Developmental regulation of V(D)J recombination and lymphocyte differentiation.

Recent insights into the mechanism of V(D)J recombination have clarified the direct role of the products of the recombination-activating genes Rag-1 and Rag-2 in site-specific DNA cleavage at recombination signal sequences and have identified components of the general DNA double-strand break repair pathway that participate in the rejoining of the Rag-1 and Rag-2-cut receptor gene segments. The V(D)J reaction is restricted to particular antigen receptor loci in a lineage-specific and stage-specific manner. This specificity appears to involve cis-regulatory elements, some of which also regulate transcription of the germline antigen receptor loci. Early developmental steps in the T and B lineages - including phenotypic differentiation, expansion of precursors, and selection processes - are effected in a stepwise fashion by signals generated, at least in part, by the products of the functionally rearranged antigen receptor genes themselves.

Genetic pathway to recurrent chromosome translocations in murine lymphoma involves V(D)J recombinase.

Chromosome translocations involving antigen receptor loci are a genetic hallmark of non-Hodgkin's lymphomas in humans. Most commonly, these translocations result in juxtaposition of the immunoglobulin heavy-chain (IgH) locus with one of several cellular proto-oncogenes, leading to deregulated oncogene expression. The V(D)J recombinase, which mediates physiologic rearrangements of antigen receptor genes, may play a mechanistic role in some lymphoma translocations, although evidence is indirect. A high incidence of B-lineage lymphomas has been observed in mice with severe combined immunodeficiency (SCID) and p53-null mutations. We show that these tumors are characteristic of the pro-B-cell stage of development and that they harbor recurrent translocations involving chromosomes 12 and 15. Fluorescence in situ hybridization (FISH) shows retention of IgH sequences on the derivative chromosome 12, implying that breakpoints involve the IgH locus. Pro-B-cell lymphomas were suppressed in SCID p53(-/-) mice by a Rag-2-null mutation, demonstrating that DNA breaks generated during V(D)J recombination are required for oncogenic transformation, and suggesting that t(12;15) arise during attempted IgH rearrangement in pro-B cells. These studies indicate that the oncogenic potential inherent in antigen receptor diversification is controlled in vivo by efficient rejoining of DNA ends generated during V(D)J recombination and an intact cellular response to DNA damage.

Analysis of ku80-mutant mice and cells with deficient levels of p53.

Absence of Ku80 results in increased sensitivity to ionizing radiation, defective lymphocyte development, early onset of an age-related phenotype, and premature replicative senescence. Here we investigate the role of p53 on the phenotype of ku80-mutant mice and cells. Reducing levels of p53 increased the cancer incidence for ku80(-/-) mice. About 20% of ku80(-/-) p53(+/-) mice developed a broad spectrum of cancer by 40 weeks and all ku80(-/-) p53(-/-) mice developed pro-B-cell lymphoma by 16 weeks. Reducing levels of p53 rescued populations of ku80(-/-) cells from replicative senescence by enabling spontaneous immortalization. The double-mutant cells are impaired for the G(1)/S checkpoint due to the p53 mutation and are hypersensitive to gamma-radiation and reactive oxygen species due to the Ku80 mutation. These data show that replicative senescence is caused by a p53-dependent cell cycle response to damaged DNA in ku80(-/-) cells and that p53 is essential for preventing very early onset of pro-B-cell lymphoma in ku80(-/-) mice.

Biology of the interleukin-2 receptor.

