Cortisone-resistant Thymocytes Research Articles

FUNCTIONAL CAPACITY OF MEMORY CELLS CD8+ UNDER LYMPHOPENIA INDUCED BY INJECTION OF HYDROCORTISONE

Background. Wide use of glucocorticoids therapy for neoplasms, autoimmune diseases and allergies is associated with suppression of adaptive immunity that requires profound study of their immunoregulatory properties and immunotoxicity. Results. In this work, using our model of selective activation of mouse CD8 + memory cells in the mixed lymphocyte reaction (MLR) in vitro, we show for the first time that intraperitoneal injection of high dose hydrocortisone (2.5 mg per animal) allows to detect memory cells in the thymus of animals immunized with allogeneic tumor cells. Similar to memory cells from other lymphoid organs, hydrocortisone-resistant thymic lymphocytes from immune animals respond on allogeneic stimulators subjected to severe heat shock and are immunologically specific to immunizing alloantigen. Thus, cortisone-resistant thymocytes are partially or completely represented by memory cells. We also show here that memory responses of heterozygotes on TCR a-chain knock-out (genetically incapable to secondary rearrangement of TCR achains) are significantly enhanced as compared with the ones of wild type mice. Conclusion. These findings allows to suggest the hypothesis according to which memory T cell clones proliferating in primary immune response migrate into thymus providing necessary microenvironment for reexpression of recombinases. After editing of genes encoding TCR achains, such T lymphocytes can return to peripheral repertoire maintaining its wideness.

Study of chemokine, produced by CD4− 8− cortisone-resistant thymocytes

Study of chemokine, produced by CD4− 8− cortisone-resistant thymocytes

Study of chemokine, produced by CD4 − 8 − cortisone-resistant thymocytes

Study of chemokine, produced by CD4 − 8 − cortisone-resistant thymocytes

Comparison of the results of interaction between lymph node-derived lymphocytes and cortisone-resistant thymocytes, and endogenous stem cells of syngeneic and allogeneic recipients

A comparative study of the interaction between lymph node lymphocytes and cortisone-resistant thymocytes, and endogenous colony-forming units was conducted using an endogenous colony-forming assay. Cortisone-resistant thymocytes induced equally efficient stimulation of the spleen endocolonization in syngeneic, semiallogeneic, and completely allogeneic recipients. However, transfer of the lymph node lymphocytes resulted in only moderate stimulation of endogenous colony-forming units in syngeneic recipients and in drastic inhibition of colony formation in F1 hybrid and allogeneic recipients. The mechanisms of the opposite effect of peripheral T lymphocytes as compared to cortisone-resistant thymocytes on endogenous stem cells are discussed.

T‐Cell Receptor Alpha Haplotype influences Vα Epitope Expression on both Cortisone‐Resistant Thymocytes and Lymph Node T Cells

We have previously described a mouse strain congenic for the T-cell receptor alpha chain locus (Tcra). The strain, B10.D2.C-Tcraa/Bo, carries the Tcraa haplotype of BALB/c instead of the Tcrab haplotype normally found in B10.D2. By comparing B10.D2.C-Tcraa and B10.D2 mice, we now show that the Tcra haplotype influences the frequencies of V alpha 8 epitope expression on T cells detected by the novel B21.14 monoclonal antibody (MoAb). This finding agrees well with our previous observation that Tcra haplotype influences the frequencies of expression of two other V alpha 8 epitopes, detected by the KT50 and KT65 MoAbs. The specificities of the three V alpha 8-specific MoAbs were compared by scatter diagram analysis of staining frequencies obtained in individual mice. The B21.14 and KT50 MoAbs appear to have very similar specificities; these two MoAbs stain slightly more alpha beta T cells than the KT65 MoAb. Investigations with another novel MoAb, the V alpha 2-specific B20.1, revealed that the Tcra haplotype also influences the frequency of V alpha 2 epitope expression. The effect of the Tcra haplotype on V alpha 2 epitope expression was evident not only among lymph node T cells but also among mature, cortisone-resistant thymocytes. Thus, the influence of the Tcra haplotype is imprinted already in the thymus. The most likely explanations for Tcra haplotype dependency of V alpha epitope expression are differing numbers of V alpha-gene segment subfamily members and/or allelic polymorphism. The significance of Tcra haplotype-dependent V alpha epitope expression is discussed.

