Differentiation of naive CTL to effector and memory CTL: correlation of effector function with phenotype and cell division

Phenotypically and functionally, the early steps of T cell differentiation are not well characterized. In addition, the effector T cell stage shares several phenotypic characteristics with memory T cells, which has made the analysis of T cell memory difficult. In this study, we have investigated in vitro and in vivo the differentiation of naive CTL into effector and memory CTL as a function of cell division using lymphocytic choriomeningitis virus-specific TCR-transgenic spleen cells labeled with the vital dye carboxyfluorescein diacetate, succinimidyl ester. The following major points emerged. 1) During the first nine cell divisions, the investigated cell surface markers were strongly modulated. 2) The TCR was stepwise down-regulated during viral infection. 3) Cytotoxic effector function was acquired within one cell division and was retained during the next four to five divisions. 4) In vitro, CTL reached a CD44highCD62L+ memory phenotype after 6-10 cell divisions and required restimulation to exert effector function. 5) Lymphocytic choriomeningitis virus memory mice contained two distinct memory populations: a CD44highCD62L- population, predominately located in the spleen and exerting rapid effector function, and a CD44highCD62L+ population found in the spleen and the lymph nodes, which had lost immediate effector function. This finding suggests that two types of memory CTL exist. The correlation between CD62L expression, effector function, and Ag persistence is discussed.     

Selective expansion of activated V delta 4+ cells during experimental infection of mice with Leishmania major

Previous work from this laboratory has revealed that infection of mice with Leishmania major leads to an expansion of gamma delta+ T cells in the spleen. Further examination of the gamma delta+ T cells expanding in infected mice has shown that the majority of these cells in the spleen, lymph nodes, blood and liver expressed the V delta 4 gene segment. Cell cycle analysis, using propidium iodide incorporation, demonstrated that while only 1% of alpha beta+ T cells in the spleen were in either S + G2/M phase, up to 10% of the gamma delta+ T cells were in cycling phase 8 weeks after infection. Comparison of the state of activation of the two populations in different organs after infection, confirmed that gamma delta+ T cells are actively dividing in lymph nodes, liver and blood, but not in the thymus or among intraepithelial lymphocytes. Examination of the expression of different activation markers on the surface of gamma delta+ T cells in the spleen of both normal and chronically infected BALB/c mice by FACS analysis, revealed increased expression of LFA-1, CD25, CD44, 4F2, CD28 and the heat-stable antigen, whereas Thy-1 and CD5 decreased. Collectively, these results suggest an oligoclonal expansion and activation of gamma delta+ T cells in response to L. major infection.     

Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated beta2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33-41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33-41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8(+) T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.     

Local immunity in lung-associated lymph nodes in a murine model of pulmonary histoplasmosis

Local immunity against acute pulmonary histoplasmosis was studied in the lung-associated lymph nodes of normal nonimmune mice infected intratracheally with live Histoplasma capsulatum yeasts. The phenotypes and distribution of cells in lung-associated lymph nodes and spleens were determined by flow cytometry. In addition, the immune responsiveness of these cells was evaluated by in vitro blastogenesis. Anti-H. capsulatum antibodies in serum and H. capsulatum antigen in tissue were measured by immunoassays. Cellular immune responses were greater in the lymph nodes than in the spleens. In lymph nodes 7 days after infection, a marked increase in the number of B lymphocytes caused the percentage to rise to 43%, compared with 26% in controls, and it remained elevated throughout the course of infection. A CD3+ cell that did not express CD4 or CD8 increased in number until it constituted 21% of lymph node cells, compared with 5% in controls, by day 14. The numbers of CD4+ and CD8+ T lymphocytes were modestly increased from days 7 to 35, but their percentages dropped because of the greater numbers of B lymphocytes and CD3+4-8- cells. Macrophages consistently constituted 2 to 3% of lymph node cells during the study. In spleens 7 days after infection, the percentage of macrophages in infected mice rose to 21%, compared with 9% in controls, but the total spleen cell number did not increase until day 14, when all cell subsets were nearly double in number. The in vitro blastogenic response of lymph node cells to H. capsulatum peaked at day 7, but spleen cell response was minimal during the course of infection. Histoplasma-specific serum immunoglobulin G antibodies reached peak levels by day 21 and remained high to the end of the study. In contrast, levels of antigen-specific immunoglobulin M antibodies were very low. These data suggest that antigen-specific immune responses occur in lung-associated lymph nodes and that this draining lymph node response may be an important component in host defense against Histoplasma lung infection.     

