Severe murine lung immunopathology elicited by the pathogenic equine herpesvirus 1 strain RacL11 correlates with early production of macrophage inflammatory proteins 1alpha, 1beta, and 2 and tumor necrosis factor alpha.

The CBA mouse model was used to investigate the immunopathology induced in the lung by the pathogenic equine herpesvirus 1 (EHV-1) strain RacL11 in comparison to infection with the attenuated vaccine candidate strain KyA. Intranasal infection with KyA resulted in almost no inflammatory infiltration in the lung. In contrast, infection with the pathogenic RacL11 strain induced a severe alveolar and interstitial inflammation, consisting primarily of lymphocytes, macrophages, and neutrophils. Infection with either EHV-1 strain resulted in the accumulation of similar numbers and ratios of CD4 and CD8 T lymphocytes in the lung and bronchoalveolar lavage (BAL) fluid. Further analysis of these T-cell populations revealed identical EHV-1-specific cytotoxic T-lymphocyte responses. RNase protection analysis of RNA isolated from the BAL fluid of RacL11-infected mice on day 3 postinfection revealed much higher levels of RNA specific for macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and MIP-2 than were observed for KyA-infected mice. Furthermore, significantly higher levels of transcripts specific for tumor necrosis factor alpha were induced on day 3 postinfection with RacL11 compared with KyA. These findings suggest that the early production of proinflammatory beta chemokines plays a major role in the severe, most often lethal, respiratory inflammatory response induced by the pathogenic EHV-1 strain RacL11.

Cytokine and adhesion molecule expression in SCID mice reconstituted with CD4+ T cells.

The objectives of this study were to quantify colonic cytokine and endothelial cell adhesion molecule (ECAM) expression in the colons of severe combined immunodeficient (SCID) mice reconstituted with different subsets of CD4+ T lymphocytes. We found that animals injected with CD45RBhigh but not CD45RBlow T cells or phosphate-buffered saline (PBS) developed clinical evidence of colitis at 6-8 weeks following reconstitution, as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of a variety of Th1 and macrophage-derived cytokines including interferon gamma, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-12, and IL-18 lymphotoxin-beta. In addition, message levels and vascular surface expression of ICAM-1, VCAM-1, and MAdCAM-1 were all significantly enhanced in the colitic SCID mice reconstituted with CD45RBhigh T cells compared with SCID mice reconstituted with PBS or CD45RBlow T cells that did not develop disease. Significant increases in some of these ECAMs were also noted in the cecum and stomach and to a lesser degree in the small bowel. Our data confirm that reconstitution of SCID mice with CD45RBhigh but not CD45RBlow T cells induces chronic colitis, and that the colonic inflammation is associated with enhanced expression of proinflammatory cytokines and different ECAMs in the colon. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RBhigh T cells enhances ECAM expression in tissues distant from the site of active inflammation.

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.

Characterization of JOK-1, a human gastric epithelial cell line.

Human gastric epithelial cells were isolated from samples of human gastric lining and immortalized with simian virus 40 (SV40) to generate the stable human gastric epithelial cell line "JOK-l." These cells express conventional epithelial markers (vimentin, cytokeratin-18, occludin, N- and E-cadherins, beta-catenin, ZO-1, ZO-2, mucin, epithelial specific antigen) as well as SV40 large T-antigen. These cells rapidly externalized E-cadherin in response to acidic medium, and exhibited epithelial-like barrier properties that are also regulated by media pH. In contrast, the kidney epithelial cell line "MDCK" also expresses several epithelial markers (vimentin, cytokeratin-18, occludin, N- and E-cadherin, beta-catenin, ZO-1, ZO-2, epithelial specific antigen), but does not express mucin, or large T-antigen. However, MDCK rapidly internalize their E-cadherin from the cell surface and increase the solute flux in an acidic medium. These data suggest that the JOK-1 cell line is a potentially useful cell line for developing models of gastric epithelial function, development, and disease.