Identification of functional T cell subsets and surface antigen changes during activation as they relate to RT6

The functional and phenotypic heterogeneity of the rat peripheral T lymphocyte antigen RT6 has been examined. The in vivo popliteal graft-vs-host reaction (GvHR), in vitro MLR, and the generation and effector populations of cytotoxic T lymphocyte were used to examine the response of RT6-positive (RT6 +) or -negative (RT6 −) subsets of CD4 and CDS T cells to alloantigen. T lymphocytes with the CD4 +RT6 + surface phenotype are necessary and sufficient for inducing a strong GvHR. T lymphocytes with the CD4 +RT6 −, CD8 +RT6 +, and CD8 +RT6 − surface phenotypes do not contribute or induce a strong GvHR. Both the CD4 +RT6 i+ and the CD4 +RT6 − T cells proliferate in the MLR assay. CTL precursors are a mixture of CD8 +RT6 + and CD8 +RT6 − phenotypes, but only cells bearing the RT6 − phenotype are potent CTL effector cells. Data presented in this paper also demonstrate that the RT6.1 alloantigen, present on the majority of rat T cells, modulates on cortisone-resistant thymocytes (CRT) and peripheral T cells. Although freshly isolated CRTs do not express the RT6.1 epitope, RT6 + cells develop when CRTs are placed into culture. Peripheral T cells that are RT6 − will also become RT6 + in culture. Stimulation of T cell cultures with a mitogen causes the loss of the RT6.1 antigen, as detected by the DS4.23 mAb. The effect is more pronounced in cultures of CRTs than peripheral T cells. Following a return to a nonactivated state, the T cells reexpress RT6. The loss and reexpression of RT6 may be related to activation and/or differentiation of T cells.

Viral infection of the thymus.

We have examined infection of the thymus during congenitally acquired chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, a classic model of antigen-specific T-cell tolerance. Our results show that (i) infection starts at the fetal stage and is maintained throughout adulthood, and (ii) this chronic infection of the thymus can be eliminated by transfer of virus-specific cytotoxic T lymphocytes (CTL) that infiltrate the thymus and clear all viral products from both medullary and cortical regions. Elimination of virus from the thymus results in abrogation of tolerance. During the fetal stage, the predominant cell type infected is the earliest precursor of T cells with a surface phenotype of Thy1+ CD4- CD8- J11d+. In the adult thymus, infection is confined primarily to the cortisone-resistant thymocytes present in the medullary region. The infected cells are CD4+ and J11d+. The presence of J11d, a marker usually associated with immature thymocytes, on infected single positive CD4+ "mature" thymocytes is intriguing and suggests that infection by this noncytolytic virus may affect development of T cells. There is minimal infection of the CD8+ medullary thymocytes or of the double positive (CD4+ CD8+) cells present in the cortex. Infection within the cortex is confined to the stromal cells. Interestingly, there is infection of the double negative (CD4- CD8-) thymocytes in the adult thymus, showing that even during adulthood the newly developing T cells are susceptible to infection by LCMV. Virus can be eliminated from the thymuses of these carrier mice by adoptive transfer of medullary region first and then from the thymic cortex. This result clearly shows the need to reevaluate the widely held notion that mature T cells are unable to reenter the thymus. In fact, in our experiments the donor T cells made up to 20 to 30% of the total cells in the thymus at 5 to 7 days after the transfer. The number of donor T cells declined as virus was eliminated from the thymus, and at 1 month posttransfer, the donor T cells were hardly detectable. The results of this study examining the dynamics of viral infection and clearance from the thymus, the primary site of T-cell development, have implications for understanding tolerance induction in chronic viral infections.