Studies of the biology of the IL-2 receptor have played a major part in establishing several of the fundamental principles that govern our current understanding of immunology. Chief among these is the contribution made by lymphokines to regulation of the interactions among vast numbers of lymphocytes, comprising a number of functionally distinct lineages. These soluble mediators likely act locally, within the context of the microanatomic organization of the primary and secondary lymphoid organs, where, in combination with signals generated by direct membrane-membrane interactions, a wide spectrum of cell fate decisions is influenced. The properties of IL-2 as a T-cell growth factor spawned the view that IL-2 worked in vivo to promote clonal T-cell expansion during immune responses. Over time, this singular view has suffered from increasing appreciation that the biologic effects of IL-2R signals are much more complex than simply mediating T-cell growth: depending on the set of conditions, IL-2R signals may also promote cell survival, effector function, and apoptosis. These sometimes contradictory effects underscore the fact that a diversity of intracellular signaling pathways are potentially activated by IL-2R. Furthermore, cell fate decisions are based on the integration of multiple signals received by a lymphocyte from the environment; IL-2R signals can thus be regarded as one input to this integration process. In part because IL-2 was first identified as a T-cell growth factor, the major focus of investigation in IL-R2 signaling has been on the mechanism of mitogenic effects in cultured cell lines. Three critical events have been identified in the generation of the IL-2R signal for cell cycle progression, including heterodimerization of the cytoplasmic domains of the IL-2R beta and gamma(c) chains, activation of the tyrosine kinase Jak3, and phosphorylation of tyrosine residues on the IL-2R beta chain. These proximal events led to the creation of an activated receptor complex, to which various cytoplasmic signaling molecules are recruited and become substrates for regulatory enzymes (especially tyrosine kinases) that are associated with the receptor. One intriguing outcome of the IL-2R signaling studies performed in cell lines is the apparent functional redundancy of the A and H regions of IL-2R beta, and their corresponding downstream pathways, with respect to the proliferative response. Why should the receptor complex induce cell proliferation through more than one mechanism or pathway? One possibility is that this redundancy is an unusual property of cultured cell lines and that primary lymphocytes require signals from both the A and the H regions of IL-2R beta for optimal proliferative responses in vivo. An alternative possibility is that the A and H regions of IL-2R beta are only redundant with respect to proliferation and that each region plays a unique and essential role in regulating other aspects of lymphocyte physiology. As examples, the A or H region could prove to be important for regulating the sensitivity of lymphocytes to AICD or for promoting the development of NK cells. These issues may be resolved by reconstituting IL-2R beta-/-mice with A-and H-deleted forms of the receptor chain and analyzing the effect on lymphocyte development and function in vivo. In addition to the redundant nature of the A and H regions, there remains a large number of biochemical activities mediated by the IL-2R for which no clear physiological role has been identified. Therefore, the circumstances are ripe for discovering new connections between molecular signaling events activated by the IL-2R and the regulation of immune physiology. Translating biochemical studies of Il-2R function into an understanding of how these signals regulate the immune system has been facilitated by the identification of natural mutations in IL-2R components in humans with immunodeficiency and by the generation of mice with targeted mutations in these gen

V(D)J rearrangement in Nijmegen breakage syndrome.

Repair of DNA double-strand breaks is essential for maintenance of genomic stability, and is specifically required for rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) loci during development of the immune system. Abnormalities in these repair processes also contribute to oncogenic chromosomal rearrangements that underlie many lymphoid malignancies. Nijmegen breakage syndrome (NBS) is a rare autosomal recessive condition characterized by immunodeficiency, radiation sensitivity, and increased predisposition to lymphoid cancers bearing oncogenic Ig and TCR locus translocations. NBS patients fail to produce nibrin, a protein required for the nuclear localization and function of a DNA repair complex that includes Mre11 and Rad50. Mre11 has biochemical properties that suggest a potential role in V(D)J recombination. We studied V(D)J recombination in NBS cells in vitro and in vivo, using cell lines and peripheral blood leukocyte DNA from NBS patients. We found that NBS cells were competent to rejoin signal substrates with normal efficiency and high fidelity. Coding substrates were similarly rejoined efficiently, and coding end structures appeared normal. In B cells from NBS patients, the spectrums of IgH CDR3 regions were diverse and normally distributed. Moreover, the lengths and composition of Igkappa VJ joins and IgH VDJ joins derived from NBS and normal subjects were indistinguishable. Our data indicate that nibrin plays no essential role in V(D)J recombination and is not required for the generation of an apparently diverse B cell repertoire.