Diminished secondary CTL response in draining lymph nodes on cutaneous challenge with herpes simplex virus.

We have shown that C57BL/6-derived CD8(+) CTL specific for an immunodominant herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) determinant express a highly conserved Vbeta10/junctional sequence combination. This extreme T cell receptor beta-chain bias can be used to track the activation of gB-specific CTL in lymph nodes draining the site of HSV-1 infection. In this report we have examined the accumulation of gB-specific CTL in the primary and secondary or recall CTL responses to HSV-1 infection. We found that gB-specific cytolytic activity present within popliteal lymph nodes draining HSV-infected foot-pads peaked at day 5 post-infection during the primary response. As found previously, this correlates with the accumulation of Vbeta10(+)CD8(+) CTL in the activated T cell subset. Lymph node-derived cytotoxicity peaked between days 3 and 4 on secondary challenge with virus and, somewhat surprisingly, was considerably below that seen in the primary response. This reduced gB-specific cytolytic activity mirrored a near absence of Vbeta10(+)CD8(+) T cell enrichment found within the draining lymph nodes during this recall response, consistent with the overall diminution of gB-specific CTL accumulation in this site. Finally, there was a second wave of biased accumulation of Vbeta10(+)CD8(+) activated T cells within the popliteal lymph nodes well after the resolution of infection in both the primary and secondary responses. These results are discussed in terms of preferential activation of virus-specific memory T cells directly in infected tissues during a secondary CTL response at the expense of draining lymphoid organs.

Phenotypic identification of antigen-dependent and antigen-independent CD8 CTL precursors in the draining lymph node during acute cutaneous herpes simplex virus type 1 infection.

Optimal immunological control of cutaneous herpes simplex virus type 1 (HSV-1) infections initiated in the hind footpad of C57BL/6 (B6, H-2b) mice is dependent upon the presence of functional HSV-1-specific T lymphocytes. The class I MHC-restricted, CD8+ T cell subpopulation is involved in the clearance of infectious HSV-1 from the skin and limiting HSV-1 replication and spread within the peripheral nervous system. However, the frequency of HSV-1-specific CTL precursors (CTLp), as a measure of potential anti-viral CD8+ T cell function, is relatively low compared with other acute viral infections. To gain insight into the basis for this low functional frequency, changes in the CD8+ T cell subpopulation phenotype associated with activation and differentiation were investigated. Analysis of the phenotypic changes showed that HSV-1-specific CTLp were found predominantly within a subpopulation of CD8+ T cells expressing high levels of CD44 (CD44high) and high levels of the IL-2 receptor alpha-chain (CD25high). A second activated subpopulation of CD8+ T cells expressing the CD44high CD25low phenotype did not contain detectable HSV-1-specific CTLp, even after the addition of HSV-1-infected stimulator cells as a source of an exogenous Ag. These data suggested that HSV-1-specific CD8+ T cells must increase expression of CD25 before attaining the potential to become CTL effector cells. These findings also indicated that the up-regulation of CD44 alone is not sufficient to identify precisely HSV-1-specific CD8+ T cells.

Phenotypic characteristics associated with the acquisition of HSV-specific CD8 T-lymphocyte-mediated cytolytic function in vitro.