A change in monokine requirements for proliferation during thymocyte maturation: Co-stimulating activity of interleukin 6 in the proliferation of LYT2 − and L3T4 − thymocytes

Interleukin 6 (IL-6) and interleukin 1 (IL-1) were compared as to their ability to induce the proliferation of distinct thymocyte subpopulations. IL-6 functions as a costimulator in IL-1- or IL-2-induced proliferation of adult double-negative (DN) thymocytes, whereas IL-6 alone failed to induce a significant level of proliferation. However, IL-6 alone induced significant proliferation of mature cortisone-resistant thymocytes, whereas IL-1 did not. Instead, IL-1 functioned as a costimulator in IL-6-induced proliferation of mature thymocytes. Finally, both IL-6 and IL-1 were capable of potentiating IL-2-induced proliferation of fetal DN thymocytes. These data suggest that the monokine requirements in thymocyte activation may vary during thymocyte maturation and that IL-6, when compared to IL-1, has a distinct effect on the proliferation of thymocytes.

Dextran sulfate induces changes in the free intracellular calcium ion concentration of a subpopulation of immature thymocytes

Previous studies have shown that certain sulfated polysaccharides, such as dextran sulfate (DxS), can induce a rapid and sustained increase in the free intracellular calcium ion concentration ([Ca2+]i) of thymocytes, whereas they cannot induce a change in the [Ca2+]i of peripheral lymphocytes. This study examined this phenomenon in more detail and clearly demonstrated that only immature thymocytes can respond to DxS. Cortisone-resistant thymocytes failed to exhibit an increase in [Ca2+]i when exposed to DxS. Furthermore, analysis of [Ca2+]i changes at the single cell level using a flow cytometer demonstrated that only a subpopulation of immature thymocytes responded to DxS. Additional flow cytometry studies revealed that DxS and Con-A induce a [Ca2+]i response in different subpopulations of thymocytes, approximately 30% of thymocytes responding to Con-A and 15-20% to DxS. Thus, responsiveness to Con-A and DxS, in terms of increases in [Ca2+]i, may represent a novel marker for two different thymocyte subpopulations. Such metabolic markers could be used to complement the conventional serological methods currently employed to define stages in thymocyte differentiation.

Thymocyte maturation following interaction with thymus-derived macrophages

Murine C57BL/6 thymocytes were cultivated together with syngeneic thymus-derived macrophages (TDMφ) for up to 96 hr to determine whether TDMφ participate in thymocyte maturation. The expression level of H-2 b and Thy-1.2 antigens served as thymocyte differentiation surface markers as analyzed by flow cytometry. Indirect immunofluorescent staining profiles of the thymocytes demonstrate a dramatic increase in H-2 b expression and a profound decrease in Thy-1.2 expression during cultivation with TDMφ. A similar phenomenon was observed when enriched populations of immature thymocytes were cocultivated with TDMφ. These changes were not observed when thymocytes were cultivated alone or with trypsin-treated TDMφ; neither were they observed when cortisone-resistant thymocytes manifesting mature characteristics were cultivated together with TDMφ. These findings suggest that interaction of thymocytes with TDMφ, involving binding and engulfment, results in the appearance of mature thymocyte subsets.

Phenotypic and functional heterogeneity of thymic and splenic lymphokine activated killer cells relative to ornithine sensitivity

We have previously reported the selective inhibition of cytotoxic T lymphocytes (CTL) by 10 m M ornithine (ORN) relative to natural killer (NK) cell-derived lymphokine activated killer cells (LAK). To determine if this were due to differences in the progenitor cells or the type of stimulus, we used cortisone-resistant thymocytes (CRT) as a source of mature T cells for induction of LAK and CTL, and compared the results with spleen. Thymic and splenic CTL precursors (CTL P) from C57B1/6 (B6) mice were CD8 +, ASGM1 −, ORN sensitive. Splenic LAK precursors (LAK P) were CD8 −, ASGM1 +, ORN resistant when assayed against both YAC-1 and P815 tumor targets. In contrast, CRT-derived LAK P were CD8 −, ASGM1 +, ORN resistant against YAC-1, whereas LAK P against P815 were CD8 +, ASGM1 +, ORN sensitive. ORN sensitivity was also observed among CTL and LAK in DBA/2 mice and was associated with CD8 + phenotype. Therefore, our initial observation of differential ORN sensitivity in CTL vs LAK was a function of the progenitor cells; furthermore, CD8 + cytolytic cells are ORN sensitive whether activated by antigen (CTL) or IL-2 (T-LAK).