Expression of lymphocyte adhesion receptors for high endothelium in primates. Anatomic partitioning and linkage to activation.

Glycoproteins of 90 to 95 kD Mr are involved in adhesion to high endothelium and migration into secondary lymphoid tissues in several species. Recent evidence indicates that in primates, one type of these molecules may be subsumed under the CD44 grouping of lymphocyte differentiation Ag. Flow cytometric analysis of circulating macaque lymphocytes for expression of these structures revealed a prominent bimodal distribution, defining two subpopulations with a 5- to 10-fold difference in immunofluorescence staining intensity. Studies of lymphocytes from different anatomic compartments revealed marked differences in the relative numbers of these two phenotypes: splenic and peripheral blood lymphocytes were mostly CD44hi, whereas thoracic duct lymphocytes, which have recently exited lymph nodes and Peyer's patches, were predominantly CD44lo, suggesting that these subsets may have different migratory behavior in vivo. Activated lymphocytes, as defined by light scatter measurements, expression of IL-2-R, and CD45R levels, were all CD44hi. Simultaneous three parameter flow cytometric determination of CD44 and CD45R expression, and cell-cycle analysis with 7-amino actinomycin D further demonstrated that intrinsically cycling cells were contained in the CD44hi, CD45R+ subset. After stimulation of CD44lo lymphocytes in vitro with cross-linked anti-CD3 antibodies and PMA, CD44 expression markedly increased. These data indicate that CD44 up-regulation is an early event in T lymphocyte activation in vivo, and support the hypothesis that patterns of lymphocyte traffic are regulated in concert with cell activation, possibly by differential expression of CD44.

Chimeric del20q in a case of chronic neutrophilic leukemia.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative disorder in which recurrent abnormalities of chromosome 20 have been reported. We report the case of a 76-year-old woman with CNL with partial deletion of the long arm of chromosome 20 in a subset of bone marrow metaphases, suggesting coexistence of a clonal stem cell disorder and normal hematopoiesis. Review of the literature suggests that such mosaicism is common in CNL, possibly accounting for the favorable prognosis observed in many patients with this disorder.

Regulation of lymphoid homeostasis by IL-2 receptor signals in vivo.

High-affinity IL-2R signals are required for peripheral lymphoid homeostasis in vivo. We found that CD25 was required for regulation of peripheral T cells in mice bearing either the DO11.10 MHC class II-restricted TCR transgene or an Iabeta-null mutation, suggesting that MHC class I- and class II-dependent T cell subsets are regulated independently by IL-2R signals. In contrast, deregulation of serum IgG1 levels in CD25-/- mice was dependent on CD4+ T cells. T cell expansion in DO11.10 CD25-/- mice was not preferential for cells escaping allelic exclusion by the TCR transgene, but was suppressed by a Rag-2-null mutation. Together, these findings suggest that endogenous TCR are required to trigger T cell expansion, but that CD25 regulates T cells activated by low-specificity signals. Expansion of DO11.10 T cells in response to cognate Ag was modestly reduced in CD25-/- T cells transferred into the normal lymphoid compartments of BALB/c mice. Moreover, activation-induced clonal contraction and apoptosis in vivo were intact in the absence of CD25. These data indicate that the regulatory role of high-affinity IL-2R signals extends beyond the control of Ag-specific responses and suggest a role for these signals in control of bystander T cell activation.

Methods of stem cell mobilization.

The rapid rescue of hematopoiesis following transplantation of peripheral blood stem cells (PBSC) has facilitated expanded application of high-dose chemotherapy regimens for several malignancies. A variety of regimens have been described to enhance the circulation of PBSC, including the use of hematopoietic growth factors, either alone or following myelosuppressive chemotherapy. Such improvements of PBSC mobilization can increase the number of hematopoietic progenitors for transplantation and reduce the number of leukapheresis procedures required. We review several approaches to the collection of PBSC, including our experience with the use of cytokines in addition to chemotherapy. In addition, we discuss the investigation of novel cytokine combinations and in vitro expansion techniques, which may lead to further improvements in both the efficiency of PBSC collection and hematopoietic recovery following transplantation.

