁺ cells that co-expressed CD29 (CD29⁺/CD4⁺) to 92±10% versus 48±15% (P=0.008) in controls and a concommittant decrease m CD45RA⁺/CD4⁺ to 16±12% versus 56±25% (P=0.008) Patients were also noted to have an increase in the percentage of CD8⁺ lymphocytes to 41±5% compared to 23±4% in controls (P=0.0004), Examination of the CD8⁺ subsets revealed a significant increase in the CD29⁺/CD8⁺ fraction to 97±3% versus 64±2% in controls (P= 0.008) and a decrease in the CD45RA⁺/CD8⁺ fraction to 36±11% versus 70±21% (P= 0.008). The number of cells co-expressing CD57 were also determined within the CD4⁺ and CD8⁺ subsets. In patients CD57⁺/CD4⁺ were increased to 29±7% versus 1±1% in controls (P=0.04), and CD57⁺/CD8⁺ to 49±12% versus 23±9% (P=0.02). Since CD29⁺ and CD57⁺ cells have a poor capability for IL-2 production and proliferation this shift in subset distribution may account for some of the defects in cellular immunity seen within the first year after allogeneic bone marrow transplantation.     

Restricted and Individual Usage of T-cell Receptor β-Gene Variables in Nickel-Induced CD4+ and CD8+ Cells

Nickel allergy is manifested as contact allergic eczema elicited by delayed-type hypersensitivity, the reaction being mediated by T lymphocytes. We examined the T-cell receptor (TCR) β-chain variable gene segment (Vβ) use of nickel-induced CD4⁺ and CD8⁺ T cells in the peripheral blood of nickel-sensitive and non-sensitized subjects. The results show that each patient had an individual Vβ repertoire overexpressed, these being in CD4⁺ cells Vβ10 and Vβ13 (in subject A); Vβ1, Vβ2, Vβ13 and Vβ21 (subject B); Vβ1 and Vβ10 (subject C); Vβ9 and Vβ19 (subject D). Thus, no single Vβ gene dominated in a majority of the CD4⁺ samples. The Vβ genes overexpressed in patient CD8⁺ nickel-induced T cells were Vβ1 (in subject A), Vβ1 (subject B), Vβ1 and Vβ2 (subject C) and Vβ7 (subject D), domination of Vβ1 being seen in most of the CD8⁺ samples (75%). No specific overexpression of any Vβ genes in the nickel-allergic subjects was found in comparison with the non-sensitized subjects. In conclusion, an individual pattern of restricted Vβ genes was induced with nickel in CD4⁺ and CD8⁺ T cells in each nickel allergy patient.     

T-Cell Receptor BJ Gene Segment Expression in Human Umbilical Cord Blood CD4+ and CD8+ T-Cell Subsets

By employing RT-PCR-based technology, followed by Southern-blot analysis, patterns of relative TCR BJ gene segment usage in human CD4⁺ and CD8⁺ umbilical cord blood T cells (UCT) from ten children were determined in relation to seven recombined TCR BV gene (sub) families (BV 3, 5S1, 6S1-3, 8, 9, 12 and 18). Normal frequency of usage of individual BJ members was observed to be extremely nonrandom. BJ usage in association with each BV was ranked and mean ranking values were calculated for individual BJs. Moreover, BJ family usage and family ranges as well as individual BJ over-representations were determined. In all these aspects of BJ exon expression, CD4⁺ and CD8⁺ UCT displayed similar distribution patterns. Comparisons of BJ usage in UCT subpopulations and in the adult peripheral blood lymphocyte (PBL) counterparts were performed and many similarities were observed. However, discrepancies in two parameters were recorded; contrary to observations in PBL, individual BJ over-representations were virtually absent in UCT, and significantly less wide BJ family ranges were demonstrated in CD8⁺ UCT relative to CD8⁺ PBL T cells. These differences support the notion that UCT are in a less dynamic state than are PBL T cells. Hence, despite the fact that PBL T cells are subjected to continuous antigenic challenge, the striking resemblance of PBL and UCT with regard to the overall individual relative usage, ranking, mean ranking and family utilisation of BJ gene segments, irrespective of the choice of recombined BV exons, may suggest a relatively nondiscriminatory role for the BJ gene product in antigen recognition as compared to those encoded by the BV, (N) and BD gene segments.     