Bacteria of Human Physiological Microflora Liberate Immunomodulating Peptides

Human isolates of Propionibacterium acnes and Staphylococcus saprophyticus could be shown to liberate low molecular weight peptides (MW less than 6.500 D) with immunomodulating activity. FACS analyses of BALB/c-mouse lymphoid cells from the thymus and spleen revealed an enhanced percentage of T-helper cells after peptide administration. Intestinal microflora decontamination of BALB/c-mice considerably reduced immune cell function and lymphatic tissue proliferation. Apparently, lack of peptide production or liberation correlated to immunosuppression. Substitution of peptides (from P. acnes or S. saprophyticus) to decontaminated mice reconstituted immune cell function and proliferation. Cortisone-resistant thymocytes were used as an experimental equivalent of functional cells in the thymus. Thus, cortisone treatment of BALB/c-mice significantly reduced the number of thymocytes, however, administration of microbial peptides restored the thymus population.

Murine thymocytes possess specific cell surface‐associated exoaminopeptidase activities: Preferential expression by immature CD4−CD8− subpopulation

Murine thymocytes are shown to possess at least three well-defined exo-N-aminopeptidase activities on their surface. One of them cleaves the prolyl bond in the synthetic dipeptide nitroanilide Gly-Pro-pNA (Km 0.95 mM and Vmax 8 nmol/h at pH 7.4 and 37 degrees C) and is specifically inhibited by phenylmethane sulfonyl fluoride, diprotin A, Gly-Pro-Ala and Gly-Pro-Gly-Gly. These data further support identification of this enzyme with a serine exopeptidase dipeptidyl peptidase IV (DPP IV), previously reported to be specific for collagen. The two other forms of N-exopeptidase activities are detected when Ala-pNA and Leu-pNA are used as substrates. Leu-aminopeptidase activity (Km 1.4 mM, Vmax 15 nmol/h) and Ala-aminopeptidase activity (Km 4.0 mM, Vmax 20 nmol/h) are inhibited by inhibitors for thiol- and trypsin-like proteinases, i.e. tosyl lysyl chloromethyl ketone, leupeptin and N-ethylmaleimide. Addition inhibition of Leu-aminopeptidase activity by peptstatin, a known inhibitor of carboxyl proteases, suggests that aminopeptidase activity detected with Leu-pNA is different in part from Ala-aminopeptidase activity. Among the various lymphoid cell populations tested, the three aminopeptidase activities are increased by three- to fourfold in the immature CD4-CD8- thymocyte subset as well as in the thymoma BW5147. In contrast, cortisone-resistant thymocytes, lymph node and spleen cells exhibit levels of activities almost similar to that of unfractionated thymocytes. During ontogeny, the levels of these activities are increased four- to sevenfold on fetal thymocytes (from days 14 to 16). Finally, when thymocytes or spleen cells are cultured with a mitogenic concentration of concanavalin A, their proliferative responses are correlated with an enhancement of the aminopeptidase activities (1.3- to 5-fold). From these results, a correlation between the presence of these peptidases on the cell surface of immature and mature lymphoid cells and biological responsiveness is suggested.

Distinct patterns of lymphokine requirement for the proliferation of various subpopulations of activated thymocytes in a single cell assay.

The frequency and capacity for clonal expansion of several murine thymocyte subpopulations responsive to various IL (fetal day 15, and adult CD4-8-, CD4+8- and CD4-8+) were investigated using a single-cell limiting-dilution cell culture system without filler cells. This assay requires the presence of PMA and ionomycin. The main conclusions of these studies are the following: 1) IL-4 is a better growth factor than IL-2 for immature thymocytes (fetal day 15 or adult CD4-8-). 2) IL-2 is a better growth factor than IL-4 for mature phenotype thymocytes (CD4+8- and CD4-8+). 3) IL-4 is a relatively poor growth factor for adult CD4-CD8- thymocytes and CD4+CD8- thymocytes, while it induced strong responses in fetal day 15 and CD4-8+ thymocytes. 4) IL-6 enhanced the response of CD4+8- thymocytes to either IL-2 or IL-4. 5) Cortisone-resistant thymocytes grown initially with IL-4 and then switched to IL-2 showed a significant decrease in cloning efficiency. No inhibitory effect was observed when cells were cultured first with IL-2 and then switched to IL-4. 6) Finally, supernatant from Con-A stimulated rat spleen cells induced maximal growth of all adult thymocyte populations tested, suggesting that unidentified thymocyte growth factor(s) remain to be characterized. These results indicate that the maturational stage of thymocytes determines their requirements for activation and proliferation.