Viral immune evasion due to persistence of activated T cells without effector function

We examined the regulation of virus-specific CD8 T cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection of mice. Our study shows that within the same persistently infected host, different mechanisms can operate to silence antiviral T cell responses; CD8 T cells specific to one dominant viral epitope were deleted, whereas CD8 T cells responding to another dominant epitope persisted indefinitely. These virus-specific CD8 T cells expressed activation markers (CD69(hi), CD44(hi), CD62Llo) and proliferated in vivo but were unable to elaborate any antiviral effector functions. This unresponsive phenotype was more pronounced under conditions of CD4 T cell deficiency, highlighting the importance of CD8- CD4 T cell collaboration in controlling persistent infections. Importantly, in the presence of CD4 T cell help, adequate CD8 effector activity was maintained and the chronic viral infection eventually resolved. The persistence of activated virus-specific CD8 T cells without effector function reveals a novel mechanism for silencing antiviral immune responses and also offers new possibilities for enhancing CD8 T cell immunity in chronically infected hosts.     

A novel glycoprotein obtained from Chlorella vulgaris strain CK22 shows antimetastatic immunopotentiation

A glycoprotein extract (CVS), derived from the unicellular green alga Chlorella vulgaris, strain CK22, exhibited a pronounced antitumor effect against both spontaneous and experimentally induced metastasis in mice. Inhibition of tumor metastasis was enhanced when intratumor administration of CVS was followed by s.c. injection of CVS. Anti-metastatic immunopotentiation was observed in euthymic mice, but not in athymic nude mice. The antitumor activity of CVS was reflected in antigen-specific, T-cell-mediated immunity. Both CD4 and CD8 T cells contributed to the antimetastatic effects, as shown by in vivo depletion experiments with anti-T-cell subset antibodies. Furthermore, CVS caused the recruitment of T cells to the regional lymph nodes and their proliferation in these organs. The CD4-positive population, following CVS injection at the time of tumor rechallenge, displayed a pronounced increase in the proportion of T cells that were CD18 bright, CD44 bright, CD25+, CD54+, CD69+ or CD71+ in the lymph nodes. Thus, CVS induces T cell activation in peripheral lymph nodes in tumor-bearing mice. We conclude that CVS augments antimetastatic immunity through T cell activation in lymphoid organs and enhances recruitment of these cells to the tumor sites. Presurgical treatment with CVS might prevent metastasis or tumor progression.     

Chicken insulin-like growth factor binding protein (IGFBP)-5: conservation of IGFBP-5 structure and expression during evolution

Virus infections cause a much more profound perturbation of the lymphoid tissue than can be accounted for by the exigencies of the antigen-specific response. The extent of this 'immunological dissonance' is seen most dramatically in mice infected with a persistent gamma-herpesvirus, MHV-68. A profile of massive, continuing proliferation of both T and B cells in the lymph nodes and spleen leads to a dramatic increase in the prevalence of a CD62Llow CD8+ T cell subset in the blood, a pattern first detected two to three weeks after intranasal exposure to the inducing virus. This syndrome, which seems identical to human infectious mononucleosis (IM), persists for a further month or more. Part of the IM-like phase of MHV-68 infection reflects the selective expansion of Vbeta4+ CD8+ T cells, with the Vbeta4 effect being apparent for several different MHC class I H-2 types but not in mice that are deficient in MHC class II glycoprotein expression. Depleting CD4(+) T helper cells in MHV-68-infected mice leads to the decreased proliferation of the CD8+ T cells in the spleen and fewer CD62Llow CD8+ T lymphocytes than would be expected in peripheral blood, but fails to diminish the prominence of the V4beta+ CD8+ population. The results so far of this unique experimental mouse model of IM suggest that both cytokine-mediated effects and a viral superantigen are operating to promote the dramatic expansion and persistence of activated CD8+ T cells in the vascular compartment.     

