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.     

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.     

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.     

Participation of Vβ4+-, Vβ7+-, and Vβ11+-T Lymphocytes in Haematogenously Acquired Staphylococcus aureus Nephritis

The most frequent pathogen in haematogenously acquired kidney infections in humans is Staphylococcus aureus . In order to characterize in situ the immunological patterns of septic nephritis we developed a murine model of this disease. A single intravenous injection of S. aureus producing toxic shock syndrome toxin-1 resulted in high frequency of inflammatory kidney lesions. Histopathologically, both focal and diffuse inflammatory infiltrates were seen in kidney cortex and medulla. Immunohistochemical evaluation revealed high numbers of Mac-1⁺ phagocytic cells as well as CD4⁺- and CD8⁺-lymphocytes. The expansion of lymphocytes carrying T-cell receptor V_β chain 4, 7, and 11 families in the kidney was observed. Our results suggest that the haematogenously acquired kidney infection by superantigen-producing staphylococci leads to migration, in situ activation and expansion of responding T-lymphocyte subsets.     

CD4− CD8− C.B-17 SCID Thymocytes Enter the CD4+ CD8+ Stage in the Presence of Neonatally Grafted T Cells

The aim of this work was to study the selection of donor T cells and their influence on thymic development in C.B-17 scid/scid (severe combined immunodeficient; SCID) mice during chronic graft-versus-host disease (GVHD). Recipient SCID mice (H-2^d), neonatally grafted with allogeneic peripheral T cells from CBA/J strain (H-2^k) of mice, only developed a mild acute GVHD, and were, at the chronic stage, devoid of pathological symptoms. Thymic cell numbers of injected mice differed from 10⁵ to 1.2 × 10⁷ at 2–3 weeks post-injection (p.i.), and from 4 × 10⁵ to 8.5 × 10⁷ at 2 months p.i. In these mice, the thymus size was correlated to the CD4^? CD8^? (double negative; DN) to CD4⁺ CD8⁺ (double positive; DP) cell ratio, where at 2 months p.i, 8 out of 16 treated SCID mice contained 5 × 10⁶ cells or more and also possessed the highest frequencies of endogenous DP cells (25–95%). In contrast to previous findings, peripheral donor T cells from allogeneic and syngeneic mice, infiltrating the host thymus, had a positive effect on the development of endogenous DP thymocytes. Furthermore, these thymocytes were developmentally blocked at the DP stage, occasionally in combination with the expression of CD25, CD44 and CD117 but in the absence of T-cell receptor (TCR) expression. Also, at this time-point, the CBA/J donor TCR Vβ repertoire was equal to that of normal CBA/J mice, but purified responding donor cells were proliferatively inhibited against H-2^d stimulators in ex vivo mixed lymphocyte cultures. In contrast, the same responders showed a pronounced proliferation against syngeneic H-2K^k stimulators, suggesting either a reversion from anergy of autoreactive CBA/J T cells or a vast expansion of multiple self-reactive T-cell clones, when parked in a milieu with a lower concentration of self-antigens.     

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.     

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.     

Induction of CD8+ CTL Recognizing Mycobacterial Peptides

Mycobacterium tuberculosis is the single, most important cause of morbidity attributable to a single infectious organism. CD8⁺ T cells play an important role in anti-tuberculous immune responses in both mice and humans. Data concerning the identity of mycobacterial antigens recognized by CD8⁺ T cells is limited; consequently, few CTL epitopes have been characterized. The authors identified allele-specific (H-2^{b and d}) MHC class I binding motifs in six prominent M. tuberculosis protein antigens (the 19 and 38 kDa lipoglycoproteins and the 10, 16, 65 and 70 kDa stress proteins). These predicted epitopes were tested for MHC binding as well as their ability to elicit peptide-specific CTL following in vivo priming. The authors were able to identify eight previously undescribed mycobacterial CTL epitopes by using spleen cells from peptide-immunized mice. In addition, CTL specific for at least one of these epitopes also recognized the naturally processed epitope presented on transfected EL4 target cells. These mycobacteria-derived CTL epitopes could be important for future analysis of the involvement of CD8⁺ T cells in M. tuberculosis infection, pathogenesis and vaccine development.     