Antibody 12‐15 cross‐reacts with mouse Feγ receptors and CD2: study of thymus expression, genetic polymorphism and biosynthesis of the CD2 protein

Previously we described a monoclonal antibody (mAb 12-15) that reacted with murine Fc receptor proteins (beta 1, beta 2 and alpha) and an undefined molecule of 37 kDa (beta 3) on certain types of cells. Here we present serological and biochemical evidence that the beta 3 chain is expressed on mouse thymocytes and that it is identical to the CD2 antigen. By immunofluorescence staining and cytofluorographic analysis greater than 95% of thymocytes were positive. Brightly staining cells coincided with cortisone-resistant thymocytes suggesting that mature thymocytes expressed higher levels of the antigen. Biosynthetic labeling of DBA/2 thymocytes with [35S]methionine showed that the size of the CD2 precursor molecule was 43 kDa which was processed to approximately 55-65 kDa in the mature molecule. mAb 12-15 was also reactive with the tunicamycin-treated form of the CD2 antigen suggesting that the cross-reactive epitope was of protein nature. Comparison of different mouse strains indicated that two molecular forms of CD2 exist. On BALB/c thymocytes, the relative mass of the native molecule was approximately 60-70 kDa (CD2.1) and slightly larger than in DBA/2 (CD2.2). Following endoglycosidase F treatment both proteins still showed a slight difference in electrophoretic mobility. Several inbred mouse strains were analyzed for expression of CD2 forms. When mAb 12-15 was used in cytotoxic T lymphocyte inhibition experiments using specific CTL and tumor target cells it was found that the antibody could specifically inhibit CTL-mediated lysis presumably by interfering with CD2 function.

Interaction between thymocytes and thymus-derived macrophages: I. Surface components participating in mutual recognition

Incubation of C57BL/6 thymus-derived macrophages (TDMφ) with syngeneic thymocytes resulted in binding of thymocytes to macrophages and rosette formation. Up to 60% of the TDMφ formed rosettes with thymocytes after 6 hr of interaction at 4 °C. Rosette formation of the immature PNA + thymocyte fraction was up to fivefold higher than that of PNA − and cortisone-resistant thymocytes. Pretreatment of PNA − thymocytes with neuraminidase enhanced thymocyte binding to macrophages up to sevenfold, whereas a marked reduction of rosette formation was seen following (1) incubation of thymocytes with tunicamycin; (2) incubation of macrophages with 20 m M d-galactose, GLCNaC, or GalNaC; (3) treatment of macrophages or thymocytes with trypsin; (4) treatment of macrophages with anti-1-A b mAb and its F(ab′) 2 fragment; (5) treatment of thymocytes with anti-Lyt-2.2 mAb; and (6) addition of EDTA and EGTA to the interacted two cell populations.

CD4 + CD8 + cells are rare among in vitro activated mouse or human T lymphocytes

The predominant cell type in the thymus expresses both of the function-associated T cell surface markers, CD4 and CD8, but CD4 + CD8 + cells are rare or absent outside the thymus. Double expression has therefore been assumed to be an indication of immaturity. However, recent reports have suggested that CD4 + CD8 + cells can appear in cultures of activated mature cells. We have therefore activated human peripheral blood lymphocytes and mouse spleen cells, lymph node cells, and cortisone-resistant thymocytes using a number of different stimulation regimes, and we have analyzed them at various times for CD4 and CD8 expression. In all cases, upon analysis of cultured cells by flow cytometry, CD4 + CD8 + cells were rare. A combination of microscopic analysis, cell sorting followed by microscopic analysis, and careful staining controls demonstrated that even when flow cytometry showed some CD4 + CD8 + cells, most of these were artifacts in the form of doublets or clumps of single positive cells or dead cells. Taking this into account, CD4 + CD8 + cells made up less than 1% in the mouse and less than 3% in the human T cell cultures at any time periods. We therefore found no evidence for the generation of large numbers of CD4 + CD8 + cells in cultures of mouse or human T cells.