Class I MHC-restricted, HSV-1-specific CD8(+) cytolytic T lymphocyte (CTL) function is rarely detected in lymphocytes isolated directly from the lymph node draining the site of infection. However, culture in vitro for 24 to 72 h in the absence of exogenous antigen results in the development of easily detectable levels of HSV-1-specific CTL effectors. The inability to detect virus-specific CTL in HSV-1-infected mice is not well understood. However, since the in vitro culture of HSV-1-immune lymphocytes results in the transition to CTL function, studies of the changes occurring to the CD8(+) T cell subpopulation may provide important insights into the development of virus-specific CTL. Therefore, the phenotypic changes taking place in the CD8(+) population of T cells from draining popliteal lymph nodes of HSV-1-infected C57BL/6 (B6) mice were investigated, focusing on changes in the expression of cell surface markers associated with T lymphocyte activation. The results demonstrate an increase in the percentage of CD8(+) T cells expressing the activation markers CD44 and CD25 in parallel with the acquisition of HSV-specific CTL effector function. Cytolytic function was found exclusively within the CD8(+) CD44(hi) CD25(hi) fraction of cells in culture, but, surprisingly, was not detectable in CD8(+) CD44(hi) CD25(lo) T cells. This suggested that the acquisition of high levels of the high-affinity IL-2 receptor was closely linked to cytolytic function and may define an important developmental stage in the transition from noncytolytic to cytolytic effector cell. In support of this, CD8(+) CD25(hi) T cells isolated from the regional lymph node exhibited direct ex vivo cytolytic function, indicating that cytolytic effector cells were present in the lymph node, but must emigrate rapidly after attaining this level of differentiation.

Effects of in utero alcohol exposure on B cell development in neonatal spleen and bone marrow.

The effects of in utero alcohol exposure on neonatal lymphopoiesis were examined in a murine model of fetal alcohol syndrome. At birth, both immature and mature B cells were decreased in the spleens of neonatal animals and these subpopulations of B cells did not recover to normal levels until 3-4 weeks of life. Pre-B cells and total B cells were decreased as well in the bone marrow of ethanol-exposed animals. By 3-4 weeks of life, the number of B cells in the bone marrow recovered to normal levels, but the pre-B cells remained below normal levels through 5 weeks of age. Furthermore, a recently described early B cell progenitor was reduced in frequency in ethanol-exposed neonates. Together, these data suggest that in utero exposure to ethanol can result in abnormalities in B cell development that may initiate at an early stage of B cell development.

T-lymphocytes contribute to hepatic leukostasis and hypoxic stress induced by gut ischemia-reperfusion.

Although neutrophils have been implicated in the hepatic injury elicited by gut ischemia/reperfusion (I/R), the contribution of other leukocyte populations to this injury process remains unclear. The objective of this study was to determine whether lymphocytes contribute to gut I/R-induced microvascular dysfunction and inflammatory responses in the liver. Intravital videomicroscopy was used to monitor leukocyte recruitment, the number of nonperfused sinusoids and pyridine nucleotide (NADH) autofluorescence in livers of wild-type, SCID, and interferon-gamma (IFN-gamma) knockout mice exposed to 15 min of gut ischemia and 1 h of reperfusion. In wild-type mice, gut I/R elicited significant increases in the number of stationary leukocytes, nonperfused sinusoids, NADH autofluorescence (indicating hypoxia), and elevated plasma alanine aminotransferase (ALT) and TNF-alpha levels. All of these responses were profoundly attenuated in SCID mice, while only some of the responses (in the midzonal region) were blunted in IFN-gamma knockout mice. Reconstitution (24 h before ischemia) of the circulating lymphocyte pool with T-cell enriched splenocytes, but not T cell deficient (from nude mice), CD4+ T-cell depleted splenocytes or splenocytes derived from IFN-gamma knockout mice, allowed the SCID mice to respond to gut I/R in a manner similar to wild-type mice. Some of the responses were restored following reconstitution with CD8+ T-cell depleted splenocytes. These findings implicate CD4+ T-lymphocytes and IFN-gamma in the hepatic microvascular dysfunction and inflammatory cell accumulation elicited by gut I/R.

Protective immunity against equine herpesvirus type-1 (EHV-1) infection in mice induced by recombinant EHV-1 gD.