Promoter element for transcription of unrearranged T-cell receptor beta-chain gene in pro-T cells.

The hallmark of T- and B-lymphocyte development is the rearrangement of variable (V), diversity (D), and joining (J) segments of T-cell receptor (TCR) and immunoglobulin (Ig) genes to generate a diverse repertoire of antigen receptor specificities in the immune system. The process of V(D)J recombination is shared in the rearrangement of all seven antigen receptor genes and is controlled by changes in chromatin structure, which regulate accessibility to the recombinase apparatus in a lineage- and stage-specific manner. These chromatin changes are linked to transcription of the locus in its unrearranged (germline) configuration. To understand how germline transcription of the TCRbeta-chain gene is regulated, we determined the structure of germline transcripts initiating near the Dbeta1 segment and identified a promoter within this region. The Dbeta1 promoter is active in the presence of the TCRbeta enhancer (Ebeta), and in this context, exhibits preferential activity in pro-T versus mature T-cell lines, as well as T- versus B-lineage specificity. These studies provide insight into the developmental regulation of TCRbeta germline transcription, one of the earliest steps in T-cell differentiation.

T cell growth cytokines cause the superinduction of molecules mediating antigen-induced T lymphocyte death.

TCR stimulation of T lymphocytes that are activated and cycling in the presence of IL-2 leads to programmed cell death. We now show that this effect is at least partly attributable to the ability of IL-2 to dramatically increase the expression of mRNAs encoding ligands and receptors that mediate apoptosis. We also found that cyclosporin was not able to fully inhibit the TCR induction of death molecule mRNAs or TCR-induced apoptosis, although it could completely turn off IL-2 expression. The effect growth cytokines was further explored in T cells derived from mice bearing a homozygous deficiency of the IL-2R alpha-chain. We found that IL-2Ralpha-/- cells were resistant to death if IL-2 was used to induce apoptosis susceptibility, but that large amounts of other T cell growth cytokines, such as IL-4 and IL-7, could induce cell cycle progression and promote TCR-induced apoptosis. However, our findings suggest that autoimmunity and lymphoproliferation in IL-2Ralpha-/- mice can result from the loss of IL-2 stimulated feedback apoptosis and that other growth cytokines are not produced at levels sufficient to compensate for this deficit.

Simian immunodeficiency virus is restricted to a subset of blood CD4+ lymphocytes that includes memory cells.

HIV and the related simian immunodeficiency virus (SIV), which causes AIDS in macaques, infect only a small percentage of CD4+ lymphocytes at any point during the disease. We have identified three distinct cellular phenotypes within the CD4+ subpopulation in macaques, based on cell surface expression of CD44 and CD45R, which putatively represent successive stages of postthymic proliferation and functional maturation. Two of these subsets, CD44hi CD45R+, which contained virtually all circulating cells in cycle, and CD44hi CD45R-, which was noncycling and has been linked to immunologic memory, were selectively depleted in SIV-infected animals at an asymptomatic stage of disease. To test whether SIV infection was restricted to cells with this phenotype in vivo, we used the polymerase chain reaction to sensitively detect SIV DNA in purified subpopulations of CD4+ lymphocytes. We found that SIV exclusively infected blood lymphocytes expressing high levels of CD44. Within this subset infection occurred not only in the fraction containing actively proliferating cells (CD45R+), but also in resting, putative memory cells (CD45R-). These data directly demonstrate that cellular maturation stages of normal postthymic T lymphocyte differentiation are important factors in permitting lentivirus infection in vivo, and that noncycling, memory T cells may be a reservoir for SIV.

Functional responses and apoptosis of CD25 (IL-2R alpha)-deficient T cells expressing a transgenic antigen receptor.