Use of an automated fluorescent microsphere method to measure regional blood flow in the fetal lamb

OBJECTIVE: To investigate the source of the expanded blood CD8+ subsets during an acute primary simian immunodeficiency virus (SIV) infection of macaques and the potential role of these cells in disease progression. DESIGN AND METHODS: The primary CD8+ lymphocytosis, which occurs at 1-2 weeks following infection with SIVsmm/PBj-14, was examined in rhesus and cynomolgus macaques. Extensive subset analysis of the expanded blood CD8+ cell pool in a rhesus macaque was compared phenotypically with those in thymus, lymph nodes, spleen, ileum and lung washouts obtained at necropsy during blood lymphocytosis. The influence of the primary CD8+ cells expansion on disease progression was assessed at days 175-679 post-infection in long-term PBj-14 survivors staged according to immunological, virological and histopathological changes in their lymphoid organs. RESULT: The very rapid and transient blood lymphocytosis following infection consisted of two distinct CD45RA(low), CD8+ and CD28-, lymphocyte function-associated antigen (LFA)-1(high), CD45RA(high), CD8+ populations. These populations were present in low levels in thymus, lymph and spleen but were highly represented in mucosal tissues, such as long washout, in which CD28- LFA-1(high) CD45RA(high) CD8+ cells comprised 86% of CD8+ cells, and gut, which was predominantly CD45RA(low) CD28- CD8+ cells. A comparison of progressor and non-progressor PBj-14-infected rhesus and cynomolgus macaques also indicated that the existence or magnitude of a blood CD8+ lymphocytosis during the acute phase of infection did not by itself appear to influence or be predictive of disease progression. CONCLUSION: The marked blood CD8+ lymphocytosis observed during acute SIV infection did not result from expansion of virus-specific precursors in peripheral lymph node and did not appear to influence the rate of disease progression. The findings provide a novel explanation for the primary CD8+ cell lymphocytosis and invoke a mechanism whereby virus-induced cytokine/chemokine production in mucosal sites initiate the transient migration of a pre-existing CD8+ population into the blood from compartments such as lung and gut. Such results suggest that the magnitude of lymphocytosis may depend on the level of viral replication in mucosal tissues and the presence of other infections, for example, cytomegalovirus.     

Differences in the kinetics of T cell accumulations in C3H/HeN (Bcg-resistant) and C57BL/6 (Bcg-susceptible) mice infected with Mycobacterium paratuberculosis

The accumulation of various T cell subsets in Bcg-susceptible (C57BL/6) and- resistant (C3H/HeN) strains of mice were compared following an intraperitoneal infection with Mycobacterium paratuberculosis. Groups of mice from both strains were killed at 3, 5, 10, 15, 30, and 150 days after infection and lymphocytes were harvested from the peritoneal exudate cells (PEC), spleen, intestinal epithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), Peyer's patches, and mesenteric lymph node (MLN) and labelled with monoclonal antibodies to CD3, CD4, CD8, gamma delta TCR, CD25, and CD44 for flow cytometric analysis. Uninfected C3H/HeN mice had higher proportions of CD4+ cells in the spleen, MLN, LPL, IEL, and Peyer's patches, while uninfected C57BL/6 mice had higher proportions of CD8+ and/or gamma delta T cells. Significant increases in accumulation of CD8+ and gamma delta T cells were detected in the peritoneum and other tissues in both strains of mice after infection. Higher CD4/CD8 ratios were observed in most lymphoid tissues of C3H/HeN mice, while increased proportions of CD8+ and/or gamma delta T cells were present in C57BL/6 mice. These results indicate that significant differences in T cell profiles exist between these two strains of mice, both inherently and in response to infection with M. paratuberculosis. Innately lower levels of CD4+ cells and/or higher percentages of CD8+ and gamma delta T cells may play a role in the increased suspectibility of C57BL/6 mice to infection with M. paratuberculosis.     

Unexpected expansions of CD8-bearing cells in old mice

As mice age, spontaneous changes occur in the receptor repertoire of their T cells. The receptor repertoire of CD4+ T cells does not change with age. By contrast, however, the percentage of alpha beta+, CD8+ T cells bearing particular V elements varies considerably between individual aged mice, although it is remarkably consistent among individual young animals within a given strain. Changes of receptor V element use among CD8+ T cells in individual mice are unpredictable. However, when a large number of mice of the same strain is analyzed, strain-specific trends in V element skewing are found. Old C3H.SW and B10.BR mice have mono- or oligoclonal expansions of CD8+ T cells. These expansions of peripheral CD8+ T cells with age are probably due to deregulation of proliferation of individual CD8+ T cells after recognition of viral or environmental Ag, accompanied, perhaps, by partial transformation of particular T cell clones. Another phenomenon documented herein is the fact that the CD4/CD8 ratio drops steadily as a function of age. Shifts in CD4/CD8 ratio were not due to increased numbers of CD8+ T cells in spleen and lymph nodes, rather the CD4+ T cells disappeared from aging mice faster than CD8+ T cells.     