The ability of recombinant preparations of equine herpesvirus type 1 (EHV-1) glycoprotein D (gD) to elicit specific antibody and T lymphocyte responses in the BALB/c mouse model of respiratory infection was investigated. Recombinant gD (rgD) expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli elicited both high titer neutralizing antibody (nAb) and CD4 T cell proliferative responses following subcutaneous or intranasal immunization, but elicited only a weak antibody response after intraperitoneal immunization. Protection against respiratory tract infection with pathogenic EHV-1 RacL11 was observed in mice immunized subcutaneously with GST-gD. Furthermore, the degree of protection correlated to the titer of nAb and the T cell response observed. Finally, GST-gD was more effective in protecting against respiratory RacL11 infection if delivered intranasally. These results confirm that gD plays an important role in eliciting the protective immune response against EHV-1 infection, and indicate that subunit vaccines containing preparations of gD may be very effective if delivered directly to the upper respiratory tract.

Interleukin 7 induces TCR gene rearrangement in adult marrow-resident murine precursor T cells.

Rearrangement of the T cell antigen receptor genes is a complex, highly regulated process. To gain a better understanding of the extracellular factors involved in the regulation of TCR beta and gamma gene rearrangement in adult murine bone marrow-resident precursor T cells, several cytokines were tested for their ability to induce gene recombination. A selected population of C58/J bone marrow cells (Thy 1(low), CD3, CD8, B220) that is enriched for pre-T cell activity was propagated in vitro in medium supplemented with IL-3 and mast cell growth factor (MGF, also referred to as stem cell factor, Steele factor and c-kit ligand). These cytokines were required for the maintenance of pre-T cell activity in culture, but had no effect on TCR gene expression. Several additional cytokines were added to the culture medium. Of all those tested, only IL-7 induced complete rearrangement of the TCR gamma locus. Complete rearrangement of the TCR beta locus was not induced under any of the culture conditions analysed here. The bone marrow cells cultured in IL-3, MGF and IL-7 did not begin to express mature T cell proteins and maintained their in vivo progenitor potential. Furthermore, IL-7 cultured bone marrow cells were capable of differentiation in vivo into all phenotypic subpopulations of T cells, without an apparent bias toward the gammadelta lineage. The data presented here suggest that TCR gamma gene rearrangement in adult pre-T cells is regulated by IL-7, but that the TCR beta locus requires additional or alternative signals for the induction of complete rearrangement.

Synthesis and processing of equine herpesvirus 1 glycoprotein D.

Previous studies (C. C. Flowers and D. J. O'Callaghan, 1992, Virology 190, 307-315) employed peptide-specific antibodies to identify the product of the glycoprotein D (gD) gene of equine herpesvirus 1 strain Kentucky A (KyA). gD polypeptides of 55 and 58 kDa were detected in EHV-1-infected L-M cells, and the 58-kDa protein was observed in the membrane fraction of EHV-1 virions. In this report, the kinetics of synthesis and processing of gD polypeptides are described. One-hour pulse-labeling of EHV-1-infected L-M cells revealed that gD proteins are first detected at 6 hr after infection and that maximal synthesis of gD occurs between 5 and 8 hr postinfection. gD polypeptides accumulate progressively with time of infection as shown by immunoprecipitation analysis of gD proteins. Pulse-chase analysis of gD revealed that the 55-kDa protein is a precursor to the 58-kDa species and that processing of all pulse-labeled precursor protein requires approximately 2.5 hr. Analysis of the carbohydrate content of gD proteins, as judged by their sensitivity to digestion with endoglycosidases, revealed that the 55-kDa gD precursor contains high-mannose N-linked oligosaccharides, while the 58-kDa gD mature polypeptide possesses complex type oligosaccharides. Expression of the mature form of gD on the cell surface, as determined by fluorescent flow cytometric analysis, is delayed compared to the accumulation of the mature form of gD within the cell. The gD ORF encodes a potential protein of 442 amino acids but analysis of the translated sequence of gD indicated that the gD polypeptide is 392 amino acids, a size predicted by previous mapping of the transcription start site of the gD mRNA. Coupled in vitro transcription/translation of a pGEM-3Z construct containing the 392-amino-acid gD ORF, in the absence or presence of canine pancreatic microsomes, demonstrated that the 43-kDa gD polypeptide undergoes processing in vitro. These studies demonstrate that the EHV-1 strain KyA gD is processed in a fashion similar to that of the gD proteins of other alphaherpesviruses.