γδ T-Cell Precursor-Derived CD4− CD8−αβ T Cells Retain γδ Cell Function

We have previously shown that some of the DNαβ⁺ T cells arising in TcRα-chain transgenic mice are of γδ T cell origin, based on phenotypic data and on their status of TcR gene rearrangements. In the present report we investigated the impact of αβ TcR expression on the functional programme of the mature γδ precursor-derived DNαβ⁺ T cells. Our results demonstrate that both their proliferative capacity and their cytokine production profile are similar to that of γδ T cells. Furthermore, both transgenic DNαβ⁺ T cells and DNγδ⁺ T cells up-regulate CD8α expression after activation, but, in contrast to CD4⁺αβ T cells, are unable to induce proliferation of naive B cells. Thus, our results suggest that the effector functions of mature T cells are determined independently of the TcR isotype, probably at an early stage of differentiation, and thereby have important implications for current models of T-cell lineage commitment.     

Activated CD3−CD16+ Natural Killer Cells Express a Subset of the Lymphokine Genes Induced in Activated αβ+ and γω+ T cells

In this study we analysed the potential of highly purified polyclonal TcR alpha beta+, TcR gamma delta + and CD3- NK cells, to produce lymphokines in response to mitogenic stimulation. RNA hybridizations were performed to detect with high sensitivity the induction of multiple lymphokine genes. Upon stimulation with lectin and phorbol ester TcR gamma delta + lymphocytes expressed the same set of lymphokine genes as the TcR alpha beta + lymphocytes expressed the same set of lymphokine genes as the TcR alpha beta + lymphocytes, which included IL-2, -3, -4, -5, GM-CSF, TNF alpha and beta, IFN gamma. In contrast, a more limited set of lymphokine genes (GM-CSF, TNF alpha and beta, IFN gamma) was induced in activated CD3- NK cells, thus indicating that this subpopulation of cells may display different regulatory functions, with respect to CD3+ T lymphocytes.     

Activation of Resting, Pure CD4+, and CD8+ Cells via CD3

We studied the requirements for secondary activation signals in pure CD4+ and CD8+ T cells after stimulation with anti-CD3 antibodies. Stimulation of CD4+ or CD8+ cells with anti-CD3 monoclonal antibodies (MoAb) bound to polystyrene monosized particles never resulted in a proliferative response. However, DNA synthesis was observed when recombinant interleukin 2 (IL-2) or other secondary signals, such as those provided by phorbol myristate acetate (PMA) or autologous accessory cells (AC), were also added. These secondary signals were not in themselves capable of inducing DNA synthesis in the absence of particle-bound anti-CD3. We also found that the signals provided by AC may be dependent on the activation state of these cells. Thus, the effects of accessory cells were enhanced by a factor present in fetal calf serum (FCS), most likely endotoxin or lipopolysaccharide (LPS), which alone, however, were not able to activate T cells, even in the presence of particle-bound anti-CD3. Recombinant IL-1 over a broad dose range was unable to replace PMA or activated AC after stimulation with particle-bound anti-CD3. Purified CD4+ and CD8+ T cells behaved identically in all the experiments, indicating that the basic mechanisms for activation in the two T-cell subsets are identical.     

CD4+, CD8+, and CD4− CD8− T Cells in CSF and Blood of Patients with Multiple Sclerosis and Tension Headache

Two-colour flow cytometric analysis was performed on paired samples of peripheral blood (PB) and cerebrospinal fluid (CSF) of patients with untreated multiple sclerosis (MS) and, for reference, subjects with muscular tension headache (TH) using anti-CD3, anti-CD4, anti-CD8, and anti-HLA-DR monoclonal antibodies in different combinations. CD4+/CD8+ T-cell ratio was increased in CSF compared to PB in both MS patients and TH subjects to a similar extent. This was mainly due to higher CD4+ T-cell levels in the CSF compartment. The proportion of HLA-DR+ T cells was higher in CSF than PB in both MS and TH; this increase of DR+ T cells in CSF was more prominent in MS. The level of CD4+ CD8+ T cells, which represent a subset of activated T cells, was not different between CSF and PB, either in MS or in TH. The proportion of CD4- CD8- T cells, which were found generally not to be blast cells, was lower in CSF compared to PB in both patient groups. However, their CSF level was higher and their PB level lower in MS compared to TH. Results point to an accumulation of activated T-helper cells in the CSF of both MS patients and healthy subjects. Fetal-type CD4- CD8- T cells bearing the unusual T-cell receptor gamma/delta seem to be selectively recruited to the CSF of MS patients.     