Recipient polyclonal B cell activation and immunoglobulin production induced by priming with a retroviral superantigen

The superantigen vSAG-7 (or MIs 1a) is a membrane glycoprotein encoded by the endogenous retrovirus mouse mammary tumor virus 7 (MMTV-7) and is highly stimulatory for V beta 6/CD4+ T cells. Priming of adult MMTV-7-negative mice with vSAG-7-expressing cells initially results in the activation of the peripheral V beta 6/CD4+ T cell compartment and is followed by T cell tolerance to the superantigen. During the course of tolerance induction the number of recipient B lymphocytes increases in the lymph nodes, but not the spleen, of vSAG-7-primed recipients. These B cells also express increased levels of class II MHC and present passively acquired superantigen. In this study we asked if these effects on the host B cell compartment are followed by the production of immunoglobulin. Priming of MMTV-7-negative BALB/c or CB.17 mice with vSAG-7-expressing cells from DBA/2 mice induced increases of both IgM and IgG2a in the serum. Use of Igh congenic CB.17 (IgMb) mice as recipients of the vSAG-7-presenting cells from DBA/2 (IgMa) donors indicated that the IgM and IgG produced were entirely of host origin. Priming with vSAG-7 also amplified (four- to fivefold) the antibody-producing cell response induced to a suboptimal dose of sheep RBC. Priming with purified B cells from vSAG-7 donors resulted in recipient V beta 6/CD4+ T cell activation and increased numbers of recipient B cells in the lymph nodes, but did not induce immunoglobulin production. In contrast, priming with purified CD8+ T cells resulted in increased quantities of serum IgM but not vSAG-7-reactive T cell activation or increased numbers of recipient B cells in the lymph nodes. These results indicate that the T cell activation and increased B cell number with upregulated class II MHC expression initially observed following vSAG-7 priming of adult MMTV-7-negative recipients are not linked to the induction of the recipient-derived immunoglobulin production.     

Role of Mitf in differentiation and transdifferentiation of chicken pigmented epithelial cell

A recombinant H-2Kb transgene, GK, containing the human HLA-G gene promoter is expressed throughout the trophoblast when inherited paternally. Male GK transgenic mice were mated with female T-cell receptor (TCR) transgenic mice to assess the effect of fetal H-2Kb expression on maternal H-2Kb-specific CD8+ T-cells during pregnancy. The number of maternal H-2Kb-specific CD8+ T-cells in spleen increased significantly (approximately 3-fold) 10 days post coitus when the GK transgene was inherited from the father. A smaller (approximately 2-fold) increase was observed in the spleen of pregnant females mated with C57BL/10 (H-2b) males. No increase was observed in mothers mated to syngeneic male mice. In both cases where expanded cohorts of maternal CD8+ T-cells were observed the amount of surface CD8 and to a lesser extent, TCR molecules was reduced. No change in the amount of surface CD44 or CD45RB was detected when levels were compared with naive T-cells from control virgin female mice. Expanded cohorts of CD8+ T-cells were also detected in para-aortic and inguinal lymph nodes draining the uterus but no changes were observed in mesenteric lymph nodes. This study concludes that maternal CD8+ T-cells are exposed to paternally inherited fetal MHC class I antigens during pregnancy. Moreover, the phenotype of the CD8+ T-cells in maternal spleen and lymph nodes that drain the uterus is not typical of activated, antigen-experienced T-cells suggesting that contact with fetal H-2Kb molecules induces a state of functional unresponsiveness.     