In utero exposure to ethanol affects postnatal development of T- and B-lymphocytes, but not natural killer cells.

The effect of intrauterine exposure to ethanol on lymphocyte development in the neonatal period was studied in C57BI/6J mice. Mice were bred, and then the female mice were assigned to 1 of 3 diet groups, 25% ethanol-derived calories (EDC), pair-fed control, or ad libitum laboratory chow. At birth, all offspring were cross-fostered to surrogate mothers who had been fed laboratory chow. At weekly intervals, the neonatal mice were weighed, and 4 mice from each group were used to assess the development of splenic lymphocytes. The total number of splenocytes was similar in all three groups at each sampling. The number of T-cells, B-cells, and natural killer (NK) cells was measured by flow cytometry. T-cells and NK cells did not vary significantly among the three diet groups. However, the total number of B-cells was decreased for the first 3 weeks of life in the ethanol-exposed animals. The function of the T-cells and B-cells was determined by assessing the response to lipopolysaccharide, pokeweed mitogen, phytohemagglutinin, and concanavalin A. The response to all four mitogens was significantly reduced in the ethanol-exposed animals and did not recover to control levels until 4-5 weeks of life. Ethanol exposure had no significant effect on the kinetics of acquisition of NK lytic function, as assessed by determining the killing of chromium-51 labeled YAC-1 tumor target cells. These data show that prenatal exposure to ethanol causes a transient immunodeficiency in some, but not all compartments of the immune system.

Expression of membrane-bound and secreted forms of equine herpesvirus 1 glycoprotein D by recombinant baculovirus.

Analyses of the synthesis and processing of recombinant full-length glycoprotein D of equine herpesvirus type 1 (EHV-1; gD392) or recombinant truncated gD (gD352) expressed in baculovirus-infected Sf9 cells revealed the following: (1) gD polypeptides encoded by both recombinant baculoviruses react with gD-specific antibodies including peptide-specific antiserum that neutralizes EHV-1 in a plaque reduction assay, (2) both the full-length recombinant gD392 and the truncated gD352 are expressed predominantly as gD species that contain high mannose-type oligosaccharides (55 kDa and 52 kDa, respectively), (3) both the full-length recombinant gD392 and the truncated gD352 are also expressed in lesser amounts as gD species that contain complex-type oligosaccharides (58 kDa and 55 kDa, respectively) as well as the unglycosylated forms of gD (43 kDa and 37 kDa, respectively), (4) flow cytometric analyses of cells expressing gD392 revealed that gD first appears on the cell surface at 24 h post infection; by 60 h, 95% of the cells express high levels of cell surface gD, (5) cells expressing gD352, in contrast to cells expressing gD392, secrete gD into the extracellular medium. This initial demonstration that immunoreactive EHV-1 glycoprotein D can be produced as a secreted polypeptide in the baculovirus system should provide reagents to assess the potential use of gD as a subunit vaccine in an animal model.

Analysis of the cytolytic T-lymphocyte response to herpes simplex virus type 1 glycoprotein B during primary and secondary infection.

The immune response to herpes simplex virus type 1 (HSV-1) infection in C57BL/6 mice includes a population of major histocompatibility complex class I-restricted cytolytic T lymphocytes (CTL) that recognize the structural glycoprotein gB. To gain insight into the importance of this CTL subpopulation in vivo, gB-specific CTL present in the regional lymph nodes after a primary infection and after a reinfection of convalescent animals were analyzed. In a primary infection, gB-specific CTL precursors (CTLp) that recognized either a cell line constitutively expressing gB or cells pulsed with the optimal Kb-restricted gB epitope 498SSIEFARL505 were present at an estimated frequency of 1/12,000 compared with a frequency of 1/3,000 for CTLp which recognized cells infected with HSV-1 itself. In convalescent mice responding to reinfection, HSV-specific CTLp were present at an estimated frequency of 1/4,000 to 1/14,000. However, gB-specific CTLp could not be detected at this site. These findings suggest that CTL specific for an immunodominant epitope contribute substantially to the primary response but may not be a component of the HSV-specific CTL population that responds rapidly to reinfection in vivo.