IL-2 was initially defined as a T lymphocyte growth factor, but recent studies have provided evidence that it may also play a role in regulating T cell differentiation, apoptosis, and tolerance. To examine the contribution of IL-2 to these processes, we have bred a class II-restricted TCR transgene into mice deficient in the alpha-chain of the IL-2R, CD25. We show that in response to Ag, T cells from these mice are unable to use IL-2 and, as a result, are less efficient at traversing the cell cycle, and proliferate less than wild-type cells. Furthermore, CD25 -/- T cells exhibit reduced survival in vitro, even in the presence of costimulatory signals. IL-4 and IL-15, a cytokine related to IL-2, enhance the survival and Ag-induced proliferation of CD25 -/- T cells. Activated CD25 -/- T cells are resistant to Fas-mediated activation-induced cell death (AICD), and this defect cannot be corrected by other cytokines. Therefore, IL-2 plays a unique role in regulating AICD, but has redundant roles in T cell survival and proliferation in vitro. The failure of AICD observed with CD25 -/- T cells may explain the unexpected observation that deficiency of IL-2 or of the alpha- or beta-chain of the IL-2R results not in immunodeficiency, but in autoimmune disease.

Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment.

Interleukin-2 receptor alpha chain (IL-2R alpha) expression occurs at specific stages of early T and B lymphocyte development and is induced upon activation of mature lymphocytes. Young mice that lack IL-2R alpha have phenotypically normal development of T and B cells. However, as adults, these mice develop massive enlargement of peripheral lymphoid organs associated with polyclonal T and B cell expansion, which, for T cells, is correlated with impaired activation-induced cell death in vivo. Older IL-2R alpha-deficient mice also develop autoimmune disorders, including hemolytic anemia and inflammatory bowel disease. Thus, IL-2R alpha is essential for regulation of both the size and content of the peripheral lymphoid compartment, probably by influencing the balance between clonal expansion and cell death following lymphocyte activation.

Association between cervical inflammation and cervical shedding of human immunodeficiency virus DNA.

A cross-sectional study was conducted among prostitutes in Nairobi, Kenya, to determine the prevalence and correlates of cervical human immunodeficiency virus (HIV) DNA. Ninety-two HIV-seropositive prostitutes were evaluated during 137 clinic visits. Cervical HIV DNA was detected by polymerase chain reaction assay in 36 (39%) women at initial visits and in 40 (44%) women at any visit. There was a significant correlation between cervical HIV and microscopic evidence of cervical inflammation (odds ratio [OR], 7.2; 95% confidence interval [CI], 2.1-24.6). Using multivariate analysis to adjust for possible confounding, the adjusted OR for the association between cervical inflammation and cervical HIV DNA was 8.7 (95% CI, 2.0-37.2). Conditions associated with cervical inflammation are associated with the detection of HIV proviral DNA. Whether such conditions lead to increased infectivity remains to be proven.

Human immunodeficiency virus infection among high-risk seronegative prostitutes in Nairobi.

To determine the frequency and duration of antibody-negative human immunodeficiency virus (HIV) infection among heterosexually exposed African women, 56 HIV-seronegative female prostitutes in Nairobi were studied. Polymerase chain reaction (PCR) was used to detect HIV DNA in peripheral blood at enrollment, and women were followed prospectively with serologic testing to determine HIV seroincidence. Six women (11%) were infected with HIV by PCR criteria at enrollment. Seroconversion occurred in 5 of these subjects within 1-12 months, while the sixth remained seronegative when last evaluated at 5 months. The cumulative annual seroconversion rate in the entire cohort was 38%. Using maximum likelihood analysis, the mean interval between HIV infection and seroconversion was estimated to be between 3 and 4 months, similar to that described for homosexual men and blood product recipients in the United States. Prolonged HIV infection in the absence of antibodies appears to be uncommon in this setting.

Selective replication of simian immunodeficiency virus in a subset of CD4+ lymphocytes.