The induction of in vivo proliferation of long-lived CD44hi CD8+ T cells after the injection of tumor cells expressing IFN-alpha1 into syngeneic mice

The tumorigenicity of transplantable tumor cells in mice is reduced by transduction with cytokine genes, including IFN-alpha and interleukin (IL) 12. Although T cells are considered important in tumor rejection, the mechanism by which genetically modified tumor cells stimulate the immune system has not been examined. In this study, the in vivo proliferation of T-cell subsets in mice transplanted with cytokine-producing syngeneic tumor cells was assessed by administering the DNA precursor bromodeoxyuridine. The injection of viable cells producing IFN-alpha or IL-12 caused a marked proliferation of CD8+ T lymphocytes in both the spleen and lymph nodes. Proliferation was most prominent among memory-phenotype CD44hi CD8+ T cells. In contrast, proliferation of CD8+ T cells did not occur in mice injected with control cells or with cells expressing IL-4, granulocyte colony-stimulating factor, or IFN-gamma. Pulse-chase studies in mice injected with IFN-alpha-producing cells showed that a proportion of proliferating CD8+ T cells survived for at least 70 days, suggesting that long-lived memory cells are induced using such an approach. In summary, these results, together with previous studies on the host immune reactivity triggered by the injection of tumor cells expressing IFN-alpha, represent a strong rationale for considering IFN-alpha as a powerful T-cell adjuvant for the generation of more effective cancer vaccines.     

Cytotoxic reactivity of gut lamina propria CD4+ alpha beta T cells in SCID mice with colitis

Polyclonal, mucosa-seeking memory/effector CD4+ T cells containing a large fraction of blasts activated in situ accumulate in the gut lamina propria of severe-combined immunodeficient (SCID) mice developing colitis after CD4+ T cell transplantation. CD4+ T cells isolated from different repopulated lymphoid tissues of transplanted SCID mice proliferate in vitro in the presence of interleukin (IL)-2 + IL-7. CD3 ligation enhances this cytokine-supported proliferation in CD4+ T cells from the spleen and the mesenteric lymph node of transplanted SCID mice; CD3 ligation suppresses the cytokine-supported proliferation in CD4+ T cells from the gut lamina propria in a cell density- and dose-dependent manner. Almost all CD4+ T cells from repopulated lymphoid tissues of transplanted SCID mice express CD95 (Fas) on the cell surface, and a large fraction of CD4+ T cells from the gut lamina propria of transplanted SCID mice express the Fas ligand on the surface. Gut lamina propria CD4+ T cells show Fas-dependent cytotoxicity. A large fraction of gut lamina propria CD4+ T cells that infiltrate the inflamed colon in transplanted SCID mice are activated in situ and many CD4+ T cells are apoptotic. Hence, a large fraction of colitis-inducing CD4+ T cells undergo activation-induced cell death in situ and can damage other cells through Fas-dependent cytotoxicity.     

Left ventricular wall thickening does occur in elite power athletes with or without anabolic steroid Use

Monoclonal antibodies (mAbs) against bovine leukocyte antigens specific for T cells (CD2, CD4, CD8 and gammadelta receptor) and B cells (surface IgM) were used in samples from one week and one-, three- and seven-month-old goats to study the evolution of lymphocyte subsets by flow cytometry in peripheral blood and the lymphoid organs: thymus, jejunal (JPP) and ileal (IPP) Peyer's patches, spleen and mesenteric lymph nodes. An increase in the values of alpha/beta receptor T cells with age was recorded whereas the gammadelta receptor T cells fell in number. In peripheral blood and in all tissues, except IPP the values for B cells (sIgM+) were low. The CD4+ and CD8+ cells predominated in JPP while B cells were the most important subpopulation in IPP. In the spleen, as in JPP, the CD4/CD8 ratio was less than one and the gammadelta T cells values were high. In mesenteric lymph nodes, CD8+ and B(sIgM) cells predominated in the youngest animals.     

Migration of lymphoid cells from vaginal epithelium to iliac lymph nodes in relation to vaginal infection by herpes simplex virus type 2

To determine whether lymphocytes and Langerhans cells in vaginal epithelium are migratory, we stained mouse vaginal epithelium, including its lymphoid cells, by intraluminal administration of H33342, a fluorescent, vital dye. Stromal staining was superficial, and no free dye reached the iliac lymph nodes. The numbers and phenotypes of H33342-stained cells that migrated from the vagina to the iliac lymph nodes during the next 48 h were determined in four groups: normal mice, mice infected intravaginally with wild-type herpes simplex virus type 2 (HSV-2), mice that were immune to vaginal HSV-2 infection, and immune mice that received vaginal challenge with HSV-2. H33342-stained cells migrated from the vaginal epithelium to the iliac lymph nodes in all groups and were mainly Thy-1.2+ cells and B220+ cells. The number of migrating Thy-1.2+ cells was similar to the sum of CD4+ and CD8+ cells in all groups and was not significantly different from the number of CD44+ cells, suggesting that most of the migrating T cells were memory cells. B lymphocytes comprised 31, 32, 43, and 68% of the migrating cells in the four groups, respectively. We found no evidence that Langerhans cells or macrophages were migrating. Thus, most MHC class II+ cells in all groups were accounted for by B cells, and migrating cells did not express B7.1 or F4/80 or exhibit indented nuclei or dendritic processes. We suggest that the migrating T cells and B cells probably belonged to a pool of lymphocytes that recirculates from blood to tissues and back to the lymph nodes via their afferent lymphatics.     