Control of acute cutaneous herpes simplex virus infection: T cell-mediated viral clearance is dependent upon interferon-gamma (IFN-gamma).

It is well established that T lymphocytes play a critical role in the control and clearance of herpes simplex virus (HSV) infections. However, the role of the CD4+ and CD8+ T cell subsets in the recovery process has not been clearly elucidated. Cutaneous HSV infection of the footpad tissue of C57BL/6 (B6) mice provides a model to determine the relative contribution of each T cell subset during the important early phase of the response to infection. In this study, we observed that the elimination of mature peripheral T lymphocytes by depletion in vivo with a combination of Cd4- and CD8-specific monoclonal antibodies prevented recovery from acute infection in this model. However, mice depleted of either the CD4+ or CD8+ subpopulation alone recovered completely, with only a slight delay in the total clearance of infectious virus. Adoptive transfer studies revealed that lymph node cells from donor mice selectively depleted of either CD4+ or CD8+ T cell subset in vivo, or from normal donors selectively depleted in vitro, were able to mediate recovery. As CD4-depleted mice fail to generate a CD8+ T cell-mediated cytolytic T lymphocyte (CTL) response, this suggested that the control of cutaneous HSV infections may be mediated by a cytokine-dependent mechanism common to both the CD4- and CD8+ T cell subpopulations. It was subsequently found that the neutralization of IFN-gamma in vivo diminished the ability of mice to clear infectious HSV from the skin, and treatment with anti-IFN-gamma in vivo ablated the ability of transferred T cells to mediate recovery. These studies suggested that IFN-gamma-mediated mechanisms play a critical role in the control of and recovery from acute cutaneous HSV infection.

The expression of CD4 by T cell precursors resident in both the thymus and the bone marrow.

A very small fraction of thymocytes has recently been identified that expresses low levels of CD4 in the absence of CD8, CD3, or a TCR. These CD4lo thymocytes appear to be the precursors of the early CD4-CD8-CD3- thymic subset and contain most of the T cell progenitor activity found within the thymus. Here, we examined adult bone marrow for the presence of a similar population of cells and found that 0.5% to 3.5% of C58/J bone marrow cells express low, but detectable levels of CD4 (CD4lo) at the cell surface in the absence of CD3. These CD4lo bone marrow cells display pre-T cell activity, in that they are able to repopulate the thymus of irradiated recipient mice after intrathymic transfer. Moreover, we found that most of pre-T cell activity found in the bone marrow is contained within the CD4lo expressing subset of marrow cells. Although the CD4lo cells found in both the thymus and bone marrow display pre-T cell activity, the CD4lo cells from these two sites showed pronounced differences with respect to their ability to respond to specific cytokine stimulation in vitro. Bone marrow-resident CD4lo cells proliferated in response to both IL-3 and mast cell growth factor in vitro, whereas CD4lo cells isolated from the thymus did not. Furthermore, CD4lo bone marrow cells, grown in media containing IL-3 and mast cell growth factor, retained their pre-T cell activity, indicating that CD4lo cells with pre-T cell capabilities were among the IL-3 and mast cell growth factor-responsive cells. These data suggest that although pre-T cells in bone marrow share the CD4lo phenotype with their intrathymic counterparts, they may be fundamentally different with respect to the environmental factors that control their growth.

Characterization of the progeny of precursor-T (pre-T) cells maintained in vitro by interleukin-3 (IL-3). Development of T-cell function in vivo.