Induction of Interleukin-2 Production in CD4+ and CD8+ T-Cell Subsets after Activation via CD3 and CD2

The activation signals necessary for interleukin-2 (IL-2) receptor induction, IL-2 production, and DNA synthesis in resting T cells were investigated. IL-2 receptors were induced after activation via CD2 or CD3 alone, while IL-2 production in both CD4+ and CD8+ T cells required activation via both CD3 and CD2. The sequence of activation signals via CD3 and CD2 was shown to be important since DNA synthesis was induced when the primary activation signal was delivered via CD3, and the CD2 signal within 8 h. In contrast, no DNA synthesis was demonstrated when the primary activation signal was delivered via CD2 and the CD3 signal later. Ciclosporin A (CyA) inhibited T-cell DNA synthesis after activation via CD2 and CD3. The inhibition seemed to be due to the prevention of IL-2 synthesis.     

Wasp venom immunotherapy induces a shift from IL-4-producing towards interferon-gamma-producing CD4+ and CD8+ T lymphocytes

BACKGROUND: Venom immunotherapy (VIT) has proven to be very effective in hymenoptera venom anaphylaxis. However, the underlying immunoregulatory mechanisms of venom immunotherapy remain poorly understood. Recent studies measured the total amount of cytokine in culture supernatans, suggesting a shift in cytokine production from Th2 to a Th1 cytokine profile during VIT. We wanted to examine the contribution of specific T lymphocyte subpopulations, which is impossible using an extracellular method to determine cytokines in supernatants. OBJECTIVE: The present study was designed to evaluate the effect of VIT on the percentages of type 1 (IL-2, interferon-gamma (IFN-gamma)) and type 2 (IL-4) CD4+ and CD8+ T lymphocytes of patients with wasp venom anaphylaxis during immunotherapy. METHODS: Peripheral blood mononuclear cells of 20 individuals with a history of wasp sting anaphylaxis and a positive serum wasp venom specific IgE were isolated and in vitro stimulated with phorbol-12-myristate-13-acetate and ionomycin before VIT, at the end of a 5-day semirush VIT and at 6 months during VIT. Three-colour flow cytometric analysis was used for intracellular cytokine (IL-2, IFN-gamma, IL-4) detection in CD4+ (CD3+CD8-) T lymphocytes and CD8+ (CD3+CD8+) T lymphocytes. RESULTS: At the end of a 5-day semirush VIT, there was a significant decrease in percentage of IL-4-producing CD4+ and CD8+ T cells, compared with cytokine-producing cells before VIT (P = 0.0002 and 0.004). After 6 months of VIT, patients showed an increased number of IL-2-producing stimulated CD4+ and CD8+ lymphocytes compared with values before VIT (P = 0.002 and P = 0.0003). A higher amount of IFN-gamma-producing stimulated CD4+ and CD8+ cells was found after 6 months of VIT (P = 0.001 and P = 0.0006). There was no correlation between cytokine-producing cells and specific IgE for wasp. CONCLUSION: Venom immunotherapy induced a shift from IL-4-producing towards IFN-gamma-producing CD4+ as well as CD8+ T lymphocytes.     

Expression of CD28 on CD8+ and CD4+ Lymphocytes During HIV Infection

CD28 interaction with B7 molecules, expressed on the membranes of antigen-presenting cells, costimulates cytokine production, T-cell proliferation and generation of cytotoxic lymphocytes. The expression of CD28 markers on CD4⁺ and CD8⁺ lymphocytes was studied in a group of subjects at various stages of HIV infection. A reduction in the percentage of CD28-bearing CD4⁺ and CD8⁺ cell subsets was observed during the asymptomatic stage of the disease. This reduction was more pronounced in AIDS than in non-AIDS patients. At the same time, an increase in the absolute CD8⁺CD28⁻ cell number (greater in stage A than in stage B and C subjects) was observed in HIV-infected patients. The finding of an altered pattern of CD28 expression on T cells might per se explain certain early defects in the cytokine pattern and in the immune response peculiar to HIV-infected patients.