Flow-microfluorometric monitoring of oligoclonal CD8+ T cell responses to an immunodominant Moloney leukemia virus-encoded epitope in vivo

The TCR repertoire of CD8+ T cells specific for Moloney murine leukemia virus (M-MuLV)-associated Ags has been investigated in vitro and in vivo. Analysis of a large panel of established CD8+ CTL clones specific for M-MuLV indicated an overwhelming bias for V beta4 in BALB/c mice and for V beta5.2 in C57BL/6 mice. These V beta biases were already detectable in mixed lymphocyte:tumor cell cultures established from virus-immune spleen cells. Furthermore, direct ex vivo analysis of PBL from BALB/c or C57BL/6 mice immunized with syngeneic M-MuLV-infected tumor cells revealed a dramatic increase in CD8+ cells expressing V beta4 or V beta5.2, respectively. M-MuLV-specific CD8+ cells with an activated (CD62L-) phenotype persisted in blood of immunized mice for at least 2 mo, and exhibited decreased TCR and CD8 levels compared with their naive counterparts. In C57BL/6 mice, most M-MuLV-specific CD8+ CTL clones and immune PBL coexpressed V alpha3.2 in association with V beta5.2. Moreover, these V beta5.2+ V alpha3.2+ cells were shown to recognize the recently described H-2Db-restricted epitope (CCLCLTVFL) encoded in the leader sequence of the M-MuLV gag polyprotein. Collectively, our data demonstrate a highly restricted TCR repertoire in the CD8+ T cell response to M-MuLV-associated Ags in vivo, and suggest the potential utility of flow-microfluorometric analysis of V beta and V alpha expression in the diagnosis and monitoring of viral infections.     

Activation and 'deletion' of self-reactive mature and immature T cells during ontogeny of Mls-1a mice: implications for neonatal tolerance induction

We assessed the kinetics of V beta 6+ T cell elimination in the lymph nodes and thymus during Mls-1a mouse ontogeny. Our results suggest that induction of tolerance to Mls-1a antigens involves mechanisms other than clonal deletion of immature T cells in the thymus. Mature CD4+CD8- (CD4SP) T cells were affected by Mls-1a antigens earlier than immature thymocyte populations. Up to 2 weeks after birth, reduced frequencies of V beta 6+ T cells were detected only in CD4SP cells from the thymus and lymph nodes, and generation of CD4SP cells in the thymus was blocked at least 1 week earlier than that of their CD4+CD8loTCRhi immature precursors. The number of V beta 6+CD4SP T cells increased during the first 2 weeks of life and remained constant thereafter. We thus found no evidence of deletion of mature V beta 6+CD4SP T cells, as the reduced frequencies in adult mice can be attributed to the dilution of previously generated cells in lymphoid organs of growing mice, which increase in cellularity after birth. V beta 6+CD4+ T cells were activated in vivo shortly after birth, as shown by a selective increase in IL-2 receptor alpha chain expression in the thymus and lymph nodes from day 0 to day 2 after birth. It is therefore likely that endogenous expression of Mls-1a antigen shortly after birth activates V beta 6+CD4SP T cells and renders them anergic. This process of tolerance induction may precede the clonal deletion of immature T cells in the thymus, described in the adult mouse.     

Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent infection with lymphocytic choriomeningitis virus

Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8+, but not of CD4+, T cells and accelerated by increasing the frequency of LCMV-specific CD8+ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8+ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8+ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P0/0 and P0/0 x TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-alpha and interferon (IFN)-gamma produced by CD8+ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-gamma-producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-gamma-producing CD8+ T cells. Thus, in the absence of IFN-gamma and/or TNF/LT-alpha, exhaustion of virus-specific T cells was not hampered.