We have recently described a bone marrow culture system which is able to maintain a portion of the precursor-T (pre-T) cell compartment of adult murine marrow in vitro, in the presence of interleukin-3 (IL-3), for at least 2 weeks. However, because growth in IL-3 might also induce the differentiation of the pre-T cells, it is necessary to determine the extent to which the developmental potential of the pre-T cells is altered during their residency in vitro. Previously, we analysed the progeny of cultured pre-T cells and compared their intrathymic development, their appearance in the periphery, and their V beta gene utilization to that of the progeny of fresh pre-T cells. Within these parameters, the cells derived from cultured marrow cells did not differ significantly from cells derived from fresh marrow cells. However, these studies did not allow us to determine the functional status of the T-cell progeny of cultured marrow. In the work presented here, we analysed the functional potential of T cells which were derived either from fresh pre-T cells or pre-T cells which had been maintained for 1 week in vitro. The T-cell mediated functions analysed included mitogen- and alloantigen-induced proliferation, IL-2 production, and generation of cytotoxic T cells. We found that the cultured pre-T cells were capable of giving rise to mature, immunocompetent T cells which did not differ significantly from the progeny of fresh pre-T cells in their functional potential.

Characterization of the progeny of pre-T cells maintained in vitro by IL-3: appearance in the periphery and V beta utilization in vivo.

We have recently demonstrated that bone marrow-resident cells, which are able to repopulate the thymus of irradiated recipient mice (pre-T cells), can be maintained in vitro for at least 2 weeks in the presence of exogenous IL-3. Because this marrow culture system can be applied to the study of early T cell differentiation, it is important to ascertain the extent to which in vitro culture of the pre-T cells might alter the T cell progeny which can develop from them. In previous work, we showed that the progeny of cultured pre-T cells appeared to develop in a kinetically normal fashion within the thymus of recipients and that the acquisition of key developmental markers (IL-2R and CD3) was identical in the progeny of fresh and cultured pre-T cells. Here, we report the results of experiments carried out to characterize the progeny of cultured pre-T cells which were found in the peripheral lymphoid tissues several weeks following intrathymic transfer to irradiated recipients. We found no remarkable differences between the progeny of cultured or fresh marrow cells with respect to the timing of their appearance in the periphery nor their expression of CD4 or CD8. By studying the patterns of utilization of five different V beta gene products by the T cells derived from fresh or cultured bone marrow, we were able to test the susceptibility of both sets of progeny to both positive and negative selection pressures during their in vivo maturation. These experiments established that the progeny of cultured marrow cells were equally susceptible to TCR repertoire selection, as were the progeny of fresh bone marrow cells, and that the process of in vitro growth did not alter the potential TCR repertoire of the pre-T cells.

Characterization of the progeny of pre-T cells maintained in vitro by IL-3: expression of the IL-2 receptor and CD3 during thymic development.

We have recently described a bone marrow culture system which is able to maintain, for at least 2 weeks, cells which have the capacity to repopulate the thymus of irradiated recipient mice (pre-T cells). Because this culture system depends upon the addition of an exogenous growth factor (IL-3) which may potentially influence the differentiation of the cultured pre-T cells, it is important to determine whether or not the progeny of cultured marrow cells are able to develop within the thymus in a kinetically normal fashion. Here we report the results of an analysis of the progeny of those cultured progenitor cells at 2, 3, and 4 weeks following intrathymic transfer. The passage of cultured donor-derived cells through critical early (expression of the IL-2 receptor) and late (expression of high levels of CD3) intrathymic events was assessed in these studies and compared with the pattern observed in the progeny of fresh bone marrow cells. The results of these studies showed that the progeny of cultured pre-T cells were able to develop expression of the IL-2 receptor and CD3 surface antigen during their residency within the thymus. In addition, both the timing and levels of expression of these surface markers were virtually identical on the progeny of fresh and cultured pre-T cells. These data suggest that cultured pre-T cells are not dramatically altered by their passage in vitro and are able to give rise to normally developing thymocytes upon in vivo transfer.