Although all CD4+ cells theoretically are at risk for infection by human immunodeficiency viruses or the related simian immunodeficiency viruses found in Old World monkeys, only a small proportion of CD4+ lymphocytes from infected individuals have detectable virus. This suggests that immunodeficiency viruses may replicate predominantly in a minor subset or activated form of CD4+ T cells, a possibility we examined in macaques infected with a simian immunodeficiency virus isolate, SIV/Mne. Macaque CD4+ lymphocytes could be divided into two subtypes that differed in their level [high (hi) or low (lo)] of expression of a class of heterotypic adhesion receptors (HARs). In blood from animals infected with SIV/Mne, HARhi CD4+ T cells were lost selectively compared to HARlo CD4+ cells and, when cultured, exhibited 50-fold more recoverable reverse transcriptase activity. The HARhi CD4+ subset was also markedly more susceptible to productive infection following exposure to SIV/Mne in vitro. Both subsets are composed primarily of small resting lymphocytes. However, HARhi cells respond differentially to mitogenic stimulation and may thus be more likely to provide the cellular factors necessary to initiate or enhance virus replication. Thus, HAR expression may prove useful both as a prognostic indicator in immunodeficiency virus infection and as a tool to analyze pathogenesis of immunodeficiency viruses.

Increased T-cell apoptosis and terminal B-cell differentiation induced by inactivation of the Ets-1 proto-oncogene.

The Ets-1 proto-oncogene is a member of a transcription factor family characterized by homology to the v-ets oncogene. In adult mice, Ets-1 is expressed predominantly in lymphoid cells where it has been implicated in regulating transcription of lymphocyte-specific genes. Following T-cell activation, the specific DNA binding activity of Ets-1 is inactivated by transient phosphorylation, suggesting a function in the transition from the resting to activated state. Ets-1 has also been suggested to cooperate with the AP-1 transcription factor complex to mediate cellular growth factor responses. Here we show, by using RAG-2-deficient blastocyst complementation, that Ets-1 deficiency has dramatic, but different, effects on development and function of T- and B-lineage cells. Ets-1-deficient T cells were present in reduced numbers and were highly susceptible to cell death in vitro. In contrast, Ets-1-deficient B cells were present in normal numbers but a large proportion were IgM plasma cells. Our data demonstrate that Ets-1 is essential for maintenance of the normal pool of resting T- and B-lineage cells.

Detection of HIV DNA in cervical and vaginal secretions. Prevalence and correlates among women in Nairobi, Kenya.

OBJECTIVE: Factors that influence heterosexual transmission of the human immunodeficiency virus (HIV), including sexually transmitted diseases, contraceptive practices, sexual practices, HIV-related immunosuppression, and presence of cervical ectopy and the penile foreskin, have been identified through cross-sectional and prospective cohort epidemiological studies. To more directly characterize factors that influence infectivity, we conducted a study of HIV shedding from the genital tract in women. DESIGN: Ninety-seven HIV-seropositive women attending a sexually transmitted disease clinic in Nairobi, Kenya, completed a questionnaire and underwent a physical examination and an evaluation for sexually transmitted diseases. Cervical and vaginal secretions were obtained for HIV DNA detection using polymerase chain reaction amplification. RESULTS: Human immunodeficiency virus DNA was detected by polymerase chain reaction in 28 (33%) of 84 cervical samples and 13 (17%) of 77 vaginal samples. The prevalence of HIV was higher in specimens from the endocervix than from the vaginal wall (P = .002), and there was no correlation between presence of virus at the two sites. After adjusting for age, cervical HIV shedding was independently associated with oral contraceptive pill use (odds ratio [OR], 11.6; 95% confidence interval [CI], 1.7 to 77.6), cervical mucopus (OR, 6.2; 95% CI, 0.9 to 41.4; P = .05), cervical ectopy (OR, 5.0; 95% CI, 1.5 to 16.9), and pregnancy (OR, 4.5; 95% CI, 1.2 to 16.3). CONCLUSIONS: Human immunodeficiency virus was detected in one third of cervical samples and one sixth of vaginal samples. The presence of HIV DNA in cervical secretions was significantly associated with oral contraceptive pill use, cervical ectopy, and pregnancy. There was a marginally significant association with cervical mucopus. The identification of factors that increase the infectivity of women suggests potential strategies for reducing heterosexual transmission of HIV.