Secretion of IL-2, IL-3, IL-4, IL-6 and GM-CSF by CD4+ and CD8+ TCRαβ+ T-Cell Clones derived early after Allogeneic Bone Marrow Transplantation

Secretion of different cytokines may be an important T-cell effector mechanism for bone marrow engraftment, graft versus host disease and graft versus leukaemia effects after allogeneic bone marrow transplantation (BMT). Cytokine secretion and autocrine proliferative capacity of T-cell clones derived from leukaemia patients 3–6 weeks after allogeneic bone marrow transplantation were investigated. Only a minority of post-transplant T-cell clones (23/120; 19%) was capable of undergoing autocrine proliferation. By contrast, 21/65 (32%) normal control clones from the marrow donors derived under the same conditions were autocrine proliferative. All clones were interleukin-2 (IL-2) responsive. A majority (12/17; 71%) of autocrine proliferating post-transplant clones secreted detectable IL-2. Compared with control clones, CD4⁺ T-cell clones derived early after BMT produced decreased levels of interleukin-4 (IL-4) and interleukin-6 (IL-6), whereas secretion of interleukin-3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) showed no significant difference. The small number ( n = 8) of post-transplant CD8⁺ clones showed decreased production of IL-3, IL-4 and IL-6 compared with control clones, but normal secretion of GM-CSF. Neither CD4⁺ nor CD8⁺ T-cell clones secreted interleukin-7 (IL-7).     

Intravenous Immunoglobulin (IVIg) Modulates the Expansion of Vβ3+ and Vβ17+ T Cells Induced by Staphylococcal Enterotoxin B Superantigen In Vitro

The authors investigated the effect of IVIg on T-cell proliferation induced by the superantigen, staphylococcal enterotoxin B (SEB). The addition of IVIg to normal PBMC stimulated with SEB resulted in a threefold increase in the proportion of CD3⁺ blast cells expressing Vβ3 and Vβ17, two subsets of T cells that selectively expand in the presence of SEB. There was no increase in the proportion of T-cell blasts expressing Vβ2, Vβ8 and Vβ13.6 antigens that do not respond to SEB in the absence of IVIg. As described previously, IVIg inhibited T-cell proliferation independently of Vβ specificity. The effects of IVIg were mediated by variable regions of immunoglobulins since they were reproduced with F(ab')₂ fragments of IVIg but not with purified Fc fragments of IgG. The observation that SEB-activated T cells are rescued from inhibition of proliferation by IVIg indicates that IVIg modulates the effects of superantigen on T cells. These results may be of relevance for understanding the mechanisms underlying the effects of IVIg in patients with diseases in which T-cell superantigens are of pathophysiological significance.     

Resistance and Susceptibility to Cyclosporin A as CD3− 4− 8− Human Thymocytes Differentiate In Vitro

Human T-cell development appears to be relatively resistant to cyclosporin A (CsA). Children exposed to CsA mw/eroaspart of kidney transplant maintenance have few abnormalities. The objective of the study described here was to analyse the effects of CsA on the development in vitro of human multinegative (MN) (CD3-4-8-) thymocytes as a model system for thymic progenitor development in vitro. MN thymocytes, prepared by depletion methods, differentiated in vitro to acquire CD3 and undergo transitions in CD45 isoform expression analogous to those postulated to occur in vivo. In this work MN thymocytes were cultured with IL-2 and on thymic epithelial cells (TEC) with or without lL-2, either in ihe presence or absence of CsA. For many thymocyte preparations, differentiation in the presence of CsA resulted in almost complete inhibition of the acquisition of CD3and ofthe low Mr isofomi CD45R0. Expression of CD45RA and of total CD45 were reduced but not eliminated and the density of CD29 was unaffected. For others, neither CD3 nor CD45 expression was affected., but selective inhibition of TCR5 expression occurred. At all doses of CsA (0.1–100 μg/ml), MN thymocytes continued to cycle indicating a CsA-resistant generative compartment. Treatment of peripheral blood T cells with CsA had no effect on surface expression of CD3 or CD45 isoforms but did reduce the amount of de novo-synthesized CD45R0 mRNA. Culture of MN thymocytes on TEC rendered them virtually resistant to the negative effects of CsA. CD3 acquisition was unhindered and total CD45 remained high, but the transition from CD45RA to CD45R0 appeared to be delayed. In the absence of TEC, expression of both TCRδ and of TCRδ was inhibited, but on TEC, TCRδ was actually up-regulated in some conditions. The effects of CsA on human thymocyte development appeared to be modulated by the physiological state of the donor and the growth conditions to which the ceils were subjected. Conditions which most closely approximated those manifest in vivo rendered thymocytes most resistant to the negative effects of CsA. The amount of CsA required to affect differentiation in vitro was significantly higher than could be attained in vivo suggesting that the immunomodulatory effects of CsA in the maintenance of organ transplants may from an as yet uncharacterized mechanism     

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.     