Lymphocyte depletion in thymic nurse cells: a tool to identify in situ lympho-epithelial complexes having thymic nurse cell characteristics.

In situ pre-existing complexes of epithelial cells and thymocytes having thymic nurse cell characteristics were visualized in the murine thymus cortex using dexamethasone as a potent killer of cortisone-sensitive thymocytes. The degradation and subsequent depletion of cortisone-sensitive thymocytes enclosed within cortical epithelial cells appeared to be paralleled by thymocyte degradation and depletion in thymic nurse cells isolated from thymic tissue fragments from dexamethasone-treated animals. This suggests that thymic nurse cells are derived from pre-existing sealed complexes of cortical epithelial cells and thymocytes. Not all thymocytes situated within in situ epithelial or thymic nurse cells complexes appear to be cortisone-sensitive: a minority of 1-2 thymocytes per complex survives the dexamethasone-treatment, thus constituting a minor subset of cortical cortisone-resistant thymocytes predominantly localized within cortical epithelial cells in situ and within thymic nurse cells derived from such structures. Cortisone resistance in thymocytes thus seems to be acquired within the cortical epithelial cell microenvironment. Cortisone-resistant thymocytes in thymic nurse cells express the phenotype of mature precursors of the T helper lineage, indicating that the in situ correlates of thymic nurse cells may play an important role in T cell maturation and selection.

Lymphokine-activated killer (LAK) cells. III. Characterization of LAK precursors and susceptible target cells within the murine thymus.

In our search for a biologic role for lymphokine-activated killers (LAK), we examined their generation from murine thymocytes. Normal adult thymocytes were capable of generating LAK upon culture with relatively large doses (500 to 1000 U/ml) of interleukin 2. Normal thymocytes were fractionated into four subsets by virtue of their co-expression of the Lyt-2 and L3T4 markers: Lyt-2+ L3T4+ (2+4+); 2+4-; 2-4+; and 2-4-. None of these subsets had any natural killer activity. Upon examining the ability of these subsets to generate LAK, it was found that the 2-4- subset was the most potent and required the smallest relative amounts of interleukin 2. In addition to lysing tumor cells, thymus-derived LAK were capable of killing "fresh" 2+4+ thymocytes. Fresh 2+4-, 2-4+, 2-4-, and cortisone-resistant thymocytes were resistant to lysis by LAK. Upon mitogen stimulation, however, the cortisone-resistant thymocytes and 2+4- thymocytes became LAK-susceptible. These data demonstrate a possible mechanism for the elimination of the 2+4+ thymocyte subset which is generally believed to be a "dead-end" population. Moreover, these data suggest a possible biologic role for LAK in the process of thymocyte maturation and intrathymic selection.

Selective down modulation of L3T4 molecules on murine thymocytes by the tumor promoter, phorbol 12-myristate 13-acetate.

Treatment of murine thymocytes, but not mature peripheral T cells, with the tumor promoter, phorbol 12-myristate 13-acetate (PMA), 3 results in a rapid disappearance of L3T4 molecules from the surface of thymocytes. The effect of PMA on L3T4 molecules persists in vitro for at least 72 hr. Down modulation of L3T4 molecules was PMA dose-dependent and temperature-dependent. L3T4 molecules on cortisone-resistant thymocytes were significantly less sensitive to the effect of PMA than were L3T4 molecules on cortisone-sensitive thymocytes. Down modulation of L3T4 molecules on thymocytes did not interfere with their capacity to respond to concanavalin A or activation signals delivered via their T cell receptors. The difference in the ability of thymocytes and peripheral T cells to respond to PMA cannot be explained by differences in the number of PMA receptors. Both thymocytes and peripheral T cells have PMA receptors in the range of 1 to 1.5 X 10(5) receptors/cell. However, there is a small difference in the affinity (Kd) of the receptors on thymocytes (Kd = 30 to 40 nM) and peripheral T cells (Kd = 10 to 15 nM). Immunofluorescent staining revealed that the down modulation of L3T4 molecules by PMA was a result of internalization of L3T4 molecules. After down modulation, L3T4 could be readily detected on the cytoplasm of thymocytes. These findings suggest that L3T4 molecules on thymocytes may be subject to different regulatory signals than L3T4 molecules on peripheral T cells.