CD3−4−8− Thymocyte Precursors with Interleukin-2 Receptors Differentiate Phenotypically in Coculture with Thymic Stromal Cells

CD3⁻4⁻8⁻ interleukin-2 receptor positive (IL-2R⁺) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL-2R⁺CD3⁻4⁻8⁻ thymocytes were obtained by positive panning of IL-2R⁺ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2½ days significant numbers of CD4⁺8⁺ and CD3⁺4⁺8⁻ cell populations developed, the latter expressing the αβ T-cell receptor (αβ-TCR). Thus heterogeneous stromal cell cultures support the development of IL-2R⁺ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal-dependent step.     

Acute Graft-versus-Host Reaction in SCID Mice Leads to an Abnormal Expansion of CD8+ Vβ14+ and a Broad Inactivation of Donor T Cells Followed by a Host-Restricted Tolerance and a Normalization of the TCR Vβ Repertoire in the Chronic Phase

The persistence and selection of allogeneic CBA/J T lymphocytes were studied during graft-versus-host (GvH) reaction in immunodeficient C. B-17 SCID (SCID) mice. After neonatal injection the donor cells primarily migrated to the spleen plus lymph nodes (SL) and the thymus of the recipients. Thirteen days post engraftment, CD8⁺ cells in SL had increased five times in cell number with an 18-fold increase of CD8⁺ Vβ14⁺ cells, paralleled by clinical signs of GvH disease (GvHD). Donor lymphocytes from these mice were proliferative unresponsive to allogeneic Balb/c or C57B1/6 SL cells, whereas 8 weeks post injection the tolerance was confined to H-2^d specific donor cells. Here, spleens had a total cell content similar to untreated SCID mice but the average percentage of donor cells had reached 25%. Moreover, the CD4/CD8 cell ratio in the donor population in SL and thymus had changed to normal and the TCR Vβ repertoire was similar to that of the originally injected cells. Following secondary transfer into syngeneic CBA/Ca nu/nu recipients donor cells regained a significant but reduced response to H-2^d stimulators indicating that the antigen specific tolerance of allogeneic donor cells in the SCID mice was due, at least in part, to a reversible state of anergy.     

Analysis and Characterization of the Interleukin 2 Receptor α and β Chain Expression inCD4−CD8− Cells

The differential effects that the binding of interleukin 2 (IL-2) to its beta or alpha beta receptors might induce in two different CD4-CD8- T-cell lines were analysed. While LD1.T3b, a double-negative T cell derived from MRL/lpr mice, constitutively expressed high levels of the IL-2R beta chain, YAC-1, a Moloney sarcoma virus-transformed CD4-CD8- T cell, expressed (as an activated T cell) the beta and alpha chains. The presence of IL-2 in the culture medium was lethal for LD1.T3b cells, while it had no effect on the growth of YAC-1 cells. IL-2 increased the expression of the beta chain and, to a lesser extent, of the alpha chain in YAC-1 cells. In addition, other markers such as CD4 and CD5 were induced by IL-2 in this cell line.     

The CD4+CD8+ and CD4+ Subsets of FOXP3+ Thymocytes Differ in their Response to Growth Factor Deprivation or Stimulation

The timing of thymic regulatory T (Treg) cell commitment remains unclear. Specifically, there is disagreement as to whether the CD4⁺CD8⁺ FOXP3⁺ thymocytes are precursors of mature CD4⁺ FOXP3⁺ Treg cells, or an independent Treg cell lineage. We reasoned that precursors should be more susceptible to apoptosis than mature Treg cells, and tested this by growth factor removal and anti-CD3 stimulation. Both treatments resulted in an increase of CD4⁺ FOXP3⁺ thymocytes, whereas the frequency of CD4⁺CD8⁺ FOXP3⁺ thymocytes decreased significantly. These changes were accompanied by an increase of annexin⁺ apoptotic cells. Both of these FOXP3⁺ subsets expressed higher levels of Bcl-2 and BIM than other thymocytes, and while in our setting expression of BIM seemed to predispose the cells to apoptosis, Bcl-2 had no apparent protective effect. These results indicate that CD4⁺CD8⁺ FOXP3⁺ thymocytes are more susceptible to apoptosis than mature CD4⁺ FOXP3⁺ Treg cells. This is consistent with the view that they are still immature and thus likely to represent a